Membranous E-cadherin Research Articles

Abstract 3403: E-cadherin and vimentin patterns by immunohistochemistry identify epithelial and mesenchymal phenotypes in lung adenocarcinoma

Abstract Purpose: To examine markers of epithelial-mesenchymal transition (EMT) in lung adenocarcinoma (ADC) defining epithelial-like (EPT) and mesenchymal-like (MES) phenotypes and correlate these with outcomes, in addition to other EMT markers. This study examined 33 patients with lung adenocarcinoma (ADC) who underwent surgical resection and later at disease progression received erlotinib. Experimental Design: All slides with tumor from resection specimens were reviewed (mean 4.8/case; range 1-9). Tumors were histologically subtyped according to recent IASLC classification. Tumor budding was graded per Ueno et al. (2002). A block containing the invasive front was selected for IHC staining with antibodies to e-cadherin (ECAD), vimentin (VIM), β-catenin (α-CAT), and SNAI1. Pattern-specific staining was evaluated at the invasive tumor front: E-cadherin-membranous, vimentin-cytoplasmic, α-CAT-membranous, SNAI1-nuclear, cytoplasmic. Complete circumferential membranous staining for e-cadherin was scored as present/absent; other markers were scored by the Allred system (0-8). PFS and OS were calculated from time of diagnosis to disease progression or death. Results: Two distinct phenotypes were identified with ECAD and VIM: EPT showing preserved membrane ECAD staining with low VIM (≥4) (N=19) and MES showing weak/absent membrane ECAD staining with high VIM (Δ5) (N=10). Median PFS for EPT group was 4.2 mo vs MES 1.6 mo; median OS for EPT group 15.5 mo vs 6.5 mo. These differences were not statistically significant. No association was found between high grade tumor budding and EPT vs MES phenotype p=0.221). Membranous α-CAT expression and nuclear SNAI1 expression showed a statistically significant correlation with EPT vs MES (β-catenin mean score 7.8 vs 6.1, p=0.0184) and (nuclear SNAI1 mean score 7.3 vs 6.1, p=0.0364). Conclusions: Epithelial and mesenchymal phenotypes can be identified by IHC for e-cadherin and vimentin. These phenotypes correlate with other EMT markers β-catenin and SNAI1 but not with tumor budding. Although differences in median PFS and OS were seen between the two groups, the number of cases is too small to identify a statistically significant difference. The results do support the rationale for defining EPT and MES groups for prognosis in a larger group of early-stage surgically resected patients. 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 3403. doi:1538-7445.AM2012-3403

Epithelial to mesenchymal transition in cutaneous squamous cell carcinoma is correlated with COX-2 expression but not with the presence of stromal macrophages or CD10-expressing cells

Epithelial to mesenchymal transition (EMT) is an intricate process by which epithelial cells loose epithelial characteristics and acquire a mesenchymal-like phenotype. EMT and cyclooxygenase 2 (COX-2) expression are related to tumor invasion and metastasis. The tumor microenvironment plays a major role in tumor progression and the induction of EMT. Here, we investigated the relationship between EMT and COX-2 expression as well as tumor-associated macrophages (TAM) and CD10-positive stromal cells during the development of cutaneous squamous neoplastic lesion. We performed immunohistochemical staining for vimentin, E-cadherin, β-catenin, COX-2, CD68, and CD10 in 41 cases of squamous cell cancers (SCC), 20 of Bowen's disease, 30 of actinic keratosis, and 30 samples of normal skin. SCC cells showed significantly increased vimentin expression and reduced expression of membranous E-cadherin and β-catenin compared with cells in precursor lesions and in normal skin. COX-2 expression was also markedly increased in SCC cells. E-cadherin expression was positively correlated with β-catenin expression and inversely correlated with COX-2 expression in SCC cells. The number of TAM and CD10-positive stromal cells increased from the normal skin to precursor lesions and SCC cells. The number of TAM and of CD10-positive stromal cells did not correlate with the expression of E-cadherin, β-catenin, COX-2, and vimentin in SCC cells. We suggest that cutaneous SCC cells show EMT, which appears to be correlated with COX-2 expression but not with stromal CD10 expression and TAM.

Epithelial Cell Rests of Malassez Contain Unique Stem Cell Populations Capable of Undergoing Epithelial–Mesenchymal Transition

The epithelial cell rests of Malassez (ERM) are odontogenic epithelial cells located within the periodontal ligament matrix. While their function is unknown, they may support tissue homeostasis and maintain periodontal ligament space or even contribute to periodontal regeneration. We investigated the notion that ERM contain a subpopulation of stem cells that could undergo epithelial-mesenchymal transition and differentiate into mesenchymal stem-like cells with multilineage potential. For this purpose, ERM collected from ovine incisors were subjected to different inductive conditions in vitro, previously developed for the characterization of bone marrow mesenchymal stromal/stem cells (BMSC). We found that ex vivo-expanded ERM expressed both epithelial (cytokeratin-8, E-cadherin, and epithelial membrane protein-1) and BMSC markers (CD44, CD29, and heat shock protein-90β). Integrin α6/CD49f could be used for the enrichment of clonogenic cell clusters [colony-forming units-epithelial cells (CFU-Epi)]. Integrin α6/CD49f-positive-selected epithelial cells demonstrated over 50- and 7-fold greater CFU-Epi than integrin α(6)/CD49f-negative cells and unfractionated cells, respectively. Importantly, ERM demonstrated stem cell-like properties in their differentiation capacity to form bone, fat, cartilage, and neural cells in vitro. When transplanted into immunocompromised mice, ERM generated bone, cementum-like and Sharpey's fiber-like structures. Additionally, gene expression studies showed that osteogenic induction of ERM triggered an epithelial-mesenchymal transition. In conclusion, ERM are unusual cells that display the morphological and phenotypic characteristics of ectoderm-derived epithelial cells; however, they also have the capacity to differentiate into a mesenchymal phenotype and thus represent a unique stem cell population within the periodontal ligament.

A novel role of Id-1 in regulation of epithelial-to-mesenchymal transition in bladder cancer

Background and objectiveInhibitor of differentiation or DNA binding -1 (Id-1) has been shown to be increased in several types of advanced cancer, and to be associated with aggressive and metastatic abilities of cancer cells. Recently, more and more evidence indicates that epithelial-to-mesenchymal transition (EMT) is an important mechanism taking place during tumor invasion and metastasis, but the molecular pathways underlying EMT have not been clearly established. This study was to investigate the expression of Id-1 in bladder cancer and its association with EMT. Materials and methodsA total of 169 tissues, consisting of 147 primary bladder cancers and 22 adjacent normal tissues were included in this study. Id-1, E-cadherin, and β-catenin were examined immunohistochemically in paraffin sections. The pBabe-Id-1 expression retroviral vector and retroviral vectors containing an Id-1-specific small interfering RNA oligonucleotides (si-Id-1) were transfected into 2 bladder cancer cell lines respectively. Then, we used Western blotting and immunofluorescent staining to detect the cellular expression of epithelial markers and mesenchymal markers. The invasion and migration ability of bladder cancer cells were identified by type I collagen invasion assay and wound closure assay. ResultsWe demonstrated that increased Id-1 expression was associated with advanced tumor stage and grade. In addition, the increased Id-1 expression in bladder tumors was also correlated with decreased membranous E-cadherin and β-catenin expression. In vitro, studies showed that inactivation of the Id-1 gene conferred morphologic transition of bladder cancer cells from a fibroblastic to epithelial appearance, and overexpression of Id-1 could lead to acquisition of a fibroblastic spindle cell phenotype accompanied by loss of cell-to-cell contacts. By Western blotting and immunofluorescent staining, we showed that the expression level of Id-1 was correlated with the expression of mesenchymal markers but was inversely correlated with the expression of epithelial markers. Moreover, results of collagen invasion and wound closure assays showed ectopic Id-1 expression led to increased ability of invasion and migration. ConclusionsOur results suggest that Id-1 may play roles in tumor progression and EMT activation in bladder cancer.

Dysregulation of Dicer1 in Beta Cells Impairs Islet Architecture and Glucose Metabolism

microRNAs (miRNAs) play important roles in pancreas development and in regulation of insulin expression in the adult. Here we show that loss of miRNAs activity in beta-cells during embryonic development results in lower beta-cell mass and in impaired glucose tolerance. Dicer1-null cells initially constitute a significant portion of the total beta-cell population. However, during postnatal development, Dicer1-null cells are depleted. Furthermore, wild-type beta cells are repopulating the islets in complex compensatory dynamics. Because loss of Dicer1 is also associated with changes in the distribution of membranous E-cadherin, we hypothesized that E-cadherin activity may play a role in beta cell survival or islet architecture. However, genetic loss of E-cadherin function does not impair islet architecture, suggesting that miRNAs likely function through other or redundant effectors in the endocrine pancreas.

Immunohistochemical expression profile of β-catenin, E-cadherin, P-cadherin, laminin-5γ2 chain, and SMAD4 in colorectal serrated adenocarcinoma

The immunohistochemical expression of cell adhesion molecules in colorectal serrated adenocarcinoma is still unknown. The immunostaining patterns of β-catenin, E-cadherin, P-cadherin, laminin 5γ2, and SMAD4 and their relationship to survival were studied in different tumor areas, namely, tumor center and invasive front, the latter comprising tumor bud and non-tumor bud clusters, as described in a previous study of 66 serrated adenocarcinomas and matched conventional carcinomas. Compared with conventional carcinomas, serrated adenocarcinomas showed significantly reduced nuclear β-catenin, membranous E-cadherin, and nuclear SMAD4 but an increased cytoplasmic expression of laminin-5γ2 at the invasive front that was particularly pronounced in the tumor buds. E-cadherin loss at the invasive front was identified as an independent prognostic factor for a poorer outcome in serrated adenocarcinoma. Serrated adenocarcinoma shows a distinct immunohistochemical profile at the invasive front compared with conventional carcinoma, which may account for its less favorable outcome. The lower frequency of nuclear β-catenin in SAC, especially in right-sided tumors, suggests that molecular mechanisms other than the canonical Wnt/β-catenin pathway may have a role in tumor bud formation.

Association of E-cadherin single nucleotide polymorphisms with the increased risk of endometriosis in Indian women

The objective of the present study was to investigate the association between gene E-cadherin single nucleotide polymorphisms (SNPs) and risk of developing endometriosis in Indian women and to evaluate the role of E-cadherin expression in the pathophysiology of endometriosis. A genetic association study was conducted in 715 endometriosis cases and 500 controls of Indian origin. We genotyped -160 C/A, +54 C/T and -347 G/GA SNPs of gene E-cadherin by PCR-sequencing and PCR-restriction fragment length polymorphism techniques. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software. In addition, to better understand genetic contributions to the pathophysiology of endometriosis, the expression pattern of E-cadherin in the endometrium of women with and without endometriosis was analyzed by western blot and immunohistochemical analysis. The frequencies of -347GA/GA (P = 0.026) and -160A/A (P = 0.0019) genotypes and -347G/-160A/+54C (P = 0.007) and -347GA/-160A/+54C (P < 0.0001) haplotypes were significantly different between patients and controls. Strong LD was observed between -347G/GA and -160C/A loci (D' = 0.64) when compared with -347G/GA and +54C/T (D' = 0.585) or -160C/A and +54C/T (D' = 0.05) loci in cases. Furthermore, increased membranous E-cadherin expression was observed in cases than in controls. The expression seems to be genotype dependent. In conclusion, the E-cadherin -347GA/GA and -160A/A genotypes and -347GA/-160A/+54C and -347G/-160A/+54C haplotypes may jointly modify the risk of endometriosis in Indian women. In addition, the differential expression of E-cadherin may play an important role in pathogenesis of endometriosis.

Activation of phosphatidylinositol 3-kinase/Akt signaling by EGF downregulates membranous E-cadherin and β-catenin and enhances invasion in nasopharyngeal carcinoma cells

Dysregulation of E-cadherin and β-catenin function in cell–cell adhesion is common in nasopharyngeal carcinoma (NPC) and correlates with metastatic disease. In this study, we examined the role of EGF-activated phosphatidylinositol 3-kinase (PI3K)-Akt signaling in E-cadherin and β-catenin regulation. We found that reduced membranous E-cadherin and β-catenin expression was positively correlated with Akt phosphorylation in NPC tissues. EGF treatment disrupted cell–cell adhesion and resulted in mesenchymal morphological features in NPC cell lines (TW01, TW04, and TW06). Western blot analysis showed that the E-cadherin protein level was partially reduced in TW04 cells only and the β-catenin levels were not considerably affected upon EGF treatment. In contrast, quantitative real-time RT-PCR showed that the E-cadherin, but not β-catenin, mRNA levels were markedly reduced by EGF in all cell lines. Immunofluorescent staining revealed that E-cadherin and β-catenin appeared to be markedly reduced on the cell surface and more localized in the cytoplasm. Inhibition of PI3K by LY294002 did not abolish the EGF-induced downregulation of E-cadherin protein or mRNA in TW04 cells but moderately increased the β-catenin protein level in TW01 cells and mRNA level in TW06 cells. However, LY294002 substantially restored or increased cell surface E-cadherin and β-catenin in all EGF-treated cell lines, in concordance with the inhibition of cell morphological changes. Moreover, LY294002 significantly blocked EGF-driven cell invasion, correlating with the elevation of membranous E-cadherin and β-catenin levels. In conclusion, EGF-induced epithelial-to-mesenchymal transition may not be only dependent on downregulation of E-cadherin protein/mRNA but also on mislocalization of E-cadherin and β-catenin. The mechanisms involved may be related, at least in part, to the PI3K-Akt pathway.

Wnt-pathway activation in intestinal-type sinonasal adenocarcinoma

Intestinal-type sinonasal adenocarcinoma (ITAC) is an epithelial cancer of the sinonasal sinuses that shows histological similarity to colorectal cancer (CRC) and share chronic inflammation as a possible etiological factor. The Wnt-pathway is one of the most important tumourigenic pathways in CRC. The aim of this study was to investigate if the Wnt-pathway is activated in ITAC. Protein expression profiles of E-cadherin, β-catenin, c-myc and cyclin D1 were analysed by immunohistochemistry in 83 samples of ITAC, organized into tissue microarray blocks. Nuclear β-catenin expression was observed in 31% of the cases and was twice as frequent in papillary/colonic ITAC compared to solid/mucinous subtypes. Loss of membranous β-catenin staining occurred in 24% and loss of membranous E-cadherin in 6% of the cases and this was more prominent in mucinous types. Strong c-myc and cyclin D1 expression was observed in 30% and 4% of the cases, respectively. Nuclear β-catenin expression was significantly related to poor clinical outcome, independent from established factors as tumour stage and histological type. The presence of nuclear β-catenin in 31% of patients with ITACs indicated that in a subset of patients, the Wnt-pathway is active and conveys a worse prognosis.

Expression of COX-2 and E-cadherin in Tunisian patients with colorectal adenocarcinoma

Cyclo-oxygenase 2 (COX-2) and E-cadherin are promising biomarkers for cancer diagnosis and therapy. The aim of this study was to examine the expression of these two proteins in primary colorectal adenocarcinomas and to investigate their association with clinicopathological characteristics including survival of patients. Immunostaining of E-cadherin and COX-2 was assessed in 70 primary colorectal adenocarcinomas from Tunisian patients. Membranous E-cadherin immunostaining and cytoplasmic COX-2 expression were observed in 74.3% and 68.6% of cases respectively. A significant association was found between COX-2 expression and age at diagnosis (P=0.02), and vessel invasion (P=0.037). The expression of E-cadherin correlated with age at diagnosis (P=0.01), and tumor size (P=0.02). In addition, by multivariate analysis, we revealed a significant association with 1-year disease free survival and a tendency with distant metastasis (P=0.017 and P=0.065 respectively). On the other hand, tumors exhibiting COX-2+/E-cadherin-profile were larger (P=0.006), and in an advanced stage (P=0.001). Survival analysis showed that COX-2 over-expression confers a reduced overall survival rate (Plog rank=0.036) and is an independent factor predictive for prognosis.

Caveolin-1 promotes pancreatic cancer cell differentiation and restores membranous E-cadherin via suppression of the epithelial-mesenchymal transition

Pancreatic cancer is one of the deadliest cancers due to early rapid metastasis and chemoresistance. Recently, epithelial to mesenchymal transition (EMT) was shown to play a key role in the pathogenesis of pancreatic cancer. To understand the role of caveolin-1 (Cav-1) in EMT, we over-expressed Cav-1 in a pancreatic cancer cell line, Panc 10.05, that does not normally express Cav-1. Here, we show that Cav-1 expression in pancreatic cancer cells induces an epithelial phenotype and promotes cell-cell contact, with increased expression of plasma membrane bound E-cadherin and beta-catenin. Mechanistically, Cav-1 induces Snail downregulation and decreased activation of AKT, MAPK and TGF-beta-Smad signaling pathways. In vitro, Cav-1 expression reduces cell migration and invasion, and attenuates doxorubicin-chemoresistance of pancreatic cancer cells. Importantly, in vivo studies revealed that Cav-1 expression greatly suppresses tumor formation in a xenograft model. Most interestingly, Panc/Cav-1 tumors displayed organized nests of differentiated cells that were totally absent in control tumors. Confirming our in vitro results, these nests of differentiated cells showed reexpression of E-cadherin and beta-catenin at the cell membrane. Thus, we provide evidence that Cav-1 functions as a crucial modulator of EMT and cell differentiation in pancreatic cancer.

Partial Mesenchymal to Epithelial Reverting Transition in Breast and Prostate Cancer Metastases

Epithelial to mesenchymal transition (EMT) is an oft-studied mechanism for the initiation of metastasis. We have recently shown that once cancer cells disseminate to a secondary organ, a mesenchymal to epithelial reverting transition (MErT) may occur, which we postulate is to enable metastatic colonization. Despite a wealth of in vitro and in vivo studies, evidence supportive of MErT in human specimens is rare and difficult to document because clinically detectable metastases are typically past the micrometastatic stage at which this transition is most likely evident. We obtained paired primary and metastatic tumors from breast and prostate cancer patients and evaluated expression of various epithelial and mesenchymal markers by immunohistochemistry. The metastases exhibited increased expression of membranous E-cadherin compared to primary tumors, consistent with EMT at the primary site and MErT at the metastatic site. However, the re-emergence of the epithelial phenotype was only partial or incomplete. Expression of epithelial markers connexins 26 and/or 43 was also increased on the majority of metastases, particularly those to the brain. Despite the upregulation of epithelial markers in metastases, expression of mesenchymal markers vimentin and FSP1 was mostly unchanged. We also examined prostate carcinoma metastases of varied sizes and found that while E-cadherin expression was increased compared to the primary lesion, the expression inversely correlated with size of the metastasis. This not only suggests that a second EMT may occur in the ectopic site for tumor growth or to seed further metastases, but also provides a basis for the failure to discern epithelial phenotypes in clinically examined macrometastases. In summary, we report increased expression of epithelial markers and persistence of mesenchymal markers consistent with a partial MErT that readily allows for a second EMT at the metastatic site. Our results suggest that cancer cells continue to display phenotypic plasticity beyond the EMT that initiates metastasis.

Decreased ezrin and paxillin expression in human urothelial bladder tumors correlate with tumor progression

ObjectivesF-actin binding proteins ezrin and paxillin are involved in cell adhesion and cell migration/invasion. The aim of the study was to investigate their role in urothelial bladder carcinogenesis. Materials and methodsExpression of ezrin and paxillin was studied by immunohistochemistry in 104 and 96 cases of urothelial bladder tumors, respectively. Correlations with clinicopathologic data and expression of p53, E-cadherin, and β-catenin were examined. ResultsPositive ezrin and paxillin protein expression was found in 99% and 93.7% of cases, respectively. Membranous expression of ezrin was significantly lower in high grade tumors and correlated with invasion. Multivariate analysis showed that ezrin is an independent predictor of muscularis propria invasion. Paxillin expression was significantly decreased in urothelial carcinomas compared with tumors of low malignant potential and low paxillin levels also correlated with advancing tumor stage and invasion. A statistically significant correlation was found between membranous ezrin and E-cadherin as well as between ezrin and paxillin expression in urothelial tumors. ConclusionsDown-regulation of ezrin and paxillin in urothelial bladder tumors is associated with aggressive tumor features and invasiveness.

Molecular Markers of Epithelial-to-Mesenchymal Transition Are Associated with Tumor Aggressiveness in Breast Carcinoma

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a transient process occurring during developmental stages and carcinogenesis, characterized by phenotypic and molecular alterations, resulting in increased invasive and metastatic capabilities of cancer cells and drug resistance. Moreover, emerging evidence suggests that EMT is associated with increased enrichment of cancer stem-like cells in neoplastic tissues. We interrogated the molecular alterations occurring in breast cancer using proposed EMT markers such as E-cadherin, vimentin, epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF) D, and nuclear factor κB (NF-κB) to decipher their roles in the EMT and breast cancer progression. METHODS: Fifty-seven invasive ductal adenocarcinomas of the breast were assessed for the expression of E-cadherin, vimentin, EGFR, NF-κB, and PDGF-D using immunohistochemical analysis. Tumors were categorized into three groups: A (ER+, and/or PR+, HER-2/neu-), B (ER+, and/or PR+, HER-2/neu+), and C (triple-negative: ER-, PR-, and HER-2/neu-). Immunostained slides were microscopically evaluated and scored using intensity (0, 1+, 2+, and 3+) and percentage of positive cells, and data were statistically analyzed. RESULTS: Membranous E-cadherin was positive in all 57 cases (100%), whereas cytoplasmic E-cadherin was predominantly positive in groups B and C compared with group A (21%, 7%, and 0%, respectively). All group A cases were negative for vimentin and EGFR. There was statistically significant increased expression of vimentin (P < .0002), EGFR (P < .0001), and NF-κB (P < .02) in triple-negative cases when compared with groups A and B. CONCLUSIONS: Vimentin, EGFR, and NF-κB were significantly increased in triple-negative tumors, which is consistent with the aggressiveness of these tumors. These markers could be useful as markers for EMT in breast cancers and may serve as predictive markers for designing customized therapy in the future.

NEDD9 and BCAR1 Negatively Regulate E-Cadherin Membrane Localization, and Promote E-Cadherin Degradation

The Cas scaffolding proteins (NEDD9/HEF1/CAS-L, BCAR1/p130Cas, EFSSIN, and HEPL/CASS4) regulate cell migration, division and survival, and are often deregulated in cancer. High BCAR1 expression is linked to poor prognosis in breast cancer patients, while upregulation of NEDD9 contributes to the metastatic behavior of melanoma and glioblastoma cells. Our recent work