Role of Epithelial-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma: Is Tumor Budding the Missing Link?

Pancreatic ductal adenocarcinoma (PDAC) ranks as the fourth commonest cause of cancer death while its incidence is increasing worldwide. For all stages, survival at 5 years is<5%. The lethal nature of pancreatic cancer is attributed to its high metastatic potential to the lymphatic system and distant organs. Lack of effective therapeutic options contributes to the high mortality rates of PDAC. Recent evidence suggests that epithelial-mesenchymal transition (EMT) plays an important role to the disease progression and development of drug resistance in PDAC. Tumor budding is thought to reflect the process of EMT which allows neoplastic epithelial cells to acquire a mesenchymal phenotype thus increasing their capacity for migration and invasion and help them become resistant to apoptotic signals. In a recent study by our own group the presence and prognostic significance of tumor budding in PDAC were investigated and an association between high-grade budding and aggressive clinicopathological features of the tumors as well as worse outcome of the patients was found. The identification of EMT phenotypic targets may help identifying new molecules so that future therapeutic strategies directed specifically against them could potentially have an impact on drug resistance and invasiveness and hence improve the prognosis of PDAC patients. The aim of this short review is to present an insight on the morphological and molecular aspects of EMT and on the factors that are involved in the induction of EMT in PDAC.

TWIST1 and TWIST2 promoter methylation and protein expression in tumor stroma influence the epithelial-mesenchymal transition-like tumor budding phenotype in colorectal cancer.

Tumor budding in colorectal cancer is likened to an epithelial-mesenchymal transition (EMT) characterized predominantly by loss of E-cadherin and up-regulation of E-cadherin repressors like TWIST1 and TWIST2. Here we investigate a possible epigenetic link between TWIST proteins and the tumor budding phenotype. TWIST1 and TWIST2 promoter methylation and protein expression were investigated in six cell lines and further correlated with tumor budding in patient cohort 1 (n = 185). Patient cohort 2 (n = 112) was used to assess prognostic effects. Laser capture microdissection (LCM) of tumor epithelium and stroma from low- and high-grade budding cancers was performed. In colorectal cancers, TWIST1 and TWIST2 expression was essentially restricted to stromal cells. LCM results of a high-grade budding case show positive TWIST1 and TWIST2 stroma and no methylation, while the low-grade budding case was characterized by negative stroma and strong hypermethylation. TWIST1 stromal cell staining was associated with adverse features like more advanced pT (p = 0.0044), lymph node metastasis (p = 0.0301), lymphatic vessel invasion (p = 0.0373), perineural invasion (p = 0.0109) and worse overall survival time (p = 0.0226). Stromal cells may influence tumor budding in colorectal cancers through expression of TWIST1. Hypermethylation of the tumor stroma may represent an alternative mechanism for regulation of TWIST1.

Accumulation of FOXP3+T-cells in the tumor microenvironment is associated with an epithelial-mesenchymal-transition-type tumor budding phenotype and is an independent prognostic factor in surgically resected pancreatic ductal adenocarcinoma.

Here we explore the role of the interplay between host immune response and epithelial-mesenchymal-transition (EMT)-Type tumor-budding on the outcome of pancreatic adenocarcinoma (PDAC).CD4+, CD8+, and FOXP3+T-cells as well as iNOS+ (M1) and CD163+- macrophages (M2) were assessed on multipunch tissue-microarrays containing 120 well-characterized PDACs, precursor lesions (PanINs) and corresponding normal tissue. Counts were normalized for the percentage of tumor/spot and associated with the clinico-pathological features, including peritumoral (PTB) and intratumoral (ITB) EMT-Type tumor-budding and outcome.Increased FOXP3+T-cell-counts and CD163-macrophages and decreased CD8+T-cell-counts were observed in PDACs compared with normal tissues and PanINs (p < 0.0001). Increased peritumoral FOXP3+T-cell-counts correlated significantly with venous invasion, distant metastasis, R1-status, high-grade ITB, PTB and independently with reduced survival. Increased intratumoral FOXP3+T-cells correlated with lymphatic invasion, N1-stage, PTB and marginally with adverse outcome. High peritumoral CD163-counts correlated with venous invasion, PTB and ITB. High intratumoral CD163-counts correlated with higher T-stage and PTB.PDAC-microenvironment displays a tumor-favoring immune-cell composition especially in the immediate environment of the tumor-buds that promotes further growth and indicates a close interaction of the immune response with the EMT-process. Increased peritumoral FOXP3+T-cell density is identified as an independent adverse prognostic factor in PDAC. Patients with phenotypically aggressive PDACs may profit from targeted immunotherapy against FOXP3.

