Role In Epithelial-mesenchymal Transition Research Articles

Regulation of microRNA expression by hepatocyte growth factor in human head and neck squamous cell carcinoma

Hepatocyte growth factor (HGF) is a multifunctional molecule that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is upregulated in various tumors including squamous cell carcinoma of the human head and neck (HNSCC), but how HGF affects the expression of downstream functional genes has not yet been elucidated in detail. In the present study, we examined the expression of microRNA (miRNA), non-coding small RNA that regulate cell proliferation and functions by interfering with the translation of target mRNA, with or without HGF stimulation in HNSCC cell line HSC3. Among several miRNAs, in which the expression was altered after HGF stimulation, we focused on miR-200c and miR-27b, both of which were drastically downregulated after HGF stimulation. Expression of ZEB1, a target mRNA for miR-200c, was upregulated 3 and 6 h after HGF stimulation, and that of E-cadherin, a downstream molecule of ZEB1, was downregulated 12 h after HGF stimulation. Expression of ST14/matriptase, an enzyme for extracellular matrix (ECM) degradation and HGF activation and a target mRNA for miR-27b, was drastically upregulated in the protein level after HGF stimulation, although it was not statistically altered in the mRNA level. These results suggest that miR-200c and miR-27b downregulated by HGF might play an important role in epithelial-mesenchymal transition mediated by ZEB1/E-cadherin and ECM degradation and HGF autoactivation mediated by ST14/matriptase, respectively. Altered expression of miRNA directly regulated by HGF might contribute enhanced progressive and invasive characteristics of HNSCC by regulating the translation of HGF-induced functional molecules.

The Role of Epithelial-Mesenchymal Transition in the Development and Progression of Bronchiolitis Obliterans Syndrome

The Role of Epithelial-Mesenchymal Transition in the Development and Progression of Bronchiolitis Obliterans Syndrome

The role of epithelial-mesenchymal transition in pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer related death in the US. Despite the advances in medical and surgical treatment, the 5-year survival rate for such cancer is only approximately 5% when considering all stages of disease. The lethal nature of pancreatic cancer stems from its high metastatic potential to the lymphatic system and distant organs. Lack of effective chemotherapies, which is believed to be due to drug-resistance, also contributes to the high mortality of pancreatic cancer. Recent evidence suggests that epithelial-mesenchymal transition of pancreatic cancer cells contributes to the development of drug resistance and an increase in invasiveness. Future strategies that specifically target against epithelial-mesenchymal transition phenotype could potentially reduce tumoral drug resistance and invasiveness and hence prolong the survival of patients with pancreatic cancer.

Knockdown of β-Catenin Through shRNA Cause a Reversal of EMT and Metastatic Phenotypes Induced by HIF-1α

Obective: Wnt/β-catenin signaling pathway regulates pattern formation during embryogenesis as well as tumor progression. Numbers of studies suggest that this signaling pathway may play an important role in Epithelial-Mesenchymal transition (EMT), however, there was no evidence that Wnt/β-catenin signaling pathway directly controlled the EMT occurrence. Our previous research has successfully proved that overexpression of hypoxia inducible factor-1α (HIF-1α) could induce EMT in LNCaP cells, but not in PC-3. Consistently, the expression of β-catenin protein increased in LNCaP/HIF-1α cells, but not in PC-3/HIF-1α. This study mainly aimed at exploring the essentiality and importance of Wnt/β-catenin signaling pathway in HIF-1α-induced EMT. Methods: Human prostate cancer cells (LNCaP) were stably transfected by recombinant plasmid pcDNA3.1(-)/HIF-1α. The positive clones were selected by G418 and confirmed through western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and indirect immunofluoesence. Then LNCaP/HIF-1α was transiently transfected with β-catenin shRNA (shRNA1 and shRNA2) and negative shRNA (shRNA-scr). The epithelial markers, mesenchymal markers, and critical proteins in Wnt/β-catenin signaling pathway were separately detected by western blot analysis. Finally, the invasive potency of cells in different transfection group was examined by Matrgel transwell assay. Result: We successfully established prostate cancer cell line LNCaP/HIF-1α and LNCaP/HIF-1α/β-catenin(-). LNCaP/HIF-1α displayed high expression of mesenchymal markers and low expression of epithelial markers. However, compared with LNCaP/HIF-1α, the epithelial marker E-cadherin was increased in LNCaP/HIF-1α/β-catenin(-), whereas the expression of mesenchymal marker N-cadherin, vimentin, MMP-2 were significantly decreased. Inhibition of Wnt signal activity through β-catenin shRNA cause a reversal of EMT induced by HIF-1α in human prostate cancer. Conclusion: Overexpression of HIF-1α stimulates the invasion potency of human prostate carcinoma cells through EMT pathway and Wnt/β-catenin signaling pathway played a vital role in this process. Wnt/β-catenin signaling pathway might be a necessary endogenous signal that directly controlled the EMT occurrence induced by HIF-1α.

