Diminished E-cadherin Expression Research Articles

HSPD1 repressed E-cadherin expression to promote cell invasion and migration for poor prognosis in oral squamous cell carcinoma

Buccal mucosa squamous cell carcinoma (BMSCC) is one of major subsites of oral cancer and is associated with a high rate of metastasis and poor prognosis. Heat shock proteins (HSPs) act as potential prognostic biomarkers in many cancer types. However, the role of HSPD1 in oral cancer, especially in BMSCC, is still unknown. Through data analysis with The Cancer Genome Atlas (TCGA), we found the association of HSPD1 gene expression with tumorigenesis and poor prognosis in oral cancer patients. Our cohort study showed that higher HSPD1 protein level was associated with tumorigenesis and poor prognosis in BMSCC patients with lymph node invasion, suggesting that HSPD1 may be involved in tumor metastasis. Moreover, knockdown of HSPD1 induced E-cadherin expression and decreased the migration and invasion of BMSCC cells. In contrast, ectopic expression of HSPD1 diminished E-cadherin expression and promoted the migration/invasion of BMSCC cells. Further, HSPD1 regulated RelA activation to repress E-cadherin expression, enhancing the migration and invasion of BMSCC cells. Furthermore, HSPD1 protein level was inversely correlated with E-cadherin protein level in tumor tissues and co-expression of high HSPD1/low E-cadherin showed a significant association with poor prognosis in BMSCC patients. Taken together, HSPD1 might repress E-cadherin expression and promote metastatic characters of BMSCC cells for poor prognosis of BMSCC patients.

Kaiso-induced intestinal inflammation is preceded by diminished E-cadherin expression and intestinal integrity.

Chronic intestinal inflammation contributes to pathologies such as inflammatory bowel disease (IBD) and colon cancer. While the precise etiology remains controversial, IBD is believed to manifest as a result of various factors. We previously reported that intestinal-specific overexpression of the transcription factor Kaiso results in an intestinal inflammatory response; however, the cause of this inflammation is unknown. To elucidate the underlying mechanism(s) of the Kaiso-mediated intestinal inflammatory phenotype, we evaluated two independent transgenic mouse lines that express varying levels of Kaiso (KaisoTg). Histological analyses of KaisoTg mice revealed intestinal damage including thickening of the mucosa, intestinal “lesions” and crypt abscesses, which are reminiscent of IBD pathology. Additionally, higher Kaiso levels induced intestinal neutrophilia as early as 12 weeks, which worsened as the mice aged. Notably, the Kaiso-induced intestinal inflammation correlated with a leaky intestinal barrier and mis-regulation of E-cadherin expression and localization. Interestingly, Kaiso overexpression resulted in reduced proliferation but enhanced migration of intestinal epithelial cells prior to the onset of inflammation. Collectively, these data suggest that Kaiso plays a role in regulating intestinal epithelial cell integrity and function, dysregulation of which contributes to a chronic inflammatory phenotype as mice age.

First experience of chick chorioallantoic membrane (CAM) assay in the clinical work flow with oral squamous cell carcinoma patients

The oral squamous cell carcinoma (OSCC) is a leading cause of death in human malignancies. The aim of this study is to integrate the CAM Assay as a reliable and good working in vivo model for the evaluation of OSCC tumor samples and its growth into the clinical work flow. Fresh human Tumor samples (OSCCs) 1×1 cm in size were cut into 350-450μm thick slices by a Vibratome and put on the prepared CAM model.After growth of the tumor tissue on the CAM, we started with topical induction of proinflammatory cytokines (TNFα) and growth factors (TGFβ). After further growth of the tumor on the assay, we explanted the tumor tissue and first performed microscopic and then immunohistochemical examinations. E-cadherin and vimentin were used as Epithelial-to-mesenchymal transition (EMT) -makers and the histologic preparations were evaluated histomorphometrically. The results were correlated with clinical parameters of the patients. Under TNFα, the small tumors (T1 / T2) show higher E-cadherin expression than larger tumors (T3 / T4). The vimentin expression under TNFα behaved in the opposite direction, at T1 / T2 the expression decreased in T3 / T4 increased. Furthermore, an increased E-cadherin expression in N0 and diminished E-cadherin expression in N1 / N2b patients could be detected depending on the N-stage of the patients. Vimentin, on the other hand, was reduced in the N0 group and expressed more frequently in the N1 / N2b group. TGFβ induction also led to increased expression of vimentin in the T3 / T4 tumors and N1 / N2b stages. By integrating a CAM assay into the clinical workflow, tumors with preserved tumor architecture can be cultured and subjected to histological and molecular biology studies. Effects on biological behavior are recognizable and demonstrable in this model. The key markers E-cadherin and vimentin alone are not sufficient to represent the complexity of the EMT in this model. Further molecular biology and signaling pathway analyzes are necessary.