REGULATION OF FOXC2 EXPRESSION AND FUNCTION DURING EPITHELIAL-MESENCHYMAL TRANSITION BY GSK-3β

Metastasis is the ultimate cause for the majority of cancer-related deaths. The forkhead box transcription factor FOXC2 is known to be involved in regulating metastasis as well as a variety of developmental processes, including the formation of lymphatic and cardiovascular systems. Previous studies have shown that FOXC2 protein is localized either in the nucleus and/or in the cytoplasm of human breast tumor cells. This pattern of localization is similar to that of another forkhead family member, FOXO3a. Additionally, localization of FOXO3a is known to be differentially regulated by upstream kinase AKT. Therefore, I investigated whether FOXC2 localization could also be regulated by upstream kinases. Analysis of FOXC2 protein sequence revealed two potential phosphorylation sites for GSK-3β. Furthermore, inhibition of GSK-3βsignificantly reduces FOXC2 protein. In addition, exposure of HMLE Twist cells expressing endogenous FOXC2 to the GSK-3β inhibitor, TWS119, results in accumulation of FOXC2 protein in the cytoplasm with concomitant decrease in the nucleus in a time-dependent manner. Furthermore, continued treatment with TWS119 eventually induces epithelial morphology and decreased stem cell properties including sphere formation in these cells. Further characterization of FOXC2- GSK-3β interaction and the associated signaling cascade are necessary to determine the effect of FOXC2 phosphorylation by GSK-3β on EMT and metastasis.

THE p63 ISOFORM ∆Np63α INHIBITS EPITHELIAL – MESENCHYMAL TRANSITION BY PROMOTING THE EXPRESSION OF MIR-205 IN HUMAN BLADDER CANCER CELLS

p63, a p53 family member, is a transcription factor that has complex roles in cancer. This study focuses on the role of the ∆Np63α isoform in bladder cancer (BC). Epithelial – mesenchymal transition (EMT) is a physiological process that plays an important part in metastasis and drug resistance. At the molecular level, EMT is characterized by the loss of the epithelial marker E-cadherin, and the acquisition of the transcriptional repressors of E-cadherin (ZEB1, ZEB2, TWIST, SNAI1 and SNAI2). Recent publications highlight the role of microRNAs belonging to the miR-200 family and miR-205 in preventing EMT through suppression of ZEB1 and ZEB2. p53, the homologue of p63, is implicated in regulating EMT by modulating the expression of miR-200c; however, the mechanisms underlying miR-205 control remain unclear. Here we show that ∆Np63α regulates the transcription of miR-205 and controls EMT in human BC cells. We observed a strong correlation between the expression of ∆Np63α, miR-205 and E-cadherin in a panel of BC cell lines (n=28) and also in bladder primary tumors from a cohort of patients (n=98). A remarkably inverse correlation is observed between ∆Np63α and ZEB1/2 in cell lines. Stable knockdown (KD) ∆Np63α in UC6, an “epithelial” BC cell line, decreased the expression of miR-205 and induced ZEB1/2 expression, the effects that were reversed by expression of exogenous miR-205. Moreover, overexpressing ∆Np63α in UC3, a “messenchymal” BC cell line, brought about opposite results, an increase in miR-205 expression and a reduction in ZEB1/2 expression. Modulation of ∆Np63α expression resulted in a parallel change in the expression of miR-205 and miR-205 “host” gene (miR-205HG). Nuclear run-on and chromatin immunoprecipitation experiments demonstrated that ∆Np63α regulates the transcription of miR-205 through controlling the recruitment of RNA Polymerase II to the promoter of miR-205HG. Interestingly, high miR-205 expression correlated with poor clinical outcome in BC patients, consistent with our recent publication highlighting the enrichment of ∆Np63 in a lethal subset of muscle invasive BC. In summary, our data present the important roles of ∆Np63α in preventing EMT mediated by miR-205. Our study also identifies miR-205 as a potential molecular marker to predict clinical outcome in BC patients.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.