The importance of integrin‐linked kinase in the regulation of bladder cancer invasion

It is important to understand the molecular mechanisms of bladder cancer progression not only to prevent cancer progression but also to detect new therapeutic targets against advanced bladder cancer. The integrin-linked kinase (ILK) is a major signaling integrator in mammalian cells and plays an important role in epithelial-mesenchymal transition (EMT) of human cancers, but its mechanisms are not completely understood. In this study, we investigated the importance and mechanisms of ILK in bladder cancer progression. When the expression of ILK in bladder cancer cell lines and N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced murine bladder cancer was evaluated, ILK has a tendency to be overexpressed in invasive cell lines and invasive BBN-induced murine bladder cancer. Overexpression of ILK in 253J bladder cancer cells suppressed E-cadherin expression, resulting in the promotion of cell invasion. Conversely, ILK knockdown by siRNA suppresses cell invasion in invasive bladder cancer cells through the regulation of E-cadherin or matrix metalloprotease 9 (MMP-9). To regulate E-cadherin expression, our results showed that the glycogen synthase kinase 3β (GSK3β)-Zeb1 pathway may play an important role downstream of ILK. Finally, the results of a human bladder tissue microarray (TMA) showed that ILK expression correlates with the invasiveness of human bladder cancer. Our study suggests that ILK is overexpressed in invasive bladder cancer and plays an important role in the EMT of bladder cancer via the control of E-cadherin and MMP-9 expression. ILK may be a new molecular target to suppress tumor progression in advanced and high-risk bladder cancer patients.

Clinicopathological Significance of ZEB1 Protein in Patients with Hepatocellular Carcinoma

ZEB1, a member of the ZFH family of proteins (zinc-finger E-box binding homeobox), plays a central role in epithelial-mesenchymal transition (EMT) during carcinogenesis. In this study, we investigated the expression of ZEB1 in patients with hepatocellular carcinoma (HCC) and its clinical effects with underlying mechanisms. Expression levels of ZEB1 were assessed by Western blot in 5 HCC cell lines and in paired cancerous and noncancerous tissues from 110 patients with HCC. Short-hairpin RNA (shRNA) interference for ZEB1 was performed in MHCC-97H cell line. ZEB1 protein was detected at a relatively high level in metastatic human HCC cell lines (MHCC-97L and MHCC-97H) when compared with that in nonmetastatic HCC cell lines (Hep3B, PLC and Huh-7). ZEB1 was expressed at high levels in 72 of 110 HCC patients (65.4%) and correlated with advanced TNM stage, tumor size >5 cm, intrahepatic metastasis, vascular invasion, and frequent early recurrence. The results of multivariate analysis revealed that ZEB1 high expression was a significant prognostic factor for poor overall and disease-free survivals. Silencing ZEB1 resulted in significant suppression of motility of MHCC-97H cell line, which was accompanied with increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal markers N-cadherin and vimentin. Furthermore, silencing ZEB1 prevented the spread of intrahepatic metastasis and increased overall survival in mouse orthotopic tumor models. This study shows that ZEB1 high expression was correlated with HCC malignant progression and subsequent poor patient survival by induction of EMT changes.

