Cadherin Switch Research Articles

Abstract 1251: Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells

Abstract NADPH oxidase 5 (Nox5), one of the seven members of the NADPH oxidase gene family that generate reactive oxygen species (ROS), has been implicated in physiological and pathophysiological processes. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas; expression in most non-malignant tissues was weak to modest. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms regulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 melanoma cells by generating stable Nox5-overexpressing clones and knockdown clones. Overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased phosphatase activity. Additionally, these cells had increased normoxic Hif-1α expression and decreased p27Kip1 expression. Conversely, knockdown of endogenous Nox5 in UACC-257 cells resulted in decreased cell growth, decreased BrdU positive cells, decreased Hif-1α expression and increased p27Kip1 expression. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in Nox5 overexpressing cells indicative of an invasive phenotype; conversely, an upregulation of E-cadherin expression in Nox5 knockdown cells was noted. Cell invasion assay through matrigel-coated membranes also demonstrated enhanced invasion by Nox5 overexpressing cells. Additionally, 3D cultures of Nox5 overexpressing cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that Nox5 expression in human melanoma UACC-257 cells could contribute to cell proliferation, invasion, amoeboid morphology, and the ability to grow in the absence of serum or growth factors in part due to the generation of high local concentrations of extracellular ROS that modulate multiple signaling networks that regulate Hif-1α, p27Kip1, and the cadherin switch. Citation Format: Smitha Antony, Yongzhong Wu, Guojian Jiang, Jennifer L. Meitzler, Han Liu, Agnes Juhasz, Jiamo Lu, Miriam R. Anver, Krishnendu K. Roy, James H. Doroshow. Expression of NADPH oxidase 5 (Nox5) modulates cellular morphology, proliferation and invasiveness of human melanoma UACC-257 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1251. doi:10.1158/1538-7445.AM2015-1251

Making a Choice: How Cadherin Switching Controls Cell Migration

Cells that undergo epithelial-to-mesenchymal transitions (EMTs) commonly switch from expressing E-cadherin to N-cadherin. But why this occurs is not well understood. In the current issue of Developmental Cell, Scarpa et al. (2015) identify a reason: cadherin switching controls Rac signaling to determine how cell locomotion is regulated by contact.

Abstract 2995: Loss of SHISA3 is an early event of the epithelial-to-mesenchymal transition associated with chemoresistance in prostate cancer

Abstract Background: Despite scientific advances over the past decade, prostate cancer still remains the fifth leading cause of death from cancer in men worldwide. Docetaxel has an established role in the treatment of metastatic castrate-resistant prostate cancer (CRPC). However, malignant cells frequently acquire Docetaxel resistance. Therefore, further research is required to understand the molecular mechanisms underlying Docetaxel-resistance, which could be helpful in formulating alternative and superior therapeutic strategies. Methods: Two Docetaxel-resistant prostate cancer cell lines (IGR-CaP1 and PC3) were obtained by continuous exposure to Docetaxel. By comparing genes and miRs expression profiles established in these two Docetaxel-resistant models, we observed an enrichment of genes involved in the epithelial-to-mesenchymal transition (EMT) in the chemoresistant models and identified new genes potentially implicated in the resistance mechanism. Results: We identified the SHISA3 gene as a highly down-regulated gene in resistant cells. This gene was originally identified as an inhibitor of Wnt and FGF signaling during development in Xenopus. We showed that SHISA3 was lost during the acquisition of resistance to Docetaxel possibly via an EMT mechanism. Knockdown of SHISA3 in the parental IGR-CaP1 and PC3 cells triggered loss of the tight junction protein Occludin, engaged the Cadherin switch and increased the migratory properties of cells in vitro. Loss of SHISA3, along with loss of E-cadherin expression and expression of the mesenchymal marker N-cadherin were also observed in Docetaxel-resistant tumors obtained from IGR-CaP1-R xenografted mice. Phylogenetic comparison of SHISA3 gene sequences suggested that it may correspond to transmembrane adapters capable of regulating the activity of membrane receptors such as growth factor receptors. We are currently studying the binding partners of SHISA3 in our models by a proteomic approach to identify the implicated signaling pathway in prostate cancer. Conclusion: Our results show that loss of SHISA3 is an early event linked to the EMT process associated with chemoresistance and suggest that SHISA3 could be a useful biomarker to identify chemoresistant cells. Moreover, identification of its mechanism of action may lead to the identification of new therapeutic targets to overcome Docetaxel resistance. Citation Format: Nicolas J-p Martin, Sophie Cotteret, Catherine Gaudin, Marine Garrido, Safae Aarab-Terrisse, Nader al Nakouzi, Lucas Gentilini, Daniel Compagno, Vasily Ogryzko, Guillaume Meurice, Karim Fizazi, Anne Chauchereau. Loss of SHISA3 is an early event of the epithelial-to-mesenchymal transition associated with chemoresistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2995. doi:10.1158/1538-7445.AM2015-2995

Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces

SummaryContact inhibition of locomotion (CIL) is the process through which cells move away from each other after cell-cell contact, and it contributes to malignant invasion and developmental migration. Various cell types exhibit CIL, whereas others remain in contact after collision and may form stable junctions. To investigate what determines this differential behavior, we study neural crest cells, a migratory stem cell population whose invasiveness has been likened to cancer metastasis. By comparing pre-migratory and migratory neural crest cells, we show that the switch from E- to N-cadherin during EMT is essential for acquisition of CIL behavior. Loss of E-cadherin leads to repolarization of protrusions, via p120 and Rac1, resulting in a redistribution of forces from intercellular tension to cell-matrix adhesions, which break down the cadherin junction. These data provide insight into the balance of physical forces that contributes to CIL in cells in vivo.

EGF Suppresses the Initiation and Drives the Reversion of TGF-β1-induced Transition in Hepatic Oval Cells Showing the Plasticity of Progenitor Cells.

Transforming growth factor-β1 (TGF-β1) induces hepatic progenitors to tumor initiating cells through epithelial-mesenchymal transition (EMT), thus raising an important drawback for stem cell-based therapy. How to block and reverse TGF-β1-induced transition is crucial for progenitors' clinical application and carcinogenic prevention. Rat adult hepatic progenitors, hepatic oval cells, experienced E-cadherin to N-cadherin switch and changed to α-smooth muscle actin (α-SMA) positive cells after TGF-β1 incubation, indicating EMT. When TGF-β1 plus EGF were co-administrated to these cells, EGF dose-dependently suppressed the cadherin switch and α-SMA expression. Interestingly, if EGF was applied to TGF-β1-pretreated cells, the cells that have experienced EMT could return to their epithelial phenotype. Abruption of EGF receptor revealed that EGF exerted its blockage and reversal effects through phosphorylation of ERK1/2 and Akt. These findings suggest an important attribute of EGF on opposing and reversing TGF-β1 effects, indicating the plasticity of hepatic progenitors.

HPV-16 E6/E7 promotes cell migration and invasion in cervical cancer via regulating cadherin switch in vitro and in vivo

Cadherin switch, as a key hallmark of epithelial-mesenchymal transition (EMT), is characterized by reduced E-cadherin expression and increased N-cadherin or P-cadherin expression, and has been implicated in many aggressive tumors, but the importance and regulatory mechanism of cadherin switch in cervical cancer have not been investigated. Our study aimed to explore the role of cadherin switch by regulation of HPV-16 E6/E7 in progression and metastasis of cervical cancer. The expressions of E-cadherin and P-cadherin were examined by immunohistochemical staining in 40 cases of high-grade cervical lesions with HPV-16 infection only in which HPV-16 E6 and E7 expression had been detected using qRT-PCR method. Through modulating E6 and E7 expression using HPV-16 E6/E7 promoter-targeting siRNAs or expressed vector in vitro, cell growth, migration, and invasion were separately tested by MTT, wound-healing and transwell invasion assays, as well as the expressions of these cadherins by western blot analyses. Finally, the expressions of these cadherins in cancerous tissues of BALB/c-nu mouse model inoculated with the stable HPV-16 E6/E7 gene silencing Siha and Caski cells were also measured by immunohistochemical staining. Pearson correlation coefficient analyses showed the strongly inverse correlation of E-cadherin expression and strongly positive correlation of P-cadherin expression with E6/E7 level in 40 cases of high-grade cervical lesions. Furthermore, the modulation of HPV-16 E6/E7 expression remarkably influenced cell proliferation, migration, and invasion, as well as the protein levels of E-cadherin and P-cadherin in cervical cell lines. Finally, the reduction of HPV-16 E6/E7 expression led to up-regulated expression of E-cadherin and down-regulated expression of P-cadherin in BALB/c-nu mouse model in vivo assay. Our results unraveled the possibility that HPV-16 E6/E7 could promote cell invasive potential via regulating cadherin switching, and consequently contribute to progression and metastasis of cervical cancer.

A switch from epithelial to mesenchymal properties correlates with lymphovascular invasion in squamous cell carcinoma of the penis

The purpose of the study was to assess the incidence and prognostic role of epithelial-to-mesenchymal-transition (EMT) in squamous cell carcinoma of the penis (SCCP).Sixty tumor specimens of surgically treated SCCP patients characterized by a central histopathologic review were stained with antibodies against E-cadherin, N-cadherin, β-catenin, and vimentin. Staining profiles were scored by two independent raters, and correlated with pertinent clinical and pathological parameters and cancer-specific mortality (CSM; median follow-up: 34 months, interquartile range: 6–60 months).Correlation statistics proved good interobserver agreement in staining evaluation (K-values between 0.62 and 1.00, all p<0.001). Based on consensus decision between both raters, 36 study cases (60%) showed a switch from E-cadherin to N-cadherin (as a hallmark of EMT), which correlated with the presence of lymphovascular invasion (ρ=0.287, p=0.026) while failing to interfere with CSM. Although cadherin switch was correlated with a loss of membranous β-catenin expression (ρ=0.629, p<0.001), none of the study cases showed nuclear β-catenin expression, and only three EMT cases (8.3%) had tumor buds revealing vimentin expression.Our data suggest that roughly half of surgically treated SCCP cases exhibit EMT, a parameter correlating with lymphovascular invasion. However, further studies are clearly needed to validate the so far unresolved possible role of cadherin switch in terms of predicting nodal spread in patients with SCCP. Moreover, the apparently complex mechanisms driving EMT in SCCP should be explored by future studies, as knowledge about these might provide a so far unexploited basis for the development of novel targeted therapies against SCCP with metastatic dissemination.

Cell adhesion and urothelial bladder cancer: the role of cadherin switching and related phenomena.

Cadherins are mediators of cell-cell adhesion in epithelial tissues. E-cadherin is a known tumour suppressor and plays a central role in suppressing the invasive phenotype of cancer cells. However, the abnormal expression of N- and P-cadherin ('cadherin switching', CS) has been shown to promote a more invasive and m̀alignant phenotype of cancer, with P-cadherin possibly acting as a key mediator of invasion and metastasis in bladder cancer. Cadherins are also implicated in numerous signalling events related to embryonic development, tissue morphogenesis and homeostasis. It is these wide ranging effects and the serious implications of CS that make the cadherin cell adhesion molecules and their related pathways strong candidate targets for the inhibition of cancer progression, including bladder cancer. This review focuses on CS in the context of bladder cancer and in particular the switch to P-cadherin expression, and discusses other related molecules and phenomena, including EpCAM and the development of the cancer stem cell phenotype.

Bone morphogenetic protein antagonist gremlin-1 regulates colon cancer progression.

Bone morphogenetic proteins (BMP) are phylogenetically conserved signaling molecules of the transforming growth factor-beta (TGF-beta) superfamily of proteins, involved in developmental and (patho)physiological processes, including cancer. BMP signaling has been regarded as tumor-suppressive in colorectal cancer (CRC) by reducing cancer cell proliferation and invasion, and by impairing epithelial-to-mesenchymal transition (EMT). Here, we mined existing proteomic repositories to explore the expression of BMPs in CRC. We found that the BMP antagonist gremlin-1 (GREM1) is secreted from heterotypic tumor-host cell interactions. We then sought to investigate whether GREM1 is contextually and mechanistically associated with EMT in CRC. Using immunohistochemistry, we showed that GREM1-expressing stromal cells harbor prominent features of myofibroblasts (i.e., cancer-associated fibroblasts), such as expression of α-smooth muscle actin and laminin-beta-1, and were in contextual proximity to invasion fronts with loss of the tight junction protein occludin and parallel nuclear accumulation of β-catenin, two prominent EMT hallmarks. Furthermore, in vitro assays demonstrated that GREM1-dependent suppression of BMP signaling results in EMT induction, characterized by cadherin switching (loss of E-cadherin-upregulation of N-cadherin) and overexpression of Snail. Collectively, our data support that GREM1 promotes the loss of cancer cell differentiation at the cancer invasion front, a mechanism that may facilitate tumor progression.

Immunohistochemical study of the expression of N-cadherin in cutaneous melanoma and in dysplastic melanocytic nevi

It has been suggested that the invasive and metastatic potential of melanoma cells reflects their ability to undergo epithelial-mesenchymal transition (EMT)-like phenotypic changes (1). Important hallmarks of EMT include the loss of E-cadherin expression and increased expression of the cell adhesion molecule N-cadherin. This cadherin switch leads melanoma cells to lose contact with keratinocytes in the epi- dermis and interact instead with stromal fibroblasts and endothelial cells, thus promoting dermal and vascular melanoma invasion (2). In melanoma, up-regulation of N-cadherin can be induced by the overexpression of the transmembrane receptor Notch1, thus providing a mechanism that underlines increased melanoma cell adhesion, survival, growth, and tumor progression when Notch signaling is activated (3). In this study, the expression of N-cadherin and Notch1 was evaluated by immuno- histochemical analysis in primary cutaneous melanomas and lymph node metastases. First, we evaluated the prognostic impact of high N-cadherin expression on sur- vival in melanoma patients. Second, we correlated the expression of N-cadherin with the full clinicopathological data of patients. Third, we investigated the relationship between the expression of N-cadherin and Notch1. Moreover, N-cadherin expression was evaluated in dysplastic melanocytic nevi and in normal skin. The results will be discussed.

N-cadherin mediates the migration of MCF-10A cells undergoing bone morphogenetic protein 4-mediated epithelial mesenchymal transition.

Epithelial-mesenchymal transition (EMT) of mammary epithelial cells is important in both normal morphogenesis of mammary glands and metastasis of breast cancer. Cadherin switching from E-cadherin to N-cadherin plays important roles in EMT. We found that cadherin switching is important in bone morphogenetic protein 4 (BMP4)-induced EMT in MCF-10A cells. BMP4 increased the phosphorylation of SMAD proteins in MCF-10A cells. Canonical BMP4 signaling decreased the expression of E-cadherin and disrupted the polarity of the tight junction protein ZO-1 in MCF-10A cells. However, the expression of N-cadherin and SNAI2 was up-regulated in BMP4-treated MCF-10A cells. MCF-10A cells that expressed N-cadherin migrated into type I collagen gels in response to BMP4 when evaluated using three-dimensional culture assays. Thus, active canonical BMP4 signaling is important for the migration and EMT of mammary epithelial cells. Moreover, the decrease in E-cadherin and/or increase in N-cadherin may be required for BMP4-induced migration and EMT.

Abstract C19: Molecular biology of lung cancer in Puerto Ricans

Abstract Lung cancer is a leading cancer killer, lung adenocarcinoma (AC) being its most common type. Several studies suggest that the frequency or nature of genetic events driving AC differ among ethnic populations. These differences impact treatment decisions as targeted agents are under development for a number of genes that drive lung cancer. For example, mutations in the epidermal growth factor receptor (EGFR) represent a driving event in a substantial number of AC patients that can be targeted with tyrosine kinase inhibitors. With the funding of a U54 Partnership grant between the Ponce School of Medicine in Ponce, PR and the Moffitt Cancer Center in Tampa, Florida we are constructing a molecular database for Puerto Rican Lung Cancer patients. Sample collection includes acquisition of fresh frozen and paraffin-embedded tissues via the U54-funded Puerto Rico tissue bank. DNA analysis of these tissues includes ancestry markers and exome sequencing of genes commonly altered in AC, including KRAS, TP53, EGFR, STK11, CDKN1C and RB1. Initial results suggest that the frequency of KRAS mutations in this population are significantly lower than those seen in a white mainland cohort, whereas the rate of EGFR and STK11 mutations is elevated. Since both EFGR and STK11 are potentially targetable mutations these data suggest that Puerto Rican AC patients would greatly benefit from genetic screening. Our U54 project also probes the role of the retinoblastoma protein (Rb) in lung cancer. Rb was the first tumor suppressor to be discovered, and yet its potency as a tumor suppressor remains only partially explained. We are interested in characterizing how Rb loss contributes to the aggressiveness of small cell lung carcinomas (SCLC). SCLC are poorly differentiated tumors with early proclivity for metastasis. Prognosis is poor with only 10-20% of patients achieving long-term disease-free intervals. Still, SCLC aggressivity remains to be molecularly explained. Interestingly, SCLC show high rates of Rb inactivation, which led us to hypothesize that in SCLC, Rb loss during early tumorigenesis facilitates both over-proliferation and early metastasis. Supporting our hypothesis, we found that the Rb-deficient SCLC cell line H187 lacks expression of E-cadherin while overexpressing N-cadherin, relative to Rb-expressing non-small cell lung carcinoma cell lines H1666 and H1650. We confirmed the Rb-positive status of H1666 and H1650 and the Rb-negative status of H187 by immunoblot. These results suggest that Rb loss in lung cancer cell lines provokes a cadherin switch indicative of an epithelial-to-mesenchymal transition. Consistently, we found overexpression of the mesenchymal marker vimentin in H187 relative to H1666 and H1650. When compared to H1666 and H1650, H187 lacked expression of the adherens junction component α-catenin and of the cytoskeletal adapter Eplin. Furthermore, H187 cells show up-regulated levels of Pak1, a Rac1 and Cdc42 effector whose up-regulation is associated to adherens junction disruption. Tiam-1, a Rac1 and Cdc42 activator, is also up-regulated in H187 relative to H1666 and H1650. Together, our data in lung cell lines suggests that Rb loss results in adherens junction alterations possibly due to transcriptional alteration of cell adhesion genes, aberrant regulation of the Rho GTPases Rac1 and Cdc42, and disruption of the cortical cytoskeleton. Based on our data, we propose that Rb loss in SCLC, not only leads to an increased proliferative capacity during early tumorigenesis, but also exacerbates early tumor aggressiveness by perturbing cellular adhesion, which facilitates tumor cell detachment and metastasis. Our ultimate goal is to link the genetic alterations we found in Puerto Ricans to Rb function, cell adhesion, and tumor aggressivity. Citation Format: Pedro G. Santiago-Cardona, Brienne E. Engel, Jonathan González-Flores, Ruth Cruz-Cosme, Teresita Muñoz-Antonia, W. Douglas Cress. Molecular biology of lung cancer in Puerto Ricans. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C19. doi:10.1158/1538-7755.DISP13-C19

Abstract 200: DDX3X induces signal switching to stem cell-specific Wnt/β-catenin signaling, resulting in EGFR-TKI resistance in lung cancer cells harboring EGFR activating mutation

Abstract [Background] The treatment targeting signal addiction caused by oncogenic driver mutation has led to unprecedented results in clinical setting; however, it is still difficult to achieve cure. Tumor diversity based on both genetic and non-genetic influences give rise of survivors as treatment-resistant cells. Most of the acquired resistance reflects the selection of the cancer cells harboring stochastic resistance-conferring genetic alterations. However, the mechanisms how the cancer cells survive until acquisition of additional mutations are unclear. We identified DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked (DDX3X), an ATP-dependent RNA helicase, was preferentially expressed in a cancer stem cell (CSC)-like population of murine melanoma. DDX3X is believed to be involved in epigenetic regulation of gene expression based on metabolism of mRNA and miRNA. [Objectives] In the current study, we examine that DDX3X affects phenotype and signal status of PC9 cells, a lung adenocarcinoma harboring EGFR exon19 deletion as an oncogenic driver mutation. [Results] DDX3X plays a role in acquisition of CSC-like properties. PC9 A-1 cells that were engineered to overexpress DDX3X acquired ability to proliferate as tumor spheres and showed up-regulated Sox2 and ALDH expression. Epithelial mesenchymal transition with cadherin switching from E-cadherin to N-cadherin accompanied with vimentin expression was detected in non-adherent A-1 population. E-cadherin+ non-adherent cells exhibited strong CD44 expression, a putative CSC marker. Surprisingly, A-1 cells exhibited almost no EGFR phosphorylation even in the presence of EGF. Knocking-down of DDX3X restored EGFR phosphorylation. On the other hand, activated nuclear translocation of β-catenin was observed. Loss of EGFR signal addiction resulted in resistance to EGFR-TKI. On contrast, PC9-A1 cells were sensitive to ICG-001, a β-catenin signal inhibitor. Further, we identified a minor non-adherent subpopulation of parental PC9 cells strongly expressed DDX3X and that they were resistant to EGFR-TKI because they did not addict to EGFR signaling. Survivor cells of parental PC9 after long term-exposure to EGFR-TKI strongly expressed DDX3X. [Conclusion] DDX3X plays a critical role in a novel EGFR-TKI resistance mechanism, signal switching, correlating with CSC transformation. DDX3X and β-catenin are likely promising target molecules to overcome EGFR-TKI resistance. Citation Format: Satoshi Shoji, Hiroshi Kagamu, Koichiro Nozaki, Natsue Igarashi, Masaaki Okajima, Satoru Miura, Satoshi Watanabe, Hrohisa Yoshizawa, Ichiei Narita. DDX3X induces signal switching to stem cell-specific Wnt/β-catenin signaling, resulting in EGFR-TKI resistance in lung cancer cells harboring EGFR activating mutation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 200. doi:10.1158/1538-7445.AM2014-200

The vitamin D analog, MART-10, represses metastasis potential via downregulation of epithelial–mesenchymal transition in pancreatic cancer cells

Pancreatic cancer (PDA) is a devastating disease and there is no effective treatment available at present. To develop new regiments against PDA is urgently needed. Previously we have shown that vitamin D analog, MART-10 (19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3), exerted potent antiproliferative effect on PDA in vitro and in vivo without causing hypercalcemia. Since metastasis is the major cause of PDA-related death, we therefore investigate the anti-metastasis effect of MART-10 on PDA. Our results showed that both 1α,25(OH)2D3 and MART-10 repressed migration and invasion of BxPC-3 and PANC cells with MART-10 much more potent than 1α,25(OH)2D3. 1α,25(OH)2D3 and MART-10 inhibited epithelial–mesenchymal transition (EMT) in pancreatic cancer cells through downregulation of Snail, Slug, and Vimentin expression in BxPC-3 and PANC cells. MART-10 further blocked cadherin switch (from E-cadherin to N-cadherin) in BxPC-3 cells. The F-actin synthesis in the cytoplasm of BxPC-3 cells was also repressed by 1α,25(OH)2D3 and MART-10 as determined by immunofluorescence stain. Both 1α,25(OH)2D3 and MART-10 decreased MMP-2 and -9 secretion in BxPC-3 cells as determined by western blot and zymography. Collectively, MART-10 should be deemed as a promising regimen against PDA.

Prognostic significance of epithelial-mesenchymal transition-related markers in extrahepatic cholangiocarcinoma: comprehensive immunohistochemical study using a tissue microarray.

Background:Epithelial–mesenchymal transition (EMT) is characterised by the loss of cell-to-cell adhesion and gaining of mesenchymal phenotypes. Epithelial–mesenchymal transition is proposed to occur in various developmental processes and cancer progression. ‘Cadherin switch', a process in which cells shift to express different isoforms of the cadherin transmembrane protein and usually refers to a switch from the expression of E-cadherin to N-cadherin, is one aspect of EMT and can have a profound effect on tumour invasion/metastasis. The aim of this study was to investigate the clinicopathological significance of EMT-related proteins and cadherin switch in extrahepatic cholangiocarcinoma (EHCC).Methods:We investigated the association between altered expression of 12 EMT-related proteins and clinical outcomes in patients with EHCC (n=117) using immunohistochemistry on tissue microarrays.Results:Univariate and multivariate analyses revealed that, in addition to N classification (P=0.0420), the expression of E-cadherin (P=0.0208), N-cadherin (P=0.0038) and S100A4 (P=0.0157) was each an independent and a significant prognostic factor. We also demonstrated that cadherin switch was independently associated with poor prognosis (P=0.0143) in patients with EHCC.Conclusions:These results may provide novel information for selection of patients with EHCC who require adjuvant therapy and strict surveillance.

The cytoplasmic domain of N-cadherin modulates MMP‑9 induction in oral squamous carcinoma cells.

Oral squamous carcinoma is the sixth most common cancer worldwide, and one of the most common cancers in developing countries. Regional and distant metastases comprise the majority of cases at initial diagnosis and correlate with poor patient outcomes. Oral epithelia is one of many tissue types to exhibit a cadherin switch during tumor progression, in which endogenous cell adhesion proteins, such as E-cadherin, give way to those of mesenchymal origin. The mesenchymal cell adhesion protein N-cadherin is found at the invading front of oral squamous carcinomas and has been strongly correlated with poor patient prognosis. The goal of the present study was to elucidate the mechanism by which N-cadherin may increase extracellular matrix-associated proteolytic activity to facilitate invasiveness in oral tumor development. The overexpression of N-cadherin in two oral squamous carcinoma cell lines increased motility, invasive capacity and synthesis of matrix metalloproteinase-9 (MMP-9) in a manner that was independent of E-cadherin downregulation. The use of EN and NE chimeric cadherin molecules with reciprocally substituted cytoplasmic domains revealed that optimal induction of MMP-9 synthesis required the cytoplasmic region, but not the extracellular region, of N-cadherin. Utilizing an N-cadherin mutant with impaired p120 binding ability, we found that such mutation resulted in a 4-fold decrease in motility compared to wild-type N-cadherin, but did not affect either MMP-9 expression or motility-normalized invasion. Overexpression of wild-type N-cadherin produced a 27-fold increase in the transcriptional activity of β-catenin, concomitant with increases in MMP-9 transcription. These results suggest that N-cadherin may promote motility and invasiveness through distinct mechanisms, and that β-catenin may be an integral mediator of N-cadherin-dependent invasive signaling in oral epithelia.

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

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.

Computational Modeling of the Interplay between Cadherin-Mediated Cell Adhesion and Wnt Signaling Pathway

Wnt signaling and cadherin-mediated adhesion have been implicated in both processes of embryonic development and the progression of carcinomas. Recent experimental studies revealed that Wnt signaling and cadherin-mediated cell adhesion have close crosstalk with each other. A comprehensive model that investigates the dynamic balance of β-catenins in Wnt signaling and cell adhesion will improve our understanding to embryonic development and carcinomas. We constructed a network model to evaluate the dynamic interplay between adhesion and Wnt signaling. The network is decomposed into three interdependent modules: the cell adhesion, the degradation circle and the transcriptional regulation. In the cell adhesion module, we consider the effect of cadherin’s lateral clustering. We found adhesion negatively contributes to Wnt signaling through competition for cytoplasmic β-catenins. In the network of degradation circle, we incorporated features from various existing models. Our simulations reproduced the most recent experimental phenomena with semi-quantitative accuracy. Finally, in the transcriptional regulation module, we developed a function selection strategy to analyze the outcomes of genetic feedback loops in modulating the gene expression of Wnt targets. The specific cellular phenomena such as cadherin switch and Axin oscillation were archived and their biological insights were discussed. Our model provides the theoretical basis of how spatial organization regulates the dynamics of cellular signaling pathways. We suggest that cell adhesion affects Wnt signaling in both negative and positive ways. Cadherins can inhibit Wnt signaling not only in a way as a stoichiometric binding partner of β-catenins that sequesters them from signaling, but also in a way through their clustering to impacts the rate at which β-catenins are involved in the destruction loop. Additionally, cadherin clustering increases the phosphorylation rate of β-catenins and promotes its signaling in nucleus.

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

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.

A cadherin switch underlies malignancy in high-grade gliomas.

Although the infiltrative behavior of malignant gliomas is one of their most critical aspects, the mechanisms underlying it have not yet been elucidated. To migrate in the brain parenchyma, malignant glioma cells need to bypass the cell-cell contact inhibitory signals. Here we propose that the blinding of cell-cell contact sensing in gliomas is caused by an unusual mechanism of cadherin switch, involving the replacement of N-cadherin with R-cadherin (Rcad) at the cell-cell junctions and the activation of ERK and p27. In our model of malignant glioma, we found that Rcad expression is necessary and sufficient to release cells from contact inhibition of proliferation, and is necessary, although not sufficient, for overriding contact inhibition of migration and for tumorigenicity. Altogether, these observations suggest that Rcad is a potential target for malignant glioma therapies.