Overexpression of GP73 promotes cell invasion, migration and metastasis by inducing epithelial-mesenchymal transition in pancreatic cancer

Pancreatic cancer is one of the most difficult clinical cases to diagnose with a very low 5-year survival rate of 5%, regardless of the advances made in both the medical and surgical treatment of the disease. One of the contributing factors for the high mortality rate seen of pancreatic cancer patients is the lack of effective chemotherapies, which is believed to be due to drug-resistance. Based on recent evidence, epithelial-mesenchymal transition (ETM) of pancreatic cancer cells has been found to be associated with the development of drug resistance and an increase in cell invasion. Therefore, we conducted the present study in order to investigate the regulatory effects of Golgi protein-73 (GP73) on PC. GP73 and EMT-related gene expressions in PC, along with the adjacent and chronic pancreatitis tissues were determined by means of RT-qPCR and Western blot analysis. Cultured PC cells were treated with pAdTrack-CMV, si-NC, GP73 overexpression, Si-GP73, Snail-siRNA and GP73 + Snail-siRNA. Cell invasion, migration and metastasis were measured in vitro and in vivo. The results revealed that the PC tissues and chronic pancreatitis tissues exhibited diminished E-cadherin expression and amplified GP73, N-cadherin, Vimentin and Snail expression. In response to GP73 gene silencing, PC cells presented with increased E-cadherin expression and decreased N-cadherin, Vimentin, Snail expression in addition to the inhibition of the number of invasive cells, tumor volume and number of liver lesions. These findings highly indicated that the overexpression of GP73 promotes cell invasion, migration and metastasis by inducing EMT in PC.

Dual role of Par-4 in abrogation of EMT and switching on Mesenchymal to Epithelial Transition (MET) in metastatic pancreatic cancer cells.

Epithelial-mesenchymal transition (EMT) is a critical event that occurs during the invasion and metastatic spread of cancer cells. Here, we conceive a dual mechanism of Par-4-mediated inhibition of EMT and induction of MET in metastatic pancreatic cancer cells. First, we demonstrate that 1,1'-β-D-glucopyranosyl-3,3'-bis(5-bromoindolyl)-octyl methane (NGD16), an N-glycosylated derivative of medicinally important phytochemical 3,3'-diindolylmethane (DIM) abrogates EMT by inducing pro-apoptotic protein Par-4. Induction of Par-4 (by NGD16 or ectopic overexpression) strongly impedes invasion with inhibition of major mesenchymal markers viz. Vimentin and Twist-1 epithelial marker- E-cadherin. Further, NGD16 triggers MET phenotypes in pancreatic cancer cells by augmenting ALK2/Smad4 signaling in a Par-4-dependent manner. Conversely, siRNA-mediated silencing of endogenous Par-4 unveil reversal of MET with diminished E-cadherin expression and invasive phenotypes. Additionally, we demonstrate that intact Smad4 is essential for Par-4-mediated maintenance of E-cadherin level in MET induced cells. Notably, we imply that Par-4 induction regulates E-cadherin levels in the pancreatic cancer cells via modulating Twist-1 promoter activity. Finally, in vivo studies with syngenic mouse metastatic pancreatic cancer model reveal that NGD16 strongly suppresses metastatic burden, ascites formation, and prolongs the overall survival of animals effectively.

Abstract 5082: Impact of HGF knockin microenvironment on epithelial-mesenchymal transition and cancer stem cells in a non-small cell lung cancer xenograft model

Abstract We previously reported the generation of rabbit monoclonal antibodies to twelve EMT (epithelial-to-mesenchymal transition) transcription factors and cancer stem cell (CSC) markers for the development of pharmacodynamic assays to inform clinical trials of new anticancer therapies (Pfister et al., AACR 2013). Here we demonstrate the functional utility of some of these reagents in detecting HGF-induced changes in EMT and CSC biology in a xenograft tumor model. Initial antibody characterization was performed in vitro and a subset [including SNAIL, SLUG, SOX9, Goosecoid (GSC), NANOG and CD133] was selected for further testing of functional utility in FFPE tissues by quantitative multiplex IFA. The antibodies were applied to xenograft tissues derived from the non-small cell lung cancer tumor line, NCI-H596, implanted in hHGFscid/scid, hHGFki/scid or hHGFki/ki mice to examine HGF-induced changes in EMT factors, CSC markers, as well as pY1235-MET expression in vivo. H596 tumors grown in either hHGFki/scid or hHGFki/ki mice exhibited enhanced EMT particularly in tumor microenvironments adjacent to mouse stroma containing the HGF knockin gene, compared to those in hHGFscid/scid mice. By quantitative immunofluorescence, H596 tumors showed increased Vimentin:E-cadherin ratio when grown in hHGFki/scid (P<0.0006) or hHGFki/ki (P<0.022) vs. HGFscid/scid mice. Moreover, significant increases in nuclear pY1235-MET, measured by%NAP (percentage nuclear area positive), were observed in H596 tumors in hHGFki/scid (P<0.0041) or hHGFki/ki (P<0.0058) vs. hHGFscid/scid. We detected varying levels of EMT/CSC marker expression, including CD44, CD133, ALDH, and GSC in the membrane or cytoplasm of noninvasive regions of H596 tumors in hHGFscid/scid mice. Sox9 was also co-expressed with GSC in some tumor cells but was predominantly in the nuclei. In tissues collected from hHGFki/scid or hHGFki/ki mice, Slug, Snail and Sox9 expression were increased in transitioning tissue regions, adjacent to HGF-containing stroma, coincident with diminished E-Cadherin expression and enhanced Vimentin expression. While Slug expression was predominantly cytoplasmic in invading tumor fronts, the expression was mutually exclusive with CD133 within non-invasive regions. Snail and Sox9 showed enhanced nuclear expression in tumor cells undergoing EMT. These markers are currently being investigated in additional tumor samples from human TNBC or CRC xenograft models treated with various drug combinations. To our knowledge, this is the first demonstration of EMT and changes in cancer stem cell biology in NSCLC induced by an HGF-knockin stromal microenvironment. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Tony Navas, Thomas D. Pfister, Scott M. Lawrence, Apurva K. Srivastava, Robert J. Kinders, Suzanne Borgel, Sergio Alcoser, Melinda G. Hollingshead, Lindsay M. Dutko, Brad A. Gouker, Donna Butcher, Elinor Ng-Eaton, Naoko Takebe, Young H. Lee, Donald P. Bottaro, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow. Impact of HGF knockin microenvironment on epithelial-mesenchymal transition and cancer stem cells in a non-small cell lung cancer xenograft model. [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 5082. doi:10.1158/1538-7445.AM2015-5082

Alterations in a defined extracellular region of the RON receptor tyrosine kinase promote RON-mediated motile and invasive phenotypes in epithelial cells

Cell migration followed by matrix invasion is a critical step required for epithelial cell differentiation, growth and survival. This study determined the RON receptor in regulation of motile-invasive phenotypes of epithelial cells. Two RON variants, RON165.e11p and RON165, with alterations in a defined extracellular domain were used as the model. RON165 is a splicing variant generated by an mRNA transcript with an in-frame deletion of 49 amino acids encoded by exon 11. In contrast, RON165.e11p was produced by a partial deletion of exon 11 with the elimination of the first 40 amino acids. Thus, RON165.e11p differs from RON165 with nine amino acids retained in the fourth immunoglobulin-plexin-transcription (IPT) domain. Biochemically, both RON165 and RON165.e11p exist as a single-chain protein, residing in the cytoplasm, and failed to mature into the two-chain receptor. Both RON165 and RON165.e11p spontaneously formed oligomers in vivo leading to constitutive phosphorylation and the activation of downstream signaling proteins. Although lacking cell-transforming activities, RON165 and RON165.e11p mediated the epithelial to mesenchymal transition with spindle-like cell morphologies, diminished E-cadherin expression, and increased N-cadherin and vimentin expression. These changes facilitated epithelial cell migration and invasion as modeled in Martin-Darby canine kidney (MDCK) cells. Moreover, expression of RON165 or RON165.e11p in breast epithelial MCF-7 cells diminished epithelial cell phenotypes and increased motile and invasive activities. Thus, alterations in the defined extracellular region result in two unique RON variants with similar biological properties. The ability of RON165 or RON165.e11p to promote motile-invasive phenotypes may represent a mechanism by which RON regulates epithelial cell phenotypes and biological activities.

Carcinogenic Hypergastrinemia: Signet-Ring Cell Carcinoma in a Patient with Multiple Endocrine Neoplasia Type 1 with Zollinger-Ellison’s Syndrome

Gastric neuroendocrine tumors are rare neoplasms that originate from gastric enterochromaffin-like (ECL) cells in the oxyntic mucosa. Gastrin and its derivates have been reported to regulate epithelial cell proliferation, migration, and differentiation. Mutations in the epithelial cadherin (E-cadherin) gene have been shown to be associated with the occurrence of diffuse gastric carcinomas in affected families. In this study we investigated the histopathological and molecular findings in the gastrointestinal wall of a patient with multiple endocrine neoplasia type 1 with malignant duodenal gastrinoma and multiple gastric ECL cell tumors, who additionally developed a signet-ring cell carcinoma of the stomach. Biopsies from the gastrointestinal tract of a patient with multiple endocrine neoplasia type 1 were immunostained for vesicular monoamine transporter-2 and E-cadherin. Nonamidated gastrin products were measured in the serum of the patient using antibodies that react with progastrin, Gly-extended, and amidated gastrins. Genetic analyses were performed to exclude germ-line mutations within the E-cadherin gene. Immunohistochemical studies of gastric ECL cell tumors showed a largely diminished E-cadherin expression in comparison to gastric surface mucosa cells and a loss of E-cadherin expression in the cells of the signet-ring carcinoma. Detailed biochemical measurements revealed progastrin concentrations that were approximately 20%, and Gly-gastrin concentrations that were approximately 10% the amidated gastrin concentrations in plasma. Molecular analyses revealed no E-cadherin germ-line mutation. Our immunohistochemical studies might suggest that the gastrinoma-associated excessive progastrin tissue concentrations led to diminished expression of E-cadherin within the gastric mucosa and promoted tumor development of a signet-ring cell carcinoma.

Methylation Patterns of the E-cadherin 5′ CpG Island Are Unstable and Reflect the Dynamic, Heterogeneous Loss of E-cadherin Expression during Metastatic Progression

Metastatic progression of most common epithelial tumors involves a heterogeneous, transient loss of expression of the homotypic cell adhesion protein, E-cadherin, rather than the uniform loss of a functional protein resulting from coding region mutation. Indeed, whereas E-cadherin loss may promote invasion, reexpression may facilitate cell survival within metastatic deposits. The mechanisms underlying such plasticity are unclear. We now show that the heterogeneous loss of E-cadherin expression in primary human breast cancers reflects a heterogeneous pattern of promoter region methylation, which begins early prior to invasion. In cultured human tumor cells, such heterogeneous methylation is dynamic, varying from allele to allele and shifting in relation to the tumor microenvironment. Following invasion in vitro, which favors diminished E-cadherin expression, the density of promoter methylation markedly increased. When these cells were cultured as spheroids, which requires homotypic cell adhesion, promoter methylation decreased dramatically, and E-cadherin was reexpressed. These data show that the methylation associated with E-cadherin loss in human breast cancer is heterogeneous and unstable and suggest that such epigenetic plasticity may contribute to the dynamic, phenotypic heterogeneity that drives metastatic progression.

E-cadherin germline mutations in familial gastric cancer.

The identification of genes predisposing to familial cancer is an essential step towards understanding the molecular events underlying tumorigenesis and is critical for the clinical management of affected families. Despite a declining incidence, gastric cancer remains a major cause of cancer death worldwide, and about 10% of cases show familial clustering. The relative contributions of inherited susceptibility and environmental effects to familial gastric cancer are poorly understood because little is known of the genetic events that predispose to gastric cancer. Here we describe the identification of the gene responsible for early-onset, histologically poorly differentiated, high grade, diffuse gastric cancer in a large kindred from New Zealand (Aotearoa). Genetic linkage analysis demonstrated significant linkage to markers flanking the gene for the calcium-dependent cell-adhesion protein E-cadherin. Sequencing of the E-cadherin gene revealed a G --> T nucleotide substitution in the donor splice consensus sequence of exon 7, leading to a truncated gene product. Diminished E-cadherin expression is associated with aggressive, poorly differentiated carcinomas. Underexpression of E-cadherin is a prognostic marker of poor clinical outcome in many tumour types, and restored expression of E-cadherin in tumour models can suppress the invasiveness of epithelial tumour cells. The role of E-cadherin in gastric cancer susceptibility was confirmed by identifying inactivating mutations in other gastric cancer families. In one family, a frameshift mutation was identified in exon 15, and in a second family a premature stop codon interrupted exon 13. These results describe, to our knowledge for the first time, a molecular basis for familial gastric cancer, and confirm the important role of E-cadherin mutations in cancer.