Abstract 3972: Regulation of microRNA expression by hepatocyte growth factorin human head and neck sqaumous cell carcinoma (HNSCC)

Abstract Introduction: Hepatocyte growth factor (HGF) is a multifunctional growth factor that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is up-regulated in various tumors including human head and neck squamous cell carcinoma (HNSCC) and its signal transduction induces tumor progression and invasion. However, how HGF affects the expression of downstream functional gene expression has not yet been elucidated in detail. MicroRNAs (miRNAs) are non-coding small RNAs that regulate posttranscriptional gene expression by interfering the translation of target mRNAs. Aberrant expression of miRNA is known to induce various human tumors including HNSCC, but the regulation of miRNA expression is still unknown. In this study, we examined the expression of miRNAs with or without HGF stimulation in human HNSCC cell line HSC3 and the expression of their putative target genes was evaluated. Methods: Human HNSCC cell line HSC3 was cultured with or without HGF for designated periods. Alteration of miRNA expression was examined by miRNA microarray analysis. Validation study of miRNAs showing altered expression was performed by quantitative RT-PCR (qRT-PCR). The expression of putative target mRNAs and their translated proteins were examined by qRT-PCR and western blot analysis, respectively. Results: The expression of several miRNAs including let-7a, miR-16, and miR-205, which are already known as tumor-suppressive miRNAs, was altered after HGF stimulation by miRNA microarray. Among them, we focused on miR-200c and miR-27b which were drastically down-regulated after HGF stimulation, because of their unique target mRNAs involving HGF-mediated functional molecules. The expression of ZEB1, a target mRNA for 200c, was up-regulated 3hr after HGF stimulation and that of E-cadherin, a downstream molecule of ZEB1, was up-regulated 12hr after HGF stimulation. The expression of ST14/matriptase, an activation enzyme of HGF and a target mRNA for miR-27b, was not statistically altered after HGF stimulation, but the protein level was drastically up-regulated 12hr after HGF stimulation. These changes were attenuated in part by the addition of oligo pre-mi27b or pre-miR200c into the culture medium. Conclusion: Our present results suggest that miR-200c down-regulated by HGF might play an important role for epithelial-mesenchymal transition (EMT), through up-regulation of ZEB1 followed by down-regulation of E-cadherin. In addition, it was also suggested that miR-27b down-regulated by HGF might induce auto-activation of HGF in HNSCC. These alterations of miRNA expression by HGF stimulation could be induce the enhanced progressive and invasive characteristics of HNSCC. This is a first report that HGF may directly regulate some miRNAs that affect the expression of HGF-mediated downstream functional molecules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3972. doi:10.1158/1538-7445.AM2011-3972

Roles of Epithelial-Mesenchymal Transition in Squamous Cell Carcinoma of the Temporal Bone

The authors recently demonstrated that extensive bone erosion correlated with a worse prognosis of the squamous cell carcinoma (SCC) of the temporal bone. The present objective was to investigate the expressions and the roles of epithelial-mesenchymal transition (EMT) in SCC of the temporal bone. Retrospective patient file review and immunohistochemical study of tissues of patients with SCC of the temporal bone. The authors examined the expressions of vimentin in 16 patients with SCC of the temporal bone by immunohistochemical analysis. As expected, vimentin was expressed on SCC of the temporal bone. We regard the patient who has vimentin-positive cells as EMT-positive patient because gain of vimentin is a hallmark in the EMT reported in several types of cancer. EMT positive tumors significantly correlated with extensive bone erosion, and extensive bone erosion correlated with a worse prognosis of the SCC of the temporal bone. The study results suggest that EMT in tumor cells becomes an important indicator of poor prognosis in SCC of the temporal bone.

Potential role for SNAIL family transcription factors in the etiology of Crohnʼs disease-associated fistulae

Fistulae represent an important clinical complication of Crohn's disease (CD). The fistula tracts are covered by flat, myofibroblast-like cells with an epithelial origin (transitional cells, TC). We recently demonstrated a role of epithelial mesenchymal transition (EMT) in the pathogenesis of CD-associated fistulae. EMT is associated with an increased migratory and invasive potential of epithelial cells in different tissues. Here we investigated whether cytokines or growth factors as well as EMT-associated SNAIL family transcription factors are expressed in CD fistulae. By immunohistochemistry we analyzed seven perianal fistulae from seven CD and two perianal fistulae from two non-inflammatory bowel disease (IBD) control patients. Hematoxylin and eosin staining or immunohistochemistry for the expression of tumor necrosis factor (TNF), TNF-receptor I (TNF-RI), SNAIL1, SLUG, fibroblast growth factors (FGF) 1, 2, 4, 7, epidermal growth factor (EGF), and TWIST were performed using standard techniques. Immunohistochemical staining of surgical specimens from CD patients revealed a strong expression of TNF and TNF-RI in and around fistula tracts. While SNAIL1 was also heavily expressed in the nuclei of TC, indicative of transcriptionally active protein, SLUG, FGF-1, and FGF-2 were detected rather in the fibrotic periphery of CD fistulae than in TC. In contrast, we did not detect considerable protein staining for FGF-4 and FGF-7 nor of EGF or the transcription factor, TWIST. Our data demonstrate that SNAIL1 and TNF are strongly expressed in TC of CD-associated fistulae. These observations support our previous data and indicate the onset of EMT-associated events in the pathogenesis of CD fistulae.

Role of LOX-1 and ROS in oxidized low-density lipoprotein induced epithelial-mesenchymal transition of NRK52E

BackgroundTo investigate the effect of oxidized low density lipoprotein receptor-1 (LOX-1) on tubular epithelial-mesenchymal transition (TEMT) induced by oxidized low-density lipoprotein (ox-LDL) and its mechanism.MethodsNRK-52E cells were incubated with ox-LDL (0, 25, 50, and 100 μg/ml) for 24 hours or pre-treated with the chemical inhibitor of the LOX-1 receptor polyinosinic acid (poly I) and carrageenan or the antioxidant N-acetyl-L-cysteine (NAC), the cells were then exposed to 50 μg/ml of ox-LDL.The expression of LOX-I, E-cadherin, α-smooth muscle actin (α-SMA) and reactive oxygen species (ROS) were analyzed by real-time PCR, western blotting analysis, immunofluorescence and confocal laser scanning microscopy.ResultsOx-LDL increased the expression of LOX-1 mRNA and protein in a dose-dependent manner from 0 to 100 μg/ml (P < 0.05). Following the increase in the LOX-1 protein level, the lipid intake, ROS generation and α-SMA expression increased; however, the E-cadherin level decreased. The pre-treatment with poly I or carrageenan or NAC significantly inhibited the LOX-1 expression, α-SMA expression, the lipid intake and ROS generation and reversed decrease of E-cadherin expression induced by ox-LDL. Meanwhile, the ROS generation were associated with a increase in the LOX-1 expression. The α-SMA expression was positively correlated with the ROS generation and LOX-1 expression, and the E-cadherin expression was negatively correlated with the ROS generation and LOX-1 expression.ConclusionsLOX-1 and ROS may play a important role in epithelial-mesenchymal transition of NRK52E induced by OX-LDL.

Review: Endothelial‐myofibroblast transition, a new player in diabetic renal fibrosis

Diabetic nephropathy (DN) is the most common cause of chronic kidney failure and end-stage renal disease in the Western world. Studies from diabetic animal models and clinical trials have shown that inhibition of the renin-angiotensin system delays the progression of advanced DN. However, a recent large-scale clinical trial has revealed that inhibition of renin-angiotensin system in early phases of DN does not slow the decline of renal function or the development of morphological lesions, suggesting that different mechanism(s) may be involved in the different stages of DN. The role of epithelial-mesenchymal transition in renal fibrosis has been intensively investigated. Recently, endothelial-mesenchymal transition, or endothelial-myofibroblast transition (EndoMT) has emerged as another mechanism involved in both developmental and pathological processes. The essential role of EndoMT in cardiac development has been thoroughly studied. EndoMT also exists and contributes to the development and progression of cardiac fibrosis, lung fibrosis, liver fibrosis and corneal fibrosis. EndoMT is a specific form of epithelial-mesenchymal transition. During EndoMT, endothelial cells lose endothelial markers and obtain mesenchymal markers. Recent evidence from our laboratory and others suggests that EndoMT plays an important role in the development of renal fibrosis in several pathological settings, including experimental DN. This review considers the evidence supporting the occurrence of EndoMT in normal development and in pathology, as well as the latest findings suggesting EndoMT contributes to fibrosis in DN. Whether experimental findings of EndoMT will be reproduced in human studies remains to be determined.

Abstract 2047: Expression of microRNAs in squamous cell carcinoma of human head and neck (HNSCC) and the esophagus (ESCC) and their regulation by hepatocyte growth factor

Abstract Introduction: MicroRNAs (miRNAs) are non-coding small RNAs that regulate cell proliferation and various cellular functions by interfering the translation of target mRNAs. Altered expression of miRNA is known to induce various human malignancies. Hepatoceyte growth factor (HGF) is a multifunctional growth factor that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is up-regulated in various tumors including squamous cell carcinoma of human head and neck (HNSCC) and the esophagus (ESCC), but how HGF affect the downstream gene expression have not yet been elucidated in detail. In this study, we examined the expression of miRNAs and their regulation by HGF in HNSCC and ESCC. Methods: Nine HNSCC, 8 ESCC and 2 normal squamous cell lines were used for cell culture. Total RNAs were extracted and used for microarray and following quantitative reverse transcription real-time PCR (qRT-PCR). Validation study was performed by qRT-PCR using total RNAs extracted from formalin-fixed paraffin-embedded human cancer tissues and paired normal tissues by laser-captured microdissection. For miRNA regulation study, HSC3 cells (HNSCC cell line) expressing MET was cultured with or without HGF stimulation, and their total RNAs were extracted and analyzed by miRNA microarray and following qRT-PCR. The expression of target mRNAs for miRNAs altered by HGF stimulation was also examined by qRT-PCR. Results: In HNSCC, ESCC, and normal squamous cell lines, miR-205 showed the highest expression in miRNA microarray analysis. High expression of miR-205 was observed in not only HNSCC and ESCC but also other organs’ SCC and adjacent normal squamous epithelia by qRT-PCR in validation study, although less expression was detected in cells and tissues other than squamous epithelia. MiR-21, which is an oncogenic miRNA in various malignancies, was also up-regulated in HNSCC and ESCC compared to paired normal squamous epithelia. By HGF stimulation, miR-200c and miR-27b were significantly down-regulated in HSC3 cells. Interestingly, the expression of ZEB1, a target mRNA for miR-200c, was up-regulated and E-cadherin, downstream molecule of ZEB1, was down-regulated in these cells after HGF stimulation. Conclusion: Our present results suggest that miRNA expression pattern and target mRNAs of HNSCC and ESCC are different from those of other organ's malignancies. MiR-205 might be a specific marker miRNA of both normal and malignant squamous epithelia, while miR-21 might be a putative oncogenic miRNA in HNSCC and ESCC. In addition, several miRNAs including miR-200c and miR-27b down-regulated by HGF stimulation might play an important role for epithelial-mesenchymal transition (EMT) followed by enhanced invasive characteristics of HNSCC and ESCC, through up-regulation of ZEB1 followed by down-regulation of E-cadherin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2047.

The Role of Epithelial-mesenchymal Transition in the Gastroenterology

The epithelial-mesenchymal transition (EMT) plays physiologic roles in the embryogenesis, wound healing, and tissue regeneration. In terms of pathological direction, it causes organ fibrosis, cancer development, progression, metastasis, and chemoresistance. Recently, the underlying mechanism of EMT and many kinds of EMT regulators have been identified. Pharmaceutical treatment strategies which target EMT pathway could be applied for the prevention of tissue fibrosis and cancer progression. In the field of gastroenterology, profuse evidences have been collected about the critical roles of EMT in cancers of the gastrointestinal tract, liver, and pancreas and hepatic fibrosis. However, EMT varies widely among cancer types, and much remains to be identified about the main regulators of EMT in a specific disease. In this review, we present recent research results regarding the roles of EMT in cancers and organic fibrosis, especially in the area of gastroenterology.

Significance of Twist expression and its association with E-cadherin in esophageal squamous cell carcinoma

BackgroundTwist is a basic helix-loop-helix (bHLH) transcriptional factor that has been identified to play an important role in epithelial-mesenchymal transition (EMT)-mediated metastasis through the regulation of E-cadherin expression. However, few authors have examined the expression of Twist and E-cadherin and their prognostic value in patients with esophageal squamous cell carcinoma (ESCC). The purpose of this study is to evaluate the clinical significance of Twist and E-cadherin expression in ESCC.MethodsWe immunohistochemically investigated the relationship between their expression and clinicopathological factors including prognosis in surgical specimens of primary tumors in 166 patients with ESCC.ResultsThe expression rate of high Twist was 42.0% and that of preserved E-cadherin was 40.4%. The expression of high Twist and reduced E-cadherin was significantly associated with depth of tumor invasion, lymph node metastasis, distant nodal metastasis, stage and lymphatic invasion, and poor prognosis. High Twist expression significantly correlated with reduced E-cadherin expression. In the preserved E-cadherin group, the 5-year survival rate was better for patients who were low for Twist expression than for those who were high for Twist expression. Multivariate analysis indicated that the combination of low Twist and preserved E-cadherin expression was an independent prognostic factor along with tumor depth, distant nodal metastasis and E-cadherin expression.ConclusionsEvaluation of Twist and E-cadherin expressions should be useful for determining tumor properties, including prognosis, in patients with ESCC.

