Concomitant expression of epithelial-mesenchymal transition biomarkers in breast ductal carcinoma: Association with progression

Epithelial to mesenchymal transition (EMT) is a process implicated in cancer progression in which the underlying cellular changes have been identified mainly using in vitro models. We determined the expression of some putative EMT biomarkers including E-cadherin, beta-catenin, zinc finger factor Snail (Snail), transforming growth factor beta 1 (TGF beta 1), TGF beta type II receptor (TBRII) and the HGF receptor (c-met) and their possible correlation to progression and overall survival in a series of breast ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDC). Biomarkers were immunohistochemically determined in 55 IDC specimens from which 21 had lymph node metastases and in 95 DCIS specimens, 46 of these cases associated to invasive carcinoma, in a tissue microarray (TMA). Positive cytoplasmic staining of TGF beta 1 (78.2%), c-met (43.6%), Snail (34.5%), TBRII (100%), membranous E-cadherin (74.5%) and membranous/cytoplasmic beta-catenin (71%) were detected in the IDC samples. Metastatic lymph node samples displayed similar frequencies. A significant increase of c-met and TGF beta 1 positivity along DCIS to IDC progression was noted but only TGF beta 1 positivity was associated with presence of lymph node metastases and advanced stages in IDC. The evaluation of the other EMT markers in DCIS did not show differences in positivity rate as compared to invasive carcinomas. DCIS either pure or associated to IDC showed similar expression of the analyzed biomarkers. All the carcinomas exhibited positive expression of TBRII. Associations between the markers, determined by Spearman`s correlation coefficient, showed a significant association between TGF beta 1 and respectively E-cadherin, beta-catenin and cmet in DCIS cases, but in invasive carcinomas only cadherin and catenin were positively correlated. Kaplan-Meier survival curves revealed that none of the EMT biomarkers analyzed were correlated with survival, which was significantly determined only by clinical and hormone receptor parameters.

The ubiquitin-proteasome system and signal transduction pathways regulating Epithelial Mesenchymal transition of cancer.

Epithelial to Mesenchymal transition (EMT) in cancer, a process permitting cancer cells to become mobile and metastatic, has a signaling hardwire forged from development. Multiple signaling pathways that regulate carcinogenesis enabling characteristics in neoplastic cells such as proliferation, resistance to apoptosis and angiogenesis are also the main players in EMT. These pathways, as almost all cellular processes, are in their turn regulated by ubiquitination and the Ubiquitin-Proteasome System (UPS). Ubiquitination is the covalent link of target proteins with the small protein ubiquitin and serves as a signal to target protein degradation by the proteasome or to other outcomes such as endocytosis, degradation by the lysosome or specification of cellular localization. This paper reviews signal transduction pathways regulating EMT and being regulated by ubiquitination.

Ubiquitination and the Ubiquitin-Proteasome System as regulators of transcription and transcription factorsin epithelial mesenchymal transition of cancer.

Epithelial to Mesenchymal Transition (EMT) in cancer is a process that allows cancer cells to detach from neighboring cells, become mobile and metastasize and shares many signaling pathways with development. Several molecular mechanisms which regulate oncogenic properties in neoplastic cells such as proliferation, resistance to apoptosis and angiogenesis through transcription factors or other mediators are also regulators of EMT. These pathways and downstream transcription factors are, in their turn, regulated by ubiquitination and the Ubiquitin-Proteasome System (UPS). Ubiquitination, the covalent link of the small 76-amino acid protein ubiquitin to target proteins, serves as a signal for protein degradation by the proteasome or for other outcomes such as endocytosis, degradation by the lysosome or directing these proteins to specific cellular compartments. This review discusses aspects of the regulation of EMT by ubiquitination and the UPS and underlines its complexity focusing on transcription and transcription factors regulating EMT and are being regulated by ubiquitination.

Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-a-induced epithelial-mesenchymal transition of MCF-7 cells

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-a (TNF-a). To evaluate the role of TNF-a in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-a -treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-a treatment. These results showed that TNF-a can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkB by the mutant IkBa also blocked the TNF-a-induced upregulation of Snail promoter activity. Thus, the activation of NFkB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-a-induced EMT. ROS caused by TNF-a seemed to play a minor role in the TNF-a-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-a - and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

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.