A novel regulatory role for tissue transglutaminase in epithelial-mesenchymal transition in cystic fibrosis

Cystic fibrosis (CF) is a genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) for which there is no overall effective treatment. Recent work indicates tissue transglutaminase (TG2) plays a pivotal intracellular role in proteostasis in CF epithelia and that the pan TG inhibitor cysteamine improves CFTR stability. Here we show TG2 has another role in CF pathology linked with TGFβ1 activation and signalling, induction of epithelial-mesenchymal transition (EMT), CFTR stability and induction of matrix deposition. We show that increased TG2 expression in normal and CF bronchial epithelial cells increases TGFβ1 levels, promoting EMT progression, and impairs tight junctions as measured by Transepithelial Electric Resistance (TEER) which can be reversed by selective inhibition of TG2 with an observed increase in CFTR stability. Our data indicate that selective inhibition of TG2 provides a potential therapeutic avenue for reducing fibrosis and increasing CFTR stability in CF.

Usual interstitial pneumonia and smoking-related interstitial fibrosis display epithelial to mesenchymal transition in fibroblastic foci

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Mutant p53 promotes epithelial-mesenchymal plasticity and enhances metastasis in mammary carcinomas of WAP-T mice.

To study the postulated mutant p53 (mutp53) "gain of function" effects in mammary tumor development, progression and metastasis, we crossed SV40 transgenic WAP-T mice with mutant p53 transgenic WAP-mutp53 mice. Compared to tumors in monotransgenic WAP-T mice, tumors in bitransgenic WAP-T x WAP-mutp53 mice showed higher tumor grading, enhanced vascularization, and significantly increased metastasis. Bitransgenic tumors revealed a gene signature associated with the oncogenic epithelial-mesenchymal transition pathway (EMT gene signature). In cultures of WAP-T tumor-derived G-2 cancer cells, which are comprised of subpopulations displaying "mesenchymal" and "epithelial" phenotypes, this EMT gene signature was associated with the "mesenchymal" compartment. Furthermore, ectopic expression of mutp53 in G-2 cells sufficed to induce a strong EMT phenotype. In contrast to these in vitro effects, monotransgenic and bitransgenic tumors were phenotypically similar suggesting that in vivo the tumor cell phenotype might be under control of the tumor microenvironment. In support, orthotopic transplantation of G-2 cells as well as of G-2 cells expressing ectopic mutp53 into syngeneic mice resulted in tumors with a predominantly epithelial phenotype, closely similar to that of endogenous primary tumors. We conclude that induction of an EMT gene signature by mutp53 in bitransgenic tumors primarily promotes tumor cell plasticity, that is, the probability of tumor cells to undergo EMT processes under appropriate stimuli, thereby possibly increasing their potential to disseminate and metastasize.

Epithelial to mesenchymal transition in the pathogenesis of uterine malignant mixed Müllerian tumours: the role of ubiquitin proteasome system and therapeutic opportunities.

Malignant mixed Müllerian tumours (malignant mixed mesodermal tumours, MMMT) of the uterus are metaplastic carcinomas with a sarcomatous component and thus they are also called carcinosarcomas. It has now been accepted that the sarcomatous component is derived from epithelial elements that have undergone metaplasia. The process that produces this metaplasia is epithelial to mesenchymal transition (EMT), which has recently been described as a neoplasia-associated programme shared with embryonic development and enabling neoplastic cells to move and metastasise. The ubiquitin proteasome system (UPS) regulates the turnover and functions of hundreds of cellular proteins. It plays important roles in EMT by being involved in the regulation of several pathways participating in the execution of this metastasis-associated programme. In this review the specifi c role of UPS in EMT of MMMT is discussed and therapeutic opportunities from UPS manipulations are proposed.

Epithelial-to-mesenchymal transition mediates docetaxel resistance and high risk of relapse in prostate cancer

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel þ androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-k B transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44 þ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44 þ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evi-dence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer.

Epithelial-to-mesenchymal transition mediates docetaxel resistance and high risk of relapse in prostate cancer

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel þ androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-k B transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44 þ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44 þ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evi-dence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer.