Low E-cadherin Expression Research Articles

PP029: E-cadherin and β-catenin immunoexpression in tongue and floor of mouth squamous cell carcinoma at initial clinical stage

Purpose To investigate the front of tumor invasion as a prognostic factor in association with E-cadherin and β-catenin immunostaining in patients with tongue and floor of mouth carcinoma (TFSCC) at clinical stages I and II. Material and methods Records from 156 patients were analyzed and submitted to bivariate (X 2 ) and survival (Kaplan–Meier) analysis. Results Caucasian (61.4%) males (75%) aged between 41 and 60 years (50.6%), who smoke (83.8%) and drink (74.8%), with tumors of the tongue (69.2%), at clinical stage II (66.4%) and pathological stage II (28.2%) were the most affected. We identified 76 cases (48.7%) of recurrence, especially in cervical lymph nodes (53.9%). One hundred and fifteen of 156 patients (73.7%) were alive. Moderately differentiated tumors (78.8%), with pattern of invasion showing small islands (79.4%), with perineural invasion of small nerves (60.9%) and strong lymphocytic infiltrate (49.4%) were predominant. The intermediate score was the most frequent (56.4%) at histologic risk assessment (HRA). Immunohistochemical analysis was performed on 131 patients. Both E-cadherin and β-catenin showed most frequently concomitant membrane and cytoplasmic expression (71.0% and 53.4%) with a diffuse distribution (61.5% and 82.1%) respectively. Tumors involving both the tongue and floor of mouth occurred only in men ( p = 0.003). Decreased E-cadherin expression was also associated with death ( p = 0.009). There were worse overall survival (OS) and disease free survival for poorly differentiated tumors in advanced pathological stage, with a high-risk AHR and low E-cadherin expression. Conclusions Demographic profile of patients with TFSCC in initial clinical stage does not differ from the classic profile for individuals with SCC of mouth. The presence of recurrence was associated with histopathological parameters of aggressiveness. There was an association between cytoplasmic expression of E-cadherin with death and worse OS rates. Even though the study population was composed by initial stage tumors, an aggressive behavior was observed.

Cdc42 is involved in basal cell carcinoma carcinogenesis

Basal cell carcinoma (BCC) is the most common type of skin cancer in older persons and is a rapidly rising incidence. E-cadherin-mediated cell-cell adhesion activates Cdc42, a Rho GTPase essential for cell polarity in numerous settings. No study has yet addressed a biological significance of Cdc42 alterations in BCC pathogenesis. Our aim was to investigate E-cadherin-dependent cell-cell contacts and Cdc42 activity in BCC formation. We evaluated E-cadherin and Cdc42 expression by immunohistochemistry and Western blot analysis in samples of 15 normal skin (NS) and 30 BCC (10 superficial, 9 nodular and 11 infiltrative subtypes). Low E-cadherin and high Cdc42 immunohistochemical expression were found in BCC samples compared with NS. E-cadherin staining was significantly reduced in infiltrative BCC compared with superficial and nodular. A significantly greater Cdc42 expression was observed in BCC compared with NS; moreover, superficial BCC had a significantly lower Cdc42 expression in respect to the other subtypes. Western blot analysis confirmed the significantly decreased E-cadherin expression in infiltrative BCC as well as Cdc42 reduction in superficial BCC in respect to the other subtypes. In BCC the increased Cdc42 in association with reduced E-cadherin might contribute to the disruption of adhesion mechanisms and to the loss of cell polarity, thus explaining a mechanism by which cancer cells can escape from the control of adjacent normal keratinocytes. Our study also showed that Cdc42 and E-cadherin expression differed according to aggressive behaviour of BCC subtypes and suggested important functions of these molecules in regulating tumour demarcation and progression.

Abstract 332: Establishment of animal model for metastasis of human lung cancers using NOJ/SCID mice.

Abstract Lung cancer is the leading cause of cancer death among malignant tumors. Lymphogenic and distant metastases were found approximately 70% at the diagnosis, and most of recurrence after surgery occurs as distant metastasis. Therefore, it is important to establish the new strategy for control of metastatic lung cancers. Since metastasis consists of several steps such as loss of cell adhesion, intravasation, survival in circulation, exit into new tissues, and colonization in a distant site, a suitable animal model is indispensable for investigation of metastasis. In addition, recent investigations revealed that biological and molecular characteristics of cancer cells were different between primary and metastatic lesions, as well as among each metastatic site. To establish the animal model reflecting human metastatic lung cancers, we used NOD/Scid/Jak3null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and DC function, and complement activation as well as T and B cell deficiencies. This animal model enabled us to engraft various human cells more easily than previous immunodeficiency mouse models. At first, twenty kinds of human lung cancer cell lines were screened by expression pattern of E-cadherin and vimentin using western blot analysis, and two cell lines with high expression of vimentin and low expression of E-cadherin were selected. The cells (1×107/mouse) were injected into the dorsal flanks, and one cell line, H1975 showed lymphogenic metastasis to axillary and abdominal lymph nodes, resulting dissemination to pleura and diaphragm with ascites. Since H1975 cells carry epidermal growth factor receptor (EGFR) mutation, L858R and T790M, we can monitor the existence of cancer cells using a sensitive detection system, mutation-biased PCR and quenched probe system (MBP-QP), which were established in our laboratory. We have also tried to establish the animal model which lung cancer cells are inoculated into lung to mimic metastasis of human lung cancers. Using these model systems, we have been performing comprehensive analysis including mRNA expression, the mutation status of lung cancer related genes in each metastatic site, and compared the phosphorylation status of 38 kinds of molecules. Our goal of this study is to establish the animal model to reproduce phenotypes of metastasis of human lung cancers for screening of anti-metastatic agents. Citation Format: Naoko Aragane, Akemi Sato, Naomi Kobayashi, Masaru Ide, Eisaburo Sueoka, Seiji Okada, Shinya Kimura. Establishment of animal model for metastasis of human lung cancers using NOJ/SCID mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 332. doi:10.1158/1538-7445.AM2013-332

Abstract 5335: miR-31 contributes to immortalization and epithelial-mesenchymal transition of oral keratinocyte.

Abstract Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression by RNA interference and crucial for tumorigenesis. Our previous studies identified miR-31 as an oncogenic miRNA of OSCC, and the increased miR-31 may be contributive to oral carcinogenic process in the early stages. This study further specified the higher miR-31 expression in dysplastic potential malignant disorder (PMD) tissues than non-dysplastic PMD tissues in oral cavity. In addition, miR-31 in the patient's saliva was significantly higher than control subjects. To elucidate the contribution of miR-31 in modulating the extended growth of normal oral keratinocyte, exogenous expression of both miR-31 and hTERT genes were introduced to normal oral keratinocyte (NOK) culture, which led to immortalization of two NOK lines, designated M31OK1 and M31OK3. These immortalized lines exhibited cellular phenotypes lying between NOKs and OSCC cell lines and diminished expression of senescence-associated β-galactosidase. Knockdown of hTERT and/or miR-31 decreased the growth and migration of both lines. M31OK1 also carries p53 gene mutation in codon 273, exhibiting the tolerance to serum-induced senescence, anchorage-independent growth and epithelial-mesenchymal transition (EMT) properties relative to NOK. However, the stemness properties were not increased in M31OK1 in relation to NOKs. Knockdown of miR-31 expression decreased the expression of vimentin and ZEB1 and slightly increased E-cadherin expression in M31OK1 cells. This was correlated with the clinical findings that high miR-31 expression was associated with higher expression of VEGF and lower expression of E-cadherin in PMD tissues. This study concluded that miR-31 may be contributive to early oral carcinogenesis by stimulating factors to facilitate cellular immortalization or by modulating the expression of EMT markers in oral keratinocytes. Citation Format: Hsi-Feng Tu, Pei-Shi Hung, Chung-Ji Liu, Shu-Chuin Lin, Kuo-Wei Chang. miR-31 contributes to immortalization and epithelial-mesenchymal transition of oral keratinocyte. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5335. doi:10.1158/1538-7445.AM2013-5335

Podoplanin-Positive Cancer-Associated Fibroblasts Could Have Prognostic Value Independent of Cancer Cell Phenotype in Stage I Lung Squamous Cell Carcinoma: Usefulness of Combining Analysis of Both Cancer Cell Phenotype and Cancer-Associated Fibroblast Phenotype

The prognostic significance of the tumor microenvironment, which is created by both cancer cells and cancer-associated fibroblasts (CAFs), has been increasingly recognized. The purpose of this study was to analyze the prognostic markers of stage I squamous cell carcinoma (SqCC), with special reference to the immunophenotypes of both cancer cells and CAFs. A total of 142 patients with stage I SqCC were included in this study. We examined the expressions of E-cadherin, laminin-5, podoplanin, c-MET, carbonic anhydrase IX (CA-IX), CD10, and CD44 in the cancer cells and those of podoplanin, CA-IX, CD10, and CD44 in the CAFs to evaluate their prognostic value. Patients with low E-cadherin expression in the cancer cells showed a significantly poorer prognosis than those with high E-cadherin expression in the cancer cells ( P , .001). On the other hand, high podoplanin expression in the CAFs was also associated with a significantly poorer prognosis ( P , .001). A multivariate analysis identified low E-cadherin expression in the cancer cells and high podoplanin expression in the CAFs as significantly independent prognostic factors for overall survival ( P 5 .013 and P 5 .0011, respectively). According to subgroup analyses combining E-cadherin expression in cancer cells and podoplanin expression in CAFs, 5-year overall survival of patients with low E-cadherin expression in the cancer cells and high podoplanin expression in the CAFs was 7.0% and showed a significantly poorer prognosis as compared with other groups ( P , .001). The current study indicates that immunophenotypes of CAFs could have a prognostic value independent of those of the cancer cells in SqCC.

Overexpression of AIB1 correlates inversely with E-cadherin expression in pancreatic adenocarcinoma and may promote lymph node metastasis

It was reported that the nuclear receptor coactivator amplified in breast cancer1 (AIB1) could regulate cancer cell invasion and migration in a nuclear receptor signaling-independent manner. Meanwhile, the process of epithelial mesenchymal transition (EMT) is critical for tumor invasion and metastasis. The present study aimed to determine the role of AIB1 and EMT markers in human pancreatic adenocarcinoma. AIB1, ZO-1, E-cadherin, vimentin, and N-cadherin protein expression in 76 pancreatic adenocarcinomas were assessed using immunohistochemistry and analyzed for clinicopathological significance. The frequency of AIB1 overexpression in pancreatic adenocarcinomas with lymph node metastasis is 68 % (19/28), which is significantly higher than in pancreatic adenocarcinomas without lymph node metastasis (42 %; 20/48). In addition, the frequency of low expression of E-cadherin in pancreatic carcinomas with lymph node metastasis (68 %; 19/28) was significantly higher than in tumors without lymph node metastasis (44 %; 21/48). Correlation analysis demonstrated that the overexpression of AIB1 was inversely correlated with low expression of E-cadherin in pancreatic adenocarcinomas. Overexpression of AIB1 might promote invasion and metastasis of cancer cells and is associated with down-regulation of E-cadherin in pancreatic adenocarcinomas.

Control of mitogenic and motogenic pathways by miR-198, diminishing hepatoma cell growth and migration

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths, worldwide. MicroRNAs, inhibiting gene expression by targeting various transcripts, are involved in genomic dysregulation during hepatocellular tumorigenesis. In previous studies, microRNA-198 (miR-198) was shown to be significantly downregulated in HCV-positive hepatocellular carcinoma (HCC). Herein, the function of miR-198 in hepatocellular carcinoma cell growth and gene expression was studied.In hepatoma cell-types with low levels of liver-specific transcription factor HNF1α indicating a low differentiation grade, miR-198 expression was most downregulated. However, miR-198 treatment did not restore the expression of the liver-specific transcription factors HNF1α or HNF4α. Importantly, overexpression of miR-198 in Pop10 hepatoma cells markedly reduced cell growth. In agreement, comprehensive gene expression profiling by microarray hybridisation and real-time quantification revealed that central signal transducers of proliferation pathways were downregulated by miR-198. In contrast, genes mediating cellular adherence were highly upregulated by miR-198. Thus, the low expression of E-cadherin and claudin-1, involved in cell adhesion and cell-cell contacts, was abolished in hepatoma cells after miR-198 overexpression. This definite induction of both proteins by miR-198 was shown to be accompanied by a significantly impaired migration activity of hepatoma Pop10 cells.In conclusion, miR-198 acts as a tumor suppressor by repression of mitogenic and motogenic pathways diminishing cell growth and migration.

Expression of E-Cadherin and Vascular Endothelial Growth Factor in Noncancerous Liver Is Associated with Recurrence of Hepatocellular Carcinoma after Curative Resection

Objectives: Hepatocellular carcinoma (HCC) frequently recurs even after curative resection. The purpose of this study was to examine how background liver affects postoperative recurrence of HCC that underwent curative resection using expression of cancer-related molecules in the adjacent noncancerous liver of HCC patients. Methods: We examined expression of E-cadherin and vascular endothelial growth factor in noncancerous liver tissues of 133 HCC patients who underwent curative resection of tumors using immunohistochemical analysis. Associations between expressions of these molecules and disease-free survival of HCC were analyzed using the Kaplan-Meier method. Results: The average period of follow-up of the patients was 6.7 years. Multivariate analyses revealed that low platelet count and negative expression of E-cadherin in adjacent noncancerous liver were significantly associated with metastatic recurrence [p = 0.017, hazard ratio (HR) = 1.31 for low platelet count, and p = 0.009, HR = 1.43 for negative expression of E-cadherin, respectively]. Conclusions: Expression levels of E-cadherin in adjacent noncancerous liver after surgical resection was associated with metastatic HCC recurrence later on. Analysis of E-cadherin expression should provide important information for predicting recurrence after curative resection of HCC.

Aberrant expression of β-catenin and E-cadherin is correlated with poor prognosis of nasopharyngeal cancer

Nasopharyngeal carcinoma has a high incidence in southern China. The Wnt/β-catenin signaling pathway plays a major role in cancer development and progression. Our current study aims to determine the clinical significance of the Wnt/β-catenin pathway components such as β-catenin, cyclooxygenase 2, cyclin D1, c-Myc, and E-cadherin in 148 nasopharyngeal carcinomas by immunohistochemistry. We found that nasopharyngeal carcinoma stage T3+T4 had significantly higher expression of β-catenin, cyclooxygenase 2, cyclin D1, and c-Myc and lower expression of E-cadherin than nasopharyngeal carcinoma stage T1+T2 (P < .001, P < .05, respectively).There was significantly higher expression of β-catenin (P = .001) and cyclooxygenase 2 (P = .003) and lower expression of E-cadherin (P = .001) in nasopharyngeal carcinoma with lymph node metastasis than in nasopharyngeal carcinoma without lymph node metastasis. The expression of β-catenin in nasopharyngeal carcinoma was positively correlated with cyclooxygenase 2 (r = 0.458, P < .0001), cyclin D1 (r = 0.700, P < .0001), and c-Myc expression (r = 0.144, P = .006) but negatively correlated with E-cadherin expression (r = -0.601, P < .0001), respectively. The univariate analysis confirmed that overexpression of β-catenin and cyclooxygenase 2 and decreased expression of E-cadherin were significantly correlated with disease-free survival (P < .01, P < .05, respectively). Overexpression of β-catenin and cyclooxygenase 2 and reduced expression of E-cadherin significantly correlated with a poor prognosis (P = .005, P = .044, P = .019, respectively) by Kaplan-Meier survival curves and the log-rank test. Multivariate analysis indicated that high expression of β-catenin and decreased expression of E-cadherin were independent prognostic factors (P = .002, P = .011, respectively) regardless of TNM stage and lymph node status. In conclusion, the aberrant high expression of β-catenin and decreased expression of E-cadherin is associated with poor prognosis in nasopharyngeal carcinoma.

CD1a‐positive Langerhans cells and their relationship with E‐cadherin in ameloblastomas and keratocystic odontogenic tumors

Ameloblastomas and keratocystic odontogenic tumors (KOTs) are lesions that are characterized by locally invasive growth and cause extensive bone destruction. In addition, it is known that E-cadherin influences the adhesion of Langerhans cells (LCs) to keratinocytes. The aim of this study was to investigate, using immunohistochemistry, the distribution of CD1a-positive cells in ameloblastomas and KOTs and their relationship with E-cadherin, in comparison to calcifying cystic odontogenic tumor (CCOT). The CD1a-positive LCs were observed in 11 ameloblastomas and KOTs. All of the cases of CCOT showed CD1a-positive LCs and a significant difference was found when this tumor was compared with ameloblastomas (P < 0.05, Mann-Whitney test). A statistically significant difference was also noted when comparing CD1a-positive LCs between CCOTs and KOTs (P < 0.05, Mann-Whitney test). Lower expression of E-cadherin in ameloblastomas (AMs) in relation to KOTs and CCOTs (P < 0.05, Fisher test) was observed. There was no correlation between E-cadherin and CD1a-positive LCs between all odontogenic tumors that were studied (P > 0.05, Spearman test). A quantitative difference of CD1a-positive cells between AMs and KOTs in comparison to CCOTs was observed. This permits to speculate that a depletion of CD1a-positive LCs might influence the local invasiveness of ameloblastomas and KOTs. Furthermore, it is suggested that E-cadherin mediates cell adhesion in these tumors.

Abstract P3-05-08: Analysis of the expression of claudin-3, -4, -7 and E-cadherin in breast cancer: are they surrogates for the claudin-low subtype?

Abstract Introduction: The molecular architecture of tight junctions (TJ) has been a subject of extensive studies in a number of malignancies. Among the TJ components, the members of the protein family claudins (CLDN) have been found to be differentially expressed correlating with histological grade (HG) in a number of tissues. Furthermore, the expression of these junctional proteins (JP) have recently been implied in the identification of the claudin-low subtype by gene expression analysis. Material and methods: 261 breast cancer specimens diagnosed between 1999 and 2002 were collected and evaluated histopathologically and immunophenotypically by a single specialist. Tissue microarrays were constructed using a TMA-builder instrument (Histopathology Ltd., Pecs, Hungary). To identify the eventual prognostic role of the TJ proteins CLDN3, −4 and −7, and adherent junction protein E-cadherin (ECDH) their immunohistochemical (IHC) expression was analysed by 3 investigators separately, and a consensus score was reached. The IHC results were compared to the patients' follow-up data applying Kaplan-Meier analysis supported by log-rank test. Furthermore, we aimed at identifying the worse prognosis tumour group based on clustering of the expression of the above mentioned JPs. Results: The expression of CLDN3 correlated with vascular invasion (VI)/p = 0.029/, necrosis (NC)/p = 0.001/, HG/p = 0.001/ and mitotic index (MI)/p = 0.022/. CLDN4 and CLDN7 correlated with NC/p = 0.001, p = 0.012, respectively/ and HG/p = 0.034, p = 0.001, respectively/, while ECDH correlated with HG/p = 0.001/ and MI/p = 0.001/. In single protein analysis, groups displaying gradually increasing CLDN3, −7 and ECDH/p = 0.017/expression (split at mean expression ± SD) showed a trend of bearing poorer prognostic features. CLDN4, on the other hand, showed different tendency: gradually increasing expression also correlated with worse relapse-free survival (RFS), while CLDN4 negative/low tumors had the poorest prognosis/p = 0.034/. In multiple protein analysis, groups were created using k-means clustering and the following cohorts were created - appearing in prognostic sequence starting with the longest RFS: ECDH-low groups with 1) low/n=31/ and 2) high/n=42/ CLDN3 expression; 3) CLDN7-low (CLDN3, -4 and ECDH-high) group/n=35/, 4) “all-high” expression group/n=70/; 5) CLDN3-low (CLDN4, -7 and ECDH-high) group/n=68/ and 6) “all-low” group/n=15/. The latter was characterised by the CLDN4-low feature as compared to the other groups. Conclusion: Expression of JPs can have prognostic relevance in breast carcinomas. The groups with low expression of ECDH have good prognosis, while gradually increasing JP expression outlines poor prognostic tumours. Strikingly the drop in all JPs expression - most likely resembling the claudin-low subtype - specifies the subgroup with the poorest outcome, which still remains an infrequent disease entity. Grant support: TÁMOP-4.2.2/B-10/1-2010-0013. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-05-08.

Abstract P3-08-02: Common variants at 10p14 and 1p11.2 display heterogeneity in breast cancer associations by E-cadherin tumor tissue expression in two independent datasets

Abstract Background: E-cadherin is a tumor suppressor gene involved in cell-cell adhesion, epithelial-to-mesenchymal transitions (EMT) and invasion. Loss of E-cadherin expression is strongly associated with lobular breast cancers, which exhibit single cell patterns of infiltration and are often estrogen receptor positive. We sought to determine if relative risk estimates for 19 established breast cancer susceptibility loci were modified by E-cadherin breast tumor tissue expression. Methods: Case-control analyses included up to 1885 invasive breast cancer cases and 2366 age and site matched controls aged 20–74 years from the Polish Breast Cancer Study (PBCS), a population based case-control study conducted in Poland from 2000–2003. Genotyping of the 19 single nucleotide polymorphisms (SNPs) was performed using TaqMan® assays. Tissue expression of E-cadherin was assessed using immunohistochemical (IHC) staining of tissue microarrays and IHC results were scored as the product of percent positive tumor cells × intensity. Tumors having a score of &amp;lt;10 were classified as E-cadherin low and those with a score ≥10 as E-cadherin high. Polytomous logistic regression models adjusted for age and study site were used to estimate odds ratios (OR) and 95% confidence intervals (95% CI) for each breast cancer subtype, defined by E-cadherin expression levels, compared to controls. Case-only data from the PBCS (N = 797) and the Study of Epidemiology and Risk Factors in Cancer Heredity, SEARCH (N = 2155) was used in logistic regression models to test for heterogeneity of SNPs by E-cadherin expression. Results: Three SNPs suggested significant heterogeneity by E-cadherin expression in the PBCS: rs2046210 at 6q25.1(ESR1) [per-allele ORs (95% CI); 1.53 (1.19–1.98) for E-cadherin low tumors and 0.99 (0.86–1.14) for E-cadherin high tumors, P-heterogeneity = 0.002]; rs1045485 at 10p14 (CASP8) [per-allele ORs (95% CI); 0.62 (0.41–0.93) for E-cadherin low tumors and 0.98 (0.81–1.18) for E-cadherin high tumors, P-heterogeneity = 0.04]; and rs11249433 at 1p11.2 (NOTCH2/FCGR1B) [per-allele ORs (95% CI); 1.29 (1.02–1.64) for E-cadherin low tumors and 1.01 (0.88–1.15) for E-cadherin high tumors, P-heterogeneity = 0.06]. Combined case-only analysis of PBCS and SEARCH for these three SNPs showed significant heterogeneity by E-cadherin expression for rs11249433 [Interaction OR (95% CI); 1.19 (1.05–1.36), P-heterogeneity = 0.007] and rs1045485 [Interaction OR (95% CI); 0.69 (0.53–0.90), P-heterogeneity = 0.007]. The association with rs2046210 [Interaction OR (95% CI); 1.12 (0.61–2.03), P-heterogeneity = 0.73] did not remain significant in combined analyses. Conclusion: Our findings provide evidence that associations for breast cancer susceptibility loci vary by E-cadherin tumor tissue expression, which has not been described previously. Specifically, our results suggest that the genetic markers rs11249433 and rs1045485 may preferentially modify risk for tumors with low or absent E-cadherin expression in two independent data sets. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-08-02.

The prognostic value of E‐cadherin in gastric cancer: A meta‐analysis

The prognostic impact of E-cadherin downregulation in gastric cancer has been assessed for years while the results are controversial and heterogeneous. We thus comprehensively reviewed the evidence for evaluation of E-cadherin expression in gastric cancer to determine this effect. We searched PubMed and Embase to identify eligible studies, and 26 studies comprising 4,383 gastric cancer patients were included to assess the association between E-cadherin immunohistochemical expression and overall survival (OS) and clinicopathological characteristics. Summary hazard ratios (HRs) or odds ratios (ORs) with 95% confidence interval (95% CI) were calculated to estimate the effect. We also performed meta-regression and subgroup analysis according to study location, publication year, number of patients, quality score of studies and cut-off value. Reduced E-cadherin expression was significantly correlated with poor OS of gastric cancer patients (HR 1.62, 95% CI 1.34-1.96). Subgroup analysis indicated that E-cadherin low-expression had an unfavorable impact on OS in Asian patients (HR 1.87, 95% CI 1.45-2.41). Moreover, downregulation of E-cadherin was significantly associated with TNM stage (OR 2.52, 95% CI 1.85-3.43), the depth of invasion (OR 2.01, 95% CI 1.39-2.90), lymph node metastasis (OR 2.39, 95% CI 1.68-3.40), distant metastasis (OR 2.23, 95% CI 1.21-4.11), grade of differentiation (OR 2.26, 95% CI 1.60-3.21), vascular invasion (OR 1.86, 95% CI 1.10-3.13) and histological type of gastric cancer (OR 4.22, 95% CI 2.96-6.02). This meta-analysis revealed that E-cadherin expression might be a predicative factor of poor prognosis for gastric cancer particularly in Asia.

Hypoxia-inducible factor 1 alpha mediates epidermal growth factor-induced down-regulation of E-cadherin expression and cell invasion in human ovarian cancer cells

Hypoxia-inducible factor 1α (HIF-1α) regulates the transcription of a number of genes under hypoxia and other extracellular stimulations. It has been shown that E-cadherin is down-regulated by epidermal growth factor receptor (EGF) stimulation, and that cells with low E-cadherin expression are more invasive. Our recent study demonstrated a novel mechanism by which EGF down-regulates E-cadherin expression through production of hydrogen peroxide (H2O2) and the activation of p38 MAPK in human ovarian cancer cells. In this study, we were interested in examining the potential role of HIF-1α in cell invasion under normoxic conditions, specifically when cells are treated with EGF, which is known to down-regulate E-cadherin and increase invasiveness. We show that EGF treatment induces HIF-1α expression in two human ovarian cancer cell lines (SKOV3 and OVCAR5), and that this effect is diminished by treatment with a membrane-permeable H2O2 scavenger, PEG-catalase. However, the induction of HIF-1α by EGF did not require the activation of p38 MAPK. Treatment with siRNA targeting HIF-1α reduces both basal and EGF-induced HIF-1α levels. Importantly, treatment with HIF-1α siRNA diminishes the up-regulation of Snail and Slug as well as the down-regulation of E-cadherin by EGF. The involvement of HIF-1α in the down-regulation of E-cadherin was confirmed with cobalt chloride (CoCl2), a hypoxia-mimetic reagent. Finally, we also show that EGF-induced cell invasion is attenuated by treatment with HIF-1α siRNA. This study demonstrates an important role for HIF-1α in mediating the effects of EGF on Snail, Slug and E-cadherin expression as well as invasiveness in human ovarian cancer cells.

Dysregulated expression of Slug, vimentin, and E‐cadherin correlates with poor clinical outcome in patients with basal‐like breast cancer

Among the different types of breast cancer, basal-like breast cancer (BLBC) has an extremely poor prognosis due to its high rate of recurrence and metastasis. The present study aimed to investigate the correlation between the expression of Slug, E-cadherin, and vimentin, and the clinicopathological characteristics and prognosis of patients with BLBC. We further inferred from these findings whether Slug leads to a poor prognosis in patients with BLBC through epithelial-mesenchymal transition (EMT). Immunohistochemistry was performed for 441 patients with breast cancer to determine the expression levels of Slug, E-cadherin, and vimentin. The correlation between protein expression and clinicopathological characteristics of patients with BLBC was evaluated, and these patients were followed up to determine survival rate. High Slug and low E-cadherin expression in BLBC patients closely correlated with histological grade, lymph node metastasis, tumor-node-metastasis (TNM) stage, and lymphatic vessel metastasis. Survival analysis revealed that the poor prognosis of BLBC is associated with TNM stage, high Slug and vimentin expression, and low E-cadherin levels. High Slug expression is closely correlated with poor prognosis in patients with BLBC. We speculate that this may be attributed to the involvement of Slug in the EMT of BLBC.

CD44s Regulates the TGF-β–Mediated Mesenchymal Phenotype and Is Associated with Poor Prognosis in Patients with Hepatocellular Carcinoma

The prognosis for individuals diagnosed with hepatocellular carcinoma (HCC) remains poor because of the high frequency of invasive tumor growth, intrahepatic spread, and extrahepatic metastasis. Here, we investigated the role of the standard isoform of CD44 (CD44s), a major adhesion molecule of the extracellular matrix and a cancer stem cell marker, in the TGF-β-mediated mesenchymal phenotype of HCC. We found that CD44s was the dominant form of CD44 mRNA expressed in HCC cells. Overexpression of CD44s promoted tumor invasiveness and increased the expression of vimentin, a mesenchymal marker, in HCC cells. Loss of CD44s abrogated these changes. Also in the setting of CD44s overexpression, treatment with TGF-β1 induced the mesenchymal phenotype of HCC cells, which was characterized by low E-cadherin and high vimentin expression. Loss of CD44s inhibited TGF-β-mediated vimentin expression, mesenchymal spindle-like morphology, and tumor invasiveness. Clinically, overexpression of CD44s was associated with low expression of E-cadherin, high expression of vimentin, a high percentage of phospho-Smad2-positive nuclei, and poor prognosis in HCC patients, including reduced disease-free and overall survival. Together, our findings suggest that CD44s plays a critical role in the TGF-β-mediated mesenchymal phenotype and therefore represents a potential therapeutic target for HCC.

Abstract B67: Increased CXCR4 expression and its nuclear localization are associated with invasion in sorafenib and sunitinib-tolerant hepatocarcinoma models

Abstract Background: Most patients treated with sunitinib and sorafenib will experience tumor stabilization and eventually develop resistance within 4–6 months after treatment initiation. Increased CXCR4 and VEGF-C expressions were correlated with lymph node metastasis in HCC patients. In addition, high CXCL12 (SDF1α) plasma levels were associated with lack of sunitinib efficacy in HCC. Objectives: The aim of this work was to investigate the role of CXCR4/CXCL12 expression in human HCC models developing tolerance to sunitinib and sorafenib. Methods: SK-HEP1 cells were exposed to increasing sunitinib or sorafenib concentrations for more than 6 months to obtain RTKi-tolerant cell lines named as SK-Suni or SK-Sora, respectively. In these cell lines, cell proliferation, clonogenic survival, mRNA levels of selected genes and invasion of cells were evaluated. Western Blot analyzed protein levels. Transient siRNA transfection was carried out to target CXCR4. In vivo sorafenib efficacy was determined in SCID mice bearing SK-HEP1 xenografts and it was administered orally (30 mg/kg/daily) starting when tumors were detectable (100–300 mm3). In tumor samples, CXCR4, vimentin, and E-cadherin expression was evaluated by qRT-PCR and immunohistochemistry. Results: Sunitinib and sorafenib displayed cytotoxic effects on SK-HEP1 parental cells with IC50 of 4.8 and 8.4 μM. SK-Suni and SK-Sora counterparts tolerated higher concentrations of sunitinib and sorafenib with IC50 of 10.5 and 11.8 μM, respectively. No cross-resistance to sunitinib or sorafenib was observed in SK-Sora or Sk-Suni, respectively. Protracted exposure to sorafenib led to a 3-fold increase mRNA expression CXCL12 and MIF in SK-Sora, whereas after long-term exposure to sunitinib CXCR4, CXCL12, and MIF mRNA levels increased in SK-Suni. Increase in CXCR4 protein levels was confirmed by Western Blot, immunofluorescence, and flow cytometry. Upon CXCL12 treatment, a prompt activation of signaling pathways involved in cytoskeleton reorganization and cell migration such as p-ERK, Cdc42, and STAT-3 were observed in SK-Suni. Interestingly, by immunofluorescence it was observed that CXCL12 treatment induced CXCR4 translocation into the nucleus in SK-Suni cells; associated with an increase Matrigel invasion compared to parental cells. In parental and suntinib/sorafenib-tolerant cells, matrigel invasion was markedly suppressed by 10 μg/ml AMD3100, a selective CXCR4 antagonist and by 2 μM Chalcone-4, a selective direct binding inhibitor of CXCL12. Small interfering RNAs were used to inhibit CXCR4 expression in cultured cells. This experiment showed that cell invasion decreased significantly in SK-Suni transfected cells compared to Sk-Suni. In SK-HEP1 xenografts treated with sorafenib for 25 days, we observed an increased CXCR4 protein expression together with changes usually associated with epithelial-to-mesenchymal transition such as high vimentin and low E-cadherin expression. Conclusions: Our data show that the CXCL12/CXCR4 axis is up-regulated and functional in sunitinib- and sorafenib- tolerant HCC cells. Inhibiting CXCR4 might counteract the increased invasion associated with tolerance to sorafenib and sunitinib in HCC patients.

Gene Expression Profiling Identifies ESRP1 as a Potential Regulator of Epithelial Mesenchymal Transition in Somatotroph Adenomas from a Large Cohort of Patients with Acromegaly

The epithelial marker E-cadherin plays a crucial role in epithelial-mesenchymal transition (EMT). Decreased protein content in somatotroph adenomas has been associated with increased tumor size, invasion, and poor response to somatostatin analog (SA) treatment, but the potential mechanisms of EMT progression in these adenomas are lacking. We hypothesized that characterization of EMT-related transcripts in somatotroph adenomas could identify novel therapeutic targets in individuals with poor response to SA treatment and provide more knowledge of the mechanism of EMT progression. Fifty-three patients with acromegaly participated in the study. We performed microarray analysis of 16 adenomas, eight with high expression and eight with low expression of E-cadherin, in order to identify EMT-related transcripts. Candidate transcripts were further explored in vivo in 53 adenomas and in vitro in a rat pituitary GH-producing cell (GH3) after exploring three models for reducing E-cadherin and inducing a mesenchymal phenotype. In vivo E-cadherin mRNA expression in tumor tissue is associated negatively with tumor size and invasiveness and positively with GH and IGF-I levels in serum and response to SA treatment. Microarray and subsequent PCR analysis identify several EMT-related genes associated with E-cadherin expression. In vitro, few of these EMT-related genes were regulated by silencing E-cadherin or by TGF-β1 treatment in GH3 cells. In contrast, silencing Esrp1 in GH3 cells regulated many of the EMT-related transcripts. These results indicate that ESRP1 could be a master regulator of the EMT process in pituitary adenomas causing acromegaly.

E-cadherin as a predictive marker of brain metastasis in non-small-cell lung cancer, and its regulation by pioglitazone in a preclinical model

It remains unclear whether patients with non-small-cell lung cancer (NSCLC) develop brain metastasis during or after standard therapy. We attempted to identify biological markers that predict brain metastasis, and investigated how to modulate expression of such markers. A case-control study of patients who were newly diagnosed with NSCLC and who had developed brain metastasis during follow-up was conducted between 2004 and 2009. These patients were compared with a control group of patients who had NSCLC but no evidence of brain metastasis. Immunohistochemical analysis of expression of Ki-67, p53, Bcl-2, Bax, vascular endothelial growth factor, epidermal growth factor receptor, caspase-3, and E-cadherin was conducted. The methylation status of the genes for O(6)-methylguanine-DNA-methyltransferase, tissue inhibitor of matrix metalloproteinase (TIMP)-2, TIMP-3, and death-associated protein-kinase was also determined, by use of a methylation-specific polymerase chain reaction. A significantly increased risk of developing brain metastasis was associated with the presence of primary tumors with low E-cadherin expression in patients with NSCLC. We also investigated the effects of pioglitazone, a peroxisome proliferator-activated receptor γ-activating drug, in tumor-bearing mouse models. We found that E-cadherin expression was proportional to pioglitazone exposure time. Interestingly, pioglitazone pretreatment before cancer cell inoculation prevented loss of E-cadherin expression and reduced expression of MMP9 and fibronectin, compared with the control group. E-cadherin expression could be a predictor of brain metastasis in patients with NSCLC. Preventive treatment with pioglitazone may be useful for modulating E-cadherin expression.

Abstract 2408: CD44s is required for a tumor-initiating stem-like mesenchymal phenotype in hepatocellular carcinoma via ligand-independent c-Met activation

Abstract c-Met, a high affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion and metastasis. Hepatocellular carcinoma (HCC) patients with active HGF/c-Met signaling have a significantly worse prognosis. CD44, the receptor for hyaluronic acid, is utilized as a surface marker to identify mesenchymal tumor-initiating stem-like cells (TISC) in several cancers. CD44v6 (spice variant 6) has been shown to promote HGF activation of c-Met. In this work, we examine the role of CD44s as a deregulator and activator of c-Met signaling in HCC. In a transcriptome profile analysis of 149 human HCC cases, we identified a significant positive correlation (R2=0.8993) between up-regulated CD44 and c-Met expression. The human HCC cell lines MHCC97-H and Huh7 were further utilized to investigate the direct relationship between c-Met and CD44s. Metastatic MHCC97-H cells are 95% CD44+ and demonstrate ligand-independent c-Met activation. CD44+/c-Met+ MHCC97-H cells demonstrate a mesenchymal phenotype with low expression of E-cadherin, high level of tumorsphere formation, increased expression of fibronectin and Zeb2 compared to Huh7 cells, which have are CD44neg/c-Metneg. In MHCC97-H cells, a direct interaction between CD44s and c-Met was demonstrated by immunoprecipitation/immuno-blot analysis. Transient and stable inhibition of c-Met in MHCC97-H cells results in increased E-cadherin, decreased CD44s, and reduced tumor-initiating characteristics and loss of tumorsphere formation. Likewise, inhibition of CD44s demonstrates similar phenotype with increased E-cadherin, decreased tumorsphere formation, and decreased c-Met activation and loss of downstream Akt and Erk phosphorylation. CD44s knockdown recovered c-Met to a regulated HGF-dependent state. Selective inhibition of either PI3K (Ly294002) or MAPK (PD98059) pathways in MHCC97-H cells demonstrate that PI3K/Akt activation signals drive CD44s expression in a feed forward fashion. Conversely, the over-expression of CD44s in Huh7 cells demonstrates a mesenchymal phenotype with fibroblastic-like cell morphology, decreased E-cadherin, increased Zeb1, increased PI3K/Akt signaling, and increased tumorsphere formation compared to control Huh7 cells. Selective inhibition of PI3K/Akt signaling in CD44+ Huh7 cells resulted in reduced CD44s expression and decreased tumorsphere formation. In conclusion, CD44s induces a mesenchymal phenotype and tumor initiating characteristics in HCC through the deregulation of c-Met and activation of a positive feedback loop through PI3K/Akt signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2408. doi:1538-7445.AM2012-2408