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Sample records for induces epithelial-to-mesenchymal transition

  1. Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition

    PubMed Central

    Wang, Wenyi; Kato, Satomi; Colvocoresses-Dodds, Jennifer; Fifadara, Nimita H.; Gauthier, Theresa W.; Helms, My N.; Carlton, David P.; Brown, Lou Ann S.

    2013-01-01

    Myofibroblast accumulation is a pathological feature of lung diseases requiring oxygen therapy. One possible source for myofibroblasts is through the epithelial-to-mesenchymal transition (EMT) of alveolar epithelial cells (AEC). To study the effects of oxygen on alveolar EMT, we used RLE-6TN and ex vivo lung slices and found that hyperoxia (85% O2, H85) decreased epithelial proteins, presurfactant protein B (pre-SpB), pro-SpC, and lamellar protein by 50% and increased myofibroblast proteins, α-smooth muscle actin (α-SMA), and vimentin by over 200% (P < 0.05). In AEC freshly isolated from H85-treated rats, mRNA for pre-SpB and pro-SpC was diminished by ∼50% and α-SMA was increased by 100% (P < 0.05). Additionally, H85 increased H2O2 content, and H2O2 (25–50 μM) activated endogenous transforming growth factor-β1 (TGF-β1), as evident by H2DCFDA immunofluorescence and ELISA (P < 0.05). Both hyperoxia and H2O2 increased SMAD3 phosphorylation (260% of control, P < 0.05). Treating cultured cells with TGF-β1 inhibitors did not prevent H85-induced H2O2 production but did prevent H85-mediated α-SMA increases and E-cadherin downregulation. Finally, to determine the role of TGF-β1 in hyperoxia-induced EMT in vivo, we evaluated AEC from H85-treated rats and found that vimentin increased ∼10-fold (P < 0.05) and that this effect was prevented by intraperitoneal TGF-β1 inhibitor SB-431542. Additionally, SB-431542 treatment attenuated changes in alveolar histology caused by hyperoxia. Our studies indicate that hyperoxia promotes alveolar EMT through a mechanism that is dependent on activation of TGF-β1 signaling. PMID:24375795

  2. Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition.

    PubMed

    Vyas-Read, Shilpa; Wang, Wenyi; Kato, Satomi; Colvocoresses-Dodds, Jennifer; Fifadara, Nimita H; Gauthier, Theresa W; Helms, My N; Carlton, David P; Brown, Lou Ann S

    2014-02-15

    Myofibroblast accumulation is a pathological feature of lung diseases requiring oxygen therapy. One possible source for myofibroblasts is through the epithelial-to-mesenchymal transition (EMT) of alveolar epithelial cells (AEC). To study the effects of oxygen on alveolar EMT, we used RLE-6TN and ex vivo lung slices and found that hyperoxia (85% O2, H85) decreased epithelial proteins, presurfactant protein B (pre-SpB), pro-SpC, and lamellar protein by 50% and increased myofibroblast proteins, α-smooth muscle actin (α-SMA), and vimentin by over 200% (P < 0.05). In AEC freshly isolated from H85-treated rats, mRNA for pre-SpB and pro-SpC was diminished by ∼50% and α-SMA was increased by 100% (P < 0.05). Additionally, H85 increased H2O2 content, and H2O2 (25-50 μM) activated endogenous transforming growth factor-β1 (TGF-β1), as evident by H2DCFDA immunofluorescence and ELISA (P < 0.05). Both hyperoxia and H2O2 increased SMAD3 phosphorylation (260% of control, P < 0.05). Treating cultured cells with TGF-β1 inhibitors did not prevent H85-induced H2O2 production but did prevent H85-mediated α-SMA increases and E-cadherin downregulation. Finally, to determine the role of TGF-β1 in hyperoxia-induced EMT in vivo, we evaluated AEC from H85-treated rats and found that vimentin increased ∼10-fold (P < 0.05) and that this effect was prevented by intraperitoneal TGF-β1 inhibitor SB-431542. Additionally, SB-431542 treatment attenuated changes in alveolar histology caused by hyperoxia. Our studies indicate that hyperoxia promotes alveolar EMT through a mechanism that is dependent on activation of TGF-β1 signaling. PMID:24375795

  3. Sox5 induces epithelial to mesenchymal transition by transactivation of Twist1

    SciTech Connect

    Pei, Xin-Hong; Lv, Xin-Quan; Li, Hui-Xiang

    2014-03-28

    Highlights: • Depletion of Sox5 inhibits breast cancer proliferation, migration, and invasion. • Sox5 transactivates Twist1 expression. • Sox5 induces epithelial to mesenchymal transition through transactivation of Twist1 expression. - Abstract: The epithelial to mesenchymal transition (EMT), a highly conserved cellular program, plays an important role in normal embryogenesis and cancer metastasis. Twist1, a master regulator of embryonic morphogenesis, is overexpressed in breast cancer and contributes to metastasis by promoting EMT. In exploring the mechanism underlying the increased Twist1 in breast cancer cells, we found that the transcription factor SRY (sex-determining region Y)-box 5(Sox5) is up-regulation in breast cancer cells and depletion of Sox5 inhibits breast cancer cell proliferation, migration, and invasion. Furthermore, depletion of Sox5 in breast cancer cells caused a dramatic decrease in Twist1 and chromosome immunoprecipitation assay showed that Sox5 can bind directly to the Twist1 promoter, suggesting that Sox5 transactivates Twist1 expression. We further demonstrated that knockdown of Sox5 up-regulated epithelial phenotype cell biomarker (E-cadherin) and down-regulated mesenchymal phenotype cell biomarkers (N-cadherin, Vimentin, and Fibronectin 1), resulting in suppression of EMT. Our study suggests that Sox5 transactivates Twist1 expression and plays an important role in the regulation of breast cancer progression.

  4. Hepatocyte nuclear factor 1 beta induces transformation and epithelial-to-mesenchymal transition.

    PubMed

    Matsui, Atsuka; Fujimoto, Jiro; Ishikawa, Kosuke; Ito, Emi; Goshima, Naoki; Watanabe, Shinya; Semba, Kentaro

    2016-04-01

    Gene amplification can be a cause of cancer, and driver oncogenes have been often identified in amplified regions. However, comprehensive analysis of other genes coamplified with an oncogene is rarely performed. We focused on the 17q12-21 amplicon, which contains ERBB2. We established a screening system for oncogenic activity with the NMuMG epithelial cell line. We identified a homeobox gene, HNF1B, as a novel cooperative transforming gene. HNF1B induced cancerous phenotypes, which were enhanced by the coexpression of ERBB2, and induced epithelial-to-mesenchymal transition and invasive phenotypes. These results suggest that HNF1B is a novel oncogene that can work cooperatively with ERBB2. PMID:27001343

  5. Lysyl oxidase promotes epithelial-to-mesenchymal transition during paraquat-induced pulmonary fibrosis.

    PubMed

    Wang, Jinfeng; Zhu, Yong; Tan, Jiuting; Meng, Xiaoxiao; Xie, Hui; Wang, Ruilan

    2016-02-01

    Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays a critical role in pulmonary fibrosis. Our previous study demonstrated that epithelial-to-mesenchymal transition (EMT) was strongly associated with paraquat (PQ) induced pulmonary fibrosis. This present study was aimed to evaluate the potential involvement of LOX on EMT in the process of pulmonary fibrosis induced by PQ. We established an in vivo rat model and an in vitro cell model induced by PQ treatment and found that LOX protein expression was significantly up-regulated and collagen deposition was enhanced in rats. The EMT process was strongly found in A549 and RLE-6TN cells after PQ exposure. After inactivating LOX with an inhibitor, pulmonary fibrosis was significantly reduced and EMT was also suppressed. Additionally, small interfering RNA (siRNA) targeting LOX was used to silence LOX expression to observe EMT in A549 cells. As a result, LOX could promote the progress of EMT, and inactivating LOX alleviated the EMT process in PQ-induced pulmonary fibrosis and mesenchymal-to-epithelial transition (MET) occurred after inactivating LOX in vitro and in vivo. In conclusion, LOX could promote the progress of EMT and inactivating LOX alleviated EMT in PQ-induced pulmonary fibrosis. Therefore, LOX could potentially be a new candidate therapeutic target for pulmonary fibrosis induced by PQ by regulating the balance between EMT and MET. PMID:26670953

  6. Metabolic reprogramming during TGFβ1-induced epithelial-to-mesenchymal transition

    PubMed Central

    Jiang, Lei; Xiao, Ling; Sugiura, Hidekazu; Huang, Xiumei; Ali, Aktar; Kuro-o, Makoto; Deberardinis, Ralph J.; Boothman, David A.

    2014-01-01

    Metastatic progression, including extravasation and micro-metastatic outgrowth, is the main cause of cancer patient death. Recent studies suggest that cancer cells reprogram their metabolism to support increased proliferation through increased glycolysis and biosynthetic activities, including lipogenesis pathways. However, metabolic changes during metastatic progression, including alterations in regulatory gene expression, remain undefined. We show that transforming growth factor beta 1 (TGFβ1) induced Epithelial-to-Mesenchymal Transition (EMT) is accompanied by coordinately reduced enzyme expression required to convert glucose into fatty acids, and concomitant enhanced respiration. Over-expressed Snail1, a transcription factor mediating TGFβ1-induced EMT, was sufficient to suppress carbohydrate-responsive-element-binding protein (ChREBP, a master lipogenic regulator), and fatty acid synthase (FASN), its effector lipogenic gene. Stable FASN knock-down was sufficient to induce EMT, stimulate migration and extravasation in vitro. FASN silencing enhanced lung metastasis and death in vivo. These data suggest that a metabolic transition that suppresses lipogenesis and favors energy production is an essential component of TGFβ1-induced EMT and metastasis. PMID:25284588

  7. PEBP4 silencing inhibits hypoxia-induced epithelial-to-mesenchymal transition in prostate cancer cells.

    PubMed

    Li, Weiping; Dong, Yongchao; Zhang, Bin; Kang, Yindong; Yang, Xukai; Wang, He

    2016-07-01

    Hypoxia induced epithelial-to-mesenchymal transition (EMT) to facilitate the tumor biology. Phosphatidylethanolamine-binding protein 4 (PEBP4) is a member of the PEBP family and has been reported to be upregulated in various cancer types. The definite function of PEBP4 in regulating the EMT of prostate cancer, however, is still unclear. Here, we examined the functional role of PEBP4 and the underlying molecular mechanisms in hypoxia-induced EMT in prostate cancer cells. Our results showed that PEBP4 mRNA and protein expression was markedly increased in the human prostate cancer tissues and cell lines. Knockdown of PEBP4 significantly inhibited hypoxia-induced migration/invasion and EMT program. Furthermore, knockdown of PEBP4 prevented hypoxia-induced the expression of p-Akt and p-mTOR in prostate cancer cells. Taken together, this study reported here provided evidence that knockdown of PEBP4 inhibited hypoxia-induced EMT in prostate cancer cells. Our study uncovered a novel role for PEBP4 in prostate cancer progression, which might support the potential for PEBP4 targeting in prostate cancer therapy. PMID:27261570

  8. Topographic confinement of epithelial clusters induces epithelial-to-mesenchymal transition in compliant matrices

    NASA Astrophysics Data System (ADS)

    Nasrollahi, Samila; Pathak, Amit

    2016-01-01

    Epithelial cells disengage from their clusters and become motile by undergoing epithelial-to-mesenchymal transition (EMT), an essential process for both embryonic development and tumor metastasis. Growing evidence suggests that high extracellular matrix (ECM) stiffness induces EMT. In reality, epithelial clusters reside in a heterogeneous microenvironment whose mechanical properties vary not only in terms of stiffness, but also topography, dimensionality, and confinement. Yet, very little is known about how various geometrical parameters of the ECM might influence EMT. Here, we adapt a hydrogel-microchannels based matrix platform to culture mammary epithelial cell clusters in ECMs of tunable stiffness and confinement. We report a previously unidentified role of ECM confinement in EMT induction. Surprisingly, confinement induces EMT even in the cell clusters surrounded by a soft matrix, which otherwise protects against EMT in unconfined environments. Further, we demonstrate that stiffness-induced and confinement-induced EMT work through cell-matrix adhesions and cytoskeletal polarization, respectively. These findings highlight that both the structure and the stiffness of the ECM can independently regulate EMT, which brings a fresh perspective to the existing paradigm of matrix stiffness-dependent dissemination and invasion of tumor cells.

  9. Topographic confinement of epithelial clusters induces epithelial-to-mesenchymal transition in compliant matrices

    PubMed Central

    Nasrollahi, Samila; Pathak, Amit

    2016-01-01

    Epithelial cells disengage from their clusters and become motile by undergoing epithelial-to-mesenchymal transition (EMT), an essential process for both embryonic development and tumor metastasis. Growing evidence suggests that high extracellular matrix (ECM) stiffness induces EMT. In reality, epithelial clusters reside in a heterogeneous microenvironment whose mechanical properties vary not only in terms of stiffness, but also topography, dimensionality, and confinement. Yet, very little is known about how various geometrical parameters of the ECM might influence EMT. Here, we adapt a hydrogel-microchannels based matrix platform to culture mammary epithelial cell clusters in ECMs of tunable stiffness and confinement. We report a previously unidentified role of ECM confinement in EMT induction. Surprisingly, confinement induces EMT even in the cell clusters surrounded by a soft matrix, which otherwise protects against EMT in unconfined environments. Further, we demonstrate that stiffness-induced and confinement-induced EMT work through cell-matrix adhesions and cytoskeletal polarization, respectively. These findings highlight that both the structure and the stiffness of the ECM can independently regulate EMT, which brings a fresh perspective to the existing paradigm of matrix stiffness-dependent dissemination and invasion of tumor cells. PMID:26728047

  10. Human RON receptor tyrosine kinase induces complete epithelial-to-mesenchymal transition but causes cellular senescence

    SciTech Connect

    Cote, Marceline; Miller, A. Dusty; Liu, Shan-Lu . E-mail: shan-lu.liu@mcgill.ca

    2007-08-17

    The RON receptor tyrosine kinase is a member of the MET proto-oncogene family and is important for cell proliferation, differentiation, and cancer development. Here, we created a series of Madin-Darby canine kidney (MDCK) epithelial cell clones that express different levels of RON, and have investigated their biological properties. While low levels of RON correlated with little morphological change in MDCK cells, high levels of RON expression constitutively led to morphological scattering or complete and stabilized epithelial-to-mesenchymal transition (EMT). Unexpectedly, MDCK clones expressing higher levels of RON exhibited retarded proliferation and senescence, despite increased motility and invasiveness. RON was constitutively tyrosine-phosphorylated in MDCK cells expressing high levels of RON and undergoing EMT, and the MAPK signaling pathway was activated. This study reveals for the first time that RON alone is sufficient to induce complete and stabilized EMT in MDCK cells, and overexpression of RON does not cause cell transformation but rather induces cell cycle arrest and senescence, leading to impaired cell proliferation.

  11. A core microRNA signature associated with inducers of the epithelial-to-mesenchymal transition.

    PubMed

    Díaz-Martín, Juan; Díaz-López, Antonio; Moreno-Bueno, Gema; Castilla, M Ángeles; Rosa-Rosa, Juan M; Cano, Amparo; Palacios, José

    2014-02-01

    Although it is becoming clear that certain miRNAs fulfil a fundamental role in the regulation of the epithelial-to-mesenchymal transition (EMT), a comprehensive study of the miRNAs associated with this process has yet to be performed. Here, we profiled the signature of miRNA expression in an in vitro model of EMT, ectopically expressing in MDCK cells one of seven EMT transcription factors (SNAI1, SNAI2, ZEB1, ZEB2, TWIST1, TWIST2 or E47) or the EMT inducer LOXL2. In this way, we identified a core subset of deregulated miRNAs that were further validated in vivo, studying endometrial carcinosarcoma (ECS), a tumour entity that represents an extreme example of phenotypic plasticity. Moreover, epigenetic silencing through DNA methylation of miRNA genes of the miR-200 family and miR-205 that are down-regulated during EMT was evident in both the in vitro (MDCK transfectants) and in vivo (ECS) models of EMT. The strong correlation between expression and DNA methylation suggests a major role for this epigenetic mark in the regulation of the miR-141-200c locus. PMID:24122292

  12. Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells

    PubMed Central

    Cohen, Evan N.; Gao, Hui; Anfossi, Simone; Mego, Michal; Reddy, Neelima G.; Debeb, Bisrat; Giordano, Antonio; Tin, Sanda; Wu, Qiong; Garza, Raul J.; Cristofanilli, Massimo; Mani, Sendurai A.; Croix, Denise A.; Ueno, Naoto T.; Woodward, Wendy A.; Luthra, Raja; Krishnamurthy, Savitri; Reuben, James M.

    2015-01-01

    Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction. PMID:26207636

  13. Negative regulation of TGFβ-induced lens epithelial to mesenchymal transition (EMT) by RTK antagonists.

    PubMed

    Zhao, Guannan; Wojciechowski, Magdalena C; Jee, Seonah; Boros, Jessica; McAvoy, John W; Lovicu, Frank J

    2015-03-01

    An eclectic range of ocular growth factors with differing actions are present within the aqueous and vitreous humors that bathe the lens. Growth factors that exert their actions via receptor tyrosine kinases (RTKs), such as FGF, play a normal regulatory role in lens; whereas other factors, such as TGFβ, can lead to an epithelial to mesenchymal transition (EMT) that underlies several forms of cataract. The respective downstream intracellular signaling pathways of these factors are in turn tightly regulated. One level of negative regulation is thought to be through RTK-antagonists, namely, Sprouty (Spry), Sef and Spred that are all expressed in the lens. In this study, we tested these different negative regulators and compared their ability to block TGFβ-induced EMT in rat lens epithelial cells. Spred expression within the rodent eye was confirmed using RT-PCR, western blotting and immunofluorescence. Rat lens epithelial explants were used to examine the morphological changes associated with TGFβ-induced EMT over 3 days of culture, as well as α-smooth muscle actin (α-sma) immunolabeling. Cells in lens epithelial explants were transfected with either a reporter (EGFP) vector (pLXSG), or with plasmids also coding for different RTK-antagonists (i.e. pLSXG-Spry1, pLSXG-Spry2, pLXSG-Sef, pLSXG-Spred1, pLSXG-Spred2, pLSXG-Spred3), before treating with TGFβ for up to 3 days. The percentages of transfected cells that underwent TGFβ-induced morphological changes consistent with an EMT were determined using cell counts and validated with a paired two-tailed t-test. Explants transfected with pLXSG demonstrated a distinct transition in cell morphology after TGFβ treatment, with ∼60% of the cells undergoing fibrotic-like cell elongation. This percentage was significantly reduced in cells overexpressing the different antagonists, indicative of a block in lens EMT. Of the antagonists tested under these in vitro conditions, Spred1 was the most potent demonstrating the

  14. MUC1 enhances invasiveness of pancreatic cancer cells by inducing epithelial to mesenchymal transition.

    PubMed

    Roy, L D; Sahraei, M; Subramani, D B; Besmer, D; Nath, S; Tinder, T L; Bajaj, E; Shanmugam, K; Lee, Y Y; Hwang, S I L; Gendler, S J; Mukherjee, P

    2011-03-24

    Increased motility and invasiveness of pancreatic cancer cells are associated with epithelial to mesenchymal transition (EMT). Snai1 and Slug are zinc-finger transcription factors that trigger this process by repressing E-cadherin and enhancing vimentin and N-cadherin protein expression. However, the mechanisms that regulate this activation in pancreatic tumors remain elusive. MUC1, a transmembrane mucin glycoprotein, is associated with the most invasive forms of pancreatic ductal adenocarcinomas (PDA). In this study, we show that over expression of MUC1 in pancreatic cancer cells triggers the molecular process of EMT, which translates to increased invasiveness and metastasis. EMT was significantly reduced when MUC1 was genetically deleted in a mouse model of PDA or when all seven tyrosines in the cytoplasmic tail of MUC1 were mutated to phenylalanine (mutated MUC1 CT). Using proteomics, RT-PCR and western blotting, we revealed a significant increase in vimentin, Slug and Snail expression with repression of E-Cadherin in MUC1-expressing cells compared with cells expressing the mutated MUC1 CT. In the cells that carried the mutated MUC1 CT, MUC1 failed to co-immunoprecipitate with β-catenin and translocate to the nucleus, thereby blocking transcription of the genes associated with EMT and metastasis. Thus, functional tyrosines are critical in stimulating the interactions between MUC1 and β-catenin and their nuclear translocation to initiate the process of EMT. This study signifies the oncogenic role of MUC1 CT and is the first to identify a direct role of the MUC1 in initiating EMT during pancreatic cancer. The data may have implications in future design of MUC1-targeted therapies for pancreatic cancer. PMID:21102519

  15. MUC1 enhances invasiveness of pancreatic cancer cells by inducing epithelial to mesenchymal transition

    PubMed Central

    Roy, Lopamudra Das; Sahraei, Mahnaz; Subramani, Durai B.; Besmer, Dahlia; Nath, Sritama; Tinder, Teresa L.; Bajaj, Ekta; Shanmugam, Kandavel; Lee, Yong Yook; Hwang, Sun IL; Gendler, Sandra J.; Mukherjee, Pinku

    2010-01-01

    Increased motility and invasiveness of pancreatic cancer cells are associated with epithelial to mesenchymal transition (EMT). Snai1 and Slug are zinc-finger transcription factors that trigger this process by repressing E-cadherin and enhancing vimentin and N-Cadherin protein expression. However, the mechanisms that regulate this activation in pancreatic tumors remain elusive. MUC1, a transmembrane mucin glycoprotein, is associated with the most invasive forms of pancreatic adenocarcinomas (PDA). In this study, we show that over expression of MUC1 in pancreatic cancer cells triggers the molecular process of EMT which translates to increased invasiveness and metastasis. EMT was significantly reduced when Muc1 was genetically deleted in a mouse model of PDA or when all seven tyrosines in the cytoplasmic tail of MUC1 were mutated to phenylalanine (mutated MUC1 CT). Using proteomics, RT-PCR, and Western blotting, we revealed a significant increase in vimentin, Slug and Snail expression with repression of E-Cadherin in MUC1-expressing cells compared to cells expressing the mutated MUC1 CT. In the cells that carried the mutated MUC1 CT, MUC1 failed to co-immunoprecipitate with β-catenin and translocate to the nucleus thereby blocking transcription of the genes associated with EMT and metastasis. Thus, functional tyrosines are critical in stimulating the interactions between MUC1 and β-catenin and their nuclear translocation to initiate the process of EMT. This study signifies the oncogenic role of MUC1 CT and is the first to identify a direct role of the MUC1 in initiating EMT during pancreatic cancer. The data may have implications in future design of MUC1-targeted therapies for pancreatic cancer. PMID:21102519

  16. Hypoxia inducible factor-1α-dependent epithelial to mesenchymal transition under hypoxic conditions in prostate cancer cells.

    PubMed

    Li, Mingchuan; Wang, Yong Xing; Luo, Yong; Zhao, Jiahui; Li, Qing; Zhang, Jiao; Jiang, Yongguang

    2016-07-01

    Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer death. Hypoxia is an environmental stimulus that plays an important role in the development and cancer progression especially for solid tumors. The key regulator under hypoxic conditions is stabilized hypoxia-inducible factor (HIF)-1α. In the present study, immune-fluorescent staining, siRNAs, qRT-PC, immunoblotting, cell migration and invasion assays were carried out to test typical epithelial to mesenchymal transition under hypoxia and the key regulators of this process in PC3, a human prostate cancer cell line. Our data demonstrated that hypoxia induces diverse molecular, phenotypic and functional changes in prostate cancer cells that are consistent with EMT. We also showed that a cell signal factor such as HIF-1α, which might be stabilized under hypoxic environment, is involved in EMT and cancer cell invasive potency. The induced hypoxia could be blocked by HIF-1α gene silencing and reoxygenation of EMT in prostate cancer cells, hypoxia partially reversed accompanied by a process of mesenchymal-epithelial reverting transition (MErT). EMT might be induced by activation of HIF-1α-dependent cell signaling in hypoxic prostate cancer cells. PMID:27108616

  17. P21 Activated Kinase-1 Mediates Transforming Growth Factor β1-Induced Prostate Cancer Cell Epithelial to Mesenchymal Transition

    PubMed Central

    Al-Azayzih, Ahmad; Gao, Fei; Somanath, Payaningal R.

    2015-01-01

    Transforming growth factor beta (TGFβ) is believed to play a dual role in prostate cancer. Molecular mechanism by which TGFβ1 suppresses early prostate tumor growth and induces epithelial-to-mesenchymal transition (EMT) in advanced stages is not known. We determined if P21-activated kinase1 (Pak1), which mediates cytoskeletal remodeling is necessary for the TGFβ1 induced prostate cancer EMT. Effects of TGFβ1 on control prostate cancer PC3 and DU145 cells and those with IPA 3 and siRNA mediated Pak1 inhibition were tested for prostate tumor xenograft in vivo and EMT in vitro. TGFβ1 inhibited PC3 tumor xenograft growth via activation of P38-MAPK and caspase-3, 9. Long-term stimulation with TGFβ1 induced PC3 and DU145 cell scattering and increased expression of EMT markers such as Snail and N-cadherin through tumor necrosis factor receptor-associated factor-6 (TRAF6)-mediated activation of Rac1/Pak1 pathway. Selective inhibition of Pak1 using IPA 3 or knockdown using siRNA both significantly inhibited TGFβ1-induced prostate cancer cell EMT and expression of mesenchymal markers. Our study demonstrated that TGFβ1 induces apoptosis and EMT in prostate cancer cells via activation of P38-MAPK and Rac1/Pak1 respectively. Our results reveal the potential therapeutic benefits of targeting TGFβ1-Pak1 pathway for advanced-stage prostate cancer. PMID:25746720

  18. Salinomycin suppresses TGF-β1-induced epithelial-to-mesenchymal transition in MCF-7 human breast cancer cells.

    PubMed

    Zhang, Chunying; Lu, Ying; Li, Qing; Mao, Jun; Hou, Zhenhuan; Yu, Xiaotang; Fan, Shujun; Li, Jiazhi; Gao, Tong; Yan, Bing; Wang, Bo; Song, Bo; Li, Lianhong

    2016-03-25

    Epithelial-to-mesenchymal transition (EMT) is the major cause of breast cancer to initiate invasion and metastasis. Salinomycin (Sal) has been found as an effective chemical compound to kill breast cancer stem cells. However, the effect of Sal on invasion and metastasis of breast cancer is unclear. In the present study, we showed that Sal reversed transforming growth factor-β1 (TGF-β1) induced invasion and metastasis accompanied with down-regulation of MMP-2 by experiments on human breast cancer cell line MCF-7. Sal was able to inhibit TGF-β1-induced EMT phenotypic transition and the activation of key signaling molecules involved in Smad (p-Smad2/3,Snail1) and non-Smad (β-catenin, p-p38 MAPK) signals which cooperatively regulate the induction of EMT. Importantly, in a series of breast cancer specimens, we found strong correlation among E-cadherin expression, β-catenin expression, and the lymph node metastatic potential of breast cancer. Our research suggests that Sal is promised to be a chemotherapeutic drug by suppressing the metastasis of breast cancer. PMID:26896736

  19. Chromatinized protein kinase C-θ directly regulates inducible genes in epithelial to mesenchymal transition and breast cancer stem cells.

    PubMed

    Zafar, Anjum; Wu, Fan; Hardy, Kristine; Li, Jasmine; Tu, Wen Juan; McCuaig, Robert; Harris, Janelle; Khanna, Kum Kum; Attema, Joanne; Gregory, Philip A; Goodall, Gregory J; Harrington, Kirsti; Dahlstrom, Jane E; Boulding, Tara; Madden, Rebecca; Tan, Abel; Milburn, Peter J; Rao, Sudha

    2014-08-01

    Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor β (TGF-β) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer. PMID:24891615

  20. Chromatinized Protein Kinase C-θ Directly Regulates Inducible Genes in Epithelial to Mesenchymal Transition and Breast Cancer Stem Cells

    PubMed Central

    Zafar, Anjum; Wu, Fan; Hardy, Kristine; Li, Jasmine; Tu, Wen Juan; McCuaig, Robert; Harris, Janelle; Khanna, Kum Kum; Attema, Joanne; Gregory, Philip A.; Goodall, Gregory J.; Harrington, Kirsti; Dahlstrom, Jane E.; Boulding, Tara; Madden, Rebecca; Tan, Abel; Milburn, Peter J.

    2014-01-01

    Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor β (TGF-β) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer. PMID:24891615

  1. Geranylgeranylacetone alleviates radiation-induced lung injury by inhibiting epithelial-to-mesenchymal transition signaling.

    PubMed

    Kim, Joong-Sun; Son, Yeonghoon; Jung, Myung-Gu; Jeong, Ye Ji; Kim, Sung-Ho; Lee, Su-Jae; Lee, Yoon-Jin; Lee, Hae-June

    2016-06-01

    Radiation-induced lung injury (RILI) involves pneumonitis and fibrosis, and results in pulmonary dysfunction. Moreover, RILI can be a fatal complication of thoracic radiotherapy. The present study investigated the protective effect of geranylgeranlyacetone (GGA), an inducer of heat shock protein (HSP)70, on RILI using a C57BL/6 mouse model of RILI developing 6 months subsequent to exposure to 12.5 Gy thoracic radiation. GGA was administered 5 times orally prior and subsequent to radiation exposure, and the results were assessed by histological analysis and western blotting. The results show that late RILI was alleviated by GGA treatment, possibly through the suppression of epithelial‑to‑mesenchymal transition (EMT) marker expression. Based on histological examination, orally administered GGA during the acute phase of radiation injury not only significantly inhibited pro‑surfactant protein C (pro‑SPC) and vimentin expression, but also preserved E‑cadherin expression 6 months after irradiation‑induced injury of the lungs. GGA induced HSP70 and inhibited EMT marker expression in L132 human lung epithelial cells following IR. These data suggest that the prevention of EMT signaling is a key cytoprotective effect in the context of RILI. Thus, HSP70‑inducing drugs, such as GGA, could be beneficial for protection against RILI. PMID:27082939

  2. ERK/GSK3ß/Snail signaling mediates radiation-induced alveolar epithelial-to-mesenchymal transition

    PubMed Central

    Nagarajan, Devipriya; Melo, Tahira; Deng, Zhiyong; Almeida, Celine; Zhao, Weiling

    2011-01-01

    Radiotherapy is one of the major treatment regimes for thoracic malignancies, but can lead to severe lung complications including pneumonitis and fibrosis. Recent studies suggest that epithelial to mesenchymal transition (EMT) plays an important role in tissue injury leading to organ fibrosis. To investigate whether radiation can induce EMT in lung epithelial cells and also understand the potential mechanism(s) associated with this change, rat alveolar type II lung epithelial RLE-6TN cells were irradiated with 8 Gy of 137Cs γ-rays. Western blot and immunofluorescence analyses revealed a time-dependent decrease in E-cadherin with a concomitant increase in α-SMA and vimentin after radiation, suggesting that the epithelial cells acquired mesenchymal-like morphology. Protein levels and nuclear translocation of Snail, the key inducer of EMT, were significantly elevated in the irradiated cells. Radiation also induced a time-dependent inactivation of glycogen synthase kinase-3β (GSK3ß), an endogenous inhibitor of Snail. A marked increase in phosphorylation of ERK1/2, but not JNKs or p38, was observed in irradiated RLE-6TN cells. Silencing ERK1/2 using siRNAs and the MEK/ERK inhibitor U0126 attenuated the radiation-induced phosphorylation of GSK3ß and altered the protein levels of Snail, α-SMA and E-cadherin in RLE-6TN cells. Pre-incubating RLE-6TN cells with N-acetyl cysteine, an antioxidant, abolished the radiation-induced phosphorylation of ERK and altered protein levels of Snail, E-cadherin and α-SMA. These findings reveal, for the first time, that radiation-induced EMT in alveolar type II epithelial cells is mediated by the ERK/GSK3ß/Snail pathway. PMID:22198183

  3. ERK/GSK3β/Snail signaling mediates radiation-induced alveolar epithelial-to-mesenchymal transition.

    PubMed

    Nagarajan, Devipriya; Melo, Tahira; Deng, Zhiyong; Almeida, Celine; Zhao, Weiling

    2012-03-15

    Radiotherapy is one of the major treatment regimes for thoracic malignancies, but can lead to severe lung complications including pneumonitis and fibrosis. Recent studies suggest that epithelial-to-mesenchymal transition (EMT) plays an important role in tissue injury leading to organ fibrosis. To investigate whether radiation can induce EMT in lung epithelial cells and also to understand the potential mechanism(s) associated with this change, rat alveolar type II lung epithelial RLE-6TN cells were irradiated with 8 Gy of (137)Cs γ-rays. Western blot and immunofluorescence analyses revealed a time-dependent decrease in E-cadherin with a concomitant increase in α-smooth muscle actin (α-SMA) and vimentin after radiation, suggesting that the epithelial cells acquired a mesenchymal-like morphology. Protein levels and nuclear translocation of Snail, the key inducer of EMT, were significantly elevated in the irradiated cells. Radiation also induced a time-dependent inactivation of glycogen synthase kinase-3β (GSK3β), an endogenous inhibitor of Snail. A marked increase in phosphorylation of ERK1/2, but not JNK or p38, was observed in irradiated RLE-6TN cells. Silencing ERK1/2 using siRNAs and the MEK/ERK inhibitor U0126 attenuated the radiation-induced phosphorylation of GSK3β and altered the protein levels of Snail, α-SMA, and E-cadherin in RLE-6TN cells. Preincubating RLE-6TN cells with N-acetylcysteine, an antioxidant, abolished the radiation-induced phosphorylation of ERK and altered protein levels of Snail, E-cadherin, and α-SMA. These findings reveal, for the first time, that radiation-induced EMT in alveolar type II epithelial cells is mediated by the ERK/GSK3β/Snail pathway. PMID:22198183

  4. TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells

    PubMed Central

    Cardenas, Horacio; Vieth, Edyta; Lee, Jiyoon; Segar, Mathew; Liu, Yunlong; Nephew, Kenneth P; Matei, Daniela

    2014-01-01

    A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer. PMID:25470663

  5. CXCL9 Regulates TGF-β1 induced Epithelial to Mesenchymal Transition in Human Alveolar Epithelial Cells

    PubMed Central

    O’Beirne, Sarah L; Walsh, Sinead M; Fabre, Aurélie; Reviriego, Carlota; Worrell, Julie C; Counihan, Ian P; Lumsden, Robert V; Cramton-Barnes, Jennifer; Belperio, John A.; Donnelly, Seamas C; Boylan, Denise; Marchal-Somme, Joëlle; Kane, Rosemary; Keane, Michael P

    2016-01-01

    Epithelial to mesenchymal transition (EMT), whereby fully differentiated epithelial cells transition to a mesenchymal phenotype has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). CXCR3 and its ligands are recognized to play a protective role in pulmonary fibrosis. In this study, we investigated the presence and extent of EMT and CXCR3 expression in human IPF surgical lung biopsies and assessed whether CXCR3 and its ligand CXCL9 modulate EMT in alveolar epithelial cells. Coexpression of the epithelial maker thyroid transcription factor-1, and mesenchymal marker α-smooth muscle actin and CXCR3 expression was examined by immunohistochemical staining of IPF surgical lung biopsies. Epithelial and mesenchymal marker expression was examined by quantitative real-time PCR, Western blotting, and immunofluorescence in human alveolar epithelial (A549) cells treated with TGF-β1 and CXCL9, whilst Smad2, Smad3, and Smad7 expression and cellular localization examined by Western blotting. We found that significantly more cells were undergoing EMT in fibrotic versus normal areas of lung in IPF surgical lung biopsy samples. CXCR3 was expressed by type II pneumocytes and fibroblasts in fibrotic areas in close proximity to cells undergoing EMT. In vitro, CXCL9 abrogated TGF-β1 induced EMT. A decrease in TGF-β1 induced phosphorylation of Smad2 and Smad3 occurred with CXCL9 treatment. This was associated with increased shuttling of Smad7 from the nucleus to the cytoplasm where it inhibits Smad phosphorylation. This suggests a role for EMT in the pathogenesis of IPF and provides a novel mechanism for the inhibitory effects of CXCL9 on TGF-β1 induced EMT. PMID:26268659

  6. Epithelial to Mesenchymal Transition induces cell cycle arrest and parenchymal damage in renal fibrosis

    PubMed Central

    Lovisa, Sara; LeBleu, Valerie S.; Tampe, Björn; Sugimoto, Hikaru; Vadnagara, Komal; Carstens, Julienne L.; Wu, Chia–Chin; Hagos, Yohannes; Burckhardt, Birgitta C.; Pentcheva–Hoang, Tsvetelina; Nischal, Hersharan; Allison, James P.; Zeisberg, Michael; Kalluri, Raghu

    2015-01-01

    Kidney fibrosis is marked by an epithelial–to–mesenchymal transition (EMT) by tubular epithelial cells (TECs). Here we find that during renal fibrosis TECs acquire a partial EMT program during which they remain associated with their basement membrane and express markers of both epithelial and mesenchymal cells. The functional consequence of EMT program during fibrotic injury is an arrest in the G2 phase of the cell cycle and lower expression of several transporters in TECs. We also found that transgenic expression of Twist or Snai1 expression is sufficient to promote prolonged TGF-β1–induced G2 arrest of TECs, limiting their potential for repair and regeneration. Also, in mouse models of experimentally-induced renal fibrosis, conditional deletion of Twist1 or Snai1 in proximal TECs resulted in inhibition of the EMT program and the maintenance of TEC integrity, while restoring proliferation, de–differentiation–associated repair and regeneration of the kidney parenchyma and attenuating interstitial fibrosis. Thus, inhibition of EMT program in TECs during chronic renal injury represents a potential anti–fibrosis therapy PMID:26236991

  7. Maresin 1 Inhibits Epithelial-to-Mesenchymal Transition in Vitro and Attenuates Bleomycin Induced Lung Fibrosis in Vivo.

    PubMed

    Wang, Yaxin; Li, Ruidong; Chen, Lin; Tan, Wen; Sun, Zhipeng; Xia, Haifa; Li, Bo; Yu, Yuan; Gong, Jie; Tang, Min; Ji, Yudong; Yuan, Shiying; Shanglong Yao; Shang, You

    2015-11-01

    Lung fibrosis is an aggressive disease with uncontrolled fibrotic response and no effective therapeutic treatment. Epithelial-to-mesenchymal transition (EMT) has been proved to be an important pathological feature in lung fibrosis. In this study, we investigated whether MaR1, a kind of proresolving lipid mediators, could inhibit TGF-β1-induced EMT in vitro and lung fibrosis in vivo. In vitro study, mouse type II alveolar epithelial cells were treated with different does of MaR1 for 30 min and were exposed to TGF-β1 for 48 h. In vivo study, C57BL/6 mice were administered bleomycin intratracheally. After 14 days, MaR1 was injected intraperitoneally daily for 7 days. In day 28, mice were sacrificed. The results demonstrate that treatment of mouse type II alveolar epithelial cells with MaR1 (10 nM) significantly prevents TGF-β1-induced fibronectin and α-SMA expression and restores E-Cadherin level. The down-regulation of profibrotic molecules of MaR1 is associated with suppression of Smad2/3 and Akt phosphorylation. In vivo, MaR1 treatment significantly prolongs survival rate and attenuates destruction of lung architecture, as well as collagen deposition after bleomycin inhalation. TGF-β1 concentration in bronchoalveolar lavage and fibrotic markers (fibronectin and α-SMA) in lung tissues are inhibited by MaR1 administration. These data indicate that MaR1 inhibits TGF-β1-induced EMT and attenuates bleomycin-induced pulmonary fibrosis. MaR1 may be a promising strategy for alleviation of lung fibrosis. PMID:26196843

  8. Correlated S-palmitoylation profiling of Snail-induced epithelial to mesenchymal transition.

    PubMed

    Hernandez, Jeannie L; Davda, Dahvid; Majmudar, Jaimeen D; Won, Sang Joon; Prakash, Ashesh; Choi, Alexandria I; Martin, Brent R

    2016-05-24

    Epithelial cells form spatially-organized adhesion complexes that establish polarity gradients, regulate cell proliferation, and direct wound healing. As cells accumulate oncogenic mutations, these key tumor suppression mechanisms are disrupted, eliminating many adhesion complexes and bypassing contact inhibition. The transcription factor Snail is often expressed in malignant cancers, where it promotes transcriptional reprogramming to drive epithelial-mesenchymal transition (EMT) and establishes a more invasive state. S-Palmitoylation describes the fatty-acyl post-translational modification of cysteine residues in proteins, and is required for membrane anchoring, trafficking, localization and function of hundreds of proteins involved in cell growth, polarity, and signaling. Since Snail-expression disrupts apico-basolateral cell polarity, we asked if Snail-dependent transformation induces proteome-wide changes in S-palmitoylation. MCF10A breast cancer cells were retrovirally transduced with Snail and correlated proteome-wide changes in protein abundance and S-palmitoylation were profiled by using stable isotope labeling in cell culture with amino acid (SILAC) mass spectrometry. This analysis identified increased levels of proteins involved in migration, glycolysis, and cell junction remodeling, and decreased levels of proteins involved in cell adhesion. Overall, protein S-palmitoylation is highly correlated with protein abundance, yet for a subset of proteins, this correlation is uncoupled. These findings suggest that Snail-overexpression affects the S-palmitoylation cycle of some proteins, which may participate in cell polarity and tumor suppression. PMID:27030425

  9. Role of hepatitis C virus induced osteopontin in epithelial to mesenchymal transition, migration and invasion of hepatocytes.

    PubMed

    Iqbal, Jawed; McRae, Steven; Mai, Thi; Banaudha, Krishna; Sarkar-Dutta, Mehuli; Waris, Gulam

    2014-01-01

    Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVβ3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection. PMID:24498111

  10. Ribosomal L22-like1 (RPL22L1) Promotes Ovarian Cancer Metastasis by Inducing Epithelial-to-Mesenchymal Transition

    PubMed Central

    Wu, Nan; Wei, Jia; Wang, Yuhui; Yan, Jinyan; Qin, Ying; Tong, Dandan; Pang, Bo; Sun, Donglin; Sun, Haiming; Yu, Yang; Sun, Wenjing; Meng, Xiangning; Zhang, Chunyu; Bai, Jing; Chen, Feng; Geng, Jingshu; Lee, Ki-Young; Fu, Songbin; Jin, Yan

    2015-01-01

    Double minute chromosomes (DMs) have important implications for cancer progression because oncogenes frequently amplified on them. We previously detected a functionally undefined gene amplified on DMs, Ribosomal L22-like1 (RPL22L1). The relationship between RPL22L1 and cancer progression is unknown. Here, RPL22L1 was characterized for its role in ovarian cancer (OC) metastasis and its underlying mechanism was examined. DNA copy number and mRNA expression of RPL22L1 in OC cells was analyzed using data obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database. An immunohistochemical analysis of clinical OC specimens was performed and the relationships between expression level and clinicopathological factors were evaluated. Additionally, in vivo and in vitro assays were performed to understand the role of RPL22L1 in OC. RPL22L1 expression was higher in OC specimens than in normal tissues, and its expression level was highly positively correlated with invasion and lymph node metastasis (P < 0.05). RPL22L1 over-expression significantly enhanced intraperitoneal xenograft tumor development in nude mice and promoted invasion and migration in vitro. Additionally, RPL22L1 knockdown remarkably inhibited UACC-1598 cells invasion and migration. Further, RPL22L1 over-expression up-regulated the mesenchymal markers vimentin, fibronectin, and α-SMA, reduced expression of the epithelial markers E-cadherin, α-catenin, and β-catenin. RPL22L1 inhibition reduced expression of vimentin and N-cadherin. These results suggest that RPL22L1 induces epithelial-to-mesenchymal transition (EMT). Our data showed that the DMs amplified gene RPL22L1 is critical in maintaining the aggressive phenotype of OC and in triggering cell metastasis by inducing EMT. It could be employed as a novel prognostic marker and/or effective therapeutic target for OC. PMID:26618703

  11. Dynamic Sialylation in Transforming Growth Factor-β (TGF-β)-induced Epithelial to Mesenchymal Transition*

    PubMed Central

    Du, Jun; Hong, Senlian; Dong, Lu; Cheng, Bo; Lin, Liang; Zhao, Bing; Chen, Ye-Guang; Chen, Xing

    2015-01-01

    Epithelial-mesenchymal transition (EMT) is a fundamental process in embryonic development and organ formation. Aberrant regulation of EMT often leads to tumor progression. Changes in cell surface sialylation have recently been implicated in mediating EMT. Herein we report the visualization of dynamic changes of sialylation and glycoproteomic analysis of newly synthesized sialylated proteins in EMT by metabolic labeling of sialylated glycans with azides, followed by click labeling with fluorophores or affinity tags. We discovered that sialylation was down-regulated during EMT but then reverted and up-regulated in the mesenchymal state after EMT, accompanied by mRNA expression level changes of genes involved in the sialic acid biosynthesis. Quantitative proteomic analysis identified a list of sialylated proteins whose biosynthesis was dynamically regulated during EMT. Sialylation of cell surface adherent receptor integrin β4 was found to be down-regulated, which may regulate integrin functions during EMT. Furthermore, a global sialylation inhibitor was used to probe the functional role of sialylation during EMT. We found that inhibition of sialylation promoted EMT. Taken together, our findings suggest the important role of sialylation in regulating EMT and imply its possible function in related pathophysiological events, such as cancer metastasis. PMID:25809486

  12. Apolipoprotein A1 Inhibits TGF-β1–Induced Epithelial-to-Mesenchymal Transition of Alveolar Epithelial Cells

    PubMed Central

    Baek, Ae Rin; Lee, Ji Min; Seo, Hyun Jung; Park, Jong Sook; Lee, June Hyuk; Jang, An Soo; Kim, Do Jin; Koh, Eun Suk; Uh, Soo Taek; Kim, Yong Hoon; Park, Choon Sik

    2016-01-01

    Background Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the accumulation of excessive fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor β1 (TGF-β1)–induced epithelial-to-mesenchymal transition (EMT) is thought to be a possible source of fibroblasts/myofibroblasts in IPF lungs. We have previously reported that apolipoprotein A1 (ApoA1) has anti-fibrotic activity in experimental lung fibrosis. In this study, we determine whether ApoA1 modulates TGF-β1–induced EMT in experimental lung fibrosis and clarify its mechanism of action. Methods The A549 alveolar epithelial cell line was treated with TGF-β1 with or without ApoA1. Morphological changes and expression of EMT-related markers, including E-cadherin, N-cadherin, and α-smooth muscle actin were evaluated. Expressions of Smad and non-Smad mediators and TGF-β1 receptor type 1 (TβRI) and type 2 (TβRII) were measured. The silica-induced lung fibrosis model was established using ApoA1 overexpressing transgenic mice. Results TGF-β1–treated A549 cells were changed to the mesenchymal morphology with less E-cadherin and more N-cadherin expression. The addition of ApoA1 inhibited the TGF-β1–induced change of the EMT phenotype. ApoA1 inhibited the TGF-β1–induced increase in the phosphorylation of Smad2 and 3 as well as that of ERK and p38 mitogen-activated protein kinase mediators. In addition, ApoA1 reduced the TGF-β1–induced increase in TβRI and TβRII expression. In a mouse model of silica-induced lung fibrosis, ApoA1 overexpression reduced the silica-mediated effects, which were increased N-cadherin and decreased E-cadherin expression in the alveolar epithelium. Conclusion Our data demonstrate that ApoA1 inhibits TGF-β1–induced EMT in experimental lung fibrosis. PMID:27433174

  13. Everolimus-induced epithelial to mesenchymal transition in immortalized human renal proximal tubular epithelial cells: key role of heparanase

    PubMed Central

    2013-01-01

    Background Everolimus (EVE) is a drug widely used in several renal transplant protocols. Although characterized by a relatively low nephrotoxicity, it may induce several adverse effects including severe fibro-interstitial pneumonitis. The exact molecular/biological mechanism associated to these pro-fibrotic effects is unknown, but epithelial to mesenchymal transition (EMT) may have a central role. Additionally, heparanase, an enzyme recently associated with the progression of chronic allograft nephropathy, could contribute to activate this machinery in renal cells. Methods Several biomolecular strategies (RT-PCR, immunofluorescence, zymography and migration assay) have been used to assess the capability of EVE (10, 100, 200 and 500 nM) to induce an in vitro heparanase-mediated EMT in wild-type (WT) and Heparanase (HPSE)-silenced immortalized human renal epithelial proximal tubular cells (HK-2). Additionally, microarray technology was used to find additional biological elements involved in EVE-induced EMT. Results Biomolecular experiments demonstrated a significant up-regulation (more than 1.5 fold increase) of several genes encoding for well known EMT markers [(alpha-smooth muscle actin (α-SMA), Vimentin (VIM), Fibronectin (FN) and matrix metalloproteinase-9 (MMP9)], enhancement of MMP9 protein level and increment of cells motility in WT HK2 cells treated with high concentrations of EVE (higher than 100 nM). Similarly, immunofluorescence analysis showed that 100 nM of EVE increased α-SMA, VIM and FN protein expression in WT HK2 cells. All these effects were absent in both HPSE- and AKT-silenced cell lines. AKT is a protein having a central role in EMT. Additionally, microarray analysis identified other 2 genes significantly up-regulated in 100 nM EVE-treated cells (p < 0.005 and FDR < 5%): transforming growth factor beta-2 (TGFβ2) and epidermal growth factor receptor (EGFR). Real-time PCR analysis validated microarray. Conclusions Our in vitro study

  14. Transcriptome profiling reveals novel gene expression signatures and regulating transcription factors of TGFβ-induced epithelial-to-mesenchymal transition.

    PubMed

    Du, Liutao; Yamamoto, Shota; Burnette, Barry L; Huang, Danshang; Gao, Kun; Jamshidi, Neema; Kuo, Michael D

    2016-08-01

    Dysregulated epithelial to mesenchymal transition (EMT) in cancer cells endows invasive and metastatic properties upon cancer cells that favor successful colonization of distal target organs and therefore play a critical role in transforming early-stage carcinomas into invasive malignancies. EMT has also been associated with tumor recurrence and drug resistance and cancer stem cell initiation. Therefore, better understanding of the mechanisms behind EMT could ultimately contribute to the development of novel prognostic approaches and individualized therapies that specifically target EMT processes. As an effort to characterize the central transcriptome changes during EMT, we have developed a Transforming growth factor (TGF)-beta-based in vitro EMT model and used it to profile EMT-related gene transcriptional changes in two different cell lines, a non-small cell lung cancer cell line H358, and a breast cell line MCF10a. After 7 days of TGF-beta/Oncostatin M (OSM) treatment, changes in cell morphology to a mesenchymal phenotype were observed as well as concordant EMT-associated changes in mRNA and protein expression. Further, increased motility was noted and flow cytometry confirmed enrichment in cancer stem cell-like populations. Microarray-based differential expression analysis identified an EMT-associated gene expression signature which was confirmed by RT-qPCR and which significantly overlapped with a previously published EMT core signature. Finally, two novel EMT-regulating transcription factors, IRF5 and LMCD1, were identified and independently validated. PMID:27318801

  15. Dynamics and plasticity of the epithelial to mesenchymal transition induced by miR-200 family inhibition

    PubMed Central

    Haraguchi, Takeshi; Kondo, Masayuki; Uchikawa, Ryo; Kobayashi, Kazuyoshi; Hiramatsu, Hiroaki; Kobayashi, Kyousuke; Chit, Ung Weng; Shimizu, Takanobu; Iba, Hideo

    2016-01-01

    Whereas miR-200 family is known to be involved in the epithelial-to-mesenchymal transition (EMT), a crucial biological process observed in normal and pathological contexts, it has been largely unclear how far the functional levels of these tiny RNAs alone can propagate the molecular events to accomplish this process within several days. By developing a potent inhibitor of miR-200 family members (TuD-141/200c), the expression of which is strictly regulatable by the Tet (tetracycline)-On system, we found using a human colorectal cell line, HCT116, that several direct gene target mRNAs (Zeb1/Zeb2, ESRP1, FN1and FHOD1) of miR-200 family were elevated with distinct kinetics. Prompt induction of the transcriptional suppressors, Zeb1/Zeb2 in turn reduced the expression levels of miR-200c/-141 locus, EpCAM, ESRP1 and E-Cad. The loss of ESRP1 subsequently switched the splicing isoforms of CD44 and p120 catenin mRNAs to mesenchymal type. Importantly, within 9 days after the release from the inhibition of miR-200 family, all of the expression changes in the 14 genes observed in this study returned to their original levels in the epithelial cells. This suggests that the inherent epithelial plasticity is supported by a weak retention of key regulatory gene expression in either the epithelial or mesenchymal states through epigenetic regulation. PMID:26887353

  16. Effects of Chinese Medicine Tong xinluo on Diabetic Nephropathy via Inhibiting TGF-β1-Induced Epithelial-to-Mesenchymal Transition

    PubMed Central

    Zhang, Na; Gao, Yanbin; Zou, Dawei; Wang, Jinyang; Li, Jiaoyang; Zhou, Shengnan; Zhu, Zhiyao; Zhao, Xuan; Xu, Liping; Zhang, Haiyan

    2014-01-01

    Diabetic nephropathy (DN) is a major cause of chronic kidney failure and characterized by interstitial and glomeruli fibrosis. Epithelial-to-mesenchymal transition (EMT) plays an important role in the pathogenesis of DN. Tong xinluo (TXL), a Chinese herbal compound, has been used in China with established therapeutic efficacy in patients with DN. To investigate the molecular mechanism of TXL improving DN, KK-Ay mice were selected as models for the evaluation of pathogenesis and treatment in DN. In vitro, TGF-β1 was used to induce EMT. Western blot (WB), immunofluorescence staining, and real-time polymerase chain reaction (RT-PCR) were applied to detect the changes of EMT markers in vivo and in vitro, respectively. Results showed the expressions of TGF-β1 and its downstream proteins smad3/p-smad3 were greatly reduced in TXL group; meantime, TXL restored the expression of smad7. As a result, the expressions of collagen IV (Col IV) and fibronectin (FN) were significantly decreased in TXL group. In vivo, 24 h-UAER (24-hour urine albumin excretion ratio) and BUN (blood urea nitrogen) were decreased and Ccr (creatinine clearance ratio) was increased in TXL group compared with DN group. In summary, the present study demonstrates that TXL successfully inhibits TGF-β1-induced epithelial-to-mesenchymal transition in DN, which may account for the therapeutic efficacy in TXL-mediated renoprotection. PMID:24864150

  17. MiRNA-21 induces epithelial to mesenchymal transition and gemcitabine resistance via the PTEN/AKT pathway in breast cancer.

    PubMed

    Wu, Zhen-Hua; Tao, Zhong-Hua; Zhang, Jian; Li, Ting; Ni, Chen; Xie, Jie; Zhang, Jin-Feng; Hu, Xi-Chun

    2016-06-01

    Acquisition of gemcitabine resistance in breast cancer has not been fully clarified. Prior studies suggest that miRNAs are important to chemoresistance in solid tumors and we confirmed that miR-21 is involved in the development of gemcitabine resistance. Epithelial-to-mesenchymal transition (EMT) and AKT pathway activation were noted to be important to this resistance as well. PTEN, a direct target gene of miR-21, was significantly downregulated in gemcitabine-resistant breast cancer cells and restoration of PTEN expression blocked miR-21-induced EMT and gemcitabine resistance. Our data offer novel insight into gemcitabine resistance in breast cancer and suggest that miR-21 may be used to predict optimal breast cancer therapy and may be a potential therapeutic target for reversing gemcitabine resistance. PMID:26666820

  18. Epithelial to mesenchymal transition is associated with rapamycin resistance

    PubMed Central

    Holder, Ashley M.; Akcakanat, Argun; Adkins, Farrell; Evans, Kurt; Chen, Huiqin; Wei, Caimiao; Milton, Denai R.; Li, Yisheng; Do, Kim-Anh; Janku, Filip; Meric-Bernstam, Funda

    2015-01-01

    Rapamycin analogues have antitumor efficacy in several tumor types, however few patients demonstrate tumor regression. Thus, there is a pressing need for markers of intrinsic response/resistance and rational combination therapies. We hypothesized that epithelial-to-mesenchymal transition (EMT) confers rapamycin resistance. We found that the epithelial marker E-cadherin protein is higher in rapamycin sensitive (RS) cells and mesenchymal breast cancer cell lines selected by transcriptional EMT signatures are less sensitive to rapamycin. MCF7 cells, transfected with constitutively active mutant Snail, had increased rapamycin resistance (RR) compared to cells transfected with wild-type Snail. Conversely, we transfected two RR mesenchymal cell lines—ACHN and MDA-MB-231—with miR-200b/c or ZEB1 siRNA to promote mesenchymal-to-epithelial transition. This induced E-cadherin expression in both cell lines, and ACHN demonstrated a significant increase in RS. Treatment of ACHN and MDA-MB-231 with trametinib modulated EMT in ACHN cells in vitro. Treatment of MDA-MB-231 and ACHN xenografts with trametinib in combination with rapamycin resulted in significant growth inhibition in both but without an apparent effect on EMT. Future studies are needed to determine whether EMT status is predictive of sensitivity to rapalogs and to determine whether combination therapy with EMT modulating agents can enhance antitumor effects of PI3K/mTOR inhibitors. PMID:25944619

  19. Vitamin D and the Epithelial to Mesenchymal Transition

    PubMed Central

    Larriba, María Jesús; García de Herreros, Antonio; Muñoz, Alberto

    2016-01-01

    Several studies support reciprocal regulation between the active vitamin D derivative 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the epithelial to mesenchymal transition (EMT). Thus, 1,25(OH)2D3 inhibits EMT via the induction of a variety of target genes that encode cell adhesion and polarity proteins responsible for the epithelial phenotype and through the repression of key EMT inducers. Both direct and indirect regulatory mechanisms mediate these effects. Conversely, certain master EMT inducers inhibit 1,25(OH)2D3 action by repressing the transcription of VDR gene encoding the high affinity vitamin D receptor that mediates 1,25(OH)2D3 effects. Consequently, the balance between the strength of 1,25(OH)2D3 signaling and the induction of EMT defines the cellular phenotype in each context. Here we review the current understanding of the genes and mechanisms involved in the interplay between 1,25(OH)2D3 and EMT. PMID:26880977

  20. MicroRNA Regulation of Epithelial to Mesenchymal Transition.

    PubMed

    Abba, Mohammed L; Patil, Nitin; Leupold, Jörg Hendrik; Allgayer, Heike

    2016-01-01

    Epithelial to mesenchymal transition (EMT) is a central regulatory program that is similar in many aspects to several steps of embryonic morphogenesis. In addition to its physiological role in tissue repair and wound healing, EMT contributes to chemo resistance, metastatic dissemination and fibrosis, amongst others. Classically, the morphological change from epithelial to mesenchymal phenotype is characterized by the appearance or loss of a group of proteins which have come to be recognized as markers of the EMT process. As with all proteins, these molecules are controlled at the transcriptional and translational level by transcription factors and microRNAs, respectively. A group of developmental transcription factors form the backbone of the EMT cascade and a large body of evidence shows that microRNAs are heavily involved in the successful coordination of mesenchymal transformation and vice versa, either by suppressing the expression of different groups of transcription factors, or otherwise acting as their functional mediators in orchestrating EMT. This article dissects the contribution of microRNAs to EMT and analyzes the molecular basis for their roles in this cellular process. Here, we emphasize their interaction with core transcription factors like the zinc finger enhancer (E)-box binding homeobox (ZEB), Snail and Twist families as well as some pluripotency transcription factors. PMID:26784241

  1. MicroRNA Regulation of Epithelial to Mesenchymal Transition

    PubMed Central

    Abba, Mohammed L.; Patil, Nitin; Leupold, Jörg Hendrik; Allgayer, Heike

    2016-01-01

    Epithelial to mesenchymal transition (EMT) is a central regulatory program that is similar in many aspects to several steps of embryonic morphogenesis. In addition to its physiological role in tissue repair and wound healing, EMT contributes to chemo resistance, metastatic dissemination and fibrosis, amongst others. Classically, the morphological change from epithelial to mesenchymal phenotype is characterized by the appearance or loss of a group of proteins which have come to be recognized as markers of the EMT process. As with all proteins, these molecules are controlled at the transcriptional and translational level by transcription factors and microRNAs, respectively. A group of developmental transcription factors form the backbone of the EMT cascade and a large body of evidence shows that microRNAs are heavily involved in the successful coordination of mesenchymal transformation and vice versa, either by suppressing the expression of different groups of transcription factors, or otherwise acting as their functional mediators in orchestrating EMT. This article dissects the contribution of microRNAs to EMT and analyzes the molecular basis for their roles in this cellular process. Here, we emphasize their interaction with core transcription factors like the zinc finger enhancer (E)-box binding homeobox (ZEB), Snail and Twist families as well as some pluripotency transcription factors. PMID:26784241

  2. Membrane type 1-matrix metalloproteinase induces epithelial-to-mesenchymal transition in esophageal squamous cell carcinoma: Observations from clinical and in vitro analyses

    PubMed Central

    Pang, Lijuan; Li, Qiuxiang; Li, Shugang; He, Jianwei; Cao, Weiwei; Lan, Jiaojiao; Sun, Bin; Zou, Hong; Wang, Chengyan; Liu, Ruixue; Wei, Cuilei; Wei, Yutao; Qi, Yan; Hu, Jianming; Liang, Weihua; Zhang, Wen Jie; Wan, Mei; Li, Feng

    2016-01-01

    Membrane type 1-matrix metalloproteinase (MT1-MMP) is associated with enhanced tumorigenicity in many cancers. A recent study has revealed that MT1-MMP induces epithelial-to-mesenchymal transition (EMT) in prostate and breast cancer cells. However, its role in esophageal squamous cell carcinoma (ESCC) has not been studied. Here, we investigated the role of MT1-MMP in the dissemination of ESCC. Expression of MT1-MMP was detected by immunohistochemistry and tissue microarray in 88 Kazakh ESCC patients. Western blotting was performed to detect endogenous and overexpressed exogenous MT1-MMP in the Eca109 and Eca9706 cell lines, respectively. Transwell assay was used to estimate MT1-MMP-induced invasion and metastasis. EMT-associated proteins were detected by immunohistochemistry and western blotting. The associations between the expression of MT1-MMP and EMT-associated proteins with clinicopathologic parameters were analyzed. Overexpression of MT1-MMP was confirmed in Kazakh ESCC patients. MT1-MMP levels were found to be correlated with the depth of tumor infiltration. MT1-MMP induced EMT in ESCC both in vivo and in vitro, N-cadherin and Vimentin expression was upregulated upon MT1-MMP transfection into cells. However, E-cadherin was found to be downregulated. MT1-MMP-induced EMT led to increase migration and invasion in ESCC cell lines. In conclusion, our results suggest that MT1-MMP promotes ESCC invasion and metastasis. PMID:26916665

  3. Endothelial cell-secreted EGF induces epithelial to mesenchymal transition and endows head and neck cancer cells with stem-like phenotype

    PubMed Central

    Zhang, Zhaocheng; Dong, Zhihong; Lauxen, Isabel S.; Filho, Manoel Sant’Ana; Nör, Jacques E.

    2014-01-01

    Emerging evidence suggests that endothelial cell-secreted factors contribute to the pathobiology of squamous cell carcinoma (SCC) by enhancing invasive migration and resistance to anoikis. Here we report that SCC cells within the perivascular niche have undergone epithelial to mesenchymal transition (EMT) in a primary human SCC of a patient that developed distant metastases. Endothelial cell-secreted EGF induced EMT of human SCC cells in vitro and also induced acquisition of a stem-like phenotype. In vivo, tumor xenografts vascularized with EGF-silenced endothelial cells exhibited a smaller fraction of cancer stem-like cells (ALDH+CD44+) and were less invasive than tumors vascularized with control endothelial cells. Collectively, these results demonstrated that endothelial cell-EGF induces EMT and an acquisition of stem-like properties by head and neck tumor cells. On this basis, we suggest that vascular endothelial cells contribute to tumor dissemination by secreting factors that endow carcinoma cells with enhanced motility and stemness. PMID:24686166

  4. miRNA-Mediated KHSRP Silencing Rewires Distinct Post-transcriptional Programs during TGF-β-Induced Epithelial-to-Mesenchymal Transition.

    PubMed

    Puppo, Margherita; Bucci, Gabriele; Rossi, Martina; Giovarelli, Matteo; Bordo, Domenico; Moshiri, Arfa; Gorlero, Franco; Gherzi, Roberto; Briata, Paola

    2016-07-26

    Epithelial-to-mesenchymal transition (EMT) confers several traits to cancer cells that are required for malignant progression. Here, we report that miR-27b-3p-mediated silencing of the single-strand RNA binding protein KHSRP is required for transforming growth factor β (TGF-β)-induced EMT in mammary gland cells. Sustained KHSRP expression limits TGF-β-dependent induction of EMT factors and cell migration, whereas its knockdown in untreated cells mimics TGF-β-induced EMT. Genome-wide sequencing analyses revealed that KHSRP controls (1) levels of mature miR-192-5p, a microRNA that targets a group of EMT factors, and (2) alternative splicing of a cohort of pre-mRNAs related to cell adhesion and motility including Cd44 and Fgfr2. KHSRP belongs to a ribonucleoprotein complex that includes hnRNPA1, and the two proteins cooperate in promoting epithelial-type exon usage of select pre-mRNAs. Thus, TGF-β-induced KHSRP silencing is central in a pathway leading to gene-expression changes that contribute to the cellular changes linked to EMT. PMID:27396342

  5. Linking epithelial-to-mesenchymal-transition and epigenetic modifications

    PubMed Central

    Stadler, Sonja C.; Allis, C. David

    2012-01-01

    Cancer, as well as other human disorders, has long been considered to result from the consequence of genetic mutations in key regulatory genes that reside in pathways controlling proliferation, cellular differentiation, DNA damage and repair. In the case of cancer, mutations are well documented to arise in key oncogenes and critically important tumor-suppressor genes as part of the disease progression process. In addition to more accepted, genetic mutations, a rapidly increasing body of evidence supports the general view that profound alterations also occur in ‘epigenes’, whose products serve to define the ‘epigenetic landscape’ of tumor cells. Aberrant changes in epigenetic mechanisms such as DNA methylation, histone modifications and expression of micro RNAs play an important role in cancer and contribute to malignant transitions. Here we review recent studies linking epigenetic mechanisms to epithelial-to-mesenchymal transition as defined in normal processes, as well as abnormal transitions that lead to oncogensis. PMID:22706095

  6. Treatment with the vascular disruptive agent OXi4503 induces an immediate and widespread epithelial to mesenchymal transition in the surviving tumor.

    PubMed

    Fifis, Theodora; Nguyen, Linh; Malcontenti-Wilson, Cathy; Chan, Lie Sam; Nunes Costa, Patricia Luiza; Daruwalla, Jurstine; Nikfarjam, Mehrdad; Muralidharan, Vijayaragavan; Waltham, Mark; Thompson, Erik W; Christophi, Christopher

    2013-10-01

    Epithelial to mesenchymal transition (EMT) is considered an important mechanism in tumor resistance to drug treatments; however, in vivo observation of this process has been limited. In this study we demonstrated an immediate and widespread EMT involving all surviving tumor cells following treatment of a mouse model of colorectal liver metastases with the vascular disruptive agent OXi4503. EMT was characterized by significant downregulation of E-cadherin, relocation and nuclear accumulation of β-catenin as well as significant upregulation of ZEB1 and vimentin. Concomitantly, significant temporal upregulation in hypoxia and the pro-angiogenic growth factors hypoxia-inducible factor 1-alpha, hepatocyte growth factor, vascular endothelial growth factor and transforming growth factor-beta were seen within the surviving tumor. The process of EMT was transient and by 5 days after treatment tumor cell reversion to epithelial morphology was evident. This reversal, termed mesenchymal to epithelial transition (MET) is a process implicated in the development of new metastases but has not been observed in vivo histologically. Similar EMT changes were observed in response to other antitumor treatments including chemotherapy, thermal ablation, and antiangiogenic treatments in our mouse colorectal metastasis model and in a murine orthotopic breast cancer model after OXi4503 treatment. These results suggest that EMT may be an early mechanism adopted by tumors in response to injury and hypoxic stress, such that inhibition of EMT in combination with other therapies could play a significant role in future cancer therapy. PMID:24403226

  7. Epithelial to mesenchymal transition (EMT) induced by bleomycin or TFG(b1)/EGF in murine induced pluripotent stem cell-derived alveolar Type II-like cells.

    PubMed

    Alipio, Zaida A; Jones, Nathan; Liao, Wenbin; Yang, Jianchang; Kulkarni, Shilpa; Sree Kumar, K; Hauer-Jensen, Martin; Ward, David C; Ma, Yupo; Fink, Louis M

    2011-09-01

    Induced pluripotent stem (iPS) cells are derived from reprogrammed somatic cells and are similar to embryonic stem (ES) cells in morphology, gene/protein expression, and pluripotency. In this study, we explored the potential of iPS cells to differentiate into alveolar Type II (ATII)-like epithelial cells. Analysis using quantitative real time polymerase chain reaction and immunofluorescence staining showed that pulmonary surfactant proteins commonly expressed by ATII cells such as surfactant protein A (SPA), surfactant protein B (SPB), and surfactant protein C (SPC) were upregulated in the differentiated cells. Microphilopodia characteristics and lamellar bodies were observed by transmission electron microscopy and lipid deposits were verified by Nile Red and Periodic Acid Schiff staining. C3 complement protein, a specific feature of ATII cells, was present at high levels in culture supernatants demonstrating functionality of these cells in culture. These data show that the differentiated cells generated from iPS cells using a culture method developed previously (Rippon et al., 2006) are ATII-like cells. To further characterize these ATII-like cells, we tested whether they could undergo epithelial to mesenchymal transition (EMT) by exposure to drugs that induce lung fibrosis in mice, such as bleomycin, and the combination of transforming growth factor beta1 (TGF(b1)) and epidermal growth factor (EGF). When the ATII-like cells were exposed to either bleomycin or a TGF(b1)-EGF cocktail, they underwent phenotypic changes including acquisition of a mesenchymal/fibroblastic morphology, upregulation of mesenchymal markers (Col1, Vim, a-Sma, and S100A4), and downregulation of surfactant proteins and E-cadherin. We have shown that ATII-like cells can be derived from skin fibroblasts and that they respond to fibrotic stimuli. These cells provide a valuable tool for screening of agents that can potentially ameliorate or prevent diseases involving lung fibrosis. PMID:21596473

  8. Epicatechin-3-gallate reverses TGF-β1-induced epithelial-to-mesenchymal transition and inhibits cell invasion and protease activities in human lung cancer cells.

    PubMed

    Huang, Shu-Fang; Horng, Chi-Ting; Hsieh, Yih-Shou; Hsieh, Yi-Hsien; Chu, Shu-Chen; Chen, Pei-Ni

    2016-08-01

    Epithelial-to-mesenchymal transition (EMT) and invasion potential have been considered as essential factors in cancer metastasis, which is the major cause of cancer death. EMT is a multi-step process that involves gain invasion, cytoskeleton change, cell adhesion, and proteolytic extracellular matrix degradation. Epicatechin-3-gallate (ECG), which is a natural polyphenolic component of green tea, elicits several antioxidant and anti-inflammatory effects. However, the effects of ECG on cancer invasion and EMT of human lung carcinoma remain unknown. We provided molecular evidence supporting the anti-metastatic effect of ECG. This compound suppressed the invasion (P < 0.001) of highly metastatic A549 cells by reducing the activities of matrix metalloproteinase-2 (P < 0.001) and urokinasetype plasminogen activator (P < 0.001). ECG also reversed the transforming growth factor (TGF)-β1-induced EMT and upregulated epithelial markers, such as E-cadherin. Conversely, ECG inhibited mesenchymal markers, such as fibronectin and p-FAK. The subcutaneous inoculation of this compound also inhibited the tumor growth of the A549 cells in vivo. Therefore, ECG may be used as an anti-cancer and anti-invasion agent for the adjuvant treatment and metastasis control of human lung cancer cells. ECG may also be administered as an effective chemopreventive agent against TGF-β1-induced EMT. PMID:27224248

  9. Mast cells induce epithelial-to-mesenchymal transition and stem cell features in human thyroid cancer cells through an IL-8-Akt-Slug pathway.

    PubMed

    Visciano, C; Liotti, F; Prevete, N; Cali', G; Franco, R; Collina, F; de Paulis, A; Marone, G; Santoro, M; Melillo, R M

    2015-10-01

    There is increasing evidence that mast cells (MCs) and their mediators are involved in the remodeling of the tumor microenvironment and promote tumor growth, angiogenesis and metastasis. We have found that an increased density of MCs in thyroid cancer (TC) correlates with enhanced invasiveness. However, the MC-derived factors responsible for this activity and the mechanisms by which they enhance TC invasiveness remain unidentified. Here, we report that MCs, when activated by TC cells, produce soluble factors that induce epithelial-to-mesenchymal transition (EMT) and stemness features of TC cells. We identified CXCL8/interleukin (IL)-8 as the main mediator contained in activated MC conditioned media (CM) capable of inducing both EMT and stemness of TC cells. Mechanistically, MC CM or exogenous IL-8 stimulated Akt phosphorylation and Slug expression in TC cells. The inhibition of the Akt pathway or depletion of the Slug transcription factor by RNA interference, reverted EMT and stemness responses. TC cells stably transfected with exogenous IL-8 underwent EMT, displayed increased stemness and enhanced tumorigenicity with respect to control cells. The analysis of TC surgical specimens by immunohistochemical analysis demonstrated a positive correlation between MC density (Tryptase(+) cells) and stemness features (OCT4 staining). Taken together, our data identify an MC-dependent IL-8-Akt-Slug pathway that sustains EMT/stemness of TC cells. The blockade of this circuit might be exploited for the therapy of advanced TC. PMID:25619830

  10. Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease.

    PubMed

    Grande, M Teresa; Sánchez-Laorden, Berta; López-Blau, Cristina; De Frutos, Cristina A; Boutet, Agnès; Arévalo, Miguel; Rowe, R Grant; Weiss, Stephen J; López-Novoa, José M; Nieto, M Angela

    2015-09-01

    Progressive kidney fibrosis contributes greatly to end-stage renal failure, and no specific treatment is available to preserve organ function. During renal fibrosis, myofibroblasts accumulate in the interstitium of the kidney, leading to massive deposition of extracellular matrix and organ dysfunction. The origin of myofibroblasts is manifold, but the contribution of an epithelial-to-mesenchymal transition (EMT) undergone by renal epithelial cells during kidney fibrosis is still debated. We show that the reactivation of Snai1 (encoding snail family zinc finger 1, known as Snail1) in mouse renal epithelial cells is required for the development of fibrosis in the kidney. Damage-mediated Snail1 reactivation induces a partial EMT in tubular epithelial cells that, without directly contributing to the myofibroblast population, relays signals to the interstitium to promote myofibroblast differentiation and fibrogenesis and to sustain inflammation. We also show that Snail1-induced fibrosis can be reversed in vivo and that obstructive nephropathy can be therapeutically ameliorated in mice by targeting Snail1 expression. These results reconcile conflicting data on the role of the EMT in renal fibrosis and provide avenues for the design of novel anti-fibrotic therapies. PMID:26236989

  11. Bleomycin (BLM) Induces Epithelial-to-Mesenchymal Transition in Cultured A549 Cells via the TGF-β/Smad Signaling Pathway

    PubMed Central

    Chen, Kui-Jun; Li, Qing; Wen, Cang-mei; Duan, Zhao-Xia; Zhang, Jie yuan; Xu, Chuan; Wang, Jian-Min

    2016-01-01

    The epithelial-to-mesenchymal transition (EMT) is a crucial cellular event in wound healing, tissue repair, and cancer progression in adult tissues, with the interactions with numerous signals. In this study, we aimed to determine whether bleomycin (BLM), an agent that causes pulmonary fibrosis, induces the EMT of the alveolar epithelial cell line A549 and investigated the possible mechanisms. We examined the EMT involved changes in cell morphology, isoform switching of the fibroblast growth factor receptor 2 (FGFR2) by alternative splicing, and expression of the phenotypic markers including E-cadherin, vimentin, and α-SMA using RT-PCR, Western blotting, and immunofluorescence assays. A TGF-β/Smad inhibitor was used to determine whether coculture with BLM would inhibit the EMT of A549 cells. The results showed that BLM induced the EMT of A549 cells possibly via the TGF-β/Smad signaling pathway, evident from the decrease in the expression of E-cadherin and increase in the expression on vimentin. PMID:27471572

  12. Augmenter of liver regeneration inhibits TGF-β1-induced renal tubular epithelial-to-mesenchymal transition via suppressing TβR II expression in vitro

    SciTech Connect

    Liao, Xiao-hui; Zhang, Ling; Chen, Guo-tao; Yan, Ru-yu; Sun, Hang; Guo, Hui; Liu, Qi

    2014-10-01

    Tubular epithelial-to-mesenchymal transition (EMT) plays a crucial role in the progression of renal tubular interstitial fibrosis (TIF), which subsequently leads to chronic kidney disease (CKD) and eventually, end-stage renal disease (ESRD). We propose that augmenter of liver regeneration (ALR), a member of the newly discovered ALR/Erv1 protein family shown to ameliorate hepatic fibrosis, plays a similar protective role in renal tubular cells and has potential as a new treatment option for CKD. Here, we showed that recombinant human ALR (rhALR) inhibits EMT in renal tubular cells by antagonizing activation of the transforming growth factor-β1 (TGF-β1) signaling pathway. Further investigation revealed that rhALR suppresses the expression of TGF-β receptor type II (TβR II) and significantly alleviates TGF-β1-induced phosphorylation of Smad2 and nuclear factor-κB (NF-κB). No apparent adverse effects were observed upon the addition of rhALR alone to cells. These findings collectively suggest that ALR plays a role in inhibiting progression of renal tubular EMT, supporting its potential utility as an effective antifibrotic strategy to reverse TIF in CKD. - Highlights: • ALR is involved in the pathological progression of renal EMT in NRK-52E cells. • ALR suppresses the expression of TβRII and the phosphorylation of Smad2 and NF-κB. • ALR plays a role in inhibiting progression of renal tubular EMT.

  13. Reversal by Growth Hormone of Homocysteine-induced Epithelial-to-Mesenchymal Transition through Membrane Raft-Redox Signaling in Podocytes

    PubMed Central

    Li, Cai-Xia; Xia, Min; Han, Wei-Qing; Li, Xiao-Xue; Zhang, Chun; Boini, Krishna M.; Liu, Xiao-Cheng; Li, Pin-Lan

    2011-01-01

    Epithelial-to-Mesenchymal Transition (EMT) is an important pathogenic mechanism mediating glomerular injury or sclerosis in a variety of renal and systemic diseases such as hyperhomocysteinemia (hHcys). The present study was designed to test whether Hcys-induced EMT in podocytes is reversed by growth hormone (GH), a hormone regulating cell differentiation and growth and to explore the cellular and molecular mechanism mediating its action. It was found that Hcys induced significant EMT in podocytes, as shown by marked decreases in slit diaphragm-associated protein P-cadherin and zonula occludens-1 as epithelial markers and by dramatic increases in the expression of mesenchymal markers, fibroblast specific protein-1 and α-smooth muscle actin, which were detected by all examinations via immunocytochemistry, real time RT-PCR and Western blot analysis. When podocytes were treated with GH at 25 ng/mL, however, Hcys failed to induce podocyte EMT. Using electromagnetic spin resonance spectrometry, Hcys-induced superoxide (O2.−) production via NADPH oxidase was found to be significantly inhibited by GH (66%). Functionally, GH was shown to substantially inhibit Hcys-induced increases in the permeability of podocyte monolayers and to block the decrease in podocin expression in these cells. In addition, NADPH oxidase subunit, gp91phox and GH receptors aggregated in membrane raft clusters, which produced O2.− in response to Hcys and could be blocked by GH, membrane raft disruptors filipin and MCD or NADPH oxidase inhibitor, apocynin. It is concluded that Hcys-induced podocyte EMT is associated with transmembrane membrane raft-redox signaling and that GH reverses this Hcys-induced EMT protecting podocytes from functional disturbance. PMID:21691087

  14. Jak2-Stat5a/b Signaling Induces Epithelial-to-Mesenchymal Transition and Stem-Like Cell Properties in Prostate Cancer.

    PubMed

    Talati, Pooja G; Gu, Lei; Ellsworth, Elyse M; Girondo, Melanie A; Trerotola, Marco; Hoang, David T; Leiby, Benjamin; Dagvadorj, Ayush; McCue, Peter A; Lallas, Costas D; Trabulsi, Edouard J; Gomella, Leonard; Aplin, Andrew E; Languino, Lucia; Fatatis, Alessandro; Rui, Hallgeir; Nevalainen, Marja T

    2015-09-01

    Active Stat5a/b predicts early recurrence and disease-specific death in prostate cancer (PC), which both typically are caused by development of metastatic disease. Herein, we demonstrate that Stat5a/b induces epithelial-to-mesenchymal transition (EMT) of PC cells, as shown by Stat5a/b regulation of EMT marker expression (Twist1, E-cadherin, N-cadherin, vimentin, and fibronectin) in PC cell lines, xenograft tumors in vivo, and patient-derived PCs ex vivo using organ explant cultures. Jak2-Stat5a/b signaling induced functional end points of EMT as well, indicated by disruption of epithelial cell monolayers and increased migration and adhesion of PC cells to fibronectin. Knockdown of Twist1 suppressed Jak2-Stat5a/b-induced EMT properties of PC cells, which were rescued by re-introduction of Twist1, indicating that Twist1 mediates Stat5a/b-induced EMT in PC cells. While promoting EMT, Jak2-Stat5a/b signaling induced stem-like properties in PC cells, such as sphere formation and expression of cancer stem cell markers, including BMI1. Mechanistically, both Twist1 and BMI1 were critical for Stat5a/b induction of stem-like features, because genetic knockdown of Twist1 suppressed Stat5a/b-induced BMI1 expression and sphere formation in stem cell culture conditions, which were rescued by re-introduction of BMI1. By using human prolactin knock-in mice, we demonstrate that prolactin-Stat5a/b signaling promoted metastases formation of PC cells in vivo. In conclusion, our data support the concept that Jak2-Stat5a/b signaling promotes metastatic progression of PC by inducing EMT and stem cell properties in PC cells. PMID:26362718

  15. Vitamin D Can Ameliorate Chlorhexidine Gluconate-Induced Peritoneal Fibrosis and Functional Deterioration through the Inhibition of Epithelial-to-Mesenchymal Transition of Mesothelial Cells

    PubMed Central

    Lee, Yi-Che; Hung, Shih-Yuan; Liou, Hung-Hsiang; Lin, Tsun-Mei; Tsai, Chu-Hung; Lin, Sheng-Hsiang; Tsai, Yau-Sheng; Chang, Min-Yu; Wang, Hsi-Hao; Ho, Li-Chun; Chen, Yi-Ting; Wu, Ching-Fang; Chen, Ho-Ching; Chen, Hsin-Pao; Liu, Kuang-Wen; Chen, Chih-I.; She, Kuan Min; Wang, Hao-Kuang; Lin, Chi-Wei; Chiou, Yuan-Yow

    2015-01-01

    Background. Peritoneal dialysis (PD) can induce fibrosis and functional alterations in PD patients' peritoneal membranes, due to long-term unphysiological dialysate exposure, partially occurring via triggering of epithelial-to-mesenchymal transition (EMT) in peritoneal mesothelial cells (MCs). Vitamin D can ameliorate these negative effects; however, the mechanism remains unexplored. Therefore, we investigated its possible links to MCs EMT inhibition. Methods. Peritoneal fibrosis was established in Sprague-Dawley rats by chlorhexidine gluconate (CG) intraperitoneal injection for 21 days, with and without 1α,25(OH)2D3 treatment. Morphological and functional evaluation and western blot analysis of EMT marker were performed upon peritoneum tissue. In vitro study was also performed in a primary human peritoneal MC culture system; MCs were incubated with transforming growth factor-β1 (TGF-β1) in the absence or presence of 1α,25(OH)2D3. EMT marker expression, migration activities, and cytoskeleton redistribution of MCs were determined. Results. 1α,25(OH)2D3 ameliorated CG-induced morphological and functional deterioration in animal model, along with CG-induced upregulation of α-SMA and downregulation of E-cadherin expression. Meanwhile, 1α,25(OH)2D3 also ameliorated TGF-β1-induced decrease in E-cadherin expression, increase in Snai1 and α-SMA expression, intracellular F-actin redistribution, and migration activity in vitro. Conclusion. 1α,25(OH)2D3 can ameliorate CG-induced peritoneal fibrosis and attenuate functional deterioration through inhibiting MC EMT. PMID:26495304

  16. Inhibition of airway epithelial-to-mesenchymal transition and fibrosis by kaempferol in endotoxin-induced epithelial cells and ovalbumin-sensitized mice.

    PubMed

    Gong, Ju-Hyun; Cho, In-Hee; Shin, Daekeun; Han, Seon-Young; Park, Sin-Hye; Kang, Young-Hee

    2014-03-01

    Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-β. In the in vitro study, we investigated whether 1-20 μM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10-20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-β1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-β-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-β entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 μM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction. PMID:24378645

  17. Hydroxysafflor Yellow A Ameliorates Renal Fibrosis by Suppressing TGF-β1-Induced Epithelial-to-Mesenchymal Transition

    PubMed Central

    Chu, Jianjie; Sun, Jin; Liu, Meiyou; Wang, Chao; Lu, Chengtao; Wen, Aidong

    2016-01-01

    Objective Renal fibrosis is the common pathological foundation of many chronic kidney diseases (CKDs). The aim of this study was to investigate whether Hydroxysafflor yellow A (HSYA) can preserve renal function by inhibiting the progression of renal fibrosis and the potential mechanisms. Methods Renal fibrosis was induced by unilateral ureteral obstruction (UUO) performed on 7-week-old C57BL/6 mice. HSYA (10, 50 and 100 mg/kg) were intragastrically administered. Sham group and model group were administered with the same volume of vehicle. Serum and kidney samples were collected 14 days after the UUO surgery. Serum biochemical indicators were measured by automatic biochemical analyzer. Histological changes were evaluated by HE and Masson staining. In vitro, the anti-fibrotic effect of HSYA was tested on human recombinant transforming growth factor-β1 (TGF-β1) stimulated HK-2 cells. The protein levels of α-SMA, collagen-I and fibronectin in kidney tissue andHK-2 cells were measured by immunohistochemistry and immunofluorescence. The protein levels of apoptosis-relative and TGF-β1/Smad3 signaling were detected by western blot. Results HSYA slowed the development of renal fibrosis both in vivo and in vitro. In UUO rats, renal function index suggested that HSYA treatment decreased the level of serum creatinine (Scr) and blood urea nitrogen (BUN) rose by UUO (P<0.05). HE staining and Masson staining demonstrated that kidney interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration were notably attenuated in the high-dose HSYA group compared with the model group. The expressions of α-SMA, collagen-I and fibronectin were decreased in the UUO kidney and HK-2 cells of the HSYA-treatment group. Moreover, HSYA reduced the apoptotic rate of HK-2 cells stimulated by TGF-β1. Further study revealed that HSYA regulated the TGF-β1/Smads signaling pathway both in kidney tissue and HK-2 cells. Conclusions These results suggested that HSYA had a protective

  18. Fibroblasts induce epithelial to mesenchymal transition in breast tumor cells which is prevented by fibroblasts treatment with histamine in high concentration.

    PubMed

    Porretti, Juliana C; Mohamad, Nora A; Martín, Gabriela A; Cricco, Graciela P

    2014-06-01

    Epithelial to mesenchymal transition (EMT) of cancer cells is an essential process in cancer progression. Cancer cells that undergone EMT loose cell-cell contacts, acquire mesenchymal properties and develop migratory and invasive abilities. In previous studies we have demonstrated that histamine may modify the invasive phenotype of pancreatic and mammary tumor cells. In this work we proposed to investigate whether histamine may also influence the interaction between tumor cells and normal fibroblasts. The potential activation of normal CCD-1059Sk fibroblasts by histamine and EMT phenotypic changes induced in MCF-7 and MDA-MB-231 breast tumor cells by the conditioned media (CM) derived from fibroblasts were evaluated. Initially, we determined the presence of H1, H2 and H4 histamine receptors and matrix metalloproteinase 2 (MMP2) mRNA in CCD-1059Sk fibroblasts. MMP2 gelatinolytic activity, cell migration and alpha-smooth muscle actin expression were increased in fibroblasts by low doses (<1μM) and decreased by high doses (20μM) of histamine. MCF-7 cells cultured with CM from fibroblasts exhibited spindle-shaped morphology, cell spreading and cytoplasmic expression of β-catenin but there was no change in MMP2 activity and cell migration. MDA-MB-231 cells cultured with CM from fibroblasts showed a more elongated phenotype, cell spreading, cytoplasmic β-catenin, increased MMP2 activity and endogenous TGF-β1 expression, and enhanced cell migration and invasion. Notably, all these features were reversed when mammary tumor cells were cultured with CM from fibroblasts treated with 20μM histamine. In conclusion, high doses of histamine may prevent the activation of fibroblasts and also avert the EMT related changes induced in epithelial tumor cells by fibroblasts CM. PMID:24685678

  19. Enhancing HOTAIR/MiR-10b Drives Normal Liver Stem Cells Toward a Tendency to Malignant Transformation Through Inducing Epithelial- to-Mesenchymal Transition.

    PubMed

    Ye, Ping; Wang, Tao; Liu, Wei-Hui; Li, Xiu-Chuan; Tang, Li-Jun; Tian, Fu-Zhou

    2015-08-01

    Previously, other groups and our team consistently have demonstrated that the possible origination of liver cancer stem cells (LCSCs) is the malignant transformation from liver normal stem cells (LNSCs). However, this complex and multi-step process is far from clear due to the accumulation of various gene dysregulations. Because non-coding RNAs (ncRNAs) could regulate multiple genes, a family of genes, and even whole chromosomes, this study further investigated the effect of dysregulated short ncRNA microRNA-10b and long ncRNA HOX transcript antisense RNA (HOTAIR) between LNSCs and LCSCs on phenotype reversion. To clarify the role of ncRNA in malignant transformation of LNSCs, we used lentivirus transduction to enhance the miR-10b and HOTAIR expression levels in our previously isolated rat LNSCs. The malignant abilities of proliferation, invasiveness, and tumorigenesis were observed and compared in cells before and after ncRNAs enhancement. After microRNA-10b and HOTAIR were enhanced separately, several cancer stem cell (CSC)-like traits appeared in these LNSCs, including in vitro-enhanced proliferative capacity, expression of putative LCSC markers, progressive invasive ability, and even in vivo aggravation into and taking the place of normal liver tissue. Furthermore, strengthened expression of these ncRNAs partially degraded E-cadherin in LNSCs, which is one of the classic markers in epithelial-to-mesenchymal transition (EMT). HOTAIR or miR-10b enhanced in LNSCs may drive the LNSCs to a tendency toward malignant transformation. This study partially uncovers the mechanism by which miR-10b or HOTAIR promotes malignant transformation of LNSCs through down-regulating E-cadherin and inducing EMT. PMID:25708830

  20. Epithelial to Mesenchymal Transition in a Clinical Perspective

    PubMed Central

    Pasquier, Jennifer; Abu-Kaoud, Nadine; Al Thani, Haya; Rafii, Arash

    2015-01-01

    Tumor growth and metastatic dissemination rely on cellular plasticity. Among the different phenotypes acquired by cancer cells, epithelial to mesenchymal transition (EMT) has been extensively illustrated. Indeed, this transition allows an epithelial polarized cell to acquire a more mesenchymal phenotype with increased mobility and invasiveness. The role of EMT is quite clear during developmental stage. In the neoplastic context in many tumors EMT has been associated with a more aggressive tumor phenotype including local invasion and distant metastasis. EMT allows the cell to invade surrounding tissues and survive in the general circulation and through a stem cell phenotype grown in the host organ. The molecular pathways underlying EMT have also been clearly defined and their description is beyond the scope of this review. Here we will summarize and analyze the attempts made to block EMT in the therapeutic context. Indeed, till today, most of the studies are made in animal models. Few clinical trials are ongoing with no obvious benefits of EMT inhibitors yet. We point out the limitations of EMT targeting such tumor heterogeneity or the dynamics of EMT during disease progression. PMID:26425122

  1. Noncoding RNAs in Tumor Epithelial-to-Mesenchymal Transition

    PubMed Central

    Lin, Ching-Wen; Lin, Pei-Ying; Yang, Pan-Chyr

    2016-01-01

    Epithelial-derived tumor cells acquire the capacity for epithelial-to-mesenchymal transition (EMT), which enables them to invade adjacent tissues and/or metastasize to distant organs. Cancer metastasis is the main cause of cancer-related death. Molecular mechanisms involved in the switch from an epithelial phenotype to mesenchymal status are complicated and are controlled by a variety of signaling pathways. Recently, a set of noncoding RNAs (ncRNAs), including miRNAs and long noncoding RNAs (lncRNAs), were found to modulate gene expressions at either transcriptional or posttranscriptional levels. These ncRNAs are involved in EMT through their interplay with EMT-related transcription factors (EMT-TFs) and EMT-associated signaling. Reciprocal regulatory interactions between lncRNAs and miRNAs further increase the complexity of the regulation of gene expression and protein translation. In this review, we discuss recent findings regarding EMT-regulating ncRNAs and their associated signaling pathways involved in cancer progression. PMID:26989421

  2. Downregulation of transcription factor Oct4 induces an epithelial-to-mesenchymal transition via enhancement of Ca{sup 2+} influx in breast cancer cells

    SciTech Connect

    Hu, Jiajia; Qin, Kunhua; Zhang, Yan; Gong, Junbo; Li, Na; Lv, Dan; Xiang, Rong; Tan, Xiaoyue

    2011-08-12

    Highlights: {yields} We examine the role of Oct4 in metastasis in cultured MCF-7 cells. {yields} The down regulation of Oct4 induces EMT and increases the capability of migration and invasion in MCF-7 cells. {yields} TGF-{beta}1 inhibits Oct4 expression in both time- and dose-dependent manners. {yields} The EMT induced by TGF-{beta}1 or down regulation of Oct4 could be abrogated by inhibitor of SOCE. {yields} The down regulation of STIM1 (one of the major components of the CRAC channel) alleviates the EMT induce by Oct4 silencing down. -- Abstract: The stem cell-related transcription factor Oct4 regulates tumor proliferation and apoptosis, but its role in tumor migration and invasion is still undefined. Here, we compared Oct4 expression in MCF-7 and MDA-MB-231 cells, two breast cancer cell lines with similar epithelial origins, but distinct invasive and metastatic characteristics. We found MCF-7 cells to express very high levels of Oct4, while no obvious expression was detected in MDA-MB-231 cells. We then downregulated Oct4 expression using small interfering RNA (siRNA) to explore its effects on migration and invasion. Transwell assays showed that silencing Oct4 in MCF-7 cells improved their migration and invasion capabilities. Reverse-transcriptase PCR and western blots showed that E-cadherin expression decreased, and {alpha}-smooth muscle actin expression increased with Oct4 downregulation, which suggests that epithelial-to-mesenchymal transition (EMT) occurred. A potent EMT stimulus, TGF-{beta}1, significantly inhibited Oct4 expression in both dose- and time course-dependent manners. Silencing Oct4 also upregulated expression of two major components of store-operated Ca{sup 2+} entry channels (SOCs), STIM1 and Orai1, and enhanced SOC-directed Ca{sup 2+} influx. Silencing STIM1 blocked the Ca{sup 2+} influx and rescued the EMT initiated by Oct4 downregulation. In conclusion, silencing Oct4 promotes invasion and metastasis in breast cancer cells by inducing EMT

  3. Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells

    PubMed Central

    Neelam, Sudha; Brooks, Morgan M.

    2015-01-01

    Purpose The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. Methods HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression. Results SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed

  4. Alisertib, an Aurora kinase A inhibitor, induces apoptosis and autophagy but inhibits epithelial to mesenchymal transition in human epithelial ovarian cancer cells

    PubMed Central

    Ding, Yong-Hui; Zhou, Zhi-Wei; Ha, Chun-Fang; Zhang, Xue-Yu; Pan, Shu-Ting; He, Zhi-Xu; Edelman, Jeffrey L; Wang, Dong; Yang, Yin-Xue; Zhang, Xueji; Duan, Wei; Yang, Tianxin; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Ovarian cancer is a leading killer of women, and no cure for advanced ovarian cancer is available. Alisertib (ALS), a selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects, and is under clinical investigation for the treatment of advanced solid tumor and hematologic malignancies. However, the role of ALS in the treatment of ovarian cancer remains unclear. This study investigated the effects of ALS on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT), and the underlying mechanisms in human epithelial ovarian cancer SKOV3 and OVCAR4 cells. Our docking study showed that ALS, MLN8054, and VX-680 preferentially bound to AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS had potent growth-inhibitory, proapoptotic, proautophagic, and EMT-inhibitory effects on SKOV3 and OVCAR4 cells. ALS arrested SKOV3 and OVCAR4 cells in G2/M phase and induced mitochondria-mediated apoptosis and autophagy in both SKOV3 and OVCAR4 cell lines in a concentration-dependent manner. ALS suppressed phosphatidylinositol 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways but activated 5′-AMP-dependent kinase, as indicated by their altered phosphorylation, contributing to the proautophagic activity of ALS. Modulation of autophagy altered basal and ALS-induced apoptosis in SKOV3 and OVCAR4 cells. Further, ALS suppressed the EMT-like phenotype in both cell lines by restoring the balance between E-cadherin and N-cadherin. ALS downregulated sirtuin 1 and pre-B cell colony enhancing factor (PBEF/visfatin) expression levels and inhibited phosphorylation of AURKA in both cell lines. These findings indicate that ALS blocks the cell cycle by G2/M phase arrest and promotes cellular apoptosis and autophagy, but inhibits EMT via phosphatidylinositol 3-kinase/Akt/mTOR-mediated and sirtuin 1-mediated pathways in human epithelial ovarian cancer

  5. Nephronophthisis-Associated CEP164 Regulates Cell Cycle Progression, Apoptosis and Epithelial-to-Mesenchymal Transition

    PubMed Central

    Slaats, Gisela G.; Le Corre, Stéphanie; Shaltiel, Indra A.; van de Hoek, Glenn; Klasson, Timothy D.; Stokman, Marijn F.; Logister, Ive; Verhaar, Marianne C.; Goldschmeding, Roel; Nguyen, Tri Q.; Drummond, Iain A.; Hildebrandt, Friedhelm; Giles, Rachel H.

    2014-01-01

    We recently reported that centrosomal protein 164 (CEP164) regulates both cilia and the DNA damage response in the autosomal recessive polycystic kidney disease nephronophthisis. Here we examine the functional role of CEP164 in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of CEP164 revealed an overall quicker cell cycle than control cells, although early S-phase was significantly longer. Follow-up FACS experiments with renal IMCD3 cells confirm that Cep164 siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type CEP164, but not disease-associated mutants. siRNA of CEP164 revealed a proliferation defect over time, as measured by CyQuant assays. The discrepancy between accelerated cell cycle and inhibited overall proliferation could be explained by induction of apoptosis and epithelial-to-mesenchymal transition. Reduction of CEP164 levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of Cep164 or overexpression of dominant negative mutant allele CEP164 Q525X induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with cep164 morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response in vivo. This study reveals a novel role for CEP164 in the pathogenesis of nephronophthisis, in which mutations cause ciliary defects coupled with DNA damage induced replicative stress, cell death, and epithelial-to-mesenchymal transition, and suggests that these events drive the characteristic fibrosis observed in nephronophthisis kidneys. PMID:25340510

  6. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

    PubMed Central

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  7. URG11 mediates hypoxia-induced epithelial-to-mesenchymal transition by modulation of E-cadherin and {beta}-catenin

    SciTech Connect

    Du, Rui; Huang, Chen; Bi, Qian; Zhai, Ying; Xia, Lin; Liu, Jie; Sun, Shiren; Fan, Daiming

    2010-01-01

    Upregulated gene 11 (URG11), recently identified as a new HBx-upregulated gene that may activate {beta}-catenin and Wnt signaling, was found to be upregulated in a human tubule cell line under low oxygen. Here, we investigated the potential role of URG11 in hypoxia-induced renal tubular epithelial-to-mesenchymal (EMT). Overexpression of URG11 in a human proximal tubule cell line (HK2) promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker E-cadherin and increased expression of the mesenchymal markers vimentin and {alpha}-SMA, while URG11 knockdown by siRNA effectively reversed hypoxia-induced EMT. URG11 promoted the expression of {beta}-catenin and increased its nuclear accumulation under normoxic conditions through transactivation of the {beta}-catenin promoter. This in turn upregulated {beta}-catenin/T-cell factor (TCF) and its downstream effector genes, vimentin, and {alpha}-SMA. In vivo, strong expression of URG11 was observed in the tubular epithelia of 5/6-nephrectomized rats, and a Western blot analysis demonstrated a close correlation between HIF-1{alpha} and URG11 protein levels. Altogether, our results indicate that URG11 mediates hypoxia-induced EMT through the suppression of E-cadherin and the activation of the {beta}-catenin/TCF pathway.

  8. Pterostilbene inhibits triple-negative breast cancer metastasis via inducing microRNA-205 expression and negatively modulates epithelial-to-mesenchymal transition.

    PubMed

    Su, Chih-Ming; Lee, Wei-Hwa; Wu, Alexander T H; Lin, Yen-Kuang; Wang, Liang-Shun; Wu, Chih-Hsiung; Yeh, Chi-Tai

    2015-06-01

    Breast cancer is the leading cause of cancer-related deaths among females in economically developing countries. Greater than 95% of breast malignancies are of epithelial origin; the induction of epithelial-to-mesenchymal transition (EMT) has been shown to initiate the metastatic process in breast carcinoma and remains the key target for drug development. Here, we examine the anti-metastatic potential of pterostilbene in modulating EMT process in breast cancer cells both in vitro and in vivo. The differential invasive ability among MCF7, Hs578t and MDA-MB-231 breast cancer cell lines were closely correlated with the expression of EMT markers, determined by Western blots and Matrigel-coated transwells assay. Pterostilbene inhibited the migratory and invasive potential of triple-negative MDA-MB-231 and Hs578t cells, accompanied by the up-regulation of E-cadherin and down-regulation of Snail, Slug, vimentin and ZEB1. Mechanistic investigations revealed a significant up-regulation of miR-205, which resulted in the reduction of Src expression in pterostilbene-treated breast cancer cells. Importantly, pterostilbene suppressed tumor growth and metastasis in MDA-MB-231-bearing NOD/SCID mice by reducing Src/Fak signaling; this observation was consistent with the negative correlations between miR-205 and Src expression in both normal and malignant breast tissues. Our findings provide supports for the usage of pterostilbene as an inhibitor of EMT process and potential candidate for adjuvant therapy. PMID:25792283

  9. Secreted Frizzled-Related Protein 4 Inhibits Glioma Stem-Like Cells by Reversing Epithelial to Mesenchymal Transition, Inducing Apoptosis and Decreasing Cancer Stem Cell Properties

    PubMed Central

    G, Bhuvanalakshmi; Arfuso, Frank; Millward, Michael; Dharmarajan, Arun; Warrier, Sudha

    2015-01-01

    The Wnt pathway is integrally involved in regulating self-renewal, proliferation, and maintenance of cancer stem cells (CSCs). We explored the effect of the Wnt antagonist, secreted frizzled-related protein 4 (sFRP4), in modulating epithelial to mesenchymal transition (EMT) in CSCs from human glioblastoma cells lines, U87 and U373. sFRP4 chemo-sensitized CSC-enriched cells to the most commonly used anti-glioblastoma drug, temozolomide (TMZ), by the reversal of EMT. Cell movement, colony formation, and invasion in vitro were suppressed by sFRP4+TMZ treatment, which correlated with the switch of expression of markers from mesenchymal (Twist, Snail, N-cadherin) to epithelial (E-cadherin). sFRP4 treatment elicited activation of the Wnt-Ca2+ pathway, which antagonizes the Wnt/ß-catenin pathway. Significantly, the chemo-sensitization effect of sFRP4 was correlated with the reduction in the expression of drug resistance markers ABCG2, ABCC2, and ABCC4. The efficacy of sFRP4+TMZ treatment was demonstrated in vivo using nude mice, which showed minimum tumor engraftment using CSCs pretreated with sFRP4+TMZ. These studies indicate that sFRP4 treatment would help to improve response to commonly used chemotherapeutics in gliomas by modulating EMT via the Wnt/ß-catenin pathway. These findings could be exploited for designing better targeted strategies to improve chemo-response and eventually eliminate glioblastoma CSCs. PMID:26030909

  10. Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β.

    PubMed

    Mele, Valentina; Muraro, Manuele G; Calabrese, Diego; Pfaff, Dennis; Amatruda, Nunzia; Amicarella, Francesca; Kvinlaug, Brynn; Bocelli-Tyndall, Chiara; Martin, Ivan; Resink, Therese J; Heberer, Michael; Oertli, Daniel; Terracciano, Luigi; Spagnoli, Giulio C; Iezzi, Giandomenica

    2014-06-01

    Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells. PMID:24214914

  11. Polo-like kinase 1 induces epithelial-to-mesenchymal transition and promotes epithelial cell motility by activating CRAF/ERK signaling

    PubMed Central

    Wu, Jianguo; Ivanov, Andrei I; Fisher, Paul B; Fu, Zheng

    2016-01-01

    Polo-like kinase 1 (PLK1) is a key cell cycle regulator implicated in the development of various cancers, including prostate cancer. However, the functions of PLK1 beyond cell cycle regulation remain poorly characterized. Here, we report that PLK1 overexpression in prostate epithelial cells triggers oncogenic transformation. It also results in dramatic transcriptional reprogramming of the cells, leading to epithelial-to-mesenchymal transition (EMT) and stimulation of cell migration and invasion. Consistently, PLK1 downregulation in metastatic prostate cancer cells enhances epithelial characteristics and inhibits cell motility. The signaling mechanisms underlying the observed cellular effects of PLK1 involve direct PLK1-dependent phosphorylation of CRAF with subsequent stimulation of the MEK1/2-ERK1/2-Fra1-ZEB1/2 signaling pathway. Our findings highlight novel non-canonical functions of PLK1 as a key regulator of EMT and cell motility in normal prostate epithelium and prostate cancer. This study also uncovers a previously unanticipated role of PLK1 as a potent activator of MAPK signaling. DOI: http://dx.doi.org/10.7554/eLife.10734.001 PMID:27003818

  12. ERK and PI3K regulate different aspects of the epithelial to mesenchymal transition of mammary tumor cells induced by truncated MUC1

    SciTech Connect

    Horn, Galit; Gaziel, Avital; Wreschner, Daniel H.; Smorodinsky, Nechama I.; Ehrlich, Marcelo

    2009-05-01

    Epithelial to mesenchymal transition (EMT) integrates changes to cell morphology and signaling pathways resulting from modifications to the cell's transcriptional response. Different combinations of stimuli ignite this process in the contexts of development or tumor progression. The human MUC1 gene encodes multiple alternatively spliced forms of a polymorphic oncoprotein that is aberrantly expressed in epithelial malignancies. MUC1 is endowed with various signaling modules and has the potential to mediate proliferative and morphological changes characteristic of the progression of epithelial tumors. The tyrosine-rich cytoplasmic domain and the heavily glycosylated extracellular domain both play a role in MUC1-mediated signal transduction. However, the attribution of function to specific domains of MUC1 is difficult due to the concomitant presence of multiple forms of the protein, which stem from alternative splicing and proteolytic cleavage. Here we show that DA3 mouse mammary tumor cells stably transfected with a truncated genomic fragment of human MUC1 undergo EMT. In their EMT, these cells demonstrate altered [i] morphology, [ii] signaling pathways and [iii] expression of epithelial and mesenchymal markers. Similarly to well characterized human breast cancer cell lines, cells transfected with truncated MUC1 show an ERK-dependent increased spreading on fibronectin, and a PI3K-dependent enhancement of their proliferative rate.

  13. Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions

    PubMed Central

    Thomson, Stuart; Buck, Elizabeth; Russo, Suzanne; Petti, Filippo; Sujka-Kwok, Izabela; Eyzaguirre, Alexandra; Rosenfeld-Franklin, Maryland; Gibson, Neil W.; Miglarese, Mark; Epstein, David; Iwata, Kenneth K.; Haley, John D.

    2008-01-01

    Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis. PMID:18236164

  14. Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition.

    PubMed

    Chang, Hongyuan; Liu, Yuwei; Xue, Mengzhu; Liu, Haiyue; Du, Shaowei; Zhang, Liwen; Wang, Peng

    2016-04-01

    Epithelial-to-mesenchymal transition (EMT) is a complex multistep process in which phenotype switches are mediated by a network of transcription factors (TFs). Systematic characterization of all dynamic TFs controlling EMT state transitions, especially for the intermediate partial-EMT state, represents a highly relevant yet largely unexplored task. Here, we performed a computational analysis that integrated time-course EMT transcriptomic data with public cistromic data and identified three synergistic master TFs (ETS2, HNF4A and JUNB) that regulate the transition through the partial-EMT state. Overexpression of these regulators predicted a poor clinical outcome, and their elimination readily abolished TGF-β-induced EMT. Importantly, these factors utilized a clique motif, physically interact and their cumulative binding generally characterized EMT-associated genes. Furthermore, analyses of H3K27ac ChIP-seq data revealed that ETS2, HNF4A and JUNB are associated with super-enhancers and the administration of BRD4 inhibitor readily abolished TGF-β-induced EMT. These findings have implications for systematic discovery of master EMT regulators and super-enhancers as novel targets for controlling metastasis. PMID:26926107

  15. Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition

    PubMed Central

    Chang, Hongyuan; Liu, Yuwei; Xue, Mengzhu; Liu, Haiyue; Du, Shaowei; Zhang, Liwen; Wang, Peng

    2016-01-01

    Epithelial-to-mesenchymal transition (EMT) is a complex multistep process in which phenotype switches are mediated by a network of transcription factors (TFs). Systematic characterization of all dynamic TFs controlling EMT state transitions, especially for the intermediate partial-EMT state, represents a highly relevant yet largely unexplored task. Here, we performed a computational analysis that integrated time-course EMT transcriptomic data with public cistromic data and identified three synergistic master TFs (ETS2, HNF4A and JUNB) that regulate the transition through the partial-EMT state. Overexpression of these regulators predicted a poor clinical outcome, and their elimination readily abolished TGF-β-induced EMT. Importantly, these factors utilized a clique motif, physically interact and their cumulative binding generally characterized EMT-associated genes. Furthermore, analyses of H3K27ac ChIP-seq data revealed that ETS2, HNF4A and JUNB are associated with super-enhancers and the administration of BRD4 inhibitor readily abolished TGF-β-induced EMT. These findings have implications for systematic discovery of master EMT regulators and super-enhancers as novel targets for controlling metastasis. PMID:26926107

  16. The Epithelial-to-Mesenchymal Transition (EMT), a Particular Case

    PubMed Central

    Cebrià-Costa, Joan Pau; Millanes-Romero, Alba; de Herreros, Antonio García; Peiró, Sandra

    2014-01-01

    Constitutive heterochromatin, an essential structure that has been conserved throughout evolution, is required to maintain genome stability. Although heterochromatin is enriched for repressive traits, it can be actively transcribed to generate thousands of noncoding RNAs that are required for correct chromatin assembly. Despite the importance of this structure, how and why heterochromatin transcription is regulated, and the proteins responsible for this regulation, remain poorly understood. Here, we summarize recent findings in heterochromatin transcription regulation during different cellular processes with a focus on the epithelial–mesenchymal transition (EMT), which elicits important changes in cell behavior, has a key role in early development, and is involved in cancer progression. PMID:27308335

  17. Epithelial to mesenchymal transition in tumor cells as consequence of phenotypic instability

    PubMed Central

    García de Herreros, Antonio

    2014-01-01

    During the last years many articles have reported epithelial-to-mesenchymal transitions (EMT) induced by a myriad of gene products either when added to the cell medium or when transfected. Molecularly the EMT is characterized by the up-regulation of transcriptional factors (EMT-TFs) repressing the epithelial gene E-cadherin, a protein essential for the maintenance of the epithelial phenotype. These EMT-TFs are subjected to a complex regulation involving binary self-stimulatory loops, allowing the possibility of the amplification of input signals. The capability of EMT-TFs to promote an EMT is controlled by E-cadherin that limits the transcription of mesenchymal genes. We discuss here the differences between normal and tumor epithelial cells; in the latter a partial inactivation of E-cadherin function enables extracellular signals to be amplified and induce an EMT. This tumor cell phenotypic instability is exacerbated in cell culture conditions. Therefore, it is likely that many of the gene products reported to control this transition act only in very specific cell tumor cell lines; thus, in cells with an unstable phenotype due to pre-existing alterations in E-cadherin safeguard mechanism. PMID:25566541

  18. Transforming growth factor-β1 induces epithelial-to-mesenchymal transition in human lung cancer cells via PI3K/Akt and MEK/Erk1/2 signaling pathways.

    PubMed

    Chen, Xiao-Feng; Zhang, Hui-Jun; Wang, Hai-Bing; Zhu, Jun; Zhou, Wen-Yong; Zhang, Hui; Zhao, Ming-Chuan; Su, Jin-Mei; Gao, Wen; Zhang, Lei; Fei, Ke; Zhang, Hong-Tao; Wang, He-Yong

    2012-04-01

    Metastasis of tumor cells is associated with epithelial-to-mesenchymal transition (EMT), which is a process whereby epithelial cells lose their polarity and acquire new features of mesenchyme. EMT has been reported to be induced by transforming growth factor-β1 (TGF-β1), but its mechanism remains elusive. In this study, we performed a study to investigate whether PI3K/Akt and MAPK/Erk1/2 signaling pathways involved in EMT in the human lung cancer A549 cells. The results showed that after treated with TGF-β1 for 48 h, A549 cells displayed more fibroblast-like shape, lost epithelial marker E-cadherin and increased mesenchymal markers Vimentin and Fibronectin. Moreover, TGF-β1-induced EMT after 48 h was accompanied by increased of cell migration and change of Akt and Erk1/2 phosphorylation. In addition, EMT was reversed by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126, which suggested that A549 cells under stimulation of TGF-β1 undergo a switch into mesenchymal cells and PI3K/Akt and MAPK/Erk1/2 signaling pathways serve to regulate TGF-β1-induced EMT of A549 cells. PMID:21713404

  19. Alveolar Epithelial Cells Undergo Epithelial-to-Mesenchymal Transition in Response to Endoplasmic Reticulum Stress*

    PubMed Central

    Tanjore, Harikrishna; Cheng, Dong-Sheng; Degryse, Amber L.; Zoz, Donald F.; Abdolrasulnia, Rasul; Lawson, William E.; Blackwell, Timothy S.

    2011-01-01

    Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis. PMID:21757695

  20. Effect of Cigarette Smoking on Epithelial to Mesenchymal Transition (EMT) in Lung Cancer

    PubMed Central

    Vu, Trung; Jin, Lin; Datta, Pran K.

    2016-01-01

    Epithelial to mesenchymal transition (EMT) is a process that allows an epithelial cell to acquire a mesenchymal phenotype through multiple biochemical changes resulting in an increased migratory capacity. During cancer progression, EMT is found to be associated with an invasive or metastatic phenotype. In this review, we focus on the discussion of recent studies about the regulation of EMT by cigarette smoking. Various groups of active compounds found in cigarette smoke such as polycyclic aromatic hydrocarbons (PAH), nicotine-derived nitrosamine ketone (NNK), and reactive oxygen specicies (ROS) can induce EMT through different signaling pathways. The links between EMT and biological responses to cigarette smoke, such as hypoxia, inflammation, and oxidative damages, are also discussed. The effect of cigarette smoke on EMT is not only limited to cancer types directly related to smoking, such as lung cancer, but has also been found in other types of cancer. Altogether, this review emphasizes the importance of understanding molecular mechanisms of the induction of EMT by cigarette smoking and will help in identifying novel small molecules for targeting EMT induced by smoking. PMID:27077888

  1. Directing epithelial to mesenchymal transition through engineered microenvironments displaying orthogonal adhesive and mechanical cues.

    PubMed

    Markowski, Marilyn C; Brown, Ashley C; Barker, Thomas H

    2012-08-01

    Cell interactions with their extracellular matrix (ECM) microenvironments play a major role in directing cellular processes that can drive wound healing and tissue regeneration but, if uncontrolled, lead to pathological progression. One such process, epithelial to mesenchymal transition (EMT), if finely controlled could have significant potential in regenerative medicine approaches. Despite recent findings that highlight the influence of biochemical and mechanical properties of the ECM on EMT, it is still unclear how these two orthogonal cues act synergistically to control epithelial cell phenotype. Here, we cultured lung epithelial cells on combinations of different mutants of fibronectin's cell binding domain that preferentially engage specific integrins and substrates of varying stiffness. Our results suggest that while stiff substrates induce spontaneous EMT, this response can be overcome by with fragments of fibronectin that support α3 and α5 integrin engagement. Furthermore, we found that substrate-induced EMT correlates with transforming growth factor beta activation by resident epithelial cells and is dependent on Rho/ROCK signaling. Suppressing cell-contractility was sufficient to maintain an epithelial phenotype. Our results suggest that integrin-specific engagement of fibronectin adhesive domains and the mechanics of the ECM act synergistically to direct EMT. PMID:22615133

  2. Epithelial-to-Mesenchymal Transition and Cancer Invasiveness: What Can We Learn from Cholangiocarcinoma?

    PubMed Central

    Brivio, Simone; Cadamuro, Massimiliano; Fabris, Luca; Strazzabosco, Mario

    2015-01-01

    In addition to its well-established role in embryo development, epithelial-to-mesenchymal transition (EMT) has been proposed as a general mechanism favoring tumor metastatization in several epithelial malignancies. Herein, we review the topic of EMT in cholangiocarcinoma (CCA), a primary liver cancer arising from the epithelial cells lining the bile ducts (cholangiocytes) and characterized by an abundant stromal reaction. CCA carries a dismal prognosis, owing to a pronounced invasiveness and scarce therapeutic opportunities. In CCA, several reports indicate that cancer cells acquire a number of EMT biomarkers and functions. These phenotypic changes are likely induced by both autocrine and paracrine signals released in the tumor microenvironment (cytokines, growth factors, morphogens) and intracellular stimuli (microRNAs, oncogenes, tumor suppressor genes) variably associated with specific disease mechanisms, including chronic inflammation and hypoxia. Nevertheless, evidence supporting a complete EMT of neoplastic cholangiocytes into stromal cells is lacking, and the gain of EMT-like changes by CCA cells rather reflects a shift towards an enhanced pro-invasive phenotype, likely induced by the tumor stroma. This concept may help to identify new biomarkers of early metastatic behavior along with potential therapeutic targets. PMID:26703747

  3. Loss of CLCA4 Promotes Epithelial-to-Mesenchymal Transition in Breast Cancer Cells

    PubMed Central

    Yu, Yang; Walia, Vijay; Elble, Randolph C.

    2013-01-01

    The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells downregulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In breast cancer, EMT facilitates invasion of surrounding tissues and correlates closely with cancer metastasis and relapse. We found previously that the candidate tumor suppressor CLCA2 is expressed in differentiated, growth-arrested mammary epithelial cells but is downregulated during tumor progression and EMT. We further demonstrated that CLCA2 is a p53-inducible proliferation-inhibitor whose loss indicates an increased risk of metastasis. We show here that another member of the CLCA gene family, CLCA4, is expressed in mammary epithelial cells and is similarly downregulated in breast tumors and in breast cancer cell lines. Like CLCA2, the gene is stress-inducible, and ectopic expression inhibits colony formation. Transcriptional profiling studies revealed that CLCA4 and CLCA2 together are markers for mammary epithelial differentiation, and both are downregulated by TGF beta. Moreover, knockdown of CLCA4 in immortalized cells by shRNAs caused downregulation of epithelial marker E-cadherin and CLCA2, while mesenchymal markers N-cadherin, vimentin, and fibronectin were upregulated. Double knockdown of CLCA2 and CLCA4 enhanced the mesenchymal profile. These findings suggest that CLCA4 and CLCA2 play complementary but distinct roles in epithelial differentiation. Clinically, low expression of CLCA4 signaled lower relapse-free survival in basal and luminal B breast cancers. PMID:24386311

  4. Involvement of epithelial-to-mesenchymal transition and associated transforming growth factor-β/Smad signaling in paraquat-induced pulmonary fibrosis.

    PubMed

    Han, Ying-Ying; Shen, Peng; Chang, Wen-Xiu

    2015-12-01

    Paraquat (PQ) is a highly toxic herbicide which is able to induce pulmonary fibrosis in humans and animals. The epithelial‑to‑mesenchymal transition (EMT) was demonstrated to be an important factor in pulmonary fibrosis. However, it has remained elusive whether PQ induces pulmonary fibrosis via EMT, which was therefore investigated in the present study. In addition, the underlying mechanisms of PQ‑induced EMT were examined in vitro. Hematoxylin and eosin staining of rat lung tissues demonstrated that PQ induced pulmonary fibrosis in vivo. Western blot analysis then revealed that the expression of epithelial cell marker E‑cadherin was significantly decreased, while the expression of mesenchymal markers α‑smooth‑muscle actin and vimentin was significantly increased in rat lung tissues and A549 cells following PQ treatment. Transforming growth factor (TGF)‑β/Smad signaling was also induced by PQ as evidenced by increased expression of TGF‑β1 and Smad2. However, PQ‑induced EMT in A549 cells was abolished by transfection with TGF‑β1‑specific small hairpin RNA. In conclusion, the present study demonstrated that PQ induced EMT in vivo and in vitro, which may be an important process in the development of PQ‑induced pulmonary fibrosis. In addition, TGF-β/Smad signaling was involved in PQ-induced EMT. PMID:26499763

  5. Substrate stiffness modulates lung cancer cell migration but not epithelial to mesenchymal transition.

    PubMed

    Shukla, V C; Higuita-Castro, N; Nana-Sinkam, P; Ghadiali, S N

    2016-05-01

    Biomechanical properties of the tumor microenvironment, including matrix/substrate stiffness, play a significant role in tumor evolution and metastasis. Epithelial to Mesenchymal Transition (EMT) is a fundamental biological process that is associated with increased cancer cell migration and invasion. The goal of this study was to investigate (1) how substrate stiffness modulates the migration behaviors of lung adenocarcinoma cells (A549) and (2) if stiffness-induced changes in cell migration correlate with biochemical markers of EMT. Collagen-coated polydimethylsiloxane (PDMS) substrates and an Ibidi migration assay were used to investigate how substrate stiffness alters the migration patterns of A549 cells. RT-PCR, western blotting and immunofluorescence were used to investigate how substrate stiffness alters biochemical markers of EMT, that is, E-cadherin and N-cadherin, and the phosphorylation of focal adhesion proteins. Increases in substrate stiffness led to slower, more directional migration but did not alter the biochemical markers of EMT. Interestingly, growth factor (i.e., Transforming Growth Factor-β) stimulation resulted in similar levels of EMT regardless of substrate stiffness. We also observed decreased levels of phosphorylated focal adhesion kinase (FAK) and paxillin on stiffer substrates which correlated with slower cell migration. These results indicate that substrate stiffness modulates lung cancer cell migration via focal adhesion signaling as opposed to EMT signaling. PMID:26779779

  6. LOXL2 catalytically inactive mutants mediate epithelial-to-mesenchymal transition.

    PubMed

    Cuevas, Eva P; Moreno-Bueno, Gema; Canesin, Giacomo; Santos, Vanesa; Portillo, Francisco; Cano, Amparo

    2014-01-01

    Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family that catalyzes the cross-linking of collagens or elastins in the extracellular matrix, thus regulating the tensile strength of tissues. However, many reports have suggested different intracellular roles for LOXL2, including the ability to regulate gene transcription and tumor progression. We previously reported that LOXL2 mediates epithelial-to-mesenchymal transition (EMT) by Snail1-dependent and independent mechanisms, related to E-cadherin silencing and downregulation of epidermal differentiation and cell polarity components, respectively. Whether or not the catalytic activity of LOXL2 is required to induce/sustain EMT is actually unknown. Here we show that LOXL2 catalytic inactive mutants collaborate with Snail1 in E-cadherin gene repression to trigger EMT and, in addition, promote FAK/Src pathway activation to support EMT. These findings reveal a non-conventional role of LOXL2 on regulating epithelial cell plasticity. PMID:24414204

  7. Abelmoschus esculentus fractions potently inhibited the pathogenic targets associated with diabetic renal epithelial to mesenchymal transition.

    PubMed

    Peng, Chiung-Huei; Chyau, Charng-Cherng; Wang, Chau-Jong; Lin, Huei-Ting; Huang, Chien-Ning; Ker, Yaw-Bee

    2016-02-01

    Although Abelmoschus esculentus (AE) is known for anti-hyperglycemia, few reports have addressed its target. Our recent studies have focused on diabetic renal epithelial to mesenchymal transition (EMT), which plays a critical role in fibrosis that accompanies increasing vimentin and suggested signals DPP-4/AT-1/TGF-β1. This study aimed to investigate whether AE is useful for preventing diabetic renal EMT. We used a succession of extractions and obtained the corresponding fractions F1-F5, each with its own individual properties: F1 inhibits high glucose-stimulated vimentin, AT-1, TGF-β1, and DPP-4, and recovers E-cadherin in tubular cells; F2 decreases high glucose-induced vimentin, AT-1 and DPP-4; F3-F5 do not reduce the expression of vimentin. Chemical analysis revealed that F1 is rich of flavonoid glycosides especially quercetin glucosides, and pentacyclic triterpene ester. F2 contains a large amount of carbohydrates and polysaccharides composed of uronic acid, galactose, glucose, myo-inositol etc. In conclusion, AE has the potential to serve as an adjuvant for diabetic nephropathy, with F1 and F2 especially deserving further investigation and development. PMID:26787242

  8. F-box proteins: Keeping the epithelial-to-mesenchymal transition (EMT) in check.

    PubMed

    Díaz, Víctor M; de Herreros, Antonio García

    2016-02-01

    F-box proteins are the key recognition subunit of multimeric E3 ubiquitin ligase complexes that participate in the proteasome degradation of specific substrates. In the last years, a discrete number of F-box proteins have been shown to regulate the epithelial-to-mesenchymal transition (EMT), a process defined by a rapid change of cell phenotype, the loss of epithelial characteristics and the acquisition of a more invasive phenotype. Specific EMT transcription factors (EMT-TFs), such as Snail, Slug, Twist and Zeb, control EMT induction both during development and in cancer. These EMT-TFs are short-lived proteins that are targeted to the proteasome system by specific F-box proteins, keeping them at low levels. F-box proteins also indirectly regulate the EMT process by controlling EMT inducers, such as Notch, c-Myc or mTOR. Here we summarize the role that these F-box proteins (Fbxw1, Fbxw7, Fbxl14, Fbxl5, Fbxo11 and Fbxo45) play in controlling EMT during development and cancer progression, a process dependent on post-translational modifications that govern their interaction with target proteins. PMID:26506454

  9. FLASH protects ZEB1 from degradation and supports cancer cells' epithelial-to-mesenchymal transition

    PubMed Central

    Abshire, C F; Carroll, J L; Dragoi, A-M

    2016-01-01

    Cancer metastasis remains a significant challenge and the leading cause of cancer-associated deaths. It is postulated that during metastasis cells undergo epithelial-to-mesenchymal transition (EMT), a process characterized by loss of cell–cell contacts and increased migratory and invasive potential. ZEB1 is one the most prominent transcriptional repressors of genes associated with EMT. We identified caspase-8-associated protein 2 (CASP8AP2 or FLASH) as a novel posttranscriptional regulator of ZEB1. Here we demonstrate that FLASH protects ZEB1 from proteasomal degradation brought by the action of the ubiquitin ligases SIAH1 and F-box protein FBXO45. As a result, loss of FLASH rapidly destabilized ZEB1 and reversed EMT cellular characteristics. Importantly, loss of FLASH blocked transforming growth factor-β-induced EMT and enhanced sensitivity to chemotherapy. Thus, we propose that FLASH–ZEB1 interplay may be a protective mechanism against ZEB1 degradation in cells undergoing EMT and may be an efficacious target for therapies aimed to block EMT progression. PMID:27526108

  10. MOF Acetylates the Histone Demethylase LSD1 to Suppress Epithelial-to-Mesenchymal Transition.

    PubMed

    Luo, Huacheng; Shenoy, Anitha K; Li, Xuehui; Jin, Yue; Jin, Lihua; Cai, Qingsong; Tang, Ming; Liu, Yang; Chen, Hao; Reisman, David; Wu, Lizi; Seto, Edward; Qiu, Yi; Dou, Yali; Casero, Robert A; Lu, Jianrong

    2016-06-21

    The histone demethylase LSD1 facilitates epithelial-to-mesenchymal transition (EMT) and tumor progression by repressing epithelial marker expression. However, little is known about how its function may be modulated. Here, we report that LSD1 is acetylated in epithelial but not mesenchymal cells. Acetylation of LSD1 reduces its association with nucleosomes, thus increasing histone H3K4 methylation at its target genes and activating transcription. The MOF acetyltransferase interacts with LSD1 and is responsible for its acetylation. MOF is preferentially expressed in epithelial cells and is downregulated by EMT-inducing signals. Expression of exogenous MOF impedes LSD1 binding to epithelial gene promoters and histone demethylation, thereby suppressing EMT and tumor invasion. Conversely, MOF depletion enhances EMT and tumor metastasis. In human cancer, high MOF expression correlates with epithelial markers and a favorable prognosis. These findings provide insight into the regulation of LSD1 and EMT and identify MOF as a critical suppressor of EMT and tumor progression. PMID:27292636

  11. FLASH protects ZEB1 from degradation and supports cancer cells' epithelial-to-mesenchymal transition.

    PubMed

    Abshire, C F; Carroll, J L; Dragoi, A-M

    2016-01-01

    Cancer metastasis remains a significant challenge and the leading cause of cancer-associated deaths. It is postulated that during metastasis cells undergo epithelial-to-mesenchymal transition (EMT), a process characterized by loss of cell-cell contacts and increased migratory and invasive potential. ZEB1 is one the most prominent transcriptional repressors of genes associated with EMT. We identified caspase-8-associated protein 2 (CASP8AP2 or FLASH) as a novel posttranscriptional regulator of ZEB1. Here we demonstrate that FLASH protects ZEB1 from proteasomal degradation brought by the action of the ubiquitin ligases SIAH1 and F-box protein FBXO45. As a result, loss of FLASH rapidly destabilized ZEB1 and reversed EMT cellular characteristics. Importantly, loss of FLASH blocked transforming growth factor-β-induced EMT and enhanced sensitivity to chemotherapy. Thus, we propose that FLASH-ZEB1 interplay may be a protective mechanism against ZEB1 degradation in cells undergoing EMT and may be an efficacious target for therapies aimed to block EMT progression. PMID:27526108

  12. The hypoxia factor Hif-1α controls neural crest chemotaxis and epithelial to mesenchymal transition

    PubMed Central

    Barriga, Elias H.; Maxwell, Patrick H.

    2013-01-01

    One of the most important mechanisms that promotes metastasis is the stabilization of Hif-1 (hypoxia-inducible transcription factor 1). We decided to test whether Hif-1α also was required for early embryonic development. We focused our attention on the development of the neural crest, a highly migratory embryonic cell population whose behavior has been likened to cancer metastasis. Inhibition of Hif-1α by antisense morpholinos in Xenopus laevis or zebrafish embryos led to complete inhibition of neural crest migration. We show that Hif-1α controls the expression of Twist, which in turn represses E-cadherin during epithelial to mesenchymal transition (EMT) of neural crest cells. Thus, Hif-1α allows cells to initiate migration by promoting the release of cell–cell adhesions. Additionally, Hif-1α controls chemotaxis toward the chemokine SDF-1 by regulating expression of its receptor Cxcr4. Our results point to Hif-1α as a novel and key regulator that integrates EMT and chemotaxis during migration of neural crest cells. PMID:23712262

  13. Kaempferol Suppresses Transforming Growth Factor-β1-Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179.

    PubMed

    Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

    2015-07-01

    Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non-small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1-induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1-mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1-mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1-induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1-mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1-induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1-induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. PMID:26297431

  14. Kaempferol Suppresses Transforming Growth Factor-β1–Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-1791

    PubMed Central

    Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

    2015-01-01

    Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non–small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1–induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1–mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1–mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1–induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1–mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1–induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1–induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. PMID:26297431

  15. Resveratrol induces chemosensitization to 5-fluorouracil through up-regulation of intercellular junctions, Epithelial-to-mesenchymal transition and apoptosis in colorectal cancer.

    PubMed

    Buhrmann, Constanze; Shayan, Parviz; Kraehe, Patricia; Popper, Bastian; Goel, Ajay; Shakibaei, Mehdi

    2015-11-01

    5-Fluorouracil (5-FU), a common chemotherapeutic agent used for the treatment of colorectal cancer (CRC), by itself has inadequate response rates; highlighting the need for novel and improved treatment regimens for these patients. Resveratrol, a naturally-occurring polyphenol, has been linked with chemosensitizing potential and anticancer properties; however, the underlying mechanisms for these effects remain poorly understood. The effect of resveratrol in parental CRC cell lines (HCT116, SW480) and their corresponding isogenic 5-FU-chemoresistant derived clones (HCT116R, SW480R) was examined by MTT assays, intercellular junction formation and apoptosis by electron- and immunoelectron microscopy, nuclear factor-kappaB (NF-κB) and NF-κB regulated gene products by western blot analysis in a 3D-alginate microenvironment. Resveratrol blocked the proliferation of all four CRC cell lines and synergized the invasion inhibitory effects of 5-FU. Interestingly, resveratrol induced a transition from 5-FU-induced formation of microvilli to a planar cell surface, which was concomitant with up-regulation of desmosomes, gap- and tight junctions (claudin-2) and adhesion molecules (E-cadherin) expression in HCT116 and HCT116R cells. Further, resveratrol significantly attenuated drug resistance through inhibition of epithelial-mesenchymal transition (EMT) factors (decreased vimentin and slug, increased E-cadherin) and down-regulation of NF-κB activation and its translocation to the nucleus and abolished NF-κB-regulated gene end-products (MMP-9, caspase-3). Moreover, this suppression was mediated through inhibition of IκBα kinase and IκBα phosphorylation and degradation. Our results demonstrate that resveratrol can potentiate the anti-tumor effects of 5-FU on CRC cells by chemosensitizing them, inhibiting an EMT phenotype via up-regulation of intercellular junctions and by down-regulation of NF-κB pathway. PMID:26310874

  16. Vertebrate limb bud formation is initiated by localized epithelial-to-mesenchymal transition.

    PubMed

    Gros, Jerome; Tabin, Clifford J

    2014-03-14

    Vertebrate limbs first emerge as small buds at specific locations along the trunk. Although a fair amount is known about the molecular regulation of limb initiation and outgrowth, the cellular events underlying these processes have remained less clear. We show that the mesenchymal limb progenitors arise through localized epithelial-to-mesenchymal transition (EMT) of the coelomic epithelium specifically within the presumptive limb fields. This EMT is regulated at least in part by Tbx5 and Fgf10, two genes known to control limb initiation. This work shows that limb buds initiate earlier than previously thought, as a result of localized EMT rather than differential proliferation rates. PMID:24626928

  17. Epithelial to mesenchymal transition-the roles of cell morphology, labile adhesion and junctional coupling.

    PubMed

    Abdulla, Tariq; Luna-Zurita, Luis; de la Pompa, José Luis; Schleich, Jean-Marc; Summers, Ron

    2013-08-01

    Epithelial to mesenchymal transition (EMT) is a fundamental process during development and disease, including development of the heart valves and tumour metastases. An extended cellular Potts model was implemented to represent the behaviour emerging from autonomous cell morphology, labile adhesion, junctional coupling and cell motility. Computer simulations normally focus on these functional changes independently whereas this model facilitates exploration of the interplay between cell shape changes, adhesion and migration. The simulation model is fitted to an in vitro model of endocardial EMT, and agrees with the finding that Notch signalling increases cell-matrix adhesion in addition to modulating cell-cell adhesion. PMID:23787029

  18. Epithelial-to-Mesenchymal Transition in Podocytes Mediated by Activation of NADPH Oxidase in Hyperhomocysteinemia

    PubMed Central

    Zhang, Chun; Xia, Min; Boini, Krishna M.; Li, Cai-Xia; Abais, Justine M.; Li, Xiao-Xue; Laperle, Laura A.; Li, Pin-Lan

    2012-01-01

    The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1) in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide (O2.−) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91phox (gp91−/−), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91+/+) mice, hHcys induced by a folate-free (FF) diet markedly enhanced expression of mesenchymal markers (FSP-1 and α-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91−/− mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91+/+ mice with hHcys were all significantly attenuated in gp91−/− mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury. PMID:21647593

  19. Neutrophil Granulocytes in Ovarian Cancer - Induction of Epithelial-To-Mesenchymal-Transition and Tumor Cell Migration

    PubMed Central

    Mayer, Christine; Darb-Esfahani, Silvia; Meyer, Anne-Sophie; Hübner, Katrin; Rom, Joachim; Sohn, Christof; Braicu, Ioana; Sehouli, Jalid; Hänsch, G. Maria; Gaida, Matthias M.

    2016-01-01

    Background: Ovarian cancer (OvCa) is a highly aggressive malignoma with a tumor-promoting microenvironment. Infiltration of polymorphonuclear neutrophils (PMN) is frequently seen, raising the question of their impact on tumor development. In that context, effects of PMN on human ovarian cancer cells were assessed. Methods: Human epithelial ovarian cancer cells were incubated with human PMN, lysate of PMN, or neutrophil elastase. Morphological alterations were observed by time-lapse video-microscopy, and the underlying molecular mechanism was analyzed by flow cytometry and Western blotting. Functional alternations were assessed by an in vitro wound healing assay. In parallel, a large cohort of n=334 primary OvCa tissue samples of various histological subtypes was histologically evaluated. Results: Co-cultivation of cancer cells with either PMN or PMN lysate causes a change of the polygonal epithelial phenotype of the cells towards a spindle shaped morphology, causing a cribriform cell growth. The PMN-induced alteration could be attributed to elastase, a major protease of PMN. Elastase-induced shape change was most likely due to the degradation of membranous E-cadherin, which results in loss of cell contacts and polarity. Moreover, in response to elastase, epithelial cytokeratins were downmodulated, in parallel with a nuclear translocation of β-catenin. These PMN-elastase induced alterations of cells are compatible with an epithelial-to-mesenchymal transition (EMT) of the cancer cells. Following EMT, the cells displayed a more migratory phenotype. In human biopsies, neutrophil infiltration was seen in 72% of the cases. PMN infiltrates were detected preferentially in areas with low E-cadherin expression. Conclusion: PMN in the microenvironment of OvCa can alter tumor cells towards a mesenchymal and migratory phenotype. PMID:27053953

  20. Prognostic implications of epithelial to mesenchymal transition related proteins (E-cadherin, Snail) and hypoxia inducible factor 1α in endometrioid endometrial carcinoma.

    PubMed

    Abouhashem, Nehal S; Ibrahim, Doaa Abdelaziz; Mohamed, Abdel Motaleb

    2016-06-01

    The epithelial-mesenchymal transition (EMT) is an important step in the invasion and metastasis of cancer. E-cadherin downregulation, which is essentially controlled by EMT-mediated proteins such as Snail, is a main molecular feature of this process. Tumor hypoxia is one of the essential biological phenomena that are associated with the development and progression of various solid tumors. Recently, hypoxia and hypoxia-inducible factor 1α (HIF-1α) signaling pathway were identified to have an essential role in the regulation of EMT phenotype. The aim of the study was to evaluate the prognostic impact of EMT-related proteins (E-cadherin, Snail) and HIF-1α in endometrioid endometrial carcinoma (EEC) among Egyptian women. Immunohistochemical evaluation of E-cadherin, Snail, and HIF-1α expression was performed using 50 cases of EEC. The relationship between protein expression and clinicopathological features was investigated. The frequency of immunopositivity for E-cadherin, Snail, and HIF-1α in our cases of EEC was 82%, 28%, and 66%, respectively. Reduced E-cadherin and increased nuclear expression of Snail as well as HIF-1α were significantly associated with histopathologic grade, clinical stage myometrial invasion, and lymph node involvement. Statistical analysis showed a positive correlation between HIF-1α overexpression and Snail upregulation (τ= +0.252, P= .025); however, E-cadherin expression level was inversely correlated with enhanced Snail expression (τ= -0.450, P< .001) as well as with HIF-1α overexpression (τ= -0.439, P< .001). The overall survival and progression-free survival were inversely related to Snail immunoreactivity and positively related to E-cadherin expression. E-cadherin and Snail have a predictive value in EEC. In conclusion, the current study reveals that both Snail and HIF-1α expressions are significantly associated with poor prognosis in EEC; however, E-cadherin expression is considered a marker of good prognosis. E-cadherin and

  1. Regulation of COX-2 expression and epithelial-to-mesenchymal transition by hypoxia-inducible factor-1α is associated with poor prognosis in hepatocellular carcinoma patients post TACE surgery

    PubMed Central

    HUANG, MINGSHENG; WANG, LONG; CHEN, JUNWEI; BAI, MINGJUN; ZHOU, CHUREN; LIU, SUJUAN; LIN, QU

    2016-01-01

    Currently, it is not entirely clear whether hypoxia-inducible factor-1α (HIF-1α) is involved in the regulation of COX-2 expression and epithelial-to-mesenchymal transition (EMT), and whether these events affect the prognosis of hepatocellular carcinoma (HCC) patients treated with transcatheter arterial chemoembolization (TACE). In this report the relationship between HIF-1α and COX-2 protein expression, EMT in tumor specimens from HCC patients after TACE surgery and the clinical significance of HIF-1α and COX-2 expression were analyzed using statistical approaches. HepG2 cells treated with CoCl2 was employed as a hypoxia cell model in vitro to study hypoxia-induced HIF-1α, COX-2 expression, and EMT alteration. The results showed that HIF-1α and COX-2 protein expression increased in HCC tissues after TACE surgery. Moreover, there was positive correlation between upregulation of HIF-1α and COX-2. Elevated expression of HIF-1α increased both Snail and Vimentin protein expression, while it reduced E-cadherin protein expression. It was further verified that hypoxia enhanced protein expression of HIF-1α and COX-2 in HepG2 cells treated with CoCl2. Upregulation of HIF-1α and COX-2, together with EMT alteration resulted in increased migration and invasion of HepG2 cells under hypoxia. In conclusion, TACE surgery results in aggravated hypoxia status, leading to increased HIF-1α protein expression in HCC tissue. To adapt to hypoxic environment, HIF-1α stimulates COX-2 protein expression and promotes EMT process in hepatocellular cancer cells, which enhances HCC invasion and metastasis, and might contribute to poor prognosis in HCC patients post TACE treatment. PMID:26984380

  2. Regulation of Epithelial-to-Mesenchymal Transition Using Biomimetic Fibrous Scaffolds.

    PubMed

    Ravikrishnan, Anitha; Ozdemir, Tugba; Bah, Mohamed; Baskerville, Karen A; Shah, S Ismat; Rajasekaran, Ayyappan K; Jia, Xinqiao

    2016-07-20

    Epithelial-to-mesenchymal transition (EMT) is a well-studied biological process that takes place during embryogenesis, carcinogenesis, and tissue fibrosis. During EMT, the polarized epithelial cells with a cuboidal architecture adopt an elongated fibroblast-like morphology. This process is accompanied by the expression of many EMT-specific molecular markers. Although the molecular mechanism leading to EMT has been well-established, the effects of matrix topography and microstructure have not been clearly elucidated. Synthetic scaffolds mimicking the meshlike structure of the basement membrane with an average fiber diameter of 0.5 and 5 μm were produced via the electrospinning of poly(ε-caprolactone) (PCL) and were used to test the significance of fiber diameter on EMT. Cell-adhesive peptide motifs were conjugated to the fiber surface to facilitate cell attachment. Madin-Darby Canine Kidney (MDCK) cells grown on these substrates showed distinct phenotypes. On 0.5 μm substrates, cells grew as compact colonies with an epithelial phenotype. On 5 μm scaffolds, cells were more individually dispersed and appeared more fibroblastic. Upon the addition of hepatocyte growth factor (HGF), an EMT inducer, cells grown on the 0.5 μm scaffold underwent pronounced scattering, as evidenced by the alteration of cell morphology, localization of focal adhesion complex, weakening of cell-cell adhesion, and up-regulation of mesenchymal markers. In contrast, HGF did not induce a pronounced scattering of MDCK cells cultured on the 5.0 μm scaffold. Collectively, our results show that the alteration of the fiber diameter of proteins found in the basement membrane may create enough disturbances in epithelial organization and scattering that might have important implications in disease progression. PMID:27322677

  3. Radical-containing ultrafine particulate matter initiates epithelial-to-mesenchymal transitions in airway epithelial cells.

    PubMed

    Thevenot, Paul T; Saravia, Jordy; Jin, Nili; Giaimo, Joseph D; Chustz, Regina E; Mahne, Sarah; Kelley, Matthew A; Hebert, Valeria Y; Dellinger, Barry; Dugas, Tammy R; Demayo, Francesco J; Cormier, Stephania A

    2013-02-01

    Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm(2)) caused substantial necrosis. At low doses (20 μg/cm(2)), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α-smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air-liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. PMID:23087054

  4. Triclosan potentiates epithelial-to-mesenchymal transition in anoikis-resistant human lung cancer cells.

    PubMed

    Winitthana, Thidarat; Lawanprasert, Somsong; Chanvorachote, Pithi

    2014-01-01

    Alteration of cancer cell toward mesenchymal phenotype has been shown to potentiate tumor aggressiveness by increasing cancer cell metastasis. Herein, we report the effect of triclosan, a widely used antibacterial agent found in many daily products, in enhancing the epithelial-to-mesenchymal transition (EMT) in aggressive anoikis resistant human H460 lung cancer cells. EMT has been long known to increase abilities of the cells to increase migration, invasion, and survival in circulating system. The present study reveals that treatment of the cancer cells with triclosan at the physiologically related concentrations significantly increased the colony number of the cancer cells assessed by tumor formation assay. Also, the mesenchymal-like morphology and decrease in cell-to-cell adhesion were observed in triclosan-treated cells. Importantly, western blot analysis revealed that triclosan-treated cells exhibited decreased E-cadherin, while the levels of EMT markers, namely N-cadherin, vimentin, snail and slug were found to be significantly up-regulated. Furthermore, EMT induced by triclosan treatment was accompanied by the activation of focal adhesion kinase/ATP dependent tyrosine kinase (FAK/Akt) and Ras-related C3 botulinum toxin substrate 1 (Rac1), which enhanced the ability of the cells to migrate and invade. In conclusion, we demonstrated for the first time that triclosan may potentiate cancer cells survival in detached condition and motility via the process of EMT. As mentioned capabilities are required for success in metastasis, the present study provides the novel toxicological information and encourages the awareness of triclosan use in cancer patients. PMID:25329306

  5. Meta-Analysis of Gene Expression Signatures Defining the Epithelial to Mesenchymal Transition during Cancer Progression

    PubMed Central

    Gröger, Christian J.; Grubinger, Markus; Waldhör, Thomas; Vierlinger, Klemens; Mikulits, Wolfgang

    2012-01-01

    The epithelial to mesenchymal transition (EMT) represents a crucial event during cancer progression and dissemination. EMT is the conversion of carcinoma cells from an epithelial to a mesenchymal phenotype that associates with a higher cell motility as well as enhanced chemoresistance and cancer stemness. Notably, EMT has been increasingly recognized as an early event of metastasis. Numerous gene expression studies (GES) have been conducted to obtain transcriptome signatures and marker genes to understand the regulatory mechanisms underlying EMT. Yet, no meta-analysis considering the multitude of GES of EMT has been performed to comprehensively elaborate the core genes in this process. Here we report the meta-analysis of 18 independent and published GES of EMT which focused on different cell types and treatment modalities. Computational analysis revealed clustering of GES according to the type of treatment rather than to cell type. GES of EMT induced via transforming growth factor-β and tumor necrosis factor-α treatment yielded uniformly defined clusters while GES of models with alternative EMT induction clustered in a more complex fashion. In addition, we identified those up- and downregulated genes which were shared between the multitude of GES. This core gene list includes well known EMT markers as well as novel genes so far not described in this process. Furthermore, several genes of the EMT-core gene list significantly correlated with impaired pathological complete response in breast cancer patients. In conclusion, this meta-analysis provides a comprehensive survey of available EMT expression signatures and shows fundamental insights into the mechanisms that are governing carcinoma progression. PMID:23251436

  6. Triclosan Potentiates Epithelial-To-Mesenchymal Transition in Anoikis-Resistant Human Lung Cancer Cells

    PubMed Central

    Winitthana, Thidarat; Lawanprasert, Somsong; Chanvorachote, Pithi

    2014-01-01

    Alteration of cancer cell toward mesenchymal phenotype has been shown to potentiate tumor aggressiveness by increasing cancer cell metastasis. Herein, we report the effect of triclosan, a widely used antibacterial agent found in many daily products, in enhancing the epithelial-to-mesenchymal transition (EMT) in aggressive anoikis resistant human H460 lung cancer cells. EMT has been long known to increase abilities of the cells to increase migration, invasion, and survival in circulating system. The present study reveals that treatment of the cancer cells with triclosan at the physiologically related concentrations significantly increased the colony number of the cancer cells assessed by tumor formation assay. Also, the mesenchymal-like morphology and decrease in cell-to-cell adhesion were observed in triclosan-treated cells. Importantly, western blot analysis revealed that triclosan-treated cells exhibited decreased E-cadherin, while the levels of EMT markers, namely N-cadherin, vimentin, snail and slug were found to be significantly up-regulated. Furthermore, EMT induced by triclosan treatment was accompanied by the activation of focal adhesion kinase/ATP dependent tyrosine kinase (FAK/Akt) and Ras-related C3 botulinum toxin substrate 1 (Rac1), which enhanced the ability of the cells to migrate and invade. In conclusion, we demonstrated for the first time that triclosan may potentiate cancer cells survival in detached condition and motility via the process of EMT. As mentioned capabilities are required for success in metastasis, the present study provides the novel toxicological information and encourages the awareness of triclosan use in cancer patients. PMID:25329306

  7. Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition

    PubMed Central

    Muir, Amanda B.; Dods, Kara; Noah, Yuli; Toltzis, Sarit; Chandramouleeswaran, Prasanna Modayur; Lee, Anna; Benitez, Alain; Bedenbaugh, Adam; Falk, Gary W.; Wells, Rebecca G.; Nakagawa, Hiroshi; Wang, Mei-Lun

    2015-01-01

    Background and Aims Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro. Methods and Results Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production. Conclusions Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis. PMID:25183431

  8. Stem cell state and the epithelial-to-mesenchymal transition: Implications for cancer therapy.

    PubMed

    Donnenberg, Vera S; Donnenberg, Albert D

    2015-06-01

    The cancer stem cell paradigm, the epithelial-to-mesenchymal transition and its converse, the mesenchymal-to-epithelial transition, have reached convergence. Implicit in this understanding is the notion that cancer cells can change state, and with such change come bidirectional alterations in motility, proliferative activity, and drug resistance. As such, tumors present a moving target for antineoplastic therapy. This article will review the evolving adult stem cell paradigm and how changes in our understanding of the bidirectional nature of cancer cell differentiation may affect the selection and timing of antineoplastic therapy. The goal is to determine how to best administer therapies potentially targeted against the cancer stem cell state in the context of established treatment regimens, and to evaluate long-term effects beyond tumor regression. PMID:25708160

  9. Epithelial to mesenchymal transition in head and neck squamous cell carcinoma.

    PubMed

    Smith, Ashley; Teknos, Theodoros N; Pan, Quintin

    2013-04-01

    Epithelial to mesenchymal transition (EMT) is a dynamic cellular process that is essential for the development of metastatic disease. During EMT, a tumor cell with epithelial characteristics transitions to a tumor cell with mesenchymal characteristics through modulation of cell polarity and adhesion. Two hallmark EMT proteins, E-Cadherin and Vimentin, are tightly controlled during EMT through multiple signal transduction pathways. Epidermal growth factor (EGF) and transforming growth factorβ (TGFβ) promote EMT by regulating a distinct set of transcription factors, including Snail and Twist. Snail, Twist, and Slug are integral to the induction of EMT through direct regulation of genes involved in cellular adhesion, migration, and invasion. This review highlights the current literature on EMT in HNSCC. Understanding the role of EMT will provide insight to the pathogenesis of disease progression and may lead to the development of novel anti-cancer therapeutics for metastatic HNSCC. PMID:23182398

  10. Pathogenesis of Type 2 Epithelial to Mesenchymal Transition (EMT) in Renal and Hepatic Fibrosis

    PubMed Central

    Tennakoon, Anusha H.; Izawa, Takeshi; Kuwamura, Mitsuru; Yamate, Jyoji

    2015-01-01

    Epithelial to mesenchymal transition (EMT), particularly, type 2 EMT, is important in progressive renal and hepatic fibrosis. In this process, incompletely regenerated renal epithelia lose their epithelial characteristics and gain migratory mesenchymal qualities as myofibroblasts. In hepatic fibrosis (importantly, cirrhosis), the process also occurs in injured hepatocytes and hepatic progenitor cells (HPCs), as well as ductular reaction-related bile epithelia. Interestingly, the ductular reaction contributes partly to hepatocarcinogenesis of HPCs, and further, regenerating cholangiocytes after injury may be derived from hepatic stellate cells via mesenchymal to epithelia transition, a reverse phenomenon of type 2 EMT. Possible pathogenesis of type 2 EMT and its differences between renal and hepatic fibrosis are reviewed based on our experimental data. PMID:26729181

  11. c-Jun N-terminal kinase 1 promotes transforming growth factor-β1-induced epithelial-to-mesenchymal transition via control of linker phosphorylation and transcriptional activity of Smad3.

    PubMed

    Velden, Jos L J van der; Alcorn, John F; Guala, Amy S; Badura, Elsbeth C H L; Janssen-Heininger, Yvonne M W

    2011-04-01

    Transforming growth factor (TGF)-β1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-β1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-β1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-β1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-β1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-β1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-β1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-β1-induced EMT. PMID:20581097

  12. Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity

    PubMed Central

    2011-01-01

    The epithelial-to-mesenchymal transition (EMT) is a critical developmental process that has recently come to the forefront of cancer biology. In breast carcinomas, acquisition of a mesenchymal-like phenotype that is reminiscent of an EMT, termed oncogenic EMT, is associated with pro-metastatic properties, including increased motility, invasion, anoikis resistance, immunosuppression and cancer stem cell characteristics. This oncogenic EMT is a consequence of cellular plasticity, which allows for interconversion between epithelial and mesenchymal-like states, and is thought to enable tumor cells not only to escape from the primary tumor, but also to colonize a secondary site. Indeed, the plasticity of cancer cells may explain the range of pro-metastatic traits conferred by oncogenic EMT, such as the recently described link between EMT and cancer stem cells and/or therapeutic resistance. Continued research into this relationship will be critical in developing drugs that block mechanisms of breast cancer progression, ultimately improving patient outcomes. PMID:22078097

  13. The roles of signaling pathways in epithelial-to-mesenchymal transition of PVR

    PubMed Central

    Chen, Zhenna; Shao, Yan

    2015-01-01

    Proliferative vitreoretinopathy (PVR) is the major cause of failure in patients undergoing surgery for rhegmatogenous retinal detachment (RRD). Characterized by the formation of an abnormal contractile membrane within the eye, PVR can cause tractional retinal redetachment. Epithelial-to-mesenchymal transition (EMT), in which epithelial cells morphologically and phenotypically transdifferentiate into mesenchymal cells, is the major pathological process implicated in PVR. Among the various cell types involved in the process, retinal pigment epithelium cells are primary contributors although, after decades of research, the mechanisms underlying EMT have remained elusive. Recently, signaling pathways, some involving growth factors, have been demonstrated to contribute to EMT. In this article, we review research to date about the roles of such signaling, including including transforming growth factor-beta-, hepatocyte growth factor-, platelet-derived growth factor-, and Notch-, Wnt/β-catenin-, and Hippo-signaling pathways, in the EMT of PVR. PMID:26109834

  14. Epicardial Epithelial-to-Mesenchymal Transition in Heart Development and Disease

    PubMed Central

    Krainock, Michael; Toubat, Omar; Danopoulos, Soula; Beckham, Allison; Warburton, David; Kim, Richard

    2016-01-01

    The epicardium is an epithelial monolayer that plays a central role in heart development and the myocardial response to injury. Recent developments in our understanding of epicardial cell biology have revealed this layer to be a dynamic participant in fundamental processes underlying the development of the embryonic ventricles, the coronary vasculature, and the cardiac valves. Likewise, recent data have identified the epicardium as an important contributor to reparative and regenerative processes in the injured myocardium. These essential functions of the epicardium rely on both non-cell autonomous and cell-autonomous mechanisms, with the latter featuring the process of epicardial Epithelial-to-Mesenchymal Transition (EMT). This review will focus on the induction and regulation of epicardial EMT, as it pertains to both cardiogenesis and the response of the myocardium to injury. PMID:26907357

  15. Controversies over the Epithelial-to-Mesenchymal Transition in Liver Fibrosis

    PubMed Central

    Taura, Kojiro; Iwaisako, Keiko; Hatano, Etsuro; Uemoto, Shinji

    2016-01-01

    Liver fibrosis is a universal consequence of chronic liver diseases. It is accompanied by activation of collagen-producing myofibroblasts, resulting in excessive deposition of extracellular matrix. The origin of myofibroblasts in the fibrotic liver has not been completely resolved and remains a matter of debate. Recently, the epithelial-to-mesenchymal transition (EMT) was proposed as one of the mechanisms that give rise to collagen-producing myofibroblasts in liver fibrosis. However, subsequent studies contradicted this hypothesis, and the EMT theory has become one of the most controversial and debatable issues in the field of liver fibrosis research. This review will summarize the existing literature on EMT in liver fibrosis and will analyze the causes for the contradictory results to draw a reasonable conclusion based on current knowledge in the field. PMID:26784242

  16. Alisertib induces cell cycle arrest and autophagy and suppresses epithelial-to-mesenchymal transition involving PI3K/Akt/mTOR and sirtuin 1-mediated signaling pathways in human pancreatic cancer cells.

    PubMed

    Wang, Feng; Li, Hai; Yan, Xiao-Gang; Zhou, Zhi-Wei; Yi, Zhi-Gang; He, Zhi-Xu; Pan, Shu-Ting; Yang, Yin-Xue; Wang, Zuo-Zheng; Zhang, Xueji; Yang, Tianxing; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Pancreatic cancer is the most aggressive cancer worldwide with poor response to current therapeutics. Alisertib (ALS), a potent and selective Aurora kinase A inhibitor, exhibits potent anticancer effects in preclinical and clinical studies; however, the effect and underlying mechanism of ALS in the pancreatic cancer treatment remain elusive. This study aimed to examine the effects of ALS on cell growth, autophagy, and epithelial-to-mesenchymal transition (EMT) and to delineate the possible molecular mechanisms in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that ALS exerted potent cell growth inhibitory, pro-autophagic, and EMT-suppressing effects in PANC-1 and BxPC-3 cells. ALS remarkably arrested PANC-1 and BxPC-3 cells in G2/M phase via regulating the expression of cyclin-dependent kinases 1 and 2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. ALS concentration-dependently induced autophagy in PANC-1 and BxPC-3 cells, which may be attributed to the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal-regulated kinases 1 and 2 (Erk1/2) but activation of 5'-AMP-dependent kinase signaling pathways. ALS significantly inhibited EMT in PANC-1 and BxPC-3 cells with an increase in the expression of E-cadherin and a decrease in N-cadherin. In addition, ALS suppressed the expression of sirtuin 1 (Sirt1) and pre-B cell colony-enhancing factor/visfatin in both cell lines with a rise in the level of acetylated p53. These findings show that ALS induces cell cycle arrest and promotes autophagic cell death but inhibits EMT in pancreatic cancer cells with the involvement of PI3K/Akt/mTOR, p38 MAPK, Erk1/2, and Sirt1-mediated signaling pathways. Taken together, ALS may represent a promising anticancer drug for pancreatic cancer treatment. More studies are warranted to investigate other molecular targets and

  17. [Inhibitory effect of Panax notoginseng saponins on alveolar epithelial to mesenchymal transition].

    PubMed

    Ren, Zhou-xin; Yu, Hai-bin; Li, Jian-sheng; Shen, Jun-ling; Li, Jun-kai; Luo, Shan

    2015-12-01

    In the study, the effects of Panax notoginseng saponins (PNS) on alveolar epithelial to mesenchymal transition (EMT) and extracellular matrix degradation were observed in a type of human alveolar epithelial cell, A549 cells, stimulated by TGF-beta1. Firstly, MTT method was applied to evaluation of cellular proliferation and found that PNS from 12.5 mg x L(-1) to 200 mg x L(-1) dosage could not inhibit significantly cellular proliferation. Then, cells were divided into five groups, normal group, TGF-beta1 group, TGF-beta1 + 50 mg x L(-1) PNS group, TGF-beta1 + 100 mg x L(-1) PNS group and TGF-beta1 + 200 mg x L(-1) PNS group. Normal cells were not stimulatec by TGF-beta1; TGF-beta1 cells were only stimulated by TGF-beta1 and the other cells were stimulated by TGF-beta1 with different doses of PNS, respectively. After stimulation, cells and supernatants were collected for assays. Cellular roundness was applied to quantitative evaluation of morphological change. Immunocytochemistry was applied to examine E-cadherion, a-SMA and FN proteins expression in the cells. Enzyme linked-immunosorbent assay was applied to MMP-9 and TIMP-1 levels. The results showed that EMT of A549 cells was induced by TGF-beta1, showing significant change of roundness, E-cadherion, alpha-SMA and FN (P < 0.05, P < 0.01). Compared to TGF-beta1, PNS significantly inhibited the changes of roundness (P < 0.05), FN and alpha-SMA (P < 0.05, P < 0.01) and not significantly inhibited the change of E-cadherion. Furthermore, MMP-9 levels were significantly increased by TGFbeta1 stimulation (P < 0.05), without significant change of TIMP-1. Compared with TGF-beta1, PNS could significantly increase MMP-9 level (P < 0.05) and decrease TIMP-1 levels (P < 0.05, P < 0.01). In conclusion, PNS could inhibit alveolar epithelial cell EMT induced by TGF-beta1, with increase of extracellular matrix degradation ability, which showed anti-fibrosis of lung ability. PMID:27141681

  18. A comprehensive DNA methylation profile of epithelial-to-mesenchymal transition.

    PubMed

    Carmona, F Javier; Davalos, Veronica; Vidal, Enrique; Gomez, Antonio; Heyn, Holger; Hashimoto, Yutaka; Vizoso, Miguel; Martinez-Cardus, Anna; Sayols, Sergi; Ferreira, Humberto J; Sánchez-Mut, Jose V; Morán, Sebastián; Margelí, Mireia; Castella, Eva; Berdasco, Maria; Stefansson, Olafur A; Eyfjord, Jorunn E; Gonzalez-Suarez, Eva; Dopazo, Joaquín; Orozco, Modesto; Gut, Ivo G; Esteller, Manel

    2014-10-01

    Epithelial-to-mesenchymal transition (EMT) is a plastic process in which fully differentiated epithelial cells are converted into poorly differentiated, migratory and invasive mesenchymal cells, and it has been related to the metastasis potential of tumors. This is a reversible process and cells can also eventually undergo mesenchymal-to-epithelial transition. The existence of a dynamic EMT process suggests the involvement of epigenetic shifts in the phenotype. Herein, we obtained the DNA methylomes at single-base resolution of Madin-Darby canine kidney cells undergoing EMT and translated the identified differentially methylated regions to human breast cancer cells undergoing a gain of migratory and invasive capabilities associated with the EMT phenotype. We noticed dynamic and reversible changes of DNA methylation, both on promoter sequences and gene-bodies in association with transcription regulation of EMT-related genes. Most importantly, the identified DNA methylation markers of EMT were present in primary mammary tumors in association with the epithelial or the mesenchymal phenotype of the studied breast cancer samples. PMID:25106427

  19. GM130 regulates epithelial-to-mesenchymal transition and invasion of gastric cancer cells via snail

    PubMed Central

    Zhao, Jianquan; Yang, Chun; Guo, Shujun; Wu, Yonggang

    2015-01-01

    Gastric cancer is one of the most common causes of digestive tract tumor. Despite of recent advances in surgical techniques and development of adjuvant therapy, the underlying mechanisms of gastric cancer remain poorly understood and relevant insight into novel treatment strategies using gene target remains incomplete. Recently, several studies report that epithelial to mesenchymal transition (EMT) is a crucial process for the invasion and metastasis of epithelial tumors; however, the molecular mechanisms underlying this transition are unknown. As a cis-Golgi matrix protein, GM130 plays an important role in cell cycle progression and transport of protein in the secretory pathway. In this study, we found that GM130 expression has a positive correlation with the pathological differentiation and tumor node metastasis (TNM) stage of gastric cancer. High GM130 expression levels also predict shorter overall survival of gastric cancer patients. RNA interference-mediated knockdown of GM130 expression increased epithelial marker (E-cadherin) and decreased mesenchymal marker (N-cadherin and vimentin) expression in gastric cancer cells, suppressing cell invasion, and tumor formation. Furthermore, we found that GM130 upregulated expression of the key EMT regulator Snail (SNAI1), which mediated EMT activation and cell invasion by GM130. Taken together, our study indicates GM130 may be a promising therapeutic biomarker for gastric cancer. PMID:26617790

  20. GM130 regulates epithelial-to-mesenchymal transition and invasion of gastric cancer cells via snail.

    PubMed

    Zhao, Jianquan; Yang, Chun; Guo, Shujun; Wu, Yonggang

    2015-01-01

    Gastric cancer is one of the most common causes of digestive tract tumor. Despite of recent advances in surgical techniques and development of adjuvant therapy, the underlying mechanisms of gastric cancer remain poorly understood and relevant insight into novel treatment strategies using gene target remains incomplete. Recently, several studies report that epithelial to mesenchymal transition (EMT) is a crucial process for the invasion and metastasis of epithelial tumors; however, the molecular mechanisms underlying this transition are unknown. As a cis-Golgi matrix protein, GM130 plays an important role in cell cycle progression and transport of protein in the secretory pathway. In this study, we found that GM130 expression has a positive correlation with the pathological differentiation and tumor node metastasis (TNM) stage of gastric cancer. High GM130 expression levels also predict shorter overall survival of gastric cancer patients. RNA interference-mediated knockdown of GM130 expression increased epithelial marker (E-cadherin) and decreased mesenchymal marker (N-cadherin and vimentin) expression in gastric cancer cells, suppressing cell invasion, and tumor formation. Furthermore, we found that GM130 upregulated expression of the key EMT regulator Snail (SNAI1), which mediated EMT activation and cell invasion by GM130. Taken together, our study indicates GM130 may be a promising therapeutic biomarker for gastric cancer. PMID:26617790

  1. The Significance of Epithelial-to-Mesenchymal Transition for Circulating Tumor Cells

    PubMed Central

    Kölbl, Alexandra C.; Jeschke, Udo; Andergassen, Ulrich

    2016-01-01

    Epithelial to mesenchymal transition (EMT) is a process involved in embryonic development, but it also plays a role in remote metastasis formation in tumor diseases. During this process cells lose their epithelial features and adopt characteristics of mesenchymal cells. Thereby single tumor cells, which dissolve from the primary tumor, are enabled to invade the blood vessels and travel throughout the body as so called “circulating tumor cells” (CTCs). After leaving the blood stream the reverse process of EMT, the mesenchymal to epithelial transition (MET) helps the cells to seed in different tissues, thereby generating the bud of metastasis formation. As metastasis is the main reason for tumor-associated death, CTCs and the EMT process are in the focus of research in recent years. This review summarizes what was already found out about the molecular mechanisms driving EMT, the consequences of EMT for tumor cell detection, and suitable markers for the detection of CTCs which underwent EMT. The research work done in this field could open new roads towards combating cancer. PMID:27529216

  2. Regulation of Myofibroblast Differentiation by miR-424 during Epithelial-to-Mesenchymal Transition

    PubMed Central

    Xiao, Xiao; Huang, Chaoqun; Zhao, Chunling; Gou, Xuxu; Senavirathna, Lakmini K.; Hinsdale, Myron; Lloyd, Pamela; Liu, Lin

    2014-01-01

    Idiopathic pulmonary fibrosis (IPF) is one of the most common and severe interstitial lung diseases. Epithelial-to-mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype. This process has been shown to contribute to IPF. MicroRNAs (miRNAs) are small non-coding RNAs of 18 to 24 nucleotides in length which regulate gene expression. Several studies have implicated miRNAs in EMT; however, specific miRNAs that regulate EMT in IPF have not yet been identified. In this study, we identified 6 up-regulated and 3 down-regulated miRNAs in a human lung epithelial cell EMT model using miRNA microarray and real-time PCR. Overexpression of one of these up-regulated miRNAs, miR-424, increased the expression of α-smooth muscle actin, an indicator of myofibroblast differentiation, but had no effects on the epithelial or mesenchymal cell markers. miR-424 enhanced the activity of the TGF-β signaling pathway, as demonstrated by a luciferase reporter assay. Further experiments showed that miR-424 decreased the protein expression of Smurf2, a negative regulator of TGF-β signaling, indicating that miR-424 exerts a forward regulatory loop in the TGF-β signaling pathway. Our results suggest that miR-424 regulates the myofibroblast differentiation during EMT by potentiating the TGF-β signaling pathway, likely through Smurf2. PMID:25524739

  3. Bioinformatic approaches to augment study of epithelial-to-mesenchymal transition in lung cancer.

    PubMed

    Beck, Tim N; Chikwem, Adaeze J; Solanki, Nehal R; Golemis, Erica A

    2014-10-01

    Bioinformatic approaches are intended to provide systems level insight into the complex biological processes that underlie serious diseases such as cancer. In this review we describe current bioinformatic resources, and illustrate how they have been used to study a clinically important example: epithelial-to-mesenchymal transition (EMT) in lung cancer. Lung cancer is the leading cause of cancer-related deaths and is often diagnosed at advanced stages, leading to limited therapeutic success. While EMT is essential during development and wound healing, pathological reactivation of this program by cancer cells contributes to metastasis and drug resistance, both major causes of death from lung cancer. Challenges of studying EMT include its transient nature, its molecular and phenotypic heterogeneity, and the complicated networks of rewired signaling cascades. Given the biology of lung cancer and the role of EMT, it is critical to better align the two in order to advance the impact of precision oncology. This task relies heavily on the application of bioinformatic resources. Besides summarizing recent work in this area, we use four EMT-associated genes, TGF-β (TGFB1), NEDD9/HEF1, β-catenin (CTNNB1) and E-cadherin (CDH1), as exemplars to demonstrate the current capacities and limitations of probing bioinformatic resources to inform hypothesis-driven studies with therapeutic goals. PMID:25096367

  4. The cytoplasmic extension of the integrin β6 subunit regulates epithelial-to-mesenchymal transition.

    PubMed

    Lee, Carlin; Lee, Casey; Lee, Stacey; Siu, Amanda; Ramos, Daniel M

    2014-02-01

    Prognosis for oral cancer patients has not improved in over 60 years due to invasion and recurrence. To understand the invasive behavior of this tumor, we evaluated the role of the αvβ6 integrin. Invasive oral SCC cells express the αvβ6 integrin, which contains an 11-amino-acid extension on its β-subunit unique to the integrin family. We determined that this β6 cytoplasmic extension regulates the composition of the intermediate filament network and the organization of signaling structures called focal contacts. The auto-phosphorylation of FAK, which is localized to focal contacts, was also regulated by the β6-cytoplasmic tail, as were the transcription factors Notch and STAT3. Lastly, we also determined that activation of MAPK required the full-length β6 integrin. Together these results indicate that the signaling critical to epithelial-to-mesenchymal transition (EMT) is regulated by the β6 integrin cytoplasmic domain. PMID:24510996

  5. Snail nuclear transport: the gateways regulating epithelial-to-mesenchymal transition?

    PubMed

    Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Mohammad, Ramzi M; Azmi, Asfar S

    2014-08-01

    Epithelial-to-mesenchymal transition (EMT) and the reverse process (MET) play central role in organ developmental biology. It is a fine tuned process that when disturbed leads to pathological conditions especially cancers with aggressive and metastatic behavior. Snail is an oncogene that has been well established to be a promoter of EMT through direct repression of epithelial morphology promoter E-cadherin. It can function in the nucleus, in the cytosol and as discovered recently, extracellularly through secretory vesicular structures. The intracellular transport of snail has for long been shown to be regulated by the nuclear pore complex. One of the Karyopherins, importin alpha, mediates snail import, while exportin 1 (Xpo1) also known as chromosome maintenance region 1 (CRM1) is its major nuclear exporter. A number of additional biological regulators are emerging that directly modulate Snail stability by altering its subcellular localization. These observations indicate that targeting the nuclear transport machinery could be an important and as of yet, unexplored avenue for therapeutic intervention against the EMT processes in cancer. In parallel, a number of novel agents that disrupt nuclear transport have recently been discovered and are being explored for their anti-cancer effects in the early clinical settings. Through this review we provide insights on the mechanisms regulating snail subcellular localization and how this impacts EMT. We discuss strategies on how the nuclear transport function can be harnessed to rein in EMT through modulation of snail signaling. PMID:24954011

  6. Snail Nuclear Transport: the Gateways Regulating Epithelial-to-Mesenchymal Transition?

    PubMed Central

    Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Mohammad, Ramzi M.; Azmi, Asfar S.

    2014-01-01

    Epithelial-to-mesenchymal transition (EMT) and the reverse process (MET) plays central role in organ developmental biology. It is a fine tuned process that when disturbed leads to pathological conditions especially cancers with aggressive and metastatic behavior. Snail is an oncogene that has been well established to be a promoter of EMT through direct repression of epithelial morphology promoter E-cadherin. It can function in the nucleus, in the cytosol and as discovered recently, extracellularly through secretory vesicular structures. The intracellular transport of snail has for long been shown to be regulated by the nuclear pore complex. One of the Karyopherins, importin alpha, mediates snail import, while importin beta/exportin 1 (Xpo1) or chromosome maintenance region 1 (CRM1) is its major nuclear exporter. A number of additional biological regulators are emerging that directly modulate Snail stability by altering its subcellular localization. These observations indicate that targeting the nuclear transport machinery could be an important and as of yet, unexplored avenue for therapeutic intervention against the EMT processes in cancer. In parallel, a number of novel agents that disrupt nuclear transport have recently been discovered and are being explored for their anti-cancer effects in the early clinical settings. Through this review we provide insights on the mechanisms regulating snail subcellular localization and how this impacts EMT. We discuss strategies on how the nuclear transport function can be harnessed to rein in EMT through modulation of snail signaling. PMID:24954011

  7. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.

    PubMed

    Fischer, Kari R; Durrans, Anna; Lee, Sharrell; Sheng, Jianting; Li, Fuhai; Wong, Stephen T C; Choi, Hyejin; El Rayes, Tina; Ryu, Seongho; Troeger, Juliane; Schwabe, Robert F; Vahdat, Linda T; Altorki, Nasser K; Mittal, Vivek; Gao, Dingcheng

    2015-11-26

    The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment. PMID:26560033

  8. CPEB1 mediates epithelial-to-mesenchyme transition and breast cancer metastasis.

    PubMed

    Nagaoka, K; Fujii, K; Zhang, H; Usuda, K; Watanabe, G; Ivshina, M; Richter, J D

    2016-06-01

    In mouse mammary epithelial cells, cytoplasmic polyadenylation element binding protein 1 (CPEB1) mediates the apical localization of ZO-1 mRNA, which encodes a critical tight junction component. In mice lacking CPEB1 and in cultured cells from which CPEB has been depleted, randomly distributed ZO-1 mRNA leads to the loss of cell polarity. We have investigated whether this diminution of polarity results in an epithelial-to-mesenchyme (EMT) transition and possible increased metastatic potential. Here, we show that CPEB1-depleted mammary epithelial cells alter their gene expression profile in a manner consistent with an EMT and also become motile, which are made particularly robust when cells are treated with transforming growth factor-β, an enhancer of EMT. CPEB1-depleted mammary cells become metastatic to the lung following injection into mouse fat pads while ectopically expressed CPEB1 prevents metastasis. Surprisingly, CPEB1 depletion causes some EMT/metastasis-related mRNAs to have shorter poly(A) tails while other mRNAs to have longer poly(A) tails. Matrix metalloproteinase 9 (MMP9) mRNA, which encodes a metastasis-promoting factor, undergoes poly(A) lengthening and enhanced translation upon CPEB reduction. Moreover, in human breast cancer cells that become progressively more metastatic, CPEB1 is reduced while MMP9 becomes more abundant. These data suggest that at least in part, CPEB1 regulation of MMP9 mRNA expression mediates metastasis of breast cancer cells. PMID:26411364

  9. An epithelial to mesenchymal transition programme does not usually drive the phenotype of invasive lobular carcinomas.

    PubMed

    McCart Reed, Amy E; Kutasovic, Jamie R; Vargas, Ana C; Jayanthan, Janani; Al-Murrani, Amel; Reid, Lynne E; Chambers, Rachael; Da Silva, Leonard; Melville, Lewis; Evans, Elizabeth; Porter, Alan; Papadimos, David; Thompson, Erik W; Lakhani, Sunil R; Simpson, Peter T

    2016-03-01

    Epithelial to mesenchymal transition (EMT) is a cellular phenotype switching phenomenon which occurs during normal development and is proposed to promote tumour cell invasive capabilities during tumour progression. Invasive lobular carcinoma (ILC) is a histological special type of breast cancer with a peculiar aetiology - the tumour cells display an invasive growth pattern, with detached, single cells or single files of cells, and a canonical feature is the loss of E-cadherin expression. These characteristics are indicative of an EMT or at the very least that they represent some plasticity between phenotypes. While some gene expression profiling data support this view, the tumour cells remain epithelial and limited immunohistochemistry data suggest that EMT markers may not feature prominently in ILC. We assessed the expression of a panel of EMT markers (fibronectin, vimentin, N-cadherin, smooth muscle actin, osteonectin, Snail, Twist) in 148 ILCs and performed a meta-analysis of publically available molecular data from 154 ILCs. Three out of 148 (2%) ILCs demonstrated an early and coordinated alteration of multiple EMT markers (down-regulation of E-cadherin, nuclear TWIST, and up-regulation of vimentin, osteonectin, and smooth muscle actin). However, the data overall do not support a role for EMT in defining the phenotypic peculiarities of the majority of ILCs. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:26510554

  10. Combinatorial Interventions Inhibit the Epithelial-to-Mesenchymal Transition and Support Hybrid Cellular Phenotypes

    NASA Astrophysics Data System (ADS)

    Zanudo, Jorge G. T.; Steinway, S. N.; Michel, P. J.; Feith, D. J.; Loughran, T. P., Jr.; Albert, Reka

    Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to leave the primary tumor site and spread to other parts of the body. The molecular network regulating EMT involves the cooperation and cross-talk between multiple signaling pathways and key transcription factors, which we incorporated into systems-level logical network model for EMT. Using the EMT network model, we investigate potential EMT-suppressing interventions by identifying which individual and combinatorial perturbations suppress the induction of EMT by TGF β, an important signal driving EMT in liver cancer. We find that all non-trivial interventions are combinatorial and involve the inhibition of the SMAD complex together with other targets, several of which we experimentally tested and validated using liver cancer cell lines. We compare the combinatorial interventions with the results from a network control method we recently developed, which allowed us to determine the specific feedback regulatory motifs through which the interventions suppress EMT. Our results also reveal that blocking certain network components gives rise to steady states that are intermediate to the epithelial and mesenchymal states, supporting the existence of hybrid epithelial-mesenchymal states. Supported by NSF Grants PHY 1205840 and IIS 1161001, and NIH Grant F30DK093234.

  11. Glycosylation in Cancer: Interplay between Multidrug Resistance and Epithelial-to-Mesenchymal Transition?

    PubMed Central

    da Fonseca, Leonardo Marques; da Silva, Vanessa Amil; Freire-de-Lima, Leonardo; Previato, José Osvaldo; Mendonça-Previato, Lucia; Capella, Márcia Alves Marques

    2016-01-01

    The expression of unusual glycan structures is a hallmark of cancer progression, and their functional roles in cancer biology have been extensively investigated in epithelial-to-mesenchymal transition (EMT) models. EMT is a physiological process involved in embryonic development and wound healing. It is characterized by loss of epithelial cell polarity and cell adhesion, permitting cell migration, and thus formation of new epithelia. However, this process is unwanted when occurring outside their physiological limit, resulting in fibrosis of organs and progression of cancer and metastasis. Several studies observed that EMT is related to the acquisition of multidrug resistance (MDR) phenotype, a condition in which cancer cells acquire resistance to multiple different drugs, which has virtually nothing in common. However, although some studies suggested interplay between these two apparently distinct phenomena, almost nothing is known about this possible relationship. A common pathway to them is the need for glycosylation, a post-translational modification that can alter biological function. Thus, this review intends to compile the main facts obtained until now in these two areas, as an effort to unravel the relationship between EMT and MDR. PMID:27446804

  12. Intratumoral heterogeneity: Clonal cooperation in epithelial-to-mesenchymal transition and metastasis

    PubMed Central

    Neelakantan, Deepika; Drasin, David J; Ford, Heide L

    2015-01-01

    Although phenotypic intratumoral heterogeneity was first described many decades ago, the advent of next-generation sequencing has provided conclusive evidence that in addition to phenotypic diversity, significant genotypic diversity exists within tumors. Tumor heterogeneity likely arises both from clonal expansions, as well as from differentiation hierarchies existent in the tumor, such as that established by cancer stem cells (CSCs) and non-CSCs. These differentiation hierarchies may arise due to genetic mutations, epigenetic alterations, or microenvironmental influences. An additional differentiation hierarchy within epithelial tumors may arise when only a few tumor cells trans-differentiate into mesenchymal-like cells, a process known as epithelial-to-mesenchymal transition (EMT). Again, this process can be influenced by both genetic and non-genetic factors. In this review we discuss the evidence for clonal interaction and cooperation for tumor maintenance and progression, particularly with respect to EMT, and further address the far-reaching effects that tumor heterogeneity may have on cancer therapy. PMID:25482627

  13. Epithelial-to-Mesenchymal Transition in Diabetic Nephropathy: Fact or Fiction?

    PubMed Central

    Loeffler, Ivonne; Wolf, Gunter

    2015-01-01

    The pathophysiology of diabetic nephropathy (DN), one of the most serious complications in diabetic patients and the leading cause of end-stage renal disease worldwide, is complex and not fully elucidated. A typical hallmark of DN is the excessive deposition of extracellular matrix (ECM) proteins in the glomerulus and in the renal tubulointerstitium, eventually leading to glomerulosclerosis and interstitial fibrosis. Although it is obvious that myofibroblasts play a major role in the synthesis and secretion of ECM, the origin of myofibroblasts in DN remains the subject of controversial debates. A number of studies have focused on epithelial-to-mesenchymal transition (EMT) as one source of matrix-generating fibroblasts in the diseased kidney. EMT is characterized by the acquisition of mesenchymal properties by epithelial cells, preferentially proximal tubular cells and podocytes. In this review we comprehensively review the literature and discuss arguments both for and against a function of EMT in renal fibrosis in DN. While the precise extent of the contribution to nephrotic fibrosis is certainly arduous to quantify, the picture that emerges from this extensive body of literature suggests EMT as a major source of myofibroblasts in DN. PMID:26473930

  14. Altered expression of epithelial-to-mesenchymal transition proteins in extraprostatic prostate cancer

    PubMed Central

    Verrill, Clare; Cerundolo, Lucia; Mckee, Chad; White, Michael; Kartsonaki, Christiana; Fryer, Eve; Morris, Emma; Brewster, Simon; Ratnayaka, Indrika; Marsden, Luke; Lilja, Hans; Muschel, Ruth; Lu, Xin; Hamdy, Freddie; Bryant, Richard J.

    2016-01-01

    Epithelial to mesenchymal transition (EMT) of cancer cells involves loss of epithelial polarity and adhesiveness, and gain of invasive and migratory mesenchymal behaviours. EMT occurs in prostate cancer (PCa) but it is unknown whether this is in specific areas of primary tumours. We examined whether any of eleven EMT-related proteins have altered expression or subcellular localisation within the extraprostatic extension component of locally advanced PCa compared with other localisations, and whether similar changes may occur in in vitro organotypic PCa cell cultures and in vivo PCa models. Expression profiles of three proteins (E-cadherin, Snail, and α-smooth muscle actin) were significantly different in extraprostatic extension PCa compared with intra-prostatic tumour, and 18/27 cases had an expression change of at least one of these three proteins. Of the three significantly altered EMT proteins in pT3 samples, one showed similar significantly altered expression patterns in in vitro organotypic culture models, and two in in vivo Pten−/− model samples. These results suggest that changes in EMT protein expression can be observed in the extraprostatic extension component of locally invasive PCa. The biology of some of these changes in protein expression may be studied in certain in vitro and in vivo PCa models. PMID:26701730

  15. Mechanisms of the epithelial-to-mesenchymal transition in sea urchin embryos

    PubMed Central

    Katow, Hideki

    2015-01-01

    Sea urchin mesenchyme is composed of the large micromere-derived spiculogenetic primary mesenchyme cells (PMC), veg2-tier macromere-derived non-spiculogenetic mesenchyme cells, the small micromere-derived germ cells, and the macro- and mesomere-derived neuronal mesenchyme cells. They are formed through the epithelial-to-mesenchymal transition (EMT) and possess multipotency, except PMCs that solely differentiate larval spicules. The process of EMT is associated with modification of epithelial cell surface property that includes loss of affinity to the apical and basal extracellular matrices, inter-epithelial cell adherens junctions and epithelial cell surface-specific proteins. These cell surface structures and molecules are endocytosed during EMT and utilized as initiators of cytoplasmic signaling pathways that often initiate protein phosphorylation to activate the gene regulatory networks. Acquisition of cell motility after EMT in these mesenchyme cells is associated with the expression of proteins such as Lefty, Snail and Seawi. Structural simplicity and genomic database of this model will further promote detailed EMT research. PMID:26716069

  16. Endocellular polyamine availability modulates epithelial-to-mesenchymal transition and unfolded protein response in MDCK cells.

    PubMed

    Prunotto, Marco; Compagnone, Alessandra; Bruschi, Maurizio; Candiano, Giovanni; Colombatto, Sebastiano; Bandino, Andrea; Petretto, Andrea; Moll, Solange; Bochaton-Piallat, Marie Luce; Gabbiani, Giulio; Dimuccio, Veronica; Parola, Maurizio; Citti, Lorenzo; Ghiggeri, Gianmarco

    2010-06-01

    Epithelial-to-mesenchymal transition (EMT) is involved in embryonic development as well as in several pathological conditions. Literature indicates that polyamine availability may affect transcription of c-myc, matrix metalloproteinase (MMP)1, MMP2, TGFbeta(1), and collagen type I mRNA. The aim of this study was to elucidate polyamines role in EMT in vitro. Madin-Darby canine kidney (MDCK) cells were subjected to experimental manipulation of intracellular levels of polyamines. Acquisition of mesenchymal phenotype was evaluated by means of immunofluorescence, western blots, and zymograms. MDCK cells were then subjected to 2D gel proteomic study and incorporation of a biotinilated polyamine (BPA). Polyamine endocellular availability modulated EMT process. Polyamine-depleted cells treated with TGFbeta(1) showed enhanced EMT with a marked decrease of E-cadherin expression at plasma membrane level and an increased expression of mesenchymal markers such as fibronectin and alpha-smooth muscle actin. Polyamine-depleted cells showed a twofold increased expression of the rough endoplasmic reticulum (ER)-stress proteins GRP78, GRP94, and HSP90 alpha/beta in 2D gels. The latter data were confirmed by western blot analysis. Administration of BPA showed that polyamines are covalently linked, within the cell, to ER-stress proteins. Intracellular polyamine availability affects EMT in MDCK cells possibly through the modulation of ER-stress protein homeostasis. PMID:20212449

  17. Regulation of heterochromatin transcription by Snail1/LOXL2 during epithelial-to-mesenchymal transition.

    PubMed

    Millanes-Romero, Alba; Herranz, Nicolás; Perrera, Valentina; Iturbide, Ane; Loubat-Casanovas, Jordina; Gil, Jesús; Jenuwein, Thomas; García de Herreros, Antonio; Peiró, Sandra

    2013-12-12

    Although heterochromatin is enriched with repressive traits, it is also actively transcribed, giving rise to large amounts of noncoding RNAs. Although these RNAs are responsible for the formation and maintenance of heterochromatin, little is known about how their transcription is regulated. Here, we show that the Snail1 transcription factor represses mouse pericentromeric transcription, acting through the H3K4 deaminase LOXL2. Since Snail1 plays a key role in the epithelial-to-mesenchymal transition (EMT), we analyzed the regulation of heterochromatin transcription in this process. At the onset of EMT, one of the major structural heterochromatin proteins, HP1α, is transiently released from heterochromatin foci in a Snail1/LOXL2-dependent manner, concomitantly with a downregulation of major satellite transcription. Moreover, preventing the downregulation of major satellite transcripts compromised the migratory and invasive behavior of mesenchymal cells. We propose that Snail1 regulates heterochromatin transcription through LOXL2, thus creating the favorable transcriptional state necessary for completing EMT. PMID:24239292

  18. Thymoquinone inhibits cancer metastasis by downregulating TWIST1 expression to reduce epithelial to mesenchymal transition

    PubMed Central

    Khan, Md. Asaduzzaman; Tania, Mousumi; Wei, Chunli; Mei, Zhiqiang; Fu, Shelly; Cheng, Jingliang; Xu, Jianming; Fu, Junjiang

    2015-01-01

    Proteins that promote epithelial to mesenchymal transition (EMT) are associated with cancer metastasis. Inhibition of EMT regulators may be a promising approach in cancer therapy. In this study, Thymoquinone (TQ) was used to treat cancer cell lines to investigate its effects on EMT-regulatory proteins and cancer metastasis. We show that TQ inhibited cancer cell growth, migration and invasion in a dose-dependent manner. At the molecular level, TQ treatment decreased the transcriptional activity of the TWIST1 promoter and the mRNA expression of TWIST1, an EMT-promoting transcription factor. Accordingly, TQ treatment also decreased the expression of TWIST1-upregulated genes such as N-Cadherin and increased the expression of TWIST1-repressed genes such as E-Cadherin, resulting in a reduction of cell migration and invasion. TQ treatment also inhibited the growth and metastasis of cancer cell-derived xenograft tumors in mice but partially attenuated the migration and invasion in TWIST1-overexpressed cell lines. Furthermore, we found that TQ treatment enhanced the promoter DNA methylation of the TWIST1 gene in BT 549 cells. Together, these results demonstrate that TQ treatment inhibits TWIST1 promoter activity and decreases its expression, leading to the inhibition of cancer cell migration, invasion and metastasis. These findings suggest TQ as a potential small molecular inhibitor of cancer growth and metastasis. PMID:26023736

  19. HMG20A is required for SNAI1-mediated epithelial to mesenchymal transition.

    PubMed

    Rivero, S; Ceballos-Chávez, M; Bhattacharya, S S; Reyes, J C

    2015-10-01

    HMG20A is a high mobility group (HMG) domain containing protein homologous to HMG20B, a core subunit of the Lys-specific demethylase 1/REST co-repressor 1 (LSD1-CoREST) histone demethylase complex. Here, we show that HMG20A can replace HMG20B and, therefore, they are mutually exclusive subunits of the complex. Both proteins interact through a coiled-coil domain with BHC80, another subunit of the LSD1-CoREST complex. To investigate the functional differences between the two proteins, we performed transcriptomic analysis of HMG20A- and HMG20B-depleted cells. Analysis of the misregulated genes in HMG20A-knockdown cells evidenced a high proportion of genes related to the epithelial-to-mesenchymal transition (EMT) process. EMT occurs during embryonic development or during the course of malignant cancer progression and consists in the dynamic and reversible transitions between epithelial and mesenchymal phenotypes. We show that HMG20A together with LSD1 are required for SNAI1-dependent repression of epithelial genes and for (transforming growth factor β) TGF-β-triggered EMT. Importantly, HMG20A-depleted cells displayed reduced binding of LSD1 to epithelial gene promoters and increased methylation of lysine 4 of histone H3, suggesting a role of HMG20A in recruiting or in stabilizing the complex at the chromatin. SNAI1 and the TGF-β-related transcription factor SMAD4 were found to be associated with the LSD1-CoREST complex containing HMG20A. Furthermore, we show that HMG20A-depleted cells displayed reduced motility and invasion activity. Finally, we show that expression of HMG20A correlates positively with mesenchymal markers and negatively with epithelial markers in human tumor samples. Taken together, our data demonstrate that HMG20A is essential for the mesenchymal phenotype. PMID:25639869

  20. Fuzheng Huayu Recipe Ameliorates Liver Fibrosis by Restoring Balance between Epithelial-to-Mesenchymal Transition and Mesenchymal-to-Epithelial Transition in Hepatic Stellate Cells

    PubMed Central

    Pan, Qin; Wang, Yu-Qin; Li, Guang-Ming; Duan, Xiao-Yan; Fan, Jian-Gao

    2015-01-01

    Activation of hepatic stellate cells (HSCs) depending on epithelial-to-mesenchymal transition (EMT) reflects the key event of liver fibrosis. Contrastively, mesenchymal-to-epithelial transition (MET) of HSCs facilitates the fibrosis resolution. Here we investigated the effect of Fuzheng Huayu (FZHY) recipe, a Chinese herbal decoction made of Radix Salviae Miltiorrhizae, Semen Persicae, Cordyceps sinensis, Pollen Pini, and Gynostemma pentaphyllum, on liver fibrosis concerning the balance of EMT and MET in HSCs. In contrast to the increased TGF-β1/BMP-7 ratio in activated HSCs, FZHY administration induced significant upregulation of BMP-7 and downregulation of TGF-β1 at both transcription and translation levels. Restoration of TGF-β1/BMP-7 ratio inhibited the expression of p38 MAPK and phosphorylated p38 MAPK, resulting in the reversal of epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) as characterized by the abolishment of EMT markers (α-SMA and desmin) and reoccurrence of MET marker (E-cadherin). In vivo treatment of FZHY recipe also demonstrated the statistical reduction of activated HSCs with EMT phenotype, which attenuated the carbon tetrachloride- (CCl4-) induced liver fibrosis in a dose-dependent manner. These findings may highlight a novel antifibrotic role of FZHY recipe on the basis of rebalancing EMT and MET in HSCs. PMID:26881209

  1. Fuzheng Huayu Recipe Ameliorates Liver Fibrosis by Restoring Balance between Epithelial-to-Mesenchymal Transition and Mesenchymal-to-Epithelial Transition in Hepatic Stellate Cells.

    PubMed

    Pan, Qin; Wang, Yu-Qin; Li, Guang-Ming; Duan, Xiao-Yan; Fan, Jian-Gao

    2015-01-01

    Activation of hepatic stellate cells (HSCs) depending on epithelial-to-mesenchymal transition (EMT) reflects the key event of liver fibrosis. Contrastively, mesenchymal-to-epithelial transition (MET) of HSCs facilitates the fibrosis resolution. Here we investigated the effect of Fuzheng Huayu (FZHY) recipe, a Chinese herbal decoction made of Radix Salviae Miltiorrhizae, Semen Persicae, Cordyceps sinensis, Pollen Pini, and Gynostemma pentaphyllum, on liver fibrosis concerning the balance of EMT and MET in HSCs. In contrast to the increased TGF-β 1/BMP-7 ratio in activated HSCs, FZHY administration induced significant upregulation of BMP-7 and downregulation of TGF-β 1 at both transcription and translation levels. Restoration of TGF-β 1/BMP-7 ratio inhibited the expression of p38 MAPK and phosphorylated p38 MAPK, resulting in the reversal of epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) as characterized by the abolishment of EMT markers (α-SMA and desmin) and reoccurrence of MET marker (E-cadherin). In vivo treatment of FZHY recipe also demonstrated the statistical reduction of activated HSCs with EMT phenotype, which attenuated the carbon tetrachloride- (CCl4-) induced liver fibrosis in a dose-dependent manner. These findings may highlight a novel antifibrotic role of FZHY recipe on the basis of rebalancing EMT and MET in HSCs. PMID:26881209

  2. The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro.

    PubMed

    Piskareva, Olga; Harvey, Harry; Nolan, John; Conlon, Ross; Alcock, Leah; Buckley, Patrick; Dowling, Paul; Henry, Michael; O'Sullivan, Finbarr; Bray, Isabella; Stallings, Raymond L

    2015-08-10

    Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma. PMID:25960282

  3. P63 regulates tubular formation via epithelial-to-mesenchymal transition

    PubMed Central

    Zhang, Y; Yan, W; Chen, X

    2016-01-01

    P63, a p53 family member, is expressed as TA and ΔN isoforms. Interestingly, both TAp63 and ΔNp63 are transcription factors, and regulate both common and distinct sets of target genes. p63 is required for survival of some epithelial cell lineages, and lack of p63 leads to loss of epidermis and other epithelia in humans and mice. Here, we explored the role of p63 isoforms in cell proliferation, migration and tubulogenesis by using Madin–Darby Canine Kidney (MDCK) tubular epithelial cells in two- or three-dimensional (2-D or 3-D) culture. We found that like downregulation of p53, downregulation of p63 and TAp63 decreases expression of growth-suppressing genes, including p21, PUMA and MIC-1, and consequently promotes cell proliferation and migration in 2-D culture. However, in 3-D culture, downregulation of p63, especially TAp63, but not p53, decapacitates MDCK cells to form a cyst structure through enhanced epithelial-to-mesenchymal transition (EMT). In contrast, downregulation of ΔNp63 inhibits MDCK cell proliferation and migration in 2-D culture, and delays but does not block MDCK cell cyst formation and tubulogenesis in 3-D culture. Consistent with this, downregulation of ΔNp63 markedly upregulates growth-suppressing genes, including p21, PUMA and MIC-1. Taken together, these data suggest that TAp63 is the major isoform required for tubulogenesis by maintaining an appropriate level of EMT, whereas ΔNp63 fine-tunes the rate of cyst formation and tubulogenesis by maintaining an appropriate expression level of genes involved in cell cycle arrest and apoptosis. PMID:23542170

  4. Inhibition of histamine receptor 3 suppresses glioblastoma tumor growth, invasion, and epithelial-to-mesenchymal transition.

    PubMed

    Lin, Jia-Ji; Zhao, Tian-Zhi; Cai, Wen-Ke; Yang, Yong-Xiang; Sun, Chao; Zhang, Zhuo; Xu, Yu-Qiao; Chang, Ting; Li, Zhu-Yi

    2015-07-10

    Histamine receptor 3 (H3R) is expressed in various tumors and correlated with malignancy and tumor proliferation. However, the role of H3R in tumor invasion and epithelial to mesenchymal transition (EMT) remains unknown. Here, we explored the H3R in the highly invasive glioblastoma (GBM) and U87MG cells. We found that H3R mRNA and protein levels were up-regulated in the GBM and glioma cell lines compared to normal brain tissue and astrocytes. In U87MG cell line, inhibition of H3R by siRNA or the antagonist ciproxifan (CPX) suppressed proliferation, invasiveness, and the expression of EMT activators (Snail, Slug and Twist). In addition, expression of epithelial markers (E-cadherin and ZO-1) was up-regulated and expression of mesenchymal markers (vimentin and N-cadherin) was down-regulated in vitro and in vivo in a xenograft model. In addition, we also showed that inhibition of H3R by siRNA or CPX inactivated the PI3K/Akt and MEK/ERK signaling pathways, while inhibition of Akt or ERK activity with antagonists or siRNAs suppressed H3R agonist (R)-(α)-(-)- methylhistamine dihydrobromide (RAMH) mediated invasion and reorganization of cadherin-household. In conclusion, overexpression of H3R is associated with glioma progression. Inhibition of H3R leads to suppressed invasion and EMT of GBM by inactivating the PI3K/Akt and MEK/ERK pathways in gliomas. PMID:25940798

  5. Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

    PubMed Central

    Jayachandran, Aparna; Anaka, Matthew; Prithviraj, Prashanth; Hudson, Christopher; McKeown, Sonja J; Lo, Pu-Han; Vella, Laura J; Goding, Colin R; Cebon, Jonathan; Behren, Andreas

    2014-01-01

    Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance. PMID:25051363

  6. Surprisal analysis characterizes the free energy time course of cancer cells undergoing epithelial-to-mesenchymal transition.

    PubMed

    Zadran, Sohila; Arumugam, Rameshkumar; Herschman, Harvey; Phelps, Michael E; Levine, R D

    2014-09-01

    The epithelial-to-mesenchymal transition (EMT) initiates the invasive and metastatic behavior of many epithelial cancers. Mechanisms underlying EMT are not fully known. Surprisal analysis of mRNA time course data from lung and pancreatic cancer cells stimulated to undergo TGF-β1-induced EMT identifies two phenotypes. Examination of the time course for these phenotypes reveals that EMT reprogramming is a multistep process characterized by initiation, maturation, and stabilization stages that correlate with changes in cell metabolism. Surprisal analysis characterizes the free energy time course of the expression levels throughout the transition in terms of two state variables. The landscape of the free energy changes during the EMT for the lung cancer cells shows a stable intermediate state. Existing data suggest this is the previously proposed maturation stage. Using a single-cell ATP assay, we demonstrate that the TGF-β1-induced EMT for lung cancer cells, particularly during the maturation stage, coincides with a metabolic shift resulting in increased cytosolic ATP levels. Surprisal analysis also characterizes the absolute expression levels of the mRNAs and thereby examines the homeostasis of the transcription system during EMT. PMID:25157127

  7. AKT1 Inhibits Epithelial-to-Mesenchymal Transition in Breast Cancer through Phosphorylation-Dependent Twist1 Degradation.

    PubMed

    Li, Chia-Wei; Xia, Weiya; Lim, Seung-Oe; Hsu, Jennifer L; Huo, Longfei; Wu, Yun; Li, Long-Yuan; Lai, Chien-Chen; Chang, Shih-Shin; Hsu, Yi-Hsin; Sun, Hui-Lung; Kim, Jongchan; Yamaguchi, Hirohito; Lee, Dung-Fang; Wang, Hongmei; Wang, Yan; Chou, Chao-Kai; Hsu, Jung-Mao; Lai, Yun-Ju; LaBaff, Adam M; Ding, Qingqing; Ko, How-Wen; Tsai, Fuu-Jen; Tsai, Chang-Hai; Hortobagyi, Gabriel N; Hung, Mien-Chie

    2016-03-15

    Epithelial-to-mesenchymal transition (EMT) is an essential physiologic process that promotes cancer cell migration, invasion, and metastasis. Several lines of evidence from both cellular and genetic studies suggest that AKT1/PKBα, but not AKT2 or AKT3, serves as a negative regulator of EMT and breast cancer metastasis. However, the underlying mechanism by which AKT1 suppresses EMT remains poorly defined. Here, we demonstrate that phosphorylation of Twist1 by AKT1 is required for β-TrCP-mediated Twist1 ubiquitination and degradation. The clinically used AKT inhibitor MK-2206, which possesses higher specificity toward AKT1, stabilized Twist1 and enhanced EMT in breast cancer cells. However, we discovered that resveratrol, a naturally occurring compound, induced β-TrCP-mediated Twist1 degradation to attenuate MK-2206-induced EMT in breast cancer cells. Taken together, our findings demonstrate that resveratrol counteracts the unexpected metastatic potential induced by anti-AKT therapy and therefore suggest that the addition of resveratrol to an anti-AKT therapeutic regimen may provide extra support for limiting EMT. PMID:26759241

  8. TSC1 activates TGF-β-Smad2/3 signaling in growth arrest and epithelial-to-mesenchymal transition.

    PubMed

    Thien, Antje; Prentzell, Mirja Tamara; Holzwarth, Birgit; Kläsener, Kathrin; Kuper, Ineke; Boehlke, Christopher; Sonntag, Annika G; Ruf, Stefanie; Maerz, Lars; Nitschke, Roland; Grellscheid, Sushma-Nagaraja; Reth, Michael; Walz, Gerd; Baumeister, Ralf; Neumann-Haefelin, Elke; Thedieck, Kathrin

    2015-03-01

    The tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling. They suppress cell growth and proliferation by acting in a heteromeric complex to inhibit the mammalian target of rapamycin complex 1 (mTORC1). In this study, we identify TSC1 as a component of the transforming growth factor β (TGF-β)-Smad2/3 pathway. Here, TSC1 functions independently of TSC2. TSC1 interacts with the TGF-β receptor complex and Smad2/3 and is required for their association with one another. TSC1 regulates TGF-β-induced Smad2/3 phosphorylation and target gene expression and controls TGF-β-induced growth arrest and epithelial-to-mesenchymal transition (EMT). Hyperactive Akt specifically activates TSC1-dependent cytostatic Smad signaling to induce growth arrest. Thus, TSC1 couples Akt activity to TGF-β-Smad2/3 signaling. This has implications for cancer treatments targeting phosphoinositide 3-kinases and Akt because they may impair tumor-suppressive cytostatic TGF-β signaling by inhibiting Akt- and TSC1-dependent Smad activation. PMID:25727005

  9. YB-1 expression promotes epithelial-to-mesenchymal transition in prostate cancer that is inhibited by a small molecule fisetin

    PubMed Central

    Khan, Mohammad Imran; Adhami, Vaqar Mustafa; Lall, Rahul Kumar; Sechi, Mario; Joshi, Dinesh C.; Haidar, Omar M.; Syed, Deeba Nadeem; Siddiqui, Imtiaz Ahmad; Chiu, Shing-Yan; Mukhtar, Hasan

    2014-01-01

    Epithelial-to-mesenchymal transition (EMT) plays an important role in prostate cancer (PCa) metastasis. The transcription/translation regulatory Y-box binding protein-1 (YB-1) is known to be associated with cancer metastasis. We observed that YB-1 expression increased with tumor grade and showed an inverse relationship with E-cadherin in a human PCa tissue array. Forced YB-1 expression induced a mesenchymal morphology that was associated with down regulation of epithelial markers. Silencing of YB-1 reversed mesenchymal features and decreased cell proliferation, migration and invasion in PCa cells. YB-1 is activated directly via Akt mediated phosphorylation at Ser102 within the cold shock domain (CSD). We next identified fisetin as an inhibitor of YB-1 activation. Computational docking and molecular dynamics suggested that fisetin binds on the residues from β1 - β4 strands of CSD, hindering Akt's interaction with YB-1. Calculated free binding energy ranged from −11.9845 to −9.6273 kcal/mol. Plasmon Surface Resonance studies showed that fisetin binds to YB-1 with an affinity of approximately 35 μM, with both slow association and dissociation. Fisetin also inhibited EGF induced YB-1 phosphorylation and markers of EMT both in vitro and in vivo. Collectively our data suggest that YB-1 induces EMT in PCa and identify fisetin as an inhibitor of its activation. PMID:24770864

  10. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells

    PubMed Central

    Yuan, Chun-Xiu; Zhou, Zhi-Wei; Yang, Yin-Xue; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Tianxing; Pan, Si-Yuan; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide, with a poor response to current chemotherapy. Danusertib is a pan-inhibitor of the Aurora kinases and a third-generation Bcr-Abl tyrosine kinase inhibitor with potent anticancer effects, but its antitumor effect and underlying mechanisms in the treatment of human gastric cancer are unknown. This study aimed to investigate the effects of danusertib on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition and the molecular mechanisms involved in human gastric cancer AGS and NCI-N78 cells. The results showed that danusertib had potent growth-inhibitory, apoptosis-inducing, and autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways as well as activation of 5′ AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression

  11. AZGP1 suppresses epithelial-to-mesenchymal transition and hepatic carcinogenesis by blocking TGFβ1-ERK2 pathways.

    PubMed

    Xu, Ming-Yi; Chen, Rong; Yu, Jing-Xia; Liu, Ting; Qu, Ying; Lu, Lun-Gen

    2016-05-01

    Zinc-α2-glycoprotein 1 (AZGP1) has been found to play important roles in TGF-β1 induced epithelial-to-mesenchymal transition (EMT). However, the mechanisms of AZGP1 inhibiting EMT and its therapeutic potential remain unknown in hepatocellular carcinoma (HCC). AZGP1, TGF-β1 or ERK2 expressions were examined in liver tissues of HCC patients and rat model. The effect of AZGP1 on EMT and crosstalking of TGFβ1-ERK2 signaling in human hepatic cancer cell was tested in vitro and in vivo. Hepatic expression of AZGP1 was nearly deficient in HCC patients and rats. It was proved that AZGP1 has the ability of down-regulating mesenchymal markers, up-regulating epithelial marker, inhibiting cell invasion and suppressing EMT in human HCC cells. The results clarified that AZGP1 has the effect on blocking TGF-β1 mediated ERK2 phosphorylation leading to depressing EMT and invasive potential in vitro. Local injection of AZGP1 mimic in vivo could significantly withhold lung metastasis in HCC. In conclusion, loss of AZGP1 could trigger EMT induced by TGFβ1-ERK2 signaling, confuse in energy metabolism, reduce cell proliferation and apoptosis, activate survival signals and promote invasion. Up-regulation of AZGP1 should be proposed to reverse EMT and might be a new promising therapy for HCC. PMID:26902423

  12. Live Imaging and Gene Expression Analysis in Zebrafish Identifies a Link between Neutrophils and Epithelial to Mesenchymal Transition

    PubMed Central

    Freisinger, Christina M.; Huttenlocher, Anna

    2014-01-01

    Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRasV12. Live imaging revealed that expression of HRasV12 in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRasV12 using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRasV12 transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRasV12 in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells. PMID:25372289

  13. Blocking the epithelial-to-mesenchymal transition pathway abrogates resistance to anti-folate chemotherapy in lung cancer

    PubMed Central

    Liang, S-Q; Marti, T M; Dorn, P; Froment, L; Hall, S R R; Berezowska, S; Kocher, G; Schmid, R A; Peng, R-W

    2015-01-01

    Anticancer therapies currently used in the clinic often can neither eradicate the tumor nor prevent disease recurrence due to tumor resistance. In this study, we showed that chemoresistance to pemetrexed, a multi-target anti-folate (MTA) chemotherapeutic agent for non-small cell lung cancer (NSCLC), is associated with a stem cell-like phenotype characterized by an enriched stem cell gene signature, augmented aldehyde dehydrogenase activity and greater clonogenic potential. Mechanistically, chemoresistance to MTA requires activation of epithelial-to-mesenchymal transition (EMT) pathway in that an experimentally induced EMT per se promotes chemoresistance in NSCLC and inhibition of EMT signaling by kaempferol renders the otherwise chemoresistant cancer cells susceptible to MTA. Relevant to the clinical setting, human primary NSCLC cells with an elevated EMT signaling feature a significantly enhanced potential to resist MTA, whereas concomitant administration of kaempferol abrogates MTA chemoresistance, regardless of whether it is due to an intrinsic or induced activation of the EMT pathway. Collectively, our findings reveal that a bona fide activation of EMT pathway is required and sufficient for chemoresistance to MTA and that kaempferol potently regresses this chemotherapy refractory phenotype, highlighting the potential of EMT pathway inhibition to enhance chemotherapeutic response of lung cancer. PMID:26181204

  14. Microenvironmental interactions and expression of molecular markers associated with epithelial-to-mesenchymal transition in colorectal carcinoma

    PubMed Central

    Lee, Sun-Jae; Yang, Chun-Seok; Kim, Dae-Dong; Kang, Yu-Na; Kwak, Sang Gyu; Park, Jae-Bok; Cho, Chang-Ho; Park, Kwan-Kyu

    2015-01-01

    The tumor microenvironment is known to play a critical role in tumor progression, invasion and metastasis. The epithelial-to-mesenchymal transition (EMT) is understood as a process of tumor invasion and metastasis. Therefore, we investigated the relation between the EMT and the microenvironment of colorectal carcinoma (CRC). The histological features and expression of EMT markers in tumor cells and surrounded stromal cells were obtained from the surgically resected tissues of 39 patients using microscopic review and immunohistochemistry. The loss of expression of E-cadherin was more prominent in the invasive front of tumor than the surface, where α-smooth muscle actin-positive carcinoma-associated fibroblasts (CAFs) are accumulated. The signaling molecules of the Wnt and TGF-β1-Smad pathway were expressed more frequently in the tumor cells and/or CAFs of the invasive margin than those of the tumor surface. The expressions of related transcription factors, such as SNAIL and ZEB1, were increased in the tumor cells and CAFs. The process of EMT may be activated in the tumor margin of CRC under the control of CAFs. Related signaling molecules and transcription factors might be induced by paracrine effects of the surrounding CAFs. PMID:26823743

  15. Black tea polyphenols reverse epithelial-to-mesenchymal transition and suppress cancer invasion and proteases in human oral cancer cells.

    PubMed

    Chang, Yu-Chao; Chen, Pei-Ni; Chu, Shu-Chen; Lin, Chin-Yin; Kuo, Wu-Hsien; Hsieh, Yih-Shou

    2012-08-29

    Epithelial-to-mesenchymal transition (EMT) in cancer cells is considered to be a prerequisite for acquiring invasive/migratory phenotype and subsequent metastasis. This study provides molecular evidence associated with the antimetastatic effect of black tea polyphenol extracts (BTE), which contain polyphenols including gallic acid, gallocatechin, catechin, epigallocatechin-3-gallate, epicatechin-3-gallate, and theaflavin 3,3'-digallate, in an an oral squamous cell culture system by showing a nearly complete inhibition on the invasion (p < 0.001) of SCC-4 cells via reduced activities of MMP-2 (p < 0.001) and u-PA (p < 0.001). Immunoblot was performed to find that BTE could induce up-regulation of epithelial markers such as E-cadherin and inhibit mesenchymal markers such as snail-1 and vimentin. BTE inhibited p-FAK and p-paxillin, indicating the anti-EMT effect of BTE in oral squamous cell carcinoma. BTE was evidenced by its inhibition of the tumor growth of SCC-4 cells via cancer cell xenografted nude mice mode. These results suggested that BTE could reduce invasion by reversing EMT in human oral cancer cells. PMID:22827697

  16. Bisphenol A modulates colorectal cancer protein profile and promotes the metastasis via induction of epithelial to mesenchymal transitions.

    PubMed

    Chen, Zhuo-Jia; Yang, Xiang-Ling; Liu, Hao; Wei, Wei; Zhang, Kun-Shui; Huang, Hong-Bin; Giesy, John P; Liu, Huan-Liang; Du, Jun; Wang, Hong-Sheng

    2015-08-01

    More and more evidences indicate that endocrine disruptor chemicals such as bisphenol A (BPA) can act as carcinogens and enhance susceptibility to tumorigenesis. Although the gut is in direct contact with orally ingested BPA, effects of BPA on occurrence and development of colorectal cancer remain an unexplored endpoint. Colorectal cancer SW480 cells treated with nanomolar (10(-8) M) or greater (10(-5) M) concentrations of BPA were compared with responses of a control group. Proteomic study revealed that more than 56 proteins were modulated following exposure to BPA, which are relevant to structure, motility and proliferation of cells, production of ATP, oxidative stress, and protein metabolism. Further studies revealed that BPA increased migration and invasion and triggered transformations from epithelial to mesenchymal transitions (EMTs) of colorectal cancer cells, which was characterized by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin with a concomitant decrease of E-cadherin. Accordingly, BPA treatment increased the expression of transcription factor Snail. Furthermore, signal AKT/GSK-3β-mediated stabilization of Snail is involved during BPA-induced EMT of colon cancer cells. Our study first demonstrated that the xenoestrogen BPA at nanomolar and greater concentrations modulates the protein profiles and promotes the metastasis of colorectal cancer cells via induction of EMT. PMID:25119493

  17. Cetuximab promotes epithelial to mesenchymal transition and cancer associated fibroblasts in patients with head and neck cancer

    PubMed Central

    Schmitz, Sandra; Bindea, Gabriela; Albu, Roxana Irina; Mlecnik, Bernhard; Machiels, Jean-Pascal

    2015-01-01

    Purpose To investigate if cetuximab induces epithelial to mesenchymal transition (EMT) and activation of cancer associated fibroblast (CAF) in the tumors of patients with squamous cell carcinoma of the head and neck (SCCHN). Methods Cetuximab was administered for two weeks prior to surgery to 20 treatment-naïve patients. Five untreated patients were included as controls. Tumor biopsies were performed at baseline and before surgery. Gene expression profiles and quantitative real-time PCR (qRT-PCR) analysis of the pre-and post-treatment biopsies were compared. To further investigate EMT and CAF, correlations between previously described EMT and CAF markers and our microarray data set were calculated. Results Gene expression profile analyses and qRT-PCR showed that some of the genes modified by cetuximab were related to CAFs and EMT (ZNF521, CXCL12, ASPN, OLFML3, OLFM1, TWIST1, LEF1, ZEB1, FAP). We identified 2 patient clusters with different EMT and CAF characteristics. Whereas one cluster showed clear upregulation of expression of genes implicated in CAF and EMT including markers of embryologic pathways like NOTCH and Wnt, the other did not. Conclusion Even if EMT and CAFs are implicated in cetuximab resistance in pre-clinical models, we demonstrate for the first time that these molecular processes may occur clinically early on. PMID:26437222

  18. Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells

    PubMed Central

    Horejs, Christine-Maria; Serio, Andrea; Purvis, Alan; Gormley, Adam J.; Bertazzo, Sergio; Poliniewicz, Anna; Wang, Alex J.; DiMaggio, Peter; Hohenester, Erhard; Stevens, Molly M.

    2014-01-01

    The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the β1-chain–derived fragment interacts via α3β1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs—key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the α3β1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9. PMID:24706882

  19. Loss of GFAT1 promotes epithelial-to-mesenchymal transition and predicts unfavorable prognosis in gastric cancer.

    PubMed

    Duan, Fangfang; Jia, Dongwei; Zhao, Junjie; Wu, Weicheng; Min, Lingqiang; Song, Shushu; Wu, Hao; Wang, Lan; Wang, Hongshan; Ruan, Yuanyuan; Gu, Jianxin

    2016-06-21

    Gastric cancer remains the third leading cause of cancer-related mortality worldwide, and invasion and metastasis of gastric cancer represent the major reason for its poor prognosis. Glutamine: fructose-6-phosphate amidotransferase 1 (GFAT1) is the first and rate-limiting enzyme of hexosamine biosynthesis pathway (HBP). Nevertheless, the role of GFAT1 in gastric cancer is little investigated. In this study, we found that the expression of GFAT1 was decreased in gastric cancer. Low expression of GFAT1 was positively associated with vessel invasion, late T stage, lymph node metastasis, distant metastasis, advanced TNM stage and poor prognosis in patients with gastric cancer. Furthermore, in vitro and in vivo studies revealed that down-regulation of GFAT1 promoted epithelial-to-mesenchymal transition (EMT) and invasive activities in gastric cancer cells through inducing the expression of TGF-β1. The GFAT1 expression also significantly correlated with EMT-related factors in gastric cancer patients. Together, these findings indicate that GFAT1 functions as a novel suppressor of EMT and tumor metastasis in gastric cancer. PMID:27509259

  20. Regulation of epithelial to mesenchymal transition in bovine conceptuses through the interaction between follistatin and activin A.

    PubMed

    Kusama, Kazuya; Bai, Rulan; Ideta, Atsushi; Aoyagi, Yoshito; Okuda, Kiyoshi; Imakawa, Kazuhiko

    2016-10-15

    Dynamic changes in bovine conceptus and endometrium occur during early gestation, in which the conceptus undergoes epithelial to mesenchymal transition (EMT) after the conceptus attachment to endometrium. To characterize EMT inducing factors, we initially undertook iTRAQ analysis with bovine uterine flushing (UF) obtained from pregnant animals on days 17 (P17: pre-attachment) and 20 (P20: post-attachment). The iTRAQ analysis demonstrated that follistatin (FST), an inhibitor of activin A, increased in P20 UF. We then found that FST decreased in P22 conceptuses, whereas elevated activin A found in P20 UF and endometria was further increased on P22. In addition, phosphorylated SMAD2 increased in P22 conceptuses. In bovine trophoblast cells, the treatment with P22 UF or activin An up-regulated EMT marker expressions, which were inhibited by FST. These results suggest that the initiation of bovine conceptus EMT could be regulated through the spatiotemporal expression of FST or activin A during the peri-attachment period. PMID:27321969

  1. Effect of CD133 overexpression on the epithelial-to-mesenchymal transition in oral cancer cell lines.

    PubMed

    Moon, YeonHee; Kim, Donghwi; Sohn, HongMoon; Lim, Wonbong

    2016-06-01

    Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world. In OSCC, CD133 promotes tumor invasion and metastasis by inducing the epithelial-to-mesenchymal transition (EMT). A small subset of cancer cells known as cancer stem cells (CSCs) are thought to give rise to differentiated tumor cells and to predict tumor recurrence and metastases, i.e., CSCs may be metastatic precursors. In this study, we show that ectopic overexpression of CD133 in OSCC cell lines KB, YD9, and YD10B cells significantly promotes the EMT and acquisition of stemness properties. CSC properties were analyzed by colony-formation assay and measurement of OCT4, SOX2, and NANOG expression, and the EMT was monitored by cell migration, a cell invasion assay, and analysis of E-cadherin, N-cadherin, and vimentin expression. CD133 overexpression led to formation of irregular spheroid colonies consistent with a stem cell phenotype and increased the expression of OCT4, SOX2, NANOG, N-cadherin, and vimentin. Taken together, these findings show that elevated levels of CD133 lead to OSCC invasiveness and metastasis, associated with the upregulation of EMT and stemness markers. PMID:27137188

  2. A systematic approach identifies FOXA1 as a key factor in the loss of epithelial traits during the epithelial-to-mesenchymal transition in lung cancer

    PubMed Central

    2013-01-01

    Background The epithelial-to-mesenchymal transition is an important mechanism in cancer metastasis. Although transcription factors including SNAIL, SLUG, and TWIST1 regulate the epithelial-to-mesenchymal transition, other unknown transcription factors could also be involved. Identification of the full complement of transcription factors is essential for a more complete understanding of gene regulation in this process. Chromatin immunoprecipitation-sequencing (ChIP-Seq) technologies have been used to detect genome-wide binding of transcription factors; here, we developed a systematic approach to integrate existing ChIP-Seq and transcriptome data. We scanned multiple transcription factors to investigate their functional impact on the epithelial-to-mesenchymal transition in the human A549 lung adenocarcinoma cell line. Results Among the transcription factors tested, impact scores identified the forkhead box protein A1 (FOXA1) as the most significant transcription factor in the epithelial-to-mesenchymal transition. FOXA1 physically associates with the promoters of its predicted target genes. Several critical epithelial-to-mesenchymal transition effectors involved in cellular adhesion and cellular communication were identified in the regulatory network of FOXA1, including FOXA2, FGA, FGB, FGG, and FGL1. The implication of FOXA1 in the epithelial-to-mesenchymal transition via its regulatory network indicates that FOXA1 may play an important role in the initiation of lung cancer metastasis. Conclusions We identified FOXA1 as a potentially important transcription factor and negative regulator in the initial stages of lung cancer metastasis. FOXA1 may modulate the epithelial-to-mesenchymal transition via its transcriptional regulatory network. Further, this study demonstrates how ChIP-Seq and expression data could be integrated to delineate the impact of transcription factors on a specific biological process. PMID:24093963

  3. Induction by transforming growth factor-β1 of epithelial to mesenchymal transition is a rare event in vitro

    PubMed Central

    Brown, Kimberly A; Aakre, Mary E; Gorska, Agnieska E; Price, James O; Eltom, Sakina E; Pietenpol, Jennifer A; Moses, Harold L

    2004-01-01

    Introduction Transforming growth factor (TGF)-β1 is proposed to inhibit the growth of epithelial cells in early tumorigenesis, and to promote tumor cell motility and invasion in the later stages of carcinogenesis through the induction of an epithelial to mesenchymal transition (EMT). EMT is a multistep process that is characterized by changes in cell morphology and dissociation of cell–cell contacts. Although there is growing interest in TGF-β1-mediated EMT, the phenotype is limited to only a few murine cell lines and mouse models. Methods To identify alternative cell systems in which to study TGF-β1-induced EMT, 18 human and mouse established cell lines and cultures of two human primary epithelial cell types were screened for TGF-β1-induced EMT by analysis of cell morphology, and localization of zonula occludens-1, E-cadherin, and F-actin. Sensitivity to TGF-β1 was also determined by [3H]thymidine incorporation, flow cytometry, phosphorylation of Smad2, and total levels of Smad2 and Smad3 in these cell lines and in six additional cancer cell lines. Results TGF-β1 inhibited the growth of most nontransformed cells screened, but many of the cancer cell lines were insensitive to the growth inhibitory effects of TGF-β1. In contrast, TGF-β1 induced Smad2 phosphorylation in the majority of cell lines, including cell lines resistant to TGF-β1-mediated cell cycle arrest. Of the cell lines screened only two underwent TGF-β1-induced EMT. Conclusion The results presented herein show that, although many cancer cell lines have lost sensitivity to the growth inhibitory effect of TGF-β1, most show evidence of TGF-β1 signal transduction, but only a few cell lines undergo TGF-β1-mediated EMT. PMID:15084245

  4. A Novel Network Integrating a miRNA-203/SNAI1 Feedback Loop which Regulates Epithelial to Mesenchymal Transition

    PubMed Central

    Moes, Michèle; Le Béchec, Antony; Crespo, Isaac; Laurini, Christina; Halavatyi, Aliaksandr; Vetter, Guillaume; del Sol, Antonio; Friederich, Evelyne

    2012-01-01

    Background The majority of human cancer deaths are caused by metastasis. The metastatic dissemination is initiated by the breakdown of epithelial cell homeostasis. During this phenomenon, referred to as epithelial to mesenchymal transition (EMT), cells change their genetic and trancriptomic program leading to phenotypic and functional alterations. The challenge of understanding this dynamic process resides in unraveling regulatory networks involving master transcription factors (e.g. SNAI1/2, ZEB1/2 and TWIST1) and microRNAs. Here we investigated microRNAs regulated by SNAI1 and their potential role in the regulatory networks underlying epithelial plasticity. Results By a large-scale analysis on epithelial plasticity, we highlighted miR-203 and its molecular link with SNAI1 and the miR-200 family, key regulators of epithelial homeostasis. During SNAI1-induced EMT in MCF7 breast cancer cells, miR-203 and miR-200 family members were repressed in a timely correlated manner. Importantly, miR-203 repressed endogenous SNAI1, forming a double negative miR203/SNAI1 feedback loop. We integrated this novel miR203/SNAI1 with the known miR200/ZEB feedback loops to construct an a priori EMT core network. Dynamic simulations revealed stable epithelial and mesenchymal states, and underscored the crucial role of the miR203/SNAI1 feedback loop in state transitions underlying epithelial plasticity. Conclusion By combining computational biology and experimental approaches, we propose a novel EMT core network integrating two fundamental negative feedback loops, miR203/SNAI1 and miR200/ZEB. Altogether our analysis implies that this novel EMT core network could function as a switch controlling epithelial cell plasticity during differentiation and cancer progression. PMID:22514743

  5. Estrogen receptor alpha mediates epithelial to mesenchymal transition, expression of specific matrix effectors and functional properties of breast cancer cells.

    PubMed

    Bouris, Panagiotis; Skandalis, Spyros S; Piperigkou, Zoi; Afratis, Nikos; Karamanou, Konstantina; Aletras, Alexios J; Moustakas, Aristidis; Theocharis, Achilleas D; Karamanos, Nikos K

    2015-04-01

    The 17β-estradiol (E2)/estrogen receptor alpha (ERα) signaling pathway is one of the most important pathways in hormone-dependent breast cancer. E2 plays pivotal roles in cancer cell growth, survival, and architecture as well as in gene expression regulatory mechanisms. In this study, we established stably transfected MCF-7 cells by knocking down the ERα gene (designated as MCF-7/SP10+ cells), using specific shRNA lentiviral particles, and compared them with the control cells (MCF-7/c). Interestingly, ERα silencing in MCF-7 cells strongly induced cellular phenotypic changes accompanied by significant changes in gene and protein expression of several markers typical of epithelial to mesenchymal transition (EMT). Notably, these cells exhibited enhanced cell proliferation, migration and invasion. Moreover, ERα suppression strongly affected the gene and protein expression of EGFR and HER2 receptor tyrosine kinases, and various extracellular matrix (ECM) effectors, including matrix metalloproteinases and their endogenous inhibitors (MMPs/TIMPs) and components of the plasminogen activation system. The action caused by E2 in MCF-7/c cells in the expression of HER2, MT1-MMP, MMP1, MMP9, uPA, tPA, and PAI-1 was abolished in MCF-7/SP10+ cells lacking ERα. These data suggested a regulatory role for the E2/ERα pathway in respect to the composition and activity of the extracellular proteolytic molecular network. Notably, loss of ERα promoted breast cancer cell migration and invasion by inducing changes in the expression levels of certain matrix macromolecules (especially uPA, tPA, PAI-1) through the EGFR-ERK signaling pathway. In conclusion, loss of ERα in breast cancer cells results in a potent EMT characterized by striking changes in the expression profile of specific matrix macromolecules highlighting the potential nodal role of matrix effectors in breast cancer endocrine resistance. PMID:25728938

  6. Honokiol confers immunogenicity by dictating calreticulin exposure, activating ER stress and inhibiting epithelial-to-mesenchymal transition.

    PubMed

    Liu, Shing-Hwa; Lee, Wen-Jane; Lai, De-Wei; Wu, Sheng-Mao; Liu, Chia-Yu; Tien, Hsing-Ru; Chiu, Chien-Shan; Peng, Yen-Chun; Jan, Yee-Jee; Chao, Te-Hsin; Pan, Hung-Chuan; Sheu, Meei-Ling

    2015-04-01

    Peritoneal dissemination is a major clinical obstacle in gastrointestinal cancer therapy, and it accounts for the majority of cancer-related mortality. Calreticulin (CRT) is over-expressed in gastric tumors and has been linked to poor prognosis. In this study, immunohistochemistry studies revealed that the up-regulation of CRT was associated with lymph node and distant metastasis in patients with gastric cancer specimens. CRT was significantly down-regulated in highly metastatic gastric cancer cell lines and metastatic animal by Honokiol-treated. Small RNA interference blocking CRT by siRNA-CRT was translocated to the cells in the early immunogenic response to Honokiol. Honokiol activated endoplasmic reticulum (ER) stress and down-regulated peroxisome proliferator-activated receptor-γ (PPARγ) activity resulting in PPARγ and CRT degradation through calpain-II activity, which could be reversed by siRNA-calpain-II. The Calpain-II/PPARγ/CRT axis and interaction evoked by Honokiol could be blocked by gene silencing or pharmacological agents. Both transforming growth factor (TGF)-β1 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced cell migration, invasion and reciprocal down-regulation of epithelial marker E-cadherin, which could be abrogated by siRNA-CRT. Moreover, Honokiol significantly suppressed MNNG-induced gastrointestinal tumor growth and over-expression of CRT in mice. Knockdown CRT in gastric cancer cells was found to effectively reduce growth ability and metastasis in vivo. The present study provides insight into the specific biological behavior of CRT in epithelial-to-mesenchymal transition (EMT) and metastasis. Taken together, our results suggest that the therapeutic inhibition of CRT by Honokiol suppresses both gastric tumor growth and peritoneal dissemination by dictating early translocation of CRT in immunogenic cell death, activating ER stress, and blocking EMT. PMID:25619450

  7. CYR61 and TAZ Upregulation and Focal Epithelial to Mesenchymal Transition May Be Early Predictors of Barrett's Esophagus Malignant Progression.

    PubMed

    Cardoso, Joana; Mesquita, Marta; Dias Pereira, António; Bettencourt-Dias, Mónica; Chaves, Paula; Pereira-Leal, José B

    2016-01-01

    Barrett's esophagus is the major risk factor for esophageal adenocarcinoma. It has a low but non-neglectable risk, high surveillance costs and no reliable risk stratification markers. We sought to identify early biomarkers, predictive of Barrett's malignant progression, using a meta-analysis approach on gene expression data. This in silico strategy was followed by experimental validation in a cohort of patients with extended follow up from the Instituto Português de Oncologia de Lisboa de Francisco Gentil EPE (Portugal). Bioinformatics and systems biology approaches singled out two candidate predictive markers for Barrett's progression, CYR61 and TAZ. Although previously implicated in other malignancies and in epithelial-to-mesenchymal transition phenotypes, our experimental validation shows for the first time that CYR61 and TAZ have the potential to be predictive biomarkers for cancer progression. Experimental validation by reverse transcriptase quantitative PCR and immunohistochemistry confirmed the up-regulation of both genes in Barrett's samples associated with high-grade dysplasia/adenocarcinoma. In our cohort CYR61 and TAZ up-regulation ranged from one to ten years prior to progression to adenocarcinoma in Barrett's esophagus index samples. Finally, we found that CYR61 and TAZ over-expression is correlated with early focal signs of epithelial to mesenchymal transition. Our results highlight both CYR61 and TAZ genes as potential predictive biomarkers for stratification of the risk for development of adenocarcinoma and suggest a potential mechanistic route for Barrett's esophagus neoplastic progression. PMID:27583562

  8. Differential Roles for DUSP Family Members in Epithelial-to-Mesenchymal Transition and Cancer Stem Cell Regulation in Breast Cancer

    PubMed Central

    Boulding, Tara; Wu, Fan; McCuaig, Robert; Dunn, Jennifer; Sutton, Christopher R.; Hardy, Kristine; Tu, Wenjuan; Bullman, Amanda; Yip, Desmond; Dahlstrom, Jane E.; Rao, Sudha

    2016-01-01

    Dual-specificity phosphatases (DUSPs) dephosphorylate threonine/serine and tyrosine residues on their substrates. Here we show that DUSP1, DUSP4, and DUSP6 are involved in epithelial-to-mesenchymal transition (EMT) and breast cancer stem cell (CSC) regulation. DUSP1, DUSP4, and DUSP6 are induced during EMT in a PKC pathway signal-mediated EMT model. We show for the first time that the key chromatin-associated kinase PKC-θ directly regulates a subset of DUSP family members. DUSP1, DUSP4, and DUSP6 globally but differentially co-exist with enhancer and permissive active histone post-translational modifications, suggesting that they play distinct roles in gene regulation in EMT/CSCs. We show that nuclear DUSP4 associates with the key acetyltransferase p300 in the context of the chromatin template and dynamically regulates the interplay between two key phosphorylation marks: the 1834 (active) and 89 (inhibitory) residues central to p300’s acetyltransferase activity. Furthermore, knockdown with small-interfering RNAs (siRNAs) shows that DUSP4 is required for maintaining H3K27ac, a mark mediated by p300. DUSP1, DUSP4, and DUSP6 knockdown with siRNAs shows that they participate in the formation of CD44hi/CD24lo/EpCAM+ breast CSCs: DUSP1 knockdown reduces CSC formation, while DUSP4 and DUSP6 knockdown enhance CSC formation. Moreover, DUSP6 is overexpressed in patient-derived HER2+ breast carcinomas compared to benign mammary tissue. Taken together, these findings illustrate novel pleiotropic roles for DUSP family members in EMT and CSC regulation in breast cancer. PMID:26859151

  9. Association between cancer stem cell-like properties and epithelial-to-mesenchymal transition in primary and secondary cancer cells.

    PubMed

    Lim, Wonbong; Kim, Hye-Eun; Kim, Young; Na, Risu; Li, Xiaojie; Jeon, Sangmi; Choi, Hongran; Kim, Okjoon

    2016-09-01

    One of the theories on cancer stem cells (CSCs) states that these cells initiate most tumors and give rise to more-or-less differentiated tumor cells. Genetic signatures of CSCs are thought to predict tumor recurrence and metastases, thus, supporting the notion that CSCs may be metastatic precursors and induce epithelial-to-mesenchymal transition (EMT). In this study, we tried to examine the association between CSCs and EMT (using specific markers) in the mucoepidermoid carcinoma cell line YD15 and its derivative cell line YD15M (lymph node metastasis). Relative protein expression levels were analyzed by western blotting, flow cytometry, and immunofluorescence assays. In addition, cell cycle assay and aldehyde dehydrogenase (ALDH) activity assay were carried out. Under growth conditions, YD15M cells formed irregular spherical colonies consistent with a stem cell phenotype. YD15M cells demonstrated the low expression of E-cadherin and β-catenin but high expression of vimentin than that in YD15 cells. In the metastatic cells (YD15M), the coexpression of vimentin and CD133 was detected. Weak proliferation based on cell cycle analysis and decreased PCNA expression was also observed. In addition, expression levels of ALDHA1, OCT4, and NANOG (CSC-like properties) were significantly increased in YD15M cells. Taken together, these findings should help to elucidate the interplay between EMT and CSC-like properties during metastasis and may provide useful information for the development of a novel classification system and therapeutic strategies against head and neck cancer. PMID:27315437

  10. Prostate-Derived ETS Factor Regulates Epithelial-to-Mesenchymal Transition through Both SLUG-Dependent and Independent Mechanisms

    PubMed Central

    Findlay, Victoria J.; Turner, David P.; Yordy, John S.; McCarragher, Brent; Shriver, Marey R.; Szalai, Gabor; Watson, Patricia M.; LaRue, Amanda C.; Moussa, Omar; Watson, Dennis K.

    2011-01-01

    The 5-year survival rate is very low when breast cancer becomes metastatic. The metastatic process is governed by a network of molecules of which SLUG is known to play a major role as a regulator of epithelial-to-mesenchymal transition (EMT). Prostate-derived ETS factor (PDEF) has been proposed as a tumor suppressor, possibly through inhibition of invasion and metastasis; therefore, understanding the mechanism of PDEF regulation may help to better understand its role in breast cancer progression. This study shows for the first time that the transcription factor SLUG is a direct target of PDEF in breast cancer. We show that the expression of PDEF is able to suppress/dampen EMT through the negative regulation of SLUG. In addition, we show that PDEF is also able to regulate downstream targets of SLUG, namely E-cadherin, in both SLUG-dependent and -independent manners, suggesting a critical role for PDEF in regulating EMT. PMID:21779485

  11. Novel roles of Src in cancer cell epithelial-to-mesenchymal transition, vascular permeability, microinvasion and metastasis.

    PubMed

    Patel, Ami; Sabbineni, Harika; Clarke, Andrea; Somanath, Payaningal R

    2016-07-15

    The Src-family kinases (SFKs), an intracellularly located group of non-receptor tyrosine kinases are involved in oncogenesis. The importance of SFKs has been implicated in the promotion of tumor cell motility, proliferation, inhibition of apoptosis, invasion and metastasis. Recent evidences indicate that specific effects of SFKs on epithelial-to-mesenchymal transition (EMT) as well as on endothelial and stromal cells in the tumor microenvironment can have profound effects on tumor microinvasion and metastasis. Although, having been studied extensively, these novel features of SFKs may contribute to greater understanding of benefits from Src inhibition in various types of cancers. Here we review the novel role of SFKs, particularly c-Src in mediating EMT, modulation of tumor endothelial-barrier, transendothelial migration (microinvasion) and metastasis of cancer cells, and discuss the utility of Src inhibitors in vascular normalization and cancer therapy. PMID:27245276

  12. Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

    PubMed

    Islam, S S; Mokhtari, R B; Noman, A S; Uddin, M; Rahman, M Z; Azadi, M A; Zlotta, A; van der Kwast, T; Yeger, H; Farhat, W A

    2016-05-01

    Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the

  13. Metformin inhibits 17β-estradiol-induced epithelial-to-mesenchymal transition via βKlotho-related ERK1/2 signaling and AMPKα signaling in endometrial adenocarcinoma cells

    PubMed Central

    Liu, Zhao; Qi, Shasha; Zhao, Xingbo; Li, Mingjiang; Ding, Sentai; Lu, Jiaju; Zhang, Hui

    2016-01-01

    The potential role of metformin in treating endometrial cancer remains to be explored. The current study investigated the role of metformin in 17β-estradiol-induced epithelial-mesenchymal transition (EMT) in endometrial adenocarcinoma cells. We found that 17β-estradiol promoted proliferation and migration, attenuated apoptosis in both estrogen receptor (ER) positive and ER negative endometrial adenocarcinoma cells (Ishikawa and KLE cells, respectively). Metformin abolished 17β-estradiol-induced cell proliferation and reversed 17β-estradiol-induced EMT in Ishikawa cells. In addition, metformin increased the expression of βKlotho, a fibroblast growth factors (FGFs) coreceptor, and decreased ERK1/2 phosphorylation in both Ishikawa and KLE cells. Decreased expression of βKlotho was noted in human endometrial adenocarcinomas, and plasmid-driven expression of βKlotho in Ishikawa cells abolished 17β-estradiol-induced EMT via inhibiting ERK1/2 signaling. βKlotho expression and metformin show synergetic effects on the proliferation and the EMT in Ishikawa cells. Furthermore, we demonstrated that the anti-EMT effects of metformin could be partly abolished by introducing Compound C, a specific AMPKα signaling inhibitor. In conclusion, metformin abolishes 17β-estradiol-induced cell proliferation and EMT in endometrial adenocarcinoma cells by upregulating βKlotho expression, inhibiting ERK1/2 signaling, and activating AMPKα signaling. Our study provides novel mechanistic insight into the anti-tumor effects of metformin. PMID:26824324

  14. Anxa2 binds to STAT3 and promotes epithelial to mesenchymal transition in breast cancer cells

    PubMed Central

    Zhang, Jie; Tian, Ran; Ji, Wei; Zhou, Yan; Yang, Yi; Song, Weijie; Zhang, Fei; Niu, Ruifang

    2015-01-01

    Overexpression of annexin A2 (Anxa2) is correlated with invasion and metastasis in breast cancer cells. In this study, breast cancer patients with upregulated Anxa2 exhibited poor overall and disease-free survival rates. Anxa2 expression was also positively correlated with the expression of epidermal growth factor receptor (EGFR) and epithelial–mesenchymal transition (EMT) markers in breast cancer tissues and cell lines. Moreover, knockdown of Anxa2 impaired EGF-induced EMT, as well as the migration and invasion of breast cancer cells in vitro. Meanwhile, Anxa2 depletion significantly ablated pulmonary metastasis in a severe combined immunodeficiency mouse model of breast cancer. Importantly, Anxa2 reduction inhibited EGF-induced activation of STAT3, which is required for EGF-induced EMT. Anxa2 directly bound to STAT3 and enhanced its transcriptional activity, thereby indicating that Anxa2 promotes EGF-induced EMT in a STAT3-dependent manner. Our findings provide clinical evidence that Anxa2 is a poor prognostic factor for breast cancer and reveal a novel mechanism through which Anxa2 promotes breast cancer metastasis. PMID:26307676

  15. Cancer development, chemoresistance, epithelial to mesenchymal transition and stem cells: A snapshot of IL-6 mediated involvement.

    PubMed

    Bharti, Rashmi; Dey, Goutam; Mandal, Mahitosh

    2016-05-28

    Interleukin-6 (IL-6) is a cytokine present in tumor microenvironment. Elevated level of IL-6 is associated with cancer cell proliferation, angiogenesis and metastasis through fueling STAT3, MAPK and Akt signaling. It promotes epithelial to mesenchymal transition (EMT) through altered expression of N-cadherin, vimentin, snail, twist and E-cadherin leading to cancer metastasis. IL-6 boosts mammosphere formation, self-renewal of stem cells, stemness properties of cancer cells and recruitment of mesenchymal stem cells. IL-6 is also a contributing factor for multidrug resistance in cancer due to gp130/MAPK/STAT3 mediated activation of transcription factors C/EBPβ/δ, overexpression of p-glycoprotein, EMT transition and expansion of stem cells. The in-depth investigation of IL-6 mediated cellular effects and its signaling pathway can provide the new window for future research and clinical development of IL-6 targeted therapy in cancer. Thus, an overview is delivered in this review deciphering the emerging aspect of the predominant influence of IL-6 in malignant transformation, EMT, cancer-associated stem cells and chemoresistance. PMID:26945971

  16. Identification of novel pathways linking epithelial-to-mesenchymal transition with resistance to HER2-targeted therapy

    PubMed Central

    Creedon, Helen; Gómez-Cuadrado, Laura; Tarnauskaitė, Žygimantė; Balla, Jozef; Canel, Marta; MacLeod, Kenneth G.; Serrels, Bryan; Fraser, Craig; Unciti-Broceta, Asier; Tracey, Natasha; Le Bihan, Thierry; Klinowska, Teresa; Sims, Andrew H.; Byron, Adam; Brunton, Valerie G.

    2016-01-01

    Resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapies in the treatment of HER2-positive breast cancer is a major clinical problem. To identify pathways linked to resistance, we generated HER2-positive breast cancer cell lines which are resistant to either lapatinib or AZD8931, two pan-HER family kinase inhibitors. Resistance was HER2 independent and was associated with epithelial-to-mesenchymal transition (EMT), resulting in increased proliferation and migration of the resistant cells. Using a global proteomics approach, we identified a novel set of EMT-associated proteins linked to HER2-independent resistance. We demonstrate that a subset of these EMT-associated genes is predictive of prognosis within the ERBB2 subtype of human breast cancers. Furthermore, targeting the EMT-associated kinases Src and Axl potently inhibited proliferation of the resistant cells, and inhibitors to these kinases may provide additional options for the treatment of HER2-independent resistance in tumors. PMID:26883193

  17. Changes in macrophage phenotype and induction of epithelial-to-mesenchymal transition genes following acute Achilles tenotomy and repair.

    PubMed

    Sugg, Kristoffer B; Lubardic, Jovan; Gumucio, Jonathan P; Mendias, Christopher L

    2014-07-01

    Tendon injuries occur frequently in physically active individuals, but the clinical outcomes for these injuries can be poor. In many injured tissues the repair process is orchestrated by two types of cells, macrophages and fibroblasts. Macrophages, which have both pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, can directly participate in tissue remodeling and direct the response of other cells through the secretion of cytokines and growth factors. In many organ systems, epithelial cells can trans-differentiate into fibroblasts, which can then regenerate damaged ECM. This process is triggered via activation of epithelial-to-mesenchymal transition (EMT) signaling programs. Most tendons are surrounded by sheets of epithelial cells, and these tissue layers could provide a source of fibroblasts to repair injured tendons. To gain greater insight into the biology of tendon repair, we performed a tenotomy and repair in Achilles tendons of adult rats and determined changes in macrophage phenotype, and ECM- and EMT-related genes over a 4-week time course. The results from this study suggest that changes in macrophage phenotype and activation of EMT-related programs likely contribute to the degradation and subsequent repair of injured tendon tissue. PMID:24700411

  18. Regulation of the epithelial to mesenchymal transition and metastasis by Raf kinase inhibitory protein-dependent Notch1 activity

    PubMed Central

    Ha, Ji Hye; Kang, Min Young; Zada, Sahib; Rha, Sun Young; Kang, Sang Soo; Kim, Hyun Joon; Park, Jae-Yong; Byun, June-Ho; Hahm, Jong Ryeal; Shin, Jeong Kyu; Jeong, Sang-Ho; Lee, Young-Joon; Kim, Deok Ryong

    2016-01-01

    Raf kinase inhibitory protein (RKIP), an endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway, has been implicated as a suppressor of metastasis and a prognostic marker in cancers. However, how RKIP acts as a suppressor during metastasis is not fully understood. Here, we show that RKIP activity in cervical and stomach cancer is inversely correlated with endogenous levels of the Notch1 intracellular domain (NICD), which stimulates the epithelial to mesenchymal transition (EMT) and metastasis. The levels of RKIP were significantly decreased in tumor tissues compared to normal tissues, whereas NICD levels were increased. Overexpression of RKIP in several cell lines resulted in a dramatic decrease of NICD and subsequent inhibition of several mesenchymal markers, such as vimentin, N-cadherin, and Snail. In contrast, knockdown of RKIP exhibited opposite results both in vitro and in vivo using mouse models. Nevertheless, knockdown of Notch1 in cancer cells had no effect on the expression of RKIP, suggesting that RKIP is likely an upstream regulator of the Notch1 pathway. We also found that RKIP directly interacts with Notch1 but has no influence on the intracellular level of the γ-secretase complex that is necessary for Notch1 activation. These data suggest that RKIP plays a distinct role in activation of Notch1 during EMT and metastasis, providing a new target for cancer treatment. PMID:26716415

  19. Changes in Macrophage Phenotype and Induction of Epithelial-to-Mesenchymal Transition Genes Following Acute Achilles Tenotomy and Repair

    PubMed Central

    Sugg, Kristoffer B; Lubardic, Jovan; Gumucio, Jonathan P; Mendias, Christopher L

    2014-01-01

    Tendon injuries occur frequently in physically active individuals, but the clinical outcomes for these injuries can be poor. In many injured tissues the repair process is orchestrated by two types of cells, macrophages and fibroblasts. Macrophages, which have both proinflammatory (M1) and antiinflammatory (M2) phenotypes, can directly participate in tissue remodeling and direct the response of other cells through the secretion of cytokines and growth factors. In many organ systems, epithelial cells can transdifferentiate into fibroblasts, which can then regenerate damaged ECM. This process is triggered via activation of epithelial-to-mesenchymal transition (EMT) signaling programs. Most tendons are surrounded by sheets of epithelial cells, and these tissue layers could provide a source of fibroblasts to repair injured tendons. To gain greater insight into the biology of tendon repair, we performed a tenotomy and repair in Achilles tendons of adult rats and determined changes in macrophage phenotype, and ECM- and EMT-related genes over a four week time course. The results from this study suggest that changes in macrophage phenotype and activation of EMT-related programs likely contribute to the degradation and subsequent repair of injured tendon tissue. PMID:24700411

  20. Regulation of the epithelial to mesenchymal transition and metastasis by Raf kinase inhibitory protein-dependent Notch1 activity.

    PubMed

    Noh, Hae Sook; Hah, Young-Sool; Ha, Ji Hye; Kang, Min Young; Zada, Sahib; Rha, Sun Young; Kang, Sang Soo; Kim, Hyun Joon; Park, Jae-Yong; Byun, June-Ho; Hahm, Jong Ryeal; Shin, Jeong Kyu; Jeong, Sang-Ho; Lee, Young-Joon; Kim, Deok Ryong

    2016-01-26

    Raf kinase inhibitory protein (RKIP), an endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway, has been implicated as a suppressor of metastasis and a prognostic marker in cancers. However, how RKIP acts as a suppressor during metastasis is not fully understood. Here, we show that RKIP activity in cervical and stomach cancer is inversely correlated with endogenous levels of the Notch1 intracellular domain (NICD), which stimulates the epithelial to mesenchymal transition (EMT) and metastasis. The levels of RKIP were significantly decreased in tumor tissues compared to normal tissues, whereas NICD levels were increased. Overexpression of RKIP in several cell lines resulted in a dramatic decrease of NICD and subsequent inhibition of several mesenchymal markers, such as vimentin, N-cadherin, and Snail. In contrast, knockdown of RKIP exhibited opposite results both in vitro and in vivo using mouse models. Nevertheless, knockdown of Notch1 in cancer cells had no effect on the expression of RKIP, suggesting that RKIP is likely an upstream regulator of the Notch1 pathway. We also found that RKIP directly interacts with Notch1 but has no influence on the intracellular level of the γ-secretase complex that is necessary for Notch1 activation. These data suggest that RKIP plays a distinct role in activation of Notch1 during EMT and metastasis, providing a new target for cancer treatment. PMID:26716415

  1. Emerging Transcriptional Mechanisms in the Regulation of Epithelial to Mesenchymal Transition and Cellular Plasticity in the Kidney

    PubMed Central

    De Chiara, Letizia; Crean, John

    2016-01-01

    Notwithstanding controversies over the role of epithelial to mesenchymal transition in the pathogenesis of renal disease, the last decade has witnessed a revolution in our understanding of the regulation of renal cell plasticity. Significant parallels undoubtedly exist between ontogenic processes and the initiation and propagation of damage in the diseased kidney as evidenced by the reactivation of developmental programmes of gene expression, in particular with respect to TGFβ superfamily signaling. Indeed, multiple signaling pathways converge on a complex transcriptional regulatory nexus that additionally involves epigenetic activator and repressor mechanisms and microRNA regulatory networks that control renal cell plasticity. It is becoming increasingly apparent that differentiated cells can acquire an undifferentiated state akin to “stemness” which is leading us towards new models of complex cell behaviors and interactions. Here we discuss the latest findings that delineate new and novel interactions between this transcriptional regulatory network and highlight a hitherto poorly recognized role for the Polycomb Repressive Complex (PRC2) in the regulation of renal cell plasticity. A comprehensive understanding of how external stimuli interact with the epigenetic control of gene expression, in normal and diseased contexts, establishes a new therapeutic paradigm to promote the resolution of renal injury and regression of fibrosis. PMID:26771648

  2. AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells.

    PubMed

    Raghavan, Cibin T; Nagaraj, Ram H

    2016-08-01

    Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis. PMID:27263094

  3. Knockdown of Human TCF4 Affects Multiple Signaling Pathways Involved in Cell Survival, Epithelial to Mesenchymal Transition and Neuronal Differentiation

    PubMed Central

    Forrest, Marc P.; Waite, Adrian J.; Martin-Rendon, Enca; Blake, Derek J.

    2013-01-01

    Haploinsufficiency of TCF4 causes Pitt-Hopkins syndrome (PTHS): a severe form of mental retardation with phenotypic similarities to Angelman, Mowat-Wilson and Rett syndromes. Genome-wide association studies have also found that common variants in TCF4 are associated with an increased risk of schizophrenia. Although TCF4 is transcription factor, little is known about TCF4-regulated processes in the brain. In this study we used genome-wide expression profiling to determine the effects of acute TCF4 knockdown on gene expression in SH-SY5Y neuroblastoma cells. We identified 1204 gene expression changes (494 upregulated, 710 downregulated) in TCF4 knockdown cells. Pathway and enrichment analysis on the differentially expressed genes in TCF4-knockdown cells identified an over-representation of genes involved in TGF-β signaling, epithelial to mesenchymal transition (EMT) and apoptosis. Among the most significantly differentially expressed genes were the EMT regulators, SNAI2 and DEC1 and the proneural genes, NEUROG2 and ASCL1. Altered expression of several mental retardation genes such as UBE3A (Angelman Syndrome), ZEB2 (Mowat-Wilson Syndrome) and MEF2C was also found in TCF4-depleted cells. These data suggest that TCF4 regulates a number of convergent signaling pathways involved in cell differentiation and survival in addition to a subset of clinically important mental retardation genes. PMID:24058414

  4. Isolating Epithelial and Epithelial-to-Mesenchymal Transition Populations from Primary Tumors by Fluorescence-Activated Cell Sorting.

    PubMed

    Aiello, Nicole M; Rhim, Andrew D; Stanger, Ben Z

    2016-01-01

    Transgenic mice that express conditional reporters allow for the isolation of specific cell lineages. These cells can be further stratified by gene expression and collected by fluorescence-activated cell sorting (FACS) for further analysis. Using Cre-recombinase (Cre) technology we have generated a transgenic mouse line termed PKCY in which all pancreatic epithelial cells and therefore all pancreatic cancer cells are constitutively labeled with yellow fluorescent protein (YFP). We have used immunofluorescent staining for E-cadherin to divide the YFP(+) tumor population into epithelial cells (E-cadherin positive) and cells that have undergone an epithelial-to-mesenchymal transition (EMT; E-cadherin negative). This protocol describes how to prepare a tumor sample for FACS, with an emphasis on separating epithelial and EMT populations. These cells can then be used for a number of applications including, but not limited to, the generation of cell lines, gene-expression analysis by quantitative polymerase chain reaction (qPCR) or RNA sequencing, DNA sequencing, chromatin immunoprecipitation, and western blots. PMID:26729901

  5. Genotypes of cancer stem cells characterized by epithelial-to-mesenchymal transition and proliferation related functions

    PubMed Central

    Hsu, Chueh-Lin; Chung, Feng-Hsiang; Chen, Chih-Hao; Hsu, Tzu-Ting; Liu, Szu-Mam; Chung, Dao-Sheng; Hsu, Ya-Fen; Chen, Chien-Lung; Ma, Nianhan; Lee, Hoong-Chien

    2016-01-01

    Cancer stem cells (CSCs), or cancer cells with stem cell-like properties, generally exhibit drug resistance and have highly potent cancer inducing capabilities. Genome-wide expression data collected at public repositories over the last few years provide excellent material for studies that can lead to insights concerning the molecular and functional characteristics of CSCs. Here, we conducted functional genomic studies of CSC based on fourteen PCA-screened high quality public CSC whole genome gene expression datasets and, as control, four high quality non-stem-like cancer cell and non-cancerous stem cell datasets from the Gene Expression Omnibus database. A total of 6,002 molecular signatures were taken from the Molecular Signatures Database and used to characterize the datasets, which, under two-way hierarchical clustering, formed three genotypes. Type 1, consisting of mainly glia CSCs, had significantly enhanced proliferation, and significantly suppressed epithelial-mesenchymal transition (EMT), related functions. Type 2, mainly breast CSCs, had significantly enhanced EMT, but not proliferation, related functions. Type 3, composed of ovarian, prostate, and colon CSCs, had significantly suppressed proliferation related functions and mixed expressions on EMT related functions. PMID:27597445

  6. Vitiligo patient-derived keratinocytes exhibit characteristics of normal wound healing via epithelial to mesenchymal transition.

    PubMed

    Banerjee, Poulomi; Venkatachalam, Sandhyaa; Mamidi, Murali Krishna; Bhonde, Ramesh; Shankar, Krupa; Pal, Rajarshi

    2015-05-01

    Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte-induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial-mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF-b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity. PMID:25690925

  7. Genotypes of cancer stem cells characterized by epithelial-to-mesenchymal transition and proliferation related functions.

    PubMed

    Hsu, Chueh-Lin; Chung, Feng-Hsiang; Chen, Chih-Hao; Hsu, Tzu-Ting; Liu, Szu-Mam; Chung, Dao-Sheng; Hsu, Ya-Fen; Chen, Chien-Lung; Ma, Nianhan; Lee, Hoong-Chien

    2016-01-01

    Cancer stem cells (CSCs), or cancer cells with stem cell-like properties, generally exhibit drug resistance and have highly potent cancer inducing capabilities. Genome-wide expression data collected at public repositories over the last few years provide excellent material for studies that can lead to insights concerning the molecular and functional characteristics of CSCs. Here, we conducted functional genomic studies of CSC based on fourteen PCA-screened high quality public CSC whole genome gene expression datasets and, as control, four high quality non-stem-like cancer cell and non-cancerous stem cell datasets from the Gene Expression Omnibus database. A total of 6,002 molecular signatures were taken from the Molecular Signatures Database and used to characterize the datasets, which, under two-way hierarchical clustering, formed three genotypes. Type 1, consisting of mainly glia CSCs, had significantly enhanced proliferation, and significantly suppressed epithelial-mesenchymal transition (EMT), related functions. Type 2, mainly breast CSCs, had significantly enhanced EMT, but not proliferation, related functions. Type 3, composed of ovarian, prostate, and colon CSCs, had significantly suppressed proliferation related functions and mixed expressions on EMT related functions. PMID:27597445

  8. Glyceollin I Reverses Epithelial to Mesenchymal Transition in Letrozole Resistant Breast Cancer through ZEB1

    PubMed Central

    Carriere, Patrick P.; Llopis, Shawn D.; Naiki, Anna C.; Nguyen, Gina; Phan, Tina; Nguyen, Mary M.; Preyan, Lynez C.; Yearby, Letitia; Pratt, Jamal; Burks, Hope; Davenport, Ian R.; Nguyen, Thu A.; Parker-Lemieux, KiTani; Payton-Stewart, Florastina; Williams, Christopher C.; Boué, Stephen M.; Burow, Matthew E.; Collins-Burow, Bridgette; Hilliard, Aaron; Davidson, A. Michael; Tilghman, Syreeta L.

    2015-01-01

    Although aromatase inhibitors are standard endocrine therapy for postmenopausal women with early-stage metastatic estrogen-dependent breast cancer, they are limited by the development of drug resistance. A better understanding of this process is critical towards designing novel strategies for disease management. Previously, we demonstrated a global proteomic signature of letrozole-resistance associated with hormone-independence, enhanced cell motility and implications of epithelial mesenchymal transition (EMT). Letrozole-resistant breast cancer cells (LTLT-Ca) were treated with a novel phytoalexin, glyceollin I, and exhibited morphological characteristics synonymous with an epithelial phenotype and decreased proliferation. Letrozole-resistance increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression (4.51-fold), while glyceollin I treatment caused a −3.39-fold reduction. Immunofluorescence analyses resulted of glyceollin I-induced increase and decrease in E-cadherin and ZEB1, respectively. In vivo studies performed in ovariectomized, female nude mice indicated that glyceollin treated tumors stained weakly for ZEB1 and N-cadherin and strongly for E-cadherin. Compared to letrozole-sensitive cells, LTLT-Ca cells displayed enhanced motility, however in the presence of glyceollin I, exhibited a 68% and 83% decrease in invasion and migration, respectively. These effects of glyceollin I were mediated in part by inhibition of ZEB1, thus indicating therapeutic potential of glyceollin I in targeting EMT in letrozole resistant breast cancer. PMID:26703648

  9. Reprogramming during epithelial to mesenchymal transition under the control of TGFβ

    PubMed Central

    Tan, E-Jean; Olsson, Anna-Karin; Moustakas, Aristidis

    2015-01-01

    Epithelial-mesenchymal transition (EMT) refers to plastic changes in epithelial tissue architecture. Breast cancer stromal cells provide secreted molecules, such as transforming growth factor β (TGFβ), that promote EMT on tumor cells to facilitate breast cancer cell invasion, stemness and metastasis. TGFβ signaling is considered to be abnormal in the context of cancer development; however, TGFβ acting on breast cancer EMT resembles physiological signaling during embryonic development, when EMT generates or patterns new tissues. Interestingly, while EMT promotes metastatic fate, successful metastatic colonization seems to require the inverse process of mesenchymal-epithelial transition (MET). EMT and MET are interconnected in a time-dependent and tissue context-dependent manner and are coordinated by TGFβ, other extracellular proteins, intracellular signaling cascades, non-coding RNAs and chromatin-based molecular alterations. Research on breast cancer EMT/MET aims at delivering biomolecules that can be used diagnostically in cancer pathology and possibly provide ideas for how to improve breast cancer therapy. PMID:25482613

  10. Sorcin silencing inhibits epithelial-to-mesenchymal transition and suppresses breast cancer metastasis in vivo.

    PubMed

    Hu, Yunhui; Li, Shuangjing; Yang, Ming; Yan, Cihui; Fan, Dongmei; Zhou, Yuan; Zhang, Yanjun; Yagüe, Ernesto; Xiong, Dongsheng

    2014-01-01

    Sorcin, a 22-kDa calcium-binding protein, renders cancer cells resistant to chemotherapeutic agents, thus playing an important role in multidrug resistance. As there is a clear association between drug resistance and an aggressive phenotype, we asked whether sorcin affects also the motility, invasion, and stem cell characteristics of cancer cells. We have used both RNA interference (transient and stable expression of hairpins) and a lentiviral expression vector to experimentally modulate sorcin expression in a variety of cells. We demonstrate that sorcin depletion in MDA-MB-231 breast cancer cells reduces the pool of CD44(+)/CD24(-) and ALDH1(high) cancer stem cells (CSCs) as well as mammosphere-forming capacity. We also observe that sorcin regulates epithelial-mesenchymal transition and CSCs partly through E-cadherin and vascular endothelial growth factor expression. This leads to the acquisition of an epithelial-like phenotype, attenuating epithelial-mesenchymal transition and suppression of metastases in nude mice. The sorcin-depleted phenotype can also be reproduced in lung adenocarcinoma A549 cells and lung fibrosarcoma HT1080 cells. In addition, overexpression of sorcin in MCF7 cells, which have low endogenous sorcin expression levels, increases their migration and invasion in vitro. This offers the rationale for the development of therapeutic strategies down-regulating sorcin expression for the treatment of cancer. PMID:24337682

  11. Loss of Trop2 Promotes Carcinogenesis and Features of Epithelial to Mesenchymal Transition in Squamous Cell Carcinoma

    PubMed Central

    Wang, Jianbo; Zhang, Kaihua; Grabowska, Dorota; Li, Aimin; Dong, Yiyu; Day, Ryan; Humphrey, Peter; Lewis, James; Kladney, Raleigh D.; Arbeit, Jeffrey M.; Weber, Jason D.; Chung, Christine H.; Michel, Loren S.