A role for epithelial-mesenchymal transition in the etiology of benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is usually described as a pathological proliferation of prostatic fibroblasts/myofibroblasts and epithelial cells. In the present study of BPH samples, we have made a morphological and immunohistochemical study of BPH prostatic sections using markers of proliferation, apoptosis, hormone receptors, and TGF-beta signaling. We found no evidence of proliferation in the stroma but in the epithelium of some ducts; 0.7% of the basal and 0.4% of luminal cells were positive for Ki67 and PCNA. Androgen receptor and estrogen receptor beta (ERbeta)1 and ERbetacx were abundant in both stromal and epithelial compartments but cells expressing ERalpha were very rare. What was very common in all BPH samples was the following: (i) regions of the ductal epithelium where the epithelial cells did not express E-cadherin, had lost their polarization, and become spindle shaped (the nuclei of these cells were strongly positive for pSmad 3 and Snail); and (ii) regions where the walls of the blood vessels were extremely thick and there was loss of endothelial layer. Loss of E-cadherin, increased pSmad 3, and high expression of Snail are all characteristic of epithelial-mesenchymal transition (EMT). We conclude that BPH is not a disease of prostatic stromal proliferation but rather of accumulation of mesenchymal-like cells derived from the prostatic epithelium and the endothelium. TGF-beta is thought to play a key role in EMT. Our data suggests that TGF-beta/Smad should be considered as targets for treatment of BPH.

Role of progesterone receptor extra-nuclear signaling in mammary epithelial cell differentiation and breast cancer progression.

Abstract Abstract #3041 PR expression is inversely associated with the stage of breast cancer and loss of PR is associated with more aggressive phenotype suggesting that PR has a protective role in established cancer. However, animal models and clinical studies showed that progesterone (PG) and PR is a risk factor for breast cancer development. The mechanism of these distinct actions of PG/PR in breast cancer is not known. PR contains a polyproline domain (PPD), which binds to the SH3 domain of c-Src. PR-SH3 interaction mediates activation of Src and MAPK and PG-mediated cell cycle progression. PR-BΔSH3, which has mutations in PPD, failed to mediate these effects. To access the biological role of PR extra-nuclear signaling mediated by PPD, PR-B, PR-BΔSH3 or vector control were stably expressed in non-transformed mammary epithelial MCF10A cell and in breast cancer cells, MCF-7 and T47DY. Cells were tested for their transformation potential by growth in soft-agar and in 3-D cultures. In MCF10A model, cells expressing PR-BΔSH3 showed an increase in both colony numbers and sizes while cells expressing PR-B showed little growth in soft-agar in the absence of PG and PG strongly inhibited colony formation. In MCF-7 cells, both cells expressing PR-B and PR-BΔSH3 formed colonies in the absence of PG and PG strongly inhibited colony formation in cells expressing PR-B but had no effects in cells expressing PR-BΔSH3. To mimic growth in vivo, cells were grown in 3D cultures. MCF-10A expressing vector control or PR-B formed polarized acini with hollow lumen while cells expressing PR-BΔSH3 formed large and filled acini. Using Ki-67 and E-cadherin immunostaining, cells expressing PR-BΔSH3 showed an increase in cells proliferation and a break down in E-caderin as compared to cells expressing PR-B or vector control. Similar changes in cells expressing PR-BΔSH3 were also found in MCF-7 model. We next determined the influence of PR PPD on cell morphology and motility in T47DY PR- cells. T47DY expressing PR-B were rounded and epithelial-like as compared to cells expressing PR-BΔSH3 or LacZ, which were large, flat and mesenchymal-like. Cells expressing PR-B showed a decrease in cell motility with few focal adhesions and were devoid of actin stress fibers, as compared to cells expressing PR-BΔSH3 and LacZ. Similar changes were also observed in MCF-7 cell series. Thus, PR PPD appears to play a role in epithelial-mesenchymal transition (EMT) of breast cancer cells. Interestingly, effects of PR-B on these cellular functions are independent of ligand, but dependent upon PR PPD. Using DNA microarrays, gene expression of T47DY expressing PR-B and PR-BΔSH3 were compared. Gene ontogeny analysis of genes regulated by PPD in unliganded receptor revealed an enrichment of genes involved in regulation of key cellular processes including cell polarity, motility, EMT, NF-KB, TGF-β, and Wnt signaling. Results from DNA microarrays demonstrate that extra-nuclear signaling of PR through PPD interactions has a profound effect on PR regulated gene in the absence of PG and may plays an important role in mediating differentiating effects of PR. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3041.