Evidence of epithelial-mesenchymal transition in canine prostate cancer metastasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epithelial-mesenchymal transition occurs in preneoplastic and neoplastic lesions of canine prostate

Background: The epithelial-mesenchymal transition (EMT) is an essential process in the tumor progression and metastasis. In human prostate carcinoma (PCa), the upregulation of cytokeratin and E-cadherin and down-regulation of vimentin have been associated with aggressive phenotype and poor prognosis. Due to the importance of canine cancer model it was evaluated the immunoexpression of AE1/AE3, E-cadherin and vimentin in canine prostatic lesions. Patients and Methods: A total of 75 prostatic tissues formalin-fixed paraffin embedded from dogs was selected: 10 normal prostatic tissues, 20 benign prostatic hyperplasia (BPH), 25 proliferative inflammatory atrophy (PIA) and 20 PCa. AE1/AE3 was detected with a monoclonal antibody (Invitrogen, 180132) at a 1:300 dilution, applied for 45 min at room temperature (RT). The antibody against Vimentin (V9, Invitrogen) and E-cadherin (NCH-38, Dako cytomatiomn) were monoclonal mouse antibodies, used at a 1:300 and 1:200, respectively, for 45 min at RT. The immunolabelling was performed by a polymer method (Histofine, Nichirei Biosciences,). A negative control was performed for all antibodies by omitting the primary antibody and substituting with Tris-buffered saline. The percentage of C-MYC, E-cadherin, and p63- positive cells per lesion was evaluated according to Prowatke et al. (2007). The samples were scored separately according to staining intensity and graded semi-quantitatively as negative, weakly positive, moderately positive, and strongly positive. The score was done in one 400 magnification field, considering only the lesion, since this was done in a TMA core of 1 mm. For statistical analyses, the immunostaining classifications were reduced to two categories: negative and positive. The negative category included negative and weakly positive staining. Chi-square or Fisher exact test was used to determine the association between the categorical variables. Results: All prostatic normal and BPH tissue were positive for cytokeratin, E-cadherin and negative for vimentin. Similarly, all PIA samples were positive for AE1/AE3. From those samples, 48% (12/25) were also positive for vimentin. 55% of PCa (11/25) was positive for vimentin and among these samples 75% (6/11) was also positive for AE1/AE3 and 45% (5/11) was negative for AE1/AE3. PIA and PCa presented a higher number of vimentin positive cells when compared with normal tissue (p=0.032). E-cadherin expression had no statistical difference among diagnosis groups, but we found a higher number of positive cases, with more than 51% of positive immunostaining in BPH and PIA (81.25% and 78.60% of the cases, respectively) than in PCa (55.55%). Conclusion: The carcinogenesis process regarding prostatic epithelial cells in dogs showed higher vimentin protein expression associated with concomitant loss of the cytokeratin and E-cadherin, similar in humans.

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.

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.

GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer.

BACKGROUND: Alterations in glucose metabolism and epithelial-mesenchymal transition (EMT) constitute two important characteristics of carcinoma progression toward invasive cancer. Despite an extensive characterization of each of them separately, the links between EMT and glucose metabolism of tumor cells remain elusive. Here we show that the neuronal glucose transporter GLUT3 contributes to glucose uptake and proliferation of lung tumor cells that have undergone an EMT. RESULTS: Using a panel of human non-small cell lung cancer (NSCLC) cell lines, we demonstrate that GLUT3 is strongly expressed in mesenchymal, but not epithelial cells, a finding corroborated in hepatoma cells. Furthermore, we identify that ZEB1 binds to the GLUT3 gene to activate transcription. Importantly, inhibiting GLUT3 expression reduces glucose import and the proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. Using a large microarray data collection of human NSCLCs, we determine that GLUT3 expression correlates with EMT markers and is prognostic of poor overall survival. CONCLUSIONS: Altogether, our results reveal that GLUT3 is a transcriptional target of ZEB1 and that this glucose transporter plays an important role in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Moreover, these findings emphasize the development of GLUT3 inhibitory drugs as a targeted therapy for the treatment of patients with poorly differentiated tumors.

Tumor budding cells, cancer stem cells and epithelial-mesenchymal transition-type cells in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with a 5-year survival rate of less than 5%. Moreover, PDAC escapes early detection and resists treatment. Multiple combinations of genetic alterations are known to occur in PDAC including mutational activation of KRAS, inactivation of p16/CDKN2A and SMAD4 (DPC4) and dysregulation of PTEN/PI3K/AKT signaling. Through their interaction with Wingless-INT pathway, the downstream molecules of these pathways have been implicated in the promotion of epithelial-mesenchymal transition (EMT). Emerging evidence has demonstrated that cancer stem cells (CSCs), small populations of which have been identified in PDAC, and EMT-type cells play critical roles in drug resistance, invasion, and metastasis in pancreatic cancer. EMT may be histologically represented by the presence of tumor budding which is described as the occurrence of single tumor cells or small clusters (<5) of dedifferentiated cells at the invasive front of gastrointestinal (including colorectal, oesophageal, gastric, and ampullary) carcinomas and is linked to poor prognosis. Tumor budding has recently been shown to occur frequently in PDAC and to be associated with adverse clinicopathological features and decreased disease-free and overall survival. The aim of this review is to present a short overview on the morphological and molecular aspects that underline the relationship between tumor budding cells, CSCs, and EMT-type cells in PDAC.

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.