    2011-01-01

    Trop2, an oncogenic cell-surface protein under investigation as a therapeutic target, is commonly overexpressed in several epithelial tumor types yet its function in tumor biology remains relatively unexplored. To investigate the role of Trop2 in epithelial carcinogenesis, we generated Trop2−/− mice, which are viable and possess a normal lifespan. Contrary to expectations, Trop2 loss fails to suppress keratinocyte transformation. Instead, ras-transformed Trop2−/− keratinocytes preferentially pass through an epithelial to mesenchymal transition (EMT) and form tumors with spindle cell histology. Furthermore, Trop2 loss renders Arf null mice susceptible to the formation of biphasic sarcomatoid carcinomas containing both squamous and spindle cell components upon carcinogen exposure in an otherwise skin-cancer resistant strain (C57Bl/6). Immortalized keratinocytes derived from Trop2−/−Arf−/− mice exhibit enhanced proliferative and migratory capacity as well as increased activation of MAPK and Src prior to transformation. The clinical relevance of these findings was supported by studying the molecular epidemiology of Trop2 in primary head and neck squamous cell carcinomas. This analysis revealed that Trop2 mRNA levels are decreased in a subset of tumors with features of EMT, and total loss of Trop2 protein expression is observed in the spindle cell component of sarcomatoid carcinomas. Therefore, while previous studies have emphasized the potential importance of Trop2 gain-of-function, these results uncover a role for Trop2 loss in tumorigenesis and the mesenchymal transdifferentiation observed in a subset of squamous cell carcinomas. PMID:21970857

  12. Ampelopsin reduces the migration and invasion of ovarian cancer cells via inhibition of epithelial-to-mesenchymal transition.

    PubMed

    Liu, Tianfeng; Liu, Peishu; Ding, Feng; Yu, Nina; Li, Shihong; Wang, Surong; Zhang, Xiaofei; Sun, Xiangxiu; Chen, Ying; Wang, Feng; Zhao, Yunhe; Li, Bo

    2015-02-01

    Ampelopsin has displayed anticancer activity in several types of cancers. However, no evidence has been reported for the direct effect of ampelopsin on ovarian cancer cell migration and invasion, and the underling mechanisms have not yet been clearly established. The aim of the present study was to investigate the influence of ampelopsin on the migration and invasion of ovarian cancer. Proliferation and viability of the ovarian cancer cells were detected by MTT assay. Migration and invasion of the cells were detected, respectively, by scratch wound healing assay and Transwell assay. The expression levels of epithelial-to-mesenchymal transition (EMT) markers were detected at the protein level after stimulation with ampelopsin. Then, the expression levels of NF-κB and p-IκBα were detected with western blot analysis. Meanwhile, an inhibitor of NF-κB was used to investigate the effect of ampelopsin. Finally, the expression of Snail was also detected. Proliferation, migration and invasion of the A2780 cells were all inhibited following the application of ampelopsin. Ampelopsin upregulated E-cadherin and downregulated N-cadherin and vimentin in a concentration- and time-dependent manner. Ampelopsin also exerted its ability to suppress the nuclear translocation of the NF-κB pathway. Administration of the inhibitor BAY11-7082 confirmed the roles of NF-κB in the expression of EMT markers and its transcription factor. These results demonstrated that ampelopsin inhibited EMT and reduced the invasion of ovarian cancer cells via the NF-κB/Snail pathway. PMID:25502786

  13. Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach

    PubMed Central

    Meshcheryakova, Anastasia; Köfeler, Harald C.; Triebl, Alexander; Mungenast, Felicitas; Heinze, Georg; Gerner, Christopher; Zimmermann, Philip; Jaritz, Markus; Mechtcheriakova, Diana

    2016-01-01

    The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery

  14. Thrombomodulin Influences the Survival of Patients with Non-Metastatic Colorectal Cancer through Epithelial-To-Mesenchymal Transition (EMT)

    PubMed Central

    Cheng, Ya-Wen; Huang, Chi-Chou; Chen, William Tzu-Liang; Ke, Tao-Wei; Wei, Po-Li

    2016-01-01

    Background Treatment resistance and metastasis are the major causes of death among patients with colorectal cancer (CRC). Approximately 20% of surgically treated patients ultimately develop metastases during the follow-up period. Currently, the TNM system is the only available prognostic test. Therefore, the identification of new markers for CRC remains important. Thrombomodulin (TM), a glycoprotein, is involved in angiogenesis and has been linked to many malignant diseases. However, the function of TM in CRC remains unclear. Methods A total of 170 patients with CRC participated in this study. TM expression was analyzed via immunohistochemistry. Univariate (Kaplan-Meier) analysis was used to analyze patient outcomes, including overall survival (OS) and disease-free survival (DFS). TM expression was manipulated using shRNA or an overexpression system. Transwell migration assays, wound healing migration assays, and the xCELLigence biosensor system were used to detect cell proliferative and migratory capacities. Results TM expression in the tumor tissues significantly and positively correlated with the DFS and OS of non-metastatic patients with CRC (ps = 0.036 and 0.0218, respectively). Suppression of TM expression increased the proliferation and migration of DLD-1 cells. TM overexpression reduced the cells’ proliferative and migratory capacities. Cyclooxygenase (COX)-2 expression was up-regulated following TM silencing. Furthermore, the association between the migration of colon cancer cells and the levels of TM and epithelial-to-mesenchymal transition (EMT) markers (fibronectin, vimentin and ezrin) was confirmed in HT29 and DLD-1 cells. Conclusions Our study demonstrates that patients with non-metastatic CRC display low TM expression in their tumors and exhibit reduced DFS and OS. The enhanced expression of mesenchymal markers and COX-2 may be involved in the mechanisms that underlie recurrence in patients with cancer displaying low TM expression. PMID:27512995

  15. miRNA-101 Suppresses Epithelial-to-Mesenchymal Transition by Targeting HMGA2 in Pancreatic Cancer Cells.

    PubMed

    Jiang, Wenli; Gu, Wen; Qiu, Ronglin; Shen, Chenglong; Wu, Yaohao; Zhang, Jie; Zhou, Jiajia; Guo, Yixing; Li, Zhixi; Deng, Jiemin; Zeng, Lexiang; Tang, Jin; Zhi, Qiaoming; Deng, Xiaogeng

    2016-01-01

    miR-101 is an outstanding tumor suppressor in various cancers, while its role in pancreatic cancer (PC) is still unknown. The aim of this study was to investigate the role of miR-101 in epithelial-to-mesenchymal transition (EMT) and its clinical relevance in PC. Our data showed that the miR-101 expression was significantly decreased in human PC tissues, compared to non-tumor counterparts (p<0.05), which was reversely correlated to clinical characteristics, including lymph node metastasis, more venous infiltration, higher expression of CA19-9 and TNM stage (p<0.05). Low miR-101 expression was also confirmed to be associated with a poorer overall survival rate in PC patients (p<0.05). We identified high-mobility group AT-hook 2 (HMGA2) gene as a putative target of miR-101 in PC by bioinformatics analysis, dual luciferase activity and western blot assay, and found that miR-101 could specifically target the HMGA2 3'-untranslated region (3'-UTR) (p<0.05). Knockdown of HMGA2 reversed EMT resembling that of miR-101 over-expression. An inverse correlation between miR-101 and HMGA2 was observed in patients with PC (p<0.05). Taken together, our findings speculated that miR-101 might act as an inhibiting factor in EMT process in PC and up-regulation of miR-101 might be considered as a potentially key molecular treatment strategy for PC patients. PMID:25968875

  16. Antrodin C inhibits epithelial-to-mesenchymal transition and metastasis of breast cancer cells via suppression of Smad2/3 and β-catenin signaling pathways.

    PubMed

    Kumar, K J Senthil; Vani, M Gokila; Chueh, Pin-Ju; Mau, Jeng-Leun; Wang, Sheng-Yang

    2015-01-01

    Epithelial-to-mesenchymal transition (EMT) is a crucial event involved metastasis of certain tumors. Thus, identifying chemical agents that can block EMT is highly warranted for the development of anti-cancer chemoprevention/chemotherapies. In this study, we found that Antrodin C (ADC), a maleimide derivative isolated from Antrodia cinnamomea health food product inhibits TGF-β1-induced EMT and breast cancer cell metastasis in vitro. Pretreatment of MCF-7 cells with ADC significantly blocked TGF-β1-induced phenotypic changes and actin cytoskeleton remodeling. In addition, ADC was able to up-regulate epithelial markers such as E-cadherin and occludin, whereas mesenchymal markers including N-cadherin and vimentin were significantly inhibited, possibly through the modulation of transcriptional regulators Smad/Smad3. ADC blocked TGF-β1-induced migration and invasion of MCF-7 cells through the down-regulation of matrix-metalloproteinases (MMP-2, -9) and urokinase plasminogen activator (uPA). The inhibition of MMPs and uPA activity by ADC was reasoned by suppression of its corresponding transcription factor β-catenin. Taken together, our data suggested that ADC attenuates the TGF-β1-induced EMT, migration and invasion of human breast carcinoma through the suppression of Smad2/3 and β-catenin signaling pathways. PMID:25658913

  17. Exosome cargo reflects TGF-β1-mediated epithelial-to-mesenchymal transition (EMT) status in A549 human lung adenocarcinoma cells.

    PubMed

    Kim, Jiyeon; Kim, Tae Yeon; Lee, Myung Shin; Mun, Ji Young; Ihm, Chunhwa; Kim, Soon Ae

    2016-09-16

    It has been suggested that tumor cells secrete exosomes to modify the local microenvironment, which then promotes intercellular communication and metastasis. Although exosomes derived from cancer cells may contribute to the epithelial-mesenchymal transition (EMT) in untransformed cells, few studies have defined exosome cargo upon induction of EMT. In this study, we investigated the changes in exosomal cargo from the epithelial to mesenchymal cell phenotype by inducing EMT with transforming growth factor (TGF)-β1 in A549 human lung adenocarcinoma cells. The protein content of the exosomes reflects the change in the cell phenotype. In addition, miR-23a was significantly enriched in the exosomes after mesenchymal transition. Following treatment of exosomes from mesenchymal cells via EMT induction with TGF-β1 to the epithelial cell type, phenotypic changes in protein expression level and cell morphology were observed. Autologous treatment of exosomes enhanced the transcriptional activity and abundance of β-catenin. Our results suggest that the exosomal protein and miRNA content reflects the physiological condition of its source and that exosomes induce phenotypic changes via autocrine signaling. PMID:27492069

  18. Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition

    PubMed Central

    Azmi, Asfar S.; Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Senapedis, William; Baloglu, Erkan; Bollig-Fischer, Aliccia; Dyson, Gregory; Kauffman, Michael; Landesman, Yosef; Shacham, Sharon; Philip, Philip A.; Mohammad, Ramzi M.

    2015-01-01

    Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (–ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15 mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore. PMID:26536918

  19. Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition.

    PubMed

    Azmi, Asfar S; Muqbil, Irfana; Wu, Jack; Aboukameel, Amro; Senapedis, William; Baloglu, Erkan; Bollig-Fischer, Aliccia; Dyson, Gregory; Kauffman, Michael; Landesman, Yosef; Shacham, Sharon; Philip, Philip A; Mohammad, Ramzi M

    2015-01-01

    Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (-ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15 mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore. PMID:26536918

  20. PAC exhibits potent anti-colon cancer properties through targeting cyclin D1 and suppressing epithelial-to-mesenchymal transition.

    PubMed

    Al-Qasem, Abeer; Al-Howail, Huda A; Al-Swailem, Mashael; Al-Mazrou, Amer; Al-Otaibi, Basem; Al-Jammaz, Ibrahim; Al-Khalaf, Huda H; Aboussekhra, Abdelilah

    2016-03-01

    Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide. Although response rates and overall survival have been improved in recent years, resistance to multiple drug combinations is inevitable. Therefore, the development of more efficient drugs, with fewer side effects is urgently needed. To this end, we have investigated in the present report the effect of PAC, a novel cucumin analogue, on CRC cells both in vitro and in vivo. We have shown that PAC induces apoptosis, mainly via the internal mitochondrial route, and inhibits cell proliferation through delaying the cell cycle at G2/M phase. Interestingly, the pro-apoptotic effect was mediated through STAT3-dependent down-regulation of cyclin D1 and its downstream target survivin. Indeed, change in the expression level of cyclin D1 modulated the expression of survivin and the response of CRC cells to PAC. Furthermore, using the ChIP assay, we have shown PAC-dependent reduction in the binding of STAT3 to the cyclin D1 promoter in vivo. Additionally, PAC suppressed the epithelial-to-mesenchymal process through down-regulating the mesenchymal markers (N-cadherin, vimentin and Twist1) and inhibiting the invasion/migration abilities of the CRC cells via repressing the pro-migration/invasion protein kinases AKT and ERK1/2. In addition, PAC inhibited tumor growth and repressed the JAK2/STAT3, AKT/mTOR and MEK/ERK pathways as well as their common downstream effectors cyclin D1 and survivin in humanized CRC xenografts. Collectively, these results indicate that PAC has potent anti-CRC effects, and therefore could constitute an effective alternative chemotherapeutic agent, which may consolidate the adjuvant treatment of colon cancer. © 2015 Wiley Periodicals, Inc. PMID:25641341

  1. β-III tubulin modulates the behavior of Snail overexpressed during the epithelial-to-mesenchymal transition in colon cancer cells.

    PubMed

    Sobierajska, Katarzyna; Wieczorek, Katarzyna; Ciszewski, Wojciech M; Sacewicz-Hofman, Izabela; Wawro, Marta E; Wiktorska, Magdalena; Boncela, Joanna; Papiewska-Pajak, Izabela; Kwasniak, Pawel; Wyroba, Elzbieta; Cierniewski, Czeslaw S; Niewiarowska, Jolanta

    2016-09-01

    Class III β-tubulin (TUBB3) is a marker of drug resistance expressed in a variety of solid tumors. Originally, it was described as an important element of chemoresistance to taxanes. Recent studies have revealed that TUBB3 is also involved in an adaptive response to a microenvironmental stressor, e.g. low oxygen levels and poor nutrient supply in some solid tumors, independently of the microtubule targeting agent. Furthermore, it has been demonstrated that TUBB3 is a marker of biological aggressiveness associated with modulation of metastatic abilities in colon cancer. The epithelial-to-mesenchymal transition (EMT) is a basic cellular process by which epithelial cells lose their epithelial behavior and become invasive cells involved in cancer metastasis. Snail is a zinc-finger transcription factor which is able to induce EMT through the repression of E-cadherin expression. In the presented studies we focused on the analysis of the TUBB3 role in EMT-induced colon adenocarcinoma cell lines HT-29 and LS180. We observed a positive correlation between Snail presence and TUBB3 upregulation in tested adenocarcinoma cell lines. The cellular and behavioral analysis revealed for the first time that elevated TUBB3 level is functionally linked to increased cell migration and invasive capability of EMT induced cells. Additionally, the post-transcriptional modifications (phosphorylation, glycosylation) appear to regulate the cellular localization of TUBB3 and its phosphorylation, observed in cytoskeleton, is probably involved in cell motility modulation. PMID:27188792

  2. Taiwan cobra cardiotoxin III suppresses EGF/EGFR-mediated epithelial-to-mesenchymal transition and invasion of human breast cancer MDA-MB-231 cells.

    PubMed

    Tsai, Pei-Chien; Fu, Yaw-Syan; Chang, Long-Sen; Lin, Shinne-Ren

    2016-03-01

    Breast cancer is a highly malignant carcinoma and most deaths of breast cancer are caused by metastasis. The epithelial-to-mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Epidermal growth factor (EGF) and its receptor, EGFR, play roles in cancer metastasis. CTX III, a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity; however, the effect of CTX III on the EMT of cancer cells remains elusive. CTX III treatment resulted in morphological changes from elongated and spindle shape to rounded and epithelial-like shape, induced upregulation of E-cadherin and concurrent downregulation of N-cadherin and Vimentin protein levels, corresponding to observed decreases in cell migration and invasion. CTX III treatment also decreased the expression of Snail and Twist in EGF-induced MDA-MB-231 cells. Concurrently, CTX III efficiently inhibited the EGFR phosphorylation and downstream activation of phosphatidylinositol 3-kinase (PI3K)/Akt and ERK1/2. The EGFR specific inhibitor AG1478 significantly suppressed ERK1/2 and Akt phosphorylation, cell migration and invasion, as well as the expressional changes associated with EMT markers in EGF-induced MDA-MB-231 cells. CTX III inhibitory effect on EGF-evoked invasion of MDA-MB-231 cells is mediated through suppressing EGF/EGFR activation and EMT process. PMID:26774845

  3. Helicobacter pylori CagA and IL-1β Promote the Epithelial-to-Mesenchymal Transition in a Nontransformed Epithelial Cell Model

    PubMed Central

    Arévalo-Romero, Haruki; Meza, Isaura; Vallejo-Flores, Gabriela

    2016-01-01

    Gastric cancer is the third cause of cancer death worldwide and infection by Helicobacter pylori (H. pylori) is considered the most important risk factor, mainly by the activity of its virulence factor CagA. H. pylori/CagA-induced chronic inflammation triggers a series of gastric lesions of increased severity, starting with gastritis and ending with cancer. IL-1β has been associated with tumor development and invasiveness in different types of cancer, including gastric cancer. Currently, it is not clear if there is an association between CagA and IL-1β at a cellular level. In this study, we analyzed the effects of IL-1β and CagA on MCF-10A nontransformed cells. We found evidence that both CagA and IL-1β trigger the initiation of the epithelial-to-mesenchymal transition characterized by β-catenin nuclear translocation, increased expression of Snail1 and ZEB1, downregulation of CDH1, and morphological changes during MCF-10A acini formation. However, only CagA induced MMP9 activity and cell invasion. Our data support that IL-1β and CagA target the β-catenin pathway, with CagA leading to acquisition of a stage related to aggressive tumors. PMID:27525003

  4. MiR-363-3p inhibits the epithelial-to-mesenchymal transition and suppresses metastasis in colorectal cancer by targeting Sox4.

    PubMed

    Hu, Fayong; Min, Jiang; Cao, Xiaonian; Liu, Liang; Ge, Zongqing; Hu, Junbo; Li, Xiaolan

    2016-05-20

    Epithelial-to-mesenchymal transition (EMT) plays an essential role in cancer invasion and metastasis and is associated with tumor recurrence in colorectal cancer (CRC). However, the mechanism that contributes to EMT have not been fully understood in CRC. In the present study, we showed that miR-363-3p was frequently down-regulated in CRC tissue specimens with lymph node metastasis. Moreover, we demonstrated that down-regulation of miR-363-3p promoted CRC cell migration and invasion, and induced EMT in vitro and in vivo, revealing that miR-363-3p playes a potential tumor-suppressive role in CRC. Analysis of the underlying mechanisms revealed that miR-363-3p could regulate Sox4 expression by directly targeting its 3'untranslated region. Down-regulation of miR-363-3p increased Sox4 expression and induced EMT, while overexpression of miR-363-3p decreased Sox4 expression. Taken together, our findings indicate that miR-363-3p is involved in CRC metastasis and functions as a tumor suppressor via negatively regulating Sox4. Therefore, the up-regulation of miR-363-3p in human CRC may have therapeutic benefits. PMID:27084453

  5. Helicobacter pylori CagA and IL-1β Promote the Epithelial-to-Mesenchymal Transition in a Nontransformed Epithelial Cell Model.

    PubMed

    Arévalo-Romero, Haruki; Meza, Isaura; Vallejo-Flores, Gabriela; Fuentes-Pananá, Ezequiel M

    2016-01-01

    Gastric cancer is the third cause of cancer death worldwide and infection by Helicobacter pylori (H. pylori) is considered the most important risk factor, mainly by the activity of its virulence factor CagA. H. pylori/CagA-induced chronic inflammation triggers a series of gastric lesions of increased severity, starting with gastritis and ending with cancer. IL-1β has been associated with tumor development and invasiveness in different types of cancer, including gastric cancer. Currently, it is not clear if there is an association between CagA and IL-1β at a cellular level. In this study, we analyzed the effects of IL-1β and CagA on MCF-10A nontransformed cells. We found evidence that both CagA and IL-1β trigger the initiation of the epithelial-to-mesenchymal transition characterized by β-catenin nuclear translocation, increased expression of Snail1 and ZEB1, downregulation of CDH1, and morphological changes during MCF-10A acini formation. However, only CagA induced MMP9 activity and cell invasion. Our data support that IL-1β and CagA target the β-catenin pathway, with CagA leading to acquisition of a stage related to aggressive tumors. PMID:27525003

  6. 14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

    PubMed

    Raychaudhuri, Kumarkrishna; Chaudhary, Neelam; Gurjar, Mansa; D'Souza, Roseline; Limzerwala, Jazeel; Maddika, Subbareddy; Dalal, Sorab N

    2016-07-29

    Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene. PMID:27261462

  7. Cadherin 6 promotes neural crest cell detachment via F-actin regulation and influences active Rho distribution during epithelial-to-mesenchymal transition

    PubMed Central

    Clay, Matthew R.; Halloran, Mary C.

    2014-01-01

    The epithelial-to-mesenchymal transition (EMT) is a complex change in cell phenotype that is important for cell migration, morphogenesis and carcinoma metastasis. Loss of epithelial cell adhesion and tight regulation of cadherin adhesion proteins are crucial for EMT. Cells undergoing EMT often display cadherin switching, where they downregulate one cadherin and induce expression of another. However, the functions of the upregulated cadherins and their effects on cell motility are poorly understood. Neural crest cells (NCCs), which undergo EMT during development, lose N-cadherin and upregulate Cadherin 6 (Cdh6) prior to EMT. Cdh6 has been suggested to suppress EMT via cell adhesion, but also to promote EMT by mediating pro-EMT signals. Here, we determine novel roles for Cdh6 in generating cell motility during EMT. We use live imaging of NCC behavior in vivo to show that Cdh6 promotes detachment of apical NCC tails, an important early step of EMT. Furthermore, we show that Cdh6 affects spatiotemporal dynamics of F-actin and active Rho GTPase, and that Cdh6 is required for accumulation of F-actin in apical NCC tails during detachment. Moreover, Cdh6 knockdown alters the subcellular distribution of active Rho, which is known to promote localized actomyosin contraction that is crucial for apical NCC detachment. Together, these data suggest that Cdh6 is an important determinant of where subcellular actomyosin forces are generated during EMT. Our results also identify mechanisms by which an upregulated cadherin can generate cell motility during EMT. PMID:24917505

  8. An hTERT/ZEB1 complex directly regulates E-cadherin to promote epithelial-to-mesenchymal transition (EMT) in colorectal cancer.

    PubMed

    Qin, Yong; Tang, Bo; Hu, Chang-Jiang; Xiao, Yu-Feng; Xie, Rui; Yong, Xin; Wu, Yu Yun; Dong, Hui; Yang, Shi-Ming

    2016-01-01

    In human cancer, high telomerase expression is correlated with tumor aggressiveness and metastatic potential. Telomerase activation occurs through telomerase reverse transcriptase (hTERT) induction, which contributes to malignant transformation by stabilizing telomeres. Previous studies have shown that hTERT can promote tumor invasion and metastasis of gastric cancer, liver cancer and esophageal cancer. Epithelial-to-mesenchymal transition (EMT), a requirement for tumor invasion and metastasis, plays a key role in cancer progression. Although hTERT promotes EMT through Wnt signaling in several cancers, it is unknown if other signaling pathways are involved. In the present study, we found that hTERT and ZEB1 form a complex, which directly binds to the E-cadherin promoter, and then inhibits E-cadherin expression and promots EMT in colorectal cancer cells. hTERT overexpression in HCT116 and SW480 cells could induce E-cadherin down-regulation. However, E-cadherin expression was recovered when ZEB1 function was impaired even during hTERT overexpression. Taken together, our findings suggest that hTERT can promote cancer metastasis by stimulating EMT through the ZEB1 pathway and therefore inhibiting them may prevent cancer progression. PMID:26540342

  9. Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model

    PubMed Central

    Gagliano, Nicoletta; Celesti, Giuseppe; Tacchini, Lorenza; Pluchino, Stefano; Sforza, Chiarella; Rasile, Marco; Valerio, Vincenza; Laghi, Luigi; Conte, Vincenzo; Procacci, Patrizia

    2016-01-01

    AIM: To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. METHODS: HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, β-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS: The E-cadherin/β-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/β-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression. CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids. PMID:27182158

  10. Lack of Radiation Dose or Quality Dependence of Epithelial-to-Mesenchymal Transition (EMT) Mediated by Transforming Growth Factor {beta}

    SciTech Connect

    Andarawewa, Kumari L.; Costes, Sylvain V.; Fernandez-Garcia, Ignacio; Chou, William S.; Ravani, Shraddha A.; Park, Howard; Barcellos-Hoff, Mary Helen

    2011-04-01

    Purpose: Epithelial-to-mesenchymal transition (EMT) is a phenotype that alters cell morphology, disrupts morphogenesis, and increases motility. Our prior studies have shown that the progeny of human mammary epithelial cells (HMECs) irradiated with 2 Gy undergoes transforming growth factor {beta} (TGF-{beta})-mediated EMT. In this study we determined whether radiation dose or quality affected TGF-{beta}-mediated EMT. Methods and Materials: HMECs were cultured on tissue culture plastic or in Matrigel (BD Biosciences, San Jose, CA) and exposed to low or high linear energy transfer (LET) and TGF-{beta} (400 pg/mL). Image analysis was used to measure membrane-associated E-cadherin, a marker of functional epithelia, or fibronectin, a product of mesenchymal cells, as a function of radiation dose and quality. Results: E-cadherin was reduced in TGF-{beta}-treated cells irradiated with low-LET radiation doses between 0.03 and 2 Gy compared with untreated, unirradiated cells or TGF-{beta} treatment alone. The radiation quality dependence of TGF-{beta}-mediated EMT was determined by use of 1 GeV/amu (gigaelectron volt / atomic mass unit) {sup 56}Fe ion particles at the National Aeronautics and Space Administration's Space Radiation Laboratory. On the basis of the relative biological effectiveness of 2 for {sup 56}Fe ion particles' clonogenic survival, TGF-{beta}-treated HMECs were irradiated with equitoxic 1-Gy {sup 56}Fe ion or 2-Gy {sup 137}Cs radiation in monolayer. Furthermore, TGF-{beta}-treated HMECs irradiated with either high- or low-LET radiation exhibited similar loss of E-cadherin and gain of fibronectin and resulted in similar large, poorly organized colonies when embedded in Matrigel. Moreover, the progeny of HMECs exposed to different fluences of {sup 56}Fe ion underwent TGF-{beta}-mediated EMT even when only one-third of the cells were directly traversed by the particle. Conclusions: Thus TGF-{beta}-mediated EMT, like other non-targeted radiation effects, is

  11. MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis

    PubMed Central

    Hur, Keun; Toiyama, Yuji; Takahashi, Masanobu; Balaguer, Francesc; Nagasaka, Takeshi; Koike, Junichi; Hemmi, Hiromichi; Koi, Minoru; Boland, C Richard; Goel, Ajay

    2013-01-01

    Objective Distant metastasis is the major cause of cancer-related death in patients with colorectal cancer (CRC). Although the microRNA-200 (miR-200) family is a crucial inhibitor of epithelial-to-mesenchymal transition (EMT) in human cancer, the role of miR-200 members in the pathogenesis of metastatic CRC has not been investigated. Design Fifty-four pairs of primary CRC and corresponding matched liver metastasis tissue specimens were analysed for expression and methylation status of the miR-200 family members. Functional analysis of miR-200c overexpression was investigated in CRC cell lines, and cells were analysed for proliferation, invasion and migration. Expression of several miR-200c target genes (ZEB1, ETS1 and FLT1) and EMT markers (E-cadherin and vimentin) in CRC cell lines and tissue specimens was validated. Results Liver metastasis tissues showed higher expression of miR-200c (primary CRC=1.31 vs. liver metastasis=1.59; p=0.0014) and miR-141 (primary CRC=0.14 vs. liver metastasis=0.17; p=0.0234) than did primary CRCs, which was significantly associated with hypomethylation of the promoter region of these miRNAs (primary CRC=61.2% vs. liver metastasis=46.7%; p<0.0001). The invasive front in primary CRC tissues revealed low miR-200c expression by in situ hybridization analysis. Transfection of miR-200c precursors resulted in enhanced cell proliferation but reduced invasion and migration behaviours in CRC cell lines. Overexpression of miR-200c in CRC cell lines caused reduced expression of putative gene targets, and resulted in increased E-cadherin and reduced vimentin expression. The associations between miR-200c, target genes and EMT markers were validated in primary CRCs and matching liver metastasis tissues. Conclusions miR-200c plays an important role in mediating EMT and metastatic behaviour in the colon. Its expression is epigenetically regulated, and miR-200c may serve as a potential diagnostic marker and therapeutic target for patients with CRC

  12. Epithelial-to-mesenchymal transition in breast cancer: a role for insulin-like growth factor I and insulin-like growth factor–binding protein 3?

    PubMed Central

    Zielinska, Hanna A; Bahl, Amit; Holly, Jeff MP; Perks, Claire M

    2015-01-01

    Evidence indicates that for most human cancers the problem is not that gene mutations occur but is more dependent upon how the body deals with damaged cells. It has been estimated that only about 1% of human cancers can be accounted for by unmistakable hereditary cancer syndromes, only up to 5% can be accounted for due to high-penetrance, single-gene mutations, and in total only 5%–15% of all cancers may have a major genetic component. The predominant contribution to the causation of most sporadic cancers is considered to be environmental factors contributing between 58% and 82% toward different cancers. A nutritionally poor lifestyle is associated with increased risk of many cancers, including those of the breast. As nutrition, energy balance, macronutrient composition of the diet, and physical activity levels are major determinants of insulin-like growth factor (IGF-I) bioactivity, it has been proposed that, at least in part, these increases in cancer risk and progression may be mediated by alterations in the IGF axis, related to nutritional lifestyle. Localized breast cancer is a manageable disease, and death from breast cancer predominantly occurs due to the development of metastatic disease as treatment becomes more complicated with poorer outcomes. In recent years, epithelial-to-mesenchymal transition has emerged as an important contributor to breast cancer progression and malignant transformation resulting in tumor cells with increased potential for migration and invasion. Furthermore, accumulating evidence suggests a strong link between components of the IGF pathway, epithelial-to-mesenchymal transition, and breast cancer mortality. Here, we highlight some recent studies highlighting the relationship between IGFs, IGF-binding protein 3, and epithelial-to-mesenchymal transition. PMID:25632238

  13. Proteomic Analysis of Epithelial to Mesenchymal Transition (EMT) Reveals Cross-talk between SNAIL and HDAC1 Proteins in Breast Cancer Cells.

    PubMed

    Palma, Camila de Souza; Grassi, Mariana Lopes; Thomé, Carolina Hassibe; Ferreira, Germano Aguiar; Albuquerque, Daniele; Pinto, Mariana Tomazini; Ferreira Melo, Fernanda Ursoli; Kashima, Simone; Covas, Dimas Tadeu; Pitteri, Sharon J; Faça, Vitor M

    2016-03-01

    Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-β, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These

  14. miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer

    PubMed Central

    Tan, Xiaohui; Fu, Yebo; Chen, Liang; Lee, Woojin; Lai, Yinglei; Rezaei, Katayoon; Tabbara, Sana; Latham, Patricia; Teal, Christine B.; Man, Yan-Gao; Siegel, Robert S.; Brem, Rachel F.; Fu, Sidney W.

    2016-01-01

    MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management. PMID:26588055

  15. Bone marrow-derived CD11b+Jagged2+ cells promote epithelial-to-mesenchymal transition and metastasization in colorectal cancer.

    PubMed

    Caiado, Francisco; Carvalho, Tânia; Rosa, Isadora; Remédio, Leonor; Costa, Ana; Matos, João; Heissig, Beate; Yagita, Hideo; Hattori, Koichi; da Silva, João Pereira; Fidalgo, Paulo; Pereira, António Dias; Dias, Sérgio

    2013-07-15

    Timely detection of colorectal cancer metastases may permit improvements in their clinical management. Here, we investigated a putative role for bone marrow-derived cells in the induction of epithelial-to-mesenchymal transition (EMT) as a marker for onset of metastasis. In ectopic and orthotopic mouse models of colorectal cancer, bone marrow-derived CD11b(Itgam)(+)Jagged2 (Jag2)(+) cells infiltrated primary tumors and surrounded tumor cells that exhibited diminished expression of E-cadherin and increased expression of vimentin, 2 hallmarks of EMT. In vitro coculture experiments showed that the bone marrow-derived CD11b(+)Jag2(+) cells induced EMT through a Notch-dependent pathway. Using neutralizing antibodies, we imposed a blockade on CD11b(+) cells' recruitment to tumors, which decreased the tumor-infiltrating CD11b(+)Jag2(+) cell population of interest, decreasing tumor growth, restoring E-cadherin expression, and delaying EMT. In support of these results, we found that peripheral blood levels of CD11b(+)Jag2(+) cells in mouse models of colorectal cancer and in a cohort of untreated patients with colorectal cancer were indicative of metastatic disease. In patients with colorectal cancer, the presence of circulating CD11b(+)Jag2(+) cells was accompanied by loss of E-cadherin in the corresponding patient tumors. Taken together, our results show that bone marrow-derived CD11b(+)Jag2(+) cells, which infiltrate primary colorectal tumors, are sufficient to induce EMT in tumor cells, thereby triggering onset of metastasis. Furthermore, they argue that quantifying circulating CD11b(+)Jag2(+) cells in patients may offer an indicator of colorectal cancer progression to metastatic levels of the disease. PMID:23722542

  16. Neuropilin-1 Promotes Epithelial-to-Mesenchymal Transition by Stimulating Nuclear Factor-Kappa B and Is Associated with Poor Prognosis in Human Oral Squamous Cell Carcinoma

    PubMed Central

    Chu, Weiming; Song, Xiaomeng; Yang, Xueming; Ma, Lu; Zhu, Jiang; He, Mengying; Wang, Zilu; Wu, Yunong

    2014-01-01

    Background The epithelial-to-mesenchymal transition (EMT) is a key process in carcinogenesis, invasion, and metastasis of oral squamous cell carcinoma (OSCC). In our previous studies, we found that neuropilin-1 (NRP1) is overexpressed in tongue squamous cell carcinoma and that this overexpression is associated with cell migration and invasion. Nuclear factor-kappa B (NF-κB) plays an essential role both in the induction and the maintenance of EMT and tumor metastasis. Therefore, we hypothesized that NRP1 induces EMT, and that NRP1-induced migration and invasion may be an important mechanism for promoting invasion and metastasis of OSCC through NF-κB activation. Methods/Results The variations in gene and protein expression and the changes in the biological behavior of OSCC cell lines transfected with a vector encoding NRP1, or the corresponding vector control, were evaluated. NRP1 overexpression promoted EMT and was associated with enhanced invasive and metastatic properties. Furthermore, the induction of EMT promoted the acquisition of some cancer stem cell (CSC)-like characteristics in OSCC cells. We addressed whether selective inhibition of NF-κB suppresses the NRP1-mediated EMT by treating cells with pyrrolidinedithiocarbamate ammonium (PDTC), an inhibitor of NF-κB. Immunohistochemical analysis of NRP1 in OSCC tissue samples further supported a key mediator role for NRP1 in tumor progression, lymph node metastasis, and indicated that NRP1 is a predictor for poor prognosis in OSCC patients. Conclusion Our results indicate that NRP1 may regulate the EMT process in OSCC cell lines through NF-κB activation, and that higher NRP1 expression levels are associated with lymph node metastasis and poor prognosis in OSCC patients. Further investigation of the role of NRP1 in tumorigenesis may help identify novel targets for the prevention and therapy of oral cancers. PMID:24999732

  17. High-Mobility Group Box 1 Mediates Epithelial-to-Mesenchymal Transition in Pulmonary Fibrosis Involving Transforming Growth Factor-β1/Smad2/3 Signaling.

    PubMed

    Li, Liu-Cheng; Li, De-Lin; Xu, Liang; Mo, Xiao-Ting; Cui, Wen-Hui; Zhao, Ping; Zhou, Wen-Cheng; Gao, Jian; Li, Jun

    2015-09-01

    Epithelial-to-mesenchymal transition (EMT) is a crucial event in the cellular origin of myofibroblasts that secrete extracellular matrix in the progression of pulmonary fibrosis (PF). High-mobility group box 1 (HMGB1) is a novel mediator of EMT. However, whether this process involves the recognized transforming growth factor-β1 (TGF-β1)/Smad signaling that also contributes to EMT in PF has not yet been elucidated. Here, we developed a model of PF induced by bleomycin (BLM) in rats and conducted several simulation experiments in A549 (human) and RLE-6TN (rat) alveolar epithelial cell (AEC) lines to unravel the role of TGF-β1/Smad2/3 signaling in HMGB1-mediated EMT. We found that the levels of serum HMGB1 and lung hydroxyproline were severely elevated after BLM administration. Moreover, the protein expression of HMGB1, TGF-β1, phosphorylated Smad2/3 (p-Smad2/3), and mesenchymal markers including α-smooth muscle actin, vimentin, and type I collagen were significantly increased with the reduced protein expression of an epithelial marker (E-cadherin) in the rat model by Western blot or immunohistochemical analysis. In addition, the uptake of both exogenous TGF-β1 and HMGB1 by AECs could induce EMT; meanwhile, HMGB1 dramatically enhanced TGF-β1 expression and triggered Smad2/3 phosphorylation. In contrast, TGF-β1 deficiency evidently ameliorated HMGB1-mediated EMT with reduced p-Smad2/3 in A549 cells. It provides new insights that HMGB1 release from injured lungs promotes AEC damage through induction of the EMT process, in which TGF-β1/Smad2/3 signaling is activated and contributes to PF. These results suggest that HMGB1 may constitute a therapeutic target for developing antifibrotic agents for abnormal lung remodeling. PMID:26126535

  18. The ethanolic extract of bark from Salix aegyptiaca L. inhibits the metastatic potential and epithelial to mesenchymal transition of colon cancer cell lines.

    PubMed

    Enayat, Shabnam; Banerjee, Sreeparna

    2014-01-01

    Willow bark extracts have been used for centuries as a natural pain killer. Recently their potential as anticancer agents has been reported. We have shown the high antioxidant activity, phenolic and flavonoid content in the ethanolic extract of bark (EEB) from Salix aegyptiaca, a species endogenous to the Middle East. We have also reported that incubation with EEB resulted in a reduction in cell proliferation through the induction of apoptosis and cell cycle arrest via the inhibition of phosphatidyl inositol 3-kinase/Protein kinase B and mitogen activated protein kinases signaling pathways as strongly as commercial inhibitors. The current study demonstrates the robust inhibition of anchorage-independent growth, motility, migration, and adhesion of colon cancer cell lines HCT-116 and HT-29 by EEB. These in vitro functional changes were accompanied by a restoration of E-cadherin expression, a reduction in EGFR, SNAI1, SNAI2, and Twist1 and the matrix metalloproteases MMP9 and MMP2. Many of these proteins are involved in the process of epithelial to mesenchymal transition, which is considered as a critical step in the progression of noninvasive tumor cells into malignant, metastatic carcinomas. We therefore propose that EEB from Salix aegyptiaca is a potent nutraceutical causing cancer chemoprevention by inhibiting epithelial to mesenchymal transition and can be consumed for its health promoting effects. PMID:25175673

  19. Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition.

    PubMed

    Li, Qisheng; Sodroski, Catherine; Lowey, Brianna; Schweitzer, Cameron J; Cha, Helen; Zhang, Fang; Liang, T Jake

    2016-07-01

    Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry. PMID:27298373

  20. Implications of the Notch1-Snail/Slug-epithelial to mesenchymal transition axis for lymph node metastasis in infiltrating ductal carcinoma.

    PubMed

    Cao, Yu-Wen; Wan, Guo-Xing; Sun, Jian-Ping; Cui, Xiao-Bin; Hu, Jian-Ming; Liang, Wei-Hua; Zheng, Yu-Qin; Li, Wen-Qin; Li, Feng

    2015-02-01

    Emerging evidence suggests that activation of the Notch1 signaling pathway inducing epithelial to mesenchymal transition (EMT) mediated by Snail/Slug promotes invasion and metastasis of breast cancer cells in vitro. However, the implication of the Notch1-Snail/Slug-EMT axis in breast cancer patients remains unclear. A total of 200 formalin-fixed paraffin-embedded samples of invasive ductal carcinoma (IDC), and 37 adjacent non-neoplastic tissue (ANNT) samples from patients who had not been treated with neoadjuvant therapy were examined. Expression of Notch1, Slug, Snail, E-cadherin, N-cadherin, and vimentin was determined by immunohistochemistry on tissue microarrays (TMAs). The correlation between protein expression and clinicopathological characteristics of breast cancer patients was also evaluated. Results showed that a significantly high percentage of cases with high expression of Notch1 (74%, 148/200), Slug (36%, 72/200), Snail (62%, 124/200), and N-cadherin (77%, 153/200) and a low percentage of cases with high expression of E-cadherin (27%, 54/200) were observed in IDC compared to those in ANNTs. High Notch1, Slug, Snail, and N-cadherin expression and low E-cadherin expression in patients with IDC were significantly correlated with lymph node metastasis. In addition, correlation analysis results revealed that high Notch1 expression was significantly associated with high Slug, Snail, and N-cadherin expression and low E-cadherin expression in IDC. Furthermore, a high Snail expression was significantly associated with low E-cadherin expression, and a high Slug expression was found to be significantly associated with increased N-cadherin expression in patients with IDC. Hence, our study suggested that the Notch1-Snail/Slug-EMT axis may be implicated in the lymph node metastasis affecting patients with IDC. PMID:25645984

  1. Plumbagin suppresses epithelial to mesenchymal transition and stemness via inhibiting Nrf2-mediated signaling pathway in human tongue squamous cell carcinoma cells

    PubMed Central

    Pan, Shu-Ting; Qin, Yiru; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Yang, Yin-Xue; Wang, Dong; Zhou, Shu-Feng; Qiu, Jia-Xuan

    2015-01-01

    Tongue squamous cell carcinoma (TSCC) is the most common malignancy in oral and maxillofacial tumors with highly metastatic characteristics. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone; PLB), a natural naphthoquinone derived from the roots of Plumbaginaceae plants, exhibits various bioactivities, including anticancer effects. However, the potential molecular targets and underlying mechanisms of PLB in the treatment of TSCC remain elusive. This study employed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic approach to investigate the molecular interactome of PLB in human TSCC cell line SCC25 and elucidate the molecular mechanisms. The proteomic data indicated that PLB inhibited cell proliferation, activated death receptor-mediated apoptotic pathway, remodeled epithelial adherens junctions pathway, and manipulated nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response signaling pathway in SCC25 cells with the involvement of a number of key functional proteins. Furthermore, we verified these protein targets using Western blotting assay. The verification results showed that PLB markedly induced cell cycle arrest at G2/M phase and extrinsic apoptosis, and inhibited epithelial to mesenchymal transition (EMT) and stemness in SCC25 cells. Of note, N-acetyl-l-cysteine (NAC) and l-glutathione (GSH) abolished the effects of PLB on cell cycle arrest, apoptosis induction, EMT inhibition, and stemness attenuation in SCC25 cells. Importantly, PLB suppressed the translocation of Nrf2 from cytosol to nucleus, resulting in an inhibition in the expression of downstream targets. Taken together, these results suggest that PLB may act as a promising anticancer compound via inhibiting Nrf2-mediated oxidative stress signaling pathway in SCC25 cells. This study provides a clue to fully identify the molecular targets and decipher the underlying mechanisms of PLB in the treatment of TSCC. PMID:26491260

  2. Phenolic Secoiridoids in Extra Virgin Olive Oil Impede Fibrogenic and Oncogenic Epithelial-to-Mesenchymal Transition: Extra Virgin Olive Oil As a Source of Novel Antiaging Phytochemicals

    PubMed Central

    Vazquez-Martin, Alejandro; Fernández-Arroyo, Salvador; Cufí, Sílvia; Oliveras-Ferraros, Cristina; Lozano-Sánchez, Jesús; Vellón, Luciano; Micol, Vicente; Joven, Jorge

    2012-01-01

    Abstract The epithelial-to-mesenchymal transition (EMT) genetic program is a molecular convergence point in the life-threatening progression of organ fibrosis and cancer toward organ failure and metastasis, respectively. Here, we employed the EMT process as a functional screen for testing crude natural extracts for accelerated drug development in fibrosis and cancer. Because extra virgin olive oil (EVOO) (i.e., the juice derived from the first cold pressing of the olives without any further refining process) naturally contains high levels of phenolic compounds associated with the health benefits derived from consuming an EVOO-rich Mediterranean diet, we have tested the ability of an EVOO-derived crude phenolic extract to regulate fibrogenic and oncogenic EMT in vitro. High-performance liquid chromatography (HPLC) coupled to time-of-flight (TOF) mass spectrometry assays revealed that the EVOO phenolic extract was mainly composed (∼70%) of two members of the secoiridoid family of complex polyphenols, namely oleuropein aglycone—the bitter principle of olives—and its derivative decarboxymethyl oleuropein aglycone. EVOO secoiridoids efficiently prevented loss of proteins associated with polarized epithelial phenotype (i.e., E-cadherin) as well as de novo synthesis of proteins associated with mesenchymal migratory morphology of transitioning cells (i.e., vimentin). The ability of EVOO to impede transforming growth factor-β (TGF-β)–induced disintegration of E-cadherin-mediated cell–cell contacts apparently occurred as a consequence of the ability of EVOO phenolics to prevent the upregulation of SMAD4—a critical mediator of TGF-β signaling—and of the SMAD transcriptional cofactor SNAIL2 (Slug)—a well-recognized epithelial repressor. Indeed, EVOO phenolics efficiently prevented crucial TGF-β–induced EMT transcriptional events, including upregulation of SNAI2, TCF4, VIM (Vimentin), FN (fibronectin), and SERPINE1 genes. While awaiting a better

  3. αB-crystallin is essential for the TGF-β2-mediated epithelial to mesenchymal transition of lens epithelial cells.

    PubMed

    Nahomi, Rooban B; Pantcheva, Mina B; Nagaraj, Ram H

    2016-05-15

    Transforming growth factor (TGF)-β2-mediated pathways play a major role in the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) during secondary cataract formation, which is also known as posterior capsule opacification (PCO). Although αB-crystallin is a major protein in LEC, its role in the EMT remains unknown. In a human LEC line (FHL124), TGF-β2 treatment resulted in changes in the EMT-associated proteins at the mRNA and protein levels. This was associated with nuclear localization of αB-crystallin, phosphorylated Smad2 (pSmad2) (S245/250/255), pSmad3 (S423/425), Smad4 and Snail and the binding of αB-crystallin to these transcription factors, all of which were reduced by the down-regulation of αB-crystallin. Expression of the functionally defective R120G mutant of αB-crystallin reduced TGF-β2-induced EMT in LECs of αB-crystallin knockout (KO) mice. Treatment of bovine lens epithelial explants and mouse LEC with TGF-β2 resulted in changes in the EMT-associated proteins at the mRNA and protein levels. This was accompanied by increase in phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) (T202/Y204), p38 MAPK (T180/Y182), protein kinase B (Akt) (S473) and Smad2 when compared with untreated cells. These changes were significantly reduced in αB-crystallin depleted or knocked out LEC. The removal of the fibre cell mass from the lens of wild-type (WT) mice resulted in the up-regulation of EMT-associated genes in the capsule-adherent epithelial cells, which was reduced in the αB-crystallin KO mice. Together, our data show that αB-crystallin plays a central role in the TGF-β2-induced EMT of LEC. αB-Crystallin could be targeted to prevent PCO and pathological fibrosis in other tissues. PMID:26987815

  4. αB-crystallin is essential for the TGF-β2-mediated epithelial to mesenchymal transition of lens epithelial cells

    PubMed Central

    Nahomi, Rooban B.; Pantcheva, Mina B.; Nagaraj, Ram H.

    2016-01-01

    Transforming growth factor (TGF)-β2-mediated pathways play a major role in the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) during secondary cataract formation, which is also known as posterior capsule opacification (PCO). Although αB-crystallin is a major protein in LEC, its role in the EMT remains unknown. In a human LEC line (FHL124), TGF-β2 treatment resulted in changes in the EMT-associated proteins at the mRNA and protein levels. This was associated with nuclear localization of αB-crystallin, phosphorylated Smad2 (pSmad2) (S245/250/255), pSmad3 (S423/425), Smad4 and Snail and the binding of αB-crystallin to these transcription factors, all of which were reduced by the down-regulation of αB-crystallin. Expression of the functionally defective R120G mutant of αB-crystallin reduced TGF-β2-induced EMT in LECs of αB-crystallin knockout (KO) mice. Treatment of bovine lens epithelial explants and mouse LEC with TGF-β2 resulted in changes in the EMT-associated proteins at the mRNA and protein levels. This was accompanied by increase in phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) (T202/Y204), p38 MAPK (T180/Y182), protein kinase B (Akt) (S473) and Smad2 when compared with untreated cells. These changes were significantly reduced in αB-crystallin depleted or knocked out LEC. The removal of the fibre cell mass from the lens of wild-type (WT) mice resulted in the up-regulation of EMT-associated genes in the capsule-adherent epithelial cells, which was reduced in the αB-crystallin KO mice. Together, our data show that αB-crystallin plays a central role in the TGF-β2-induced EMT of LEC. αB-Crystallin could be targeted to prevent PCO and pathological fibrosis in other tissues. PMID:26987815

  5. Afatinib resistance in non-small cell lung cancer involves the PI3K/AKT and MAPK/ERK signalling pathways and epithelial-to-mesenchymal transition.

    PubMed

    Coco, Simona; Truini, Anna; Alama, Angela; Dal Bello, Maria Giovanna; Venè, Roberta; Garuti, Anna; Carminati, Enrico; Rijavec, Erika; Genova, Carlo; Barletta, Giulia; Sini, Claudio; Ballestrero, Alberto; Boccardo, Francesco; Grossi, Francesco

    2015-09-01

    The epidermal growth factor receptor (EGFR) signalling is one of the most deregulated pathways in non-small cell lung cancer (NSCLC). Recently, the development of novel irreversible tyrosine kinase inhibitors (TKI), such as afatinib, has significantly improved the survival of advanced NSCLC patients harbouring activated EGFR mutations. However, treatment with TKI is not always curative due to the development of resistance. In the present study, we investigated the sensitivity to afatinib in two NSCLC EGFR mutated cell lines (NCI-H1650 and NCI-H1975) by expression profile analysis of 92 genes involved in the EGF pathway. Thereafter, the established afatinib resistant clones were evaluated at different biological levels: genomic, by array comparative genomic hybridisation (aCGH) and deep sequencing; transcriptomic, by quantitative polymerase chain reaction (qPCR) and proteomic, by Western blot and immunofluorescence. The baseline gene expression of the two cell lines revealed that NCI-H1650, the less afatinib-responsive cell, showed activation of two main EGFR downstream pathways such as PI3K/AKT and PLCγ/PKC axes. Analysis of the afatinib-resistant cells showed PI3K/AKT and MAPK/ERK pathways activation together with a biological switch from an epithelial-to-mesenchymal phenotype might confer afatinib-resistant properties to this cell line. Our data suggest that the activation of EGFR-dependent downstream pathways might be involved in the occurrence of resistance to afatinib assuming that the EGFR mutational status should not be exclusively considered when selecting TKI treatments. In particular, the epithelial-to-mesenchymal transition might provide a new basis for understanding afatinib resistance. PMID:25341405

  6. Tension Monitoring during Epithelial-to-Mesenchymal Transition Links the Switch of Phenotype to Expression of Moesin and Cadherins in NMuMG Cells

    PubMed Central

    Schneider, David; Baronsky, Thilo; Pietuch, Anna; Rother, Jan; Oelkers, Marieelen; Fichtner, Dagmar; Wedlich, Doris; Janshoff, Andreas

    2013-01-01

    Structural alterations during epithelial-to-mesenchymal transition (EMT) pose a substantial challenge to the mechanical response of cells and are supposed to be key parameters for an increased malignancy during metastasis. Herein, we report that during EMT, apical tension of the epithelial cell line NMuMG is controlled by cell-cell contacts and the architecture of the underlying actin structures reflecting the mechanistic interplay between cellular structure and mechanics. Using force spectroscopy we find that tension in NMuMG cells slightly increases 24 h after EMT induction, whereas upon reaching the final mesenchymal-like state characterized by a complete loss of intercellular junctions and a concerted down-regulation of the adherens junction protein E-cadherin, the overall tension becomes similar to that of solitary adherent cells and fibroblasts. Interestingly, the contribution of the actin cytoskeleton on apical tension increases significantly upon EMT induction, most likely due to the formation of stable and highly contractile stress fibers which dominate the elastic properties of the cells after the transition. The structural alterations lead to the formation of single, highly motile cells rendering apical tension a good indicator for the cellular state during phenotype switching. In summary, our study paves the way towards a more profound understanding of cellular mechanics governing fundamental morphological programs such as the EMT. PMID:24339870

  7. Determination of a Comprehensive Alternative Splicing Regulatory Network and Combinatorial Regulation by Key Factors during the Epithelial-to-Mesenchymal Transition.

    PubMed

    Yang, Yueqin; Park, Juw Won; Bebee, Thomas W; Warzecha, Claude C; Guo, Yang; Shang, Xuequn; Xing, Yi; Carstens, Russ P

    2016-06-01

    The epithelial-to-mesenchymal transition (EMT) is an essential biological process during embryonic development that is also implicated in cancer metastasis. While the transcriptional regulation of EMT has been well studied, the role of alternative splicing (AS) regulation in EMT remains relatively uncharacterized. We previously showed that the epithelial cell-type-specific proteins epithelial splicing regulatory proteins 1 (ESRP1) and ESRP2 are important for the regulation of many AS events that are altered during EMT. However, the contributions of the ESRPs and other splicing regulators to the AS regulatory network in EMT require further investigation. Here, we used a robust in vitro EMT model to comprehensively characterize splicing switches during EMT in a temporal manner. These investigations revealed that the ESRPs are the major regulators of some but not all AS events during EMT. We determined that the splicing factor RBM47 is downregulated during EMT and also regulates numerous transcripts that switch splicing during EMT. We also determined that Quaking (QKI) broadly promotes mesenchymal splicing patterns. Our study highlights the broad role of posttranscriptional regulation during the EMT and the important role of combinatorial regulation by different splicing factors to fine tune gene expression programs during these physiological and developmental transitions. PMID:27044866

  8. Neuropilin-2 expression promotes TGF-β1-mediated epithelial to mesenchymal transition in colorectal cancer cells.

    PubMed

    Grandclement, Camille; Pallandre, Jean René; Valmary Degano, Séverine; Viel, Erika; Bouard, Adeline; Balland, Jérémy; Rémy-Martin, Jean-Paul; Simon, Benoit; Rouleau, Alain; Boireau, Wilfrid; Klagsbrun, Michael; Ferrand, Christophe; Borg, Christophe

    2011-01-01

    Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-β1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-β1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFβ-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFβ-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-β1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors. PMID:21747928

  9. Neuropilin-2 Expression Promotes TGF-β1-Mediated Epithelial to Mesenchymal Transition in Colorectal Cancer Cells

    PubMed Central

    Grandclement, Camille; Pallandre, Jean René; Valmary Degano, Séverine; Viel, Erika; Bouard, Adeline; Balland, Jérémy; Rémy-Martin, Jean-Paul; Simon, Benoit; Rouleau, Alain; Boireau, Wilfrid; Klagsbrun, Michael; Ferrand, Christophe; Borg, Christophe

    2011-01-01

    Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-β1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-β1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFβ-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFβ-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-β1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors. PMID:21747928

  10. Plumbagin inhibits prostate carcinogenesis in intact and castrated PTEN knockout mice via targeting PKCε, Stat3 and epithelial to mesenchymal transition markers

    PubMed Central

    Hafeez, Bilal Bin; Fischer, Joseph W.; Singh, Ashok; Zhong, Weixiong; Mustafa, Ala; Meske, Louise; Sheikhani, Mohammad Ozair; Verma, Ajit Kumar

    2015-01-01

    Prostate cancer (PCa) continues to remain the most common cancer and the second leading cause of cancer-related deaths in American males. The Pten deletions and/or mutations are frequently observed in both primary prostate cancers and metastatic prostate tissue samples. Pten deletion in prostate epithelium in mice results in prostatic intraepithelial neoplasia (PIN), followed by progression to invasive adenocarcinoma. The Pten conditional knockout mice (Ptenloxp/loxp:PB-Cre4) ((Pten-KO) ) provide a unique preclinical model to evaluate agents for efficacy for both the prevention and treatment of prostate cancer (PCa). We present here for the first time that dietary plumbagin (PL), a medicinal plant-derived naphthoquinone (200 or 500 ppm) inhibits tumor development in intact as well as castrated Pten-KO mice. PL has shown no signs of toxicity at either of these doses. PL treatment resulted in a decrease expression of PKCε, AKT, Stat3 and COX2 compared to the control mice. PL treatment also inhibited the expression of vimentin and slug, the markers of epithelial to mesenchymal transition (EMT) in prostate tumors. In summary, the results indicate that dietary PL inhibits growth of both primary and castration resistant prostate cancer (CRPC) in Pten-KO mice, possibly via inhibition of PKCε, Stat3, AKT, and EMT markers (vimentin and slug), which are linked to the induction and progression of PCa. PMID:25627799

  11. Small molecule/ML327 mediated transcriptional de-repression of E-cadherin and inhibition of epithelial-to-mesenchymal transition

    PubMed Central

    An, Hanbing; Stoops, Sydney L.; Deane, Natasha G.; Zhu, Jing; Zi, Jinghuan; Weaver, Connie; Waterson, Alex G.; Zijlstra, Andries; Lindsley, Craig W.; Beauchamp, Robert Daniel

    2015-01-01

    Transcriptional repression of E-cadherin is a hallmark of Epithelial-to-Mesenchymal Transition (EMT) and is associated with cancer cell invasion and metastasis. Understanding the mechanisms underlying E-cadherin repression during EMT may provide insights into the development of novel targeted therapeutics for cancer. Here, we report on the chemical probe, ML327, which de-represses E-cadherin transcription, partially reverses EMT, and inhibits cancer cell invasiveness and tumor cell migration in vitro and in vivo. Induction of E-cadherin mRNA expression by ML327 treatment does not require de novo protein synthesis. RNA sequencing analysis revealed that ML327 treatment significantly alters expression of over 2,500 genes within three hours in the presence of the translational inhibitor, cycloheximide. Network analysis reveals Hepatocyte Nuclear Factor 4-alpha (HNF4α) as the most significant upstream transcriptional regulator of multiple genes whose expressions were altered by ML327 treatment. Further, small interfering RNA-mediated depletion of HNF4α markedly attenuates the E-cadherin expression response to ML327. In summary, ML327 represents a valuable tool to understand mechanisms of EMT and may provide the basis for a novel targeted therapeutic strategy for carcinomas. PMID:26082441

  12. LincHOTAIR epigenetically silences miR34a by binding to PRC2 to promote the epithelial-to-mesenchymal transition in human gastric cancer

    PubMed Central

    Liu, Y-w; Sun, M; Xia, R; Zhang, E-b; Liu, X-h; Zhang, Z-h; Xu, T-p; De, W; Liu, B-r; Wang, Z-x

    2015-01-01

    lncRNAs play important roles in the epigenetic regulation of carcinogenesis and progression. Previous studies suggest that HOTAIR contributes to gastric cancer (GC) development, and the overexpression of HOTAIR predicts a poor prognosis. In this study, we found that HOTAIR was more highly expressed in diffuse-type GC than in intestinal type (P=0.048). In the diffuse type, there is significant relationship between HOTAIR expression and DFS (P<0.001). CDH1 was downregulated in diffuse-type GC tissues (P=0.0007) and showed a negative relationship with HOTAIR (r2=0.154, P=0.0354). In addition, HOTAIR knockdown significantly repressed migration, invasion and metastasis both in vitro and vivo and reversed the epithelial-to-mesenchymal transition in GC cells. We also showed that HOTAIR recruiting and binding to PRC2 epigenetically represses miR34a, which controls the targets C-Met (HGF/C-Met/Snail pathway) and Snail, thus contributing to GC cell-EMT process and accelerating tumor metastasis. Moreover, it is demonstrated that HOTAIR crosstalk with microRNAs during epigenetic regulation. Our results suggest that HOTAIR acts as an EMT regulator and may be a candidate prognostic biomarker and a target for new therapies in GC patients. PMID:26136075

  13. Hibiscus sabdariffa polyphenols alleviate insulin resistance and renal epithelial to mesenchymal transition: a novel action mechanism mediated by type 4 dipeptidyl peptidase.

    PubMed

    Peng, Chiung-Huei; Yang, Yi-Sun; Chan, Kuei-Chuan; Wang, Chau-Jong; Chen, Mu-Lin; Huang, Chien-Ning

    2014-10-01

    The epithelial to mesenchymal transition (EMT) is important in renal fibrosis. Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1 (S307)) is a hallmark of insulin resistance. We report that polyphenol extracts of Hibiscus sabdariffa (HPE) ameliorate diabetic nephropathy and EMT. Recently it has been observed that type 4 dipeptidyl peptidase (DPP-4) inhibitor linagliptin is effective for treating type 2 diabetes and albuminuria. We investigated if DPP-4 and insulin resistance are involved in renal EMT and explored the role of HPE. In high glucose-stimulated tubular cells, HPE, like linagliptin, inhibited DPP-4 activation, thereby regulating vimentin (EMT marker) and IRS-1 (S307). IRS-1 knockdown revealed its essential role in mediating downstream EMT. In type 2 diabetic rats, pIRS-1 (S307) abundantly surrounds the tubular region, with increased vimentin in kidney. Both the expressions were reduced by HPE. In conclusion, HPE exerts effects similar to those of linagliptin, which improves insulin resistance and EMT, and could be an adjuvant to prevent diabetic nephropathy. PMID:25226384

  14. Reprogramming of cell junction modules during stepwise epithelial to mesenchymal transition and accumulation of malignant features in vitro in a prostate cell model.

    PubMed

    Ke, Xi-song; Li, Wen-cheng; Hovland, Randi; Qu, Yi; Liu, Run-hui; McCormack, Emmet; Thorsen, Frits; Olsen, Jan Roger; Molven, Anders; Kogan-Sakin, Ira; Rotter, Varda; Akslen, Lars A; Oyan, Anne Margrete; Kalland, Karl-Henning

    2011-01-15

    Epithelial to mesenchymal transition (EMT) is pivotal in tumor metastasis. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to cells with mesenchymal phenotype (EPT1) without malignant transformation. To promote prostate cell transformation, cells were maintained in saturation density cultures to select for cells overriding quiescence. Foci formed repeatedly following around 8 weeks in confluent EPT1 monolayers. Only later passage EPT1, but not EP156T cells of any passage, could form foci. Cells isolated from the foci were named EPT2 and formed robust colonies in soft agar, a malignant feature present neither in EP156T nor in EPT1 cells. EPT2 cells showed additional malignant traits in vitro, including higher ability to proliferate following confluence, higher resistance to apoptosis and lower dependence on exogenous growth factors than EP156T and EPT1 cells. Microarray profiling identified gene sets, many of which belong to cell junction modules, that changed expression from EP156T to EPT1 cells and continued to change from EPT1 to EPT2 cells. Our findings provide a novel stepwise cell culture model in which EMT emerges independently of transformation and is associated with subsequent accumulation of malignant features in prostate cells. Reprogramming of cell junction modules is involved in both steps. PMID:20969863

  15. Suppression of Epithelial-to-Mesenchymal Transitioning Enhances Ex Vivo Reprogramming of Human Exocrine Pancreatic Tissue Toward Functional Insulin-Producing β-Like Cells

    PubMed Central

    Lima, Maria João; Muir, Kenneth R.; Docherty, Hilary M.; Drummond, Robert; McGowan, Neil W.A.; Forbes, Shareen; Heremans, Yves; Houbracken, Isabelle; Ross, James A.; Forbes, Stuart J.; Ravassard, Philippe; Heimberg, Harry; Casey, John; Docherty, Kevin

    2013-01-01

    Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer. PMID:23610058

  16. An emerging role of the cellular prion protein as a modulator of a morphogenetic program underlying epithelial-to-mesenchymal transition.

    PubMed

    Mehrabian, Mohadeseh; Ehsani, Sepehr; Schmitt-Ulms, Gerold

    2014-01-01

    Knowledge of phenotypic changes the cellular prion protein (PrP(C)) contributes to may provide novel avenues for understanding its function. Here we consider data from functional knockout/down studies and protein-protein interaction analyses from the perspective of PrP's relationship to its ancestral ZIP metal ion transporting proteins. When approached in this manner, a role of PrP(C) as a modulator of a complex morphogenetic program that underlies epithelial-to-mesenchymal transition (EMT) emerges. To execute EMT, cells have to master the challenge to shift from cell-cell to cell-substrate modes of adherence. During this process, cell-cell junctions stabilized by E-cadherins are replaced by focal adhesions that mediate cell-substrate contacts. A similar reprogramming occurs during distinct organogenesis events that have been shown to rely on ZIP transporters. A model is presented that sees ZIP transporters, and possibly also PrP(C), affect this balance of adherence modes at both the transcriptional and post-translational levels. PMID:25453033

  17. An emerging role of the cellular prion protein as a modulator of a morphogenetic program underlying epithelial-to-mesenchymal transition

    PubMed Central

    Mehrabian, Mohadeseh; Ehsani, Sepehr; Schmitt-Ulms, Gerold

    2014-01-01

    Knowledge of phenotypic changes the cellular prion protein (PrPC) contributes to may provide novel avenues for understanding its function. Here we consider data from functional knockout/down studies and protein–protein interaction analyses from the perspective of PrP's relationship to its ancestral ZIP metal ion transporting proteins. When approached in this manner, a role of PrPC as a modulator of a complex morphogenetic program that underlies epithelial-to-mesenchymal transition (EMT) emerges. To execute EMT, cells have to master the challenge to shift from cell-cell to cell-substrate modes of adherence. During this process, cell-cell junctions stabilized by E-cadherins are replaced by focal adhesions that mediate cell-substrate contacts. A similar reprogramming occurs during distinct organogenesis events that have been shown to rely on ZIP transporters. A model is presented that sees ZIP transporters, and possibly also PrPC, affect this balance of adherence modes at both the transcriptional and post-translational levels. PMID:25453033

  18. Targeting the SIN3A-PF1 interaction inhibits epithelial to mesenchymal transition and maintenance of a stem cell phenotype in triple negative breast cancer

    PubMed Central

    Bansal, Nidhi; Petrie, Kevin; Leibovitch, Boris A.; Howell, Louise; Gil, Veronica; Sbirkov, Yordan; Lee, EunJee; Wexler, Joanna; Ariztia, Edgardo V.; Sharma, Rajal; Zhu, Jun; Bernstein, Emily; Zhou, Ming-Ming; Zelent, Arthur; Farias, Eduardo; Waxman, Samuel

    2015-01-01

    Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic α-helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial-to-mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment also led to a reduction in primary tumor growth and disseminated metastatic disease in vivo. In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting. PMID:26460951

  19. Aquaporin 9 is down-regulated in hepatocellular carcinoma and its over-expression suppresses hepatoma cell invasion through inhibiting epithelial-to-mesenchymal transition.

    PubMed

    Zhang, Wen-Guang; Li, Chuan-Fei; Liu, Min; Chen, Xiao-Feng; Shuai, Kai; Kong, Xin; Lv, Lin; Mei, Zhe-Chuan

    2016-08-10

    Aquaporin 9 (AQP9) is the main aquaglyceroporin in the liver. Few studies have been performed regarding the role of AQP9 in hepatocellular carcinoma (HCC). Here, we report the expression and function of AQP9 in HCC tissues and cell lines. We found that AQP9 mRNA and protein levels were down-regulated in HCC tissues and human hepatoma cell lines compared to the para-cancer normal liver tissues and normal hepatocyte line, respectively. In a human HCC SMMC-7721 cell line, over-expression of AQP9 suppressed cell invasion in vitro and xenograft tumor growth in vivo. AQP9 over-expression increased the expression of E-cadherin and decreased the expression of N-cadherin in SMMC-7721 cells and xenografted tumors, which was correlated with decreased levels of phosphoinositide 3-kinase (PI3K) and p-Akt. Conversely, using siRNA to knock down AQP9 over-expression could reverse the phenotype caused by AQP9 over-expression. Our findings suggest that AQP9 is down-regulated in hepatocellular carcinoma and its over-expression suppresses hepatoma cell invasion through inhibiting epithelial-to-mesenchymal transition. PMID:27216981

  20. The lncRNA H19 promotes epithelial to mesenchymal transition by functioning as miRNA sponges in colorectal cancer

    PubMed Central

    Liang, Wei-Cheng; Fu, Wei-Ming; Wong, Cheuk-Wa; Wang, Yan; Wang, Wei-Mao; Hu, Guo-Xin; Zhang, Li; Xiao, Li-Jia; Wan, David Chi-Cheong; Zhang, Jin-Fang; Waye, Mary Miu-Yee

    2015-01-01

    Recently, the long non-coding RNA (lncRNA) H19 has been identified as an oncogenic gene in multiple cancer types and elevated expression of H19 was tightly linked to tumorigenesis and cancer progression. However, the molecular basis for this observation has not been characterized in colorectal cancer (CRC) especially during epithelial to mesenchymal transition (EMT) progression. In our studies, H19 was characterized as a novel regulator of EMT in CRC. We found that H19 was highly expressed in mesenchymal-like cancer cells and primary CRC tissues. Stable expression of H19 significantly promotes EMT progression and accelerates in vivo and in vitro tumor growth. Furthermore, by using bioinformatics study and RNA immunoprecipitation combined with luciferase reporter assays, we demonstrated that H19 functioned as a competing endogenous RNA (ceRNA) for miR-138 and miR-200a, antagonized their functions and led to the de-repression of their endogenous targets Vimentin, ZEB1, and ZEB2, all of which were core marker genes for mesenchymal cells. Taken together, these observations imply that the lncRNA H19 modulated the expression of multiple genes involved in EMT by acting as a competing endogenous RNA, which may build up the missing link between the regulatory miRNA network and EMT progression. PMID:26068968

  1. Association of the epithelial-to-mesenchymal transition phenotype with responsiveness to the p21-activated kinase inhibitor, PF-3758309, in colon cancer models

    PubMed Central

    Pitts, Todd M.; Kulikowski, Gillian N.; Tan, Aik-Choon; Murray, Brion W.; Arcaroli, John J.; Tentler, John J.; Spreafico, Anna; Selby, Heather M.; Kachaeva, Maria I.; McPhillips, Kelly L.; Britt, Blair C.; Bradshaw-Pierce, Erica L.; Messersmith, Wells A.; Varella-Garcia, Marileila; Eckhardt, S. Gail

    2013-01-01

    The p21-activated kinase (PAK) family of serine/threonine kinases, which are overexpressed in several cancer types, are critical mediators of cell survival, motility, mitosis, transcription, and translation. In the study presented here, we utilized a panel of colorectal cancer (CRC) cell lines to identify potential biomarkers of sensitivity or resistance that may be used to individualize therapy to the PAK inhibitor PF-03758309. We observed a wide range of proliferative responses in the CRC cell lines exposed to PF-03758309, this response was recapitulated in other phenotypic assays such as anchorage-independent growth, three-dimensional (3D) tumor spheroid formation, and migration. Interestingly, we observed that cells most sensitive to PF-03758309 exhibited up-regulation of genes associated with a mesenchymal phenotype (CALD1, VIM, ZEB1) and cells more resistant had an up-regulation of genes associated with an epithelial phenotype (CLDN2, CDH1, CLDN3, CDH17) allowing us to derive an epithelial-to-mesenchymal transition (EMT) gene signature for this agent. We assessed the functional role of EMT-associated genes in mediating responsiveness to PF-3758309, by targeting known genes and transcriptional regulators of EMT. We observed that suppression of genes associated with the mesenchymal phenotype conferred resistance to PF-3758309, in vitro and in vivo. These results indicate that PAK inhibition is associated with a unique response phenotype in CRC and that further studies should be conducted to facilitate both patient selection and rational combination strategies with these agents. PMID:23543898

  2. The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition

    PubMed Central

    Tan, E-Jean; Kahata, Kaoru; Idås, Oskar; Thuault, Sylvie; Heldin, Carl-Henrik; Moustakas, Aristidis

    2015-01-01

    The loss of the tumour suppressor E-cadherin (Cdh1) is a key event during tumourigenesis and epithelial–mesenchymal transition (EMT). Transforming growth factor-β (TGFβ) triggers EMT by inducing the expression of non-histone chromatin protein High Mobility Group A2 (HMGA2). We have previously shown that HMGA2, together with Smads, regulate a network of EMT-transcription factors (EMT-TFs) like Snail1, Snail2, ZEB1, ZEB2 and Twist1, most of which are well-known repressors of the Cdh1 gene. In this study, we show that the Cdh1 promoter is hypermethylated and epigenetically silenced in our constitutive EMT cell model, whereby HMGA2 is ectopically expressed in mammary epithelial NMuMG cells and these cells are highly motile and invasive. Furthermore, HMGA2 remodels the chromatin to favour binding of de novo DNA methyltransferase 3A (DNMT3A) to the Cdh1 promoter. E-cadherin expression could be restored after treatment with the DNA de-methylating agent 5-aza-2′-deoxycytidine. Here, we describe a new epigenetic role for HMGA2, which follows the actions that HMGA2 initiates via the EMT-TFs, thus achieving sustained silencing of E-cadherin expression and promoting tumour cell invasion. PMID:25492890

  3. The endoplasmic reticulum may be an Achilles' heel of cancer cells that have undergone an epithelial-to-mesenchymal transition

    PubMed Central

    Feng, Yu-xiong; Sokol, Ethan S; Gupta, Piyush B

    2014-01-01

    In a recent report published in Cancer Discovery we identified a novel vulnerability of cancer cells that have undergone an epithelial–mesenchymal transition (EMT) and established that the PERK branch of the unfolded protein response is constitutively activated upon EMT. In this commentary, we summarize and provide context for our findings.

  4. Loss of breast epithelial marker hCLCA2 promotes epithelial-to-mesenchymal transition and indicates higher risk of metastasis.

    PubMed

    Walia, V; Yu, Y; Cao, D; Sun, M; McLean, J R; Hollier, B G; Cheng, J; Mani, S A; Rao, K; Premkumar, L; Elble, R C

    2012-04-26

    Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of epithelial-to-mesenchymal transition by cell dilution, TGFβ or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral small hairpin RNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18-year period among breast cancer patients compared with lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells. PMID:21909135

  5. Prostate-derived ets factor represses tumorigenesis and modulates epithelial-to-mesenchymal transition in bladder carcinoma cells.

    PubMed

    Tsui, Ke-Hung; Lin, Yu-Hsiang; Chung, Li-Chuan; Chuang, Sung-Ting; Feng, Tsui-Hsia; Chiang, Kun-Chun; Chang, Phei-Lang; Yeh, Chi-Ju; Juang, Horng-Heng

    2016-05-28

    Prostate-derived Ets (E-twenty six) factor (PDEF), an epithelium-specific member of the Ets family of transcription factors, has been shown to play a role in suppressing the development of many epithelium-derived cancers such as prostate and breast cancer. It is not clear, however, whether PDEF is involved in the development or progression of bladder cancer. In a comparison between normal urothelium and bladder tumor tissue, we identified significant decreases of PDEF in the tumor tissue. Further, the immunohistochemistry assays indicated a significantly higher immunostaining of PDEF in low-grade bladder tumors. Additionally, the highly differentiated transitional-cell bladder carcinoma RT-4 cells expressed significantly more PDEF levels than the bladder carcinoma HT1376 and the T24 cells. Ectopic overexpression of PDEF attenuated proliferation, invasion, and tumorigenesis of bladder carcinoma cells in vitro and in vivo. PDEF enhanced the expression levels of mammary serine protease inhibitor (MASPIN), N-myc downstream regulated gene 1 (NDRG1), KAI1, and B-cell translocation gene 2 (BTG2). PDEF modulated epithelial-mesenchymal-transition (EMT) by upregulating E-cadherin expression and downregulating the expression of N-cadherin, SNAIL, SLUG, and vimentin, leading to lower migration and invasion abilities of bladder carcinoma cells. Filamentous actin (F-actin) polarization and remodeling were observed in PDEF-knockdown RT-4 cells. Our results suggest that PDEF gene expression is associated with the extent of bladder neoplasia and PDEF modulated the expressions of EMT-related genes. The induction of BTG2, NDRG1, MASPIN, and KAI1 gene expressions by PDEF may explain the inhibitory functions of PDEF on the proliferation, invasion, and tumorigenesis in bladder carcinoma cells. PMID:26965996

  6. Embigin is overexpressed in pancreatic ductal adenocarcinoma and regulates cell motility through epithelial to mesenchymal transition via the TGF-β pathway.

    PubMed

    Jung, Dawoon E; Kim, Jeong Mi; Kim, Chanyang; Song, Si Young

    2016-05-01

    Embigin is a member of the immunoglobulin superfamily and encodes a transmembrane glycoprotein. There have been reports of Embigin involvement in neuromuscular junction formation and plasticity; however, the molecular functions of Embigin in other organs are unknown. Our aim was to investigate the possible role of Embigin in pancreatic cancer. In pancreatic ductal adenocarcinoma tissues, Embigin expression was higher than that in normal pancreatic tissues. Immunohistochemical analysis revealed expression of Embigin in pancreatic cancer cells, as well as expression of monocarboxylate transporter 2 (MCT2) in cancer tissues. To gain further insight, we transfected BxPC-3 and HPAC pancreatic cancer cells with siRNA or shRNA targeting Embigin and observed reductions in cell proliferation, migration, invasion, wound healing, and reduced levels of matrix metalloproteinases-2 and -9. Silencing of Embigin increased intracellular L-lactate concentration by 1.5-fold and decreased MCT2 levels at the plasma membrane. Furthermore, Embigin silencing led to a reduced expression of PI3K, GSK3-β, and Snail/Slug. Upon treating BxPC-3 cells with transforming growth factor-β (TGF-β), we observed elevated expression of Snail/Slug, Embigin, and Vimentin; meanwhile, when treating cells with SB-216763, a GSK3-β inhibitor, we noted decreases in GSK3-β, Snail/Slug, and Embigin expression, suggesting that the TGF-β signaling cascade, comprising PI3K, GSK3-β, Snail/Slug, and Embigin signals, mediates epithelial to mesenchymal transition (EMT) in pancreatic cancer cells. These findings indicate the involvement of Embigin in EMT in pancreatic cancer progression and suggest Embigin as a putative target for the detection and/or treatment of pancreatic cancer. © 2015 Wiley Periodicals, Inc. PMID:25773908

  7. Long non-coding RNA Linc00152 is involved in cell cycle arrest, apoptosis, epithelial to mesenchymal transition, cell migration and invasion in gastric cancer

    PubMed Central

    Zhao, Jing; Liu, Yongchao; Zhang, Wenhong; Zhou, Zhongwen; Wu, Jing; Cui, Peng; Zhang, Ying; Huang, Guangjian

    2015-01-01

    Gastric cancer remains a serious threat to public health with high incidence and mortality worldwide. Accumulating evidence demonstrates that long non-coding RNAs (lncRNAs) play important roles in regulating gene expression and are involved in various pathological processes, including gastric cancer. To investigate the possible role of dysregulated lncRNAs in gastric cancer development, we performed lncRNA microarray and identified 3141 significantly differentially expressed lncRNAs in gastric cancer tissues. Next, some of deregulated lncRNAs were validated among about 60 paired gastric cancer specimens such as Linc00261, DKFZP434K028, RPL34-AS1, H19, HOTAIR and Linc00152. Our results found that the decline of DKFZP434K028 and RPL34-AS1, and the increased expression of Linc00152 positively correlated with larger tumor size. The high expression levels of HOTAIR were associated with lymphatic metastasis and poor differentiation. Since the biological roles of Linc00152 are largely unknown in gastric cancer pathogenesis, we assessed its functions by silencing its up-regulation in gastric cancer cells. We found that Linc00152 knockdown could inhibit cell proliferation and colony formation, promote cell cycle arrest at G1 phase, trigger late apoptosis, reduce the epithelial to mesenchymal transition (EMT) program, and suppress cell migration and invasion. Taken together, we delineate the gastric cancer lncRNA signature and demonstrate the oncogenic functions of Linc00152. These findings may have implications for developing lncRNA-based biomarkers for diagnosis and therapeutics for gastric cancer. PMID:26237576

  8. Syndecan-1 up-regulates microRNA-331-3p and mediates epithelial-to-mesenchymal transition in prostate cancer.

    PubMed

    Fujii, Tomomi; Shimada, Keiji; Tatsumi, Yoshihiro; Tanaka, Nobumichi; Fujimoto, Kiyohide; Konishi, Noboru

    2016-09-01

    MicroRNAs (miRNAs) are small noncoding RNAs with a length of approximately 19-24 nucleotides that regulate gene expression through translational inhibition and contribute to the progression of various tumors including prostate cancer. Aberrant expression of miRNAs has been implicated in the progression and metastasis of prostate cancer. The present study aimed to investigate whether miR-331-3p controlled by syndecan-1 positively affects the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-331-3p upregulated mesenchymal markers such as vimentin, N-cadherin, and snail and downregulated epithelial markers such as E-cadherin and desmoplakin in the prostate cancer cell line PC3. We identified Neuropilin 2 and nucleus accumbens-associated protein 1 as putative target molecules in silico, as they were closely associated with the expression of miR-331-3p and TGF-β/Smad 4 signals. In situ hybridization and immunohistochemistry of radical prostatectomy samples revealed miR-331-3p in cancer cells with high Gleason patterns, in which EMT was demonstrated by decreased E-cadherin, and increased vimentin staining. Syndecan-1 gene silencing decreased levels of Dicer, which is involved in miRNA maturation. MiR-331-3p-mediated miRNA maturation and enhanced EMT via effects on TGF-β/Smad 4 and Dicer are essential for the development of prostate cancer mediated by syndecan-1. © 2015 Wiley Periodicals, Inc. PMID:26259043

  9. MicroRNA-27a-3p regulates epithelial to mesenchymal transition via targeting YAP1 in oral squamous cell carcinoma cells.

    PubMed

    Zeng, Guang; Xun, Wenxing; Wei, Kewen; Yang, Yongjin; Shen, Huan

    2016-09-01

    MicroRNAs (miRNAs) are small non-coding RNAs frequently dysregulated in human malignancies. Here, we profiled isolated cells from freshly resected tumors from oral squamous cell carcinoma (OSCC) patients and OSCC cell lines using a SYBR Green-based qPCR miRNA array to identify the expression change of the miRNAs. Based on the microarray data and clincopathological factor analysis of 50 OSCC patients related to these miRNAs, miR-27a-3p was selected as a putative miRNA which might play important role in OSCC progression. By bioinformatics analysis and dual-luciferase reporter assay, we found that YAP1 (Yes-associated protein-1) was a direct target gene of miR-27a-3p. Intriguingly, increased expression of miR-27a-3p could significantly decrease the expression level of YAP1 as well as several epithelial to mesenchymal transition (EMT)-related molecules in OSCC cell lines, including Twist and Snail. Then, follow-up studies revealed that miR-27a-3p expression was able to downregulate the EMT-related molecules effectively, which might be involved in the regulation of Sox2 via the YAP1-OCT4-Sox2 signaling axis. In summary, this study found that miR-27a-3p could inhibit the YAP1 directly by post-transcriptionally silencing and potentially suppress EMT process, suggesting that miR‑27a-3p might play pivotal roles in effectively manipulating the invasion and metastasis in oral squamous cell carcinoma cells through the EMT inhibition. PMID:27432214

  10. The role of epithelial to mesenchymal transition in resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer

    PubMed Central

    Jakobsen, Kristine Raaby; Demuth, Christina; Sorensen, Boe Sandahl

    2016-01-01

    Inhibition of the epidermal growth factor receptor (EGFR) is an important strategy when treating non-small cell lung cancer (NSCLC) patients. However, intrinsic resistance or development of resistance during the course of treatment constitutes a major challenge. The knowledge on EGFR-directed tyrosine kinase inhibitors (TKIs) and their biological effect keeps increasing. Within the group of patients with EGFR mutations some benefit to a much higher degree than others, and for patients lacking EGFR mutations a subset experience an effect. Up to 70% of patients with EGFR mutations and 10–20% of patients without EGFR mutations initially respond to the EGFR-TKI erlotinib, but there is a severe absence of good prognostic markers. Despite initial effect, all patients acquire resistance to EGFR-TKIs. Multiple mechanisms have implications in resistance development, but much is still to be explored. Epithelial to mesenchymal transition (EMT) is a transcriptionally regulated phenotypic shift rendering cells more invasive and migratory. Within the EMT process lays a need for external or internal stimuli to give rise to changes in central signaling pathways. Expression of mesenchymal markers correlates to a bad prognosis and an inferior response to EGFR-TKIs in NSCLC due to the contribution to a resistant phenotype. A deeper understanding of the role of EMT in NSCLC and especially in EGFR-TKI resistance-development constitute one opportunity to improve the benefit of TKI treatment for the individual patient. Many scientific studies have linked the EMT process to EGFR-TKI resistance in NSCLC and our aim is to review the role of EMT in both intrinsic and acquired resistance to EGFR-TKIs. PMID:27186512