Small C-terminal Domain Phosphatase Enhances Snail Activity through Dephosphorylation

Down-regulation of E-cadherin plays an important role in epithelial-mesenchymal transition (EMT), which is critical in normal development and disease states such as tissue fibrosis and metastasis. Snail, a key transcription repressor of E-cadherin, is a labile protein with a short half-life and is regulated through phosphorylation, ubiquitination, and degradation. Previously, we showed that GSK-3beta phosphorylated two stretches of serine residues within the nuclear export signal and the destruction box of Snail, provoking its cytoplasmic export for ubiquitin-mediated proteasome degradation. However, the mechanism of Snail dephosphorylation and the identity of the Snail-specific phosphatase remain elusive. Using a functional genomic screening, we found that the small C-terminal domain phosphatase (SCP) is a specific phosphatase for Snail. SCP interacted and co-localized with Snail in the nucleus. We also found that SCP expression induced Snail dephosphorylation and stabilization in vitro and in vivo. However, a catalytically inactive mutant of SCP had no effect on Snail. Furthermore, we found that Snail stabilization induced by SCP enhanced snail activity in the suppression of E-cadherin and increased cell migration. Thus, our findings indicate that SCP functions as a Snail phosphatase to control its phosphorylation and stabilization, and our study provides novel insights for the regulation of Snail during EMT and cancer metastasis.

Evidence for a role of epithelial mesenchymal transition during pathogenesis of fistulae in Crohnʼs disease

The pathogenesis of fistulae in Crohn's disease (CD) patients is barely understood. We recently showed that more than two-thirds of CD fistulae are covered with flat, mesenchymal-like cells (transitional cells [TC]) forming a patchy basement membrane. Epithelial-to-mesenchymal transition (EMT) is a process of reprogramming epithelial cells, allowing them to migrate more effectively and giving epithelial cells an "invasive" potential. EMT has been suggested to be crucial in fibrosis found in different tissues and diseases. We therefore investigated whether EMT could be involved in the pathogenesis of fistulae formation in CD. In all, 18 perianal fistulae, 2 enteroenteric, and 1 enterovesical fistulae from 17 CD patients were analyzed. In addition 2 perianal fistulae of non-CD patients were studied. Hematoxylin and eosin staining, immunohistochemistry for the expression of cytokeratins 8 and 20, beta6-integrin, E-cadherin, beta-catenin, vimentin, and TGF-beta1 and 2 were performed according to standard techniques. The TC covering perianal or enteroenteric fistulae were strongly positive for cytokeratins 8 and 20 but negative for vimentin, indicating their epithelial origin. beta6-Integrin and TGF-beta had the highest staining intensities in the transitional zone between the epithelium and the TC. Expression of junctional proteins such as E-cadherin was reduced in TC as compared to regular fistulae epithelium. In addition, a translocation of beta-catenin from the membrane to the cytoplasm was observed. Our data for the first time indicate an expression pattern of epithelial and mesenchymal markers in TC associated with fistulae formation that is characteristic for EMT. Studying the pathways of EMT during intestinal fistulae formation may help to develop new therapeutic strategies.

シスプラチンによる腎間質線維化へのEpithelial-mesenchymal transitionの関与

シスプラチンによる腎間質線維化へのEpithelial-mesenchymal transitionの関与

The Pathogenesis of Pterygium: Current Concepts and Their Therapeutic Implications

Pterygium is a disease of the ocular surface that is associated with chronic UV exposure and is characterized by proliferation, inflammatory infiltrates, fibrosis, angiogenesis and extracellular matrix breakdown. Although pterygium is not fully understood, significant progress has been made toward understanding the mechanisms involved in its pathogenesis. In this review, we provide an update on the signaling pathways activated by UV light that result in induction of mediators responsible for the growth of pterygium. We also review the recent genetic studies on hereditary factors and provide a brief overview of the role of epithelial mesenchymal transition, bone marrow progenitor cells, and neuronal signals that may also contribute to the pathogenesis of pterygium. Therapeutic options for pterygium are discussed based on the mechanisms that perpetuate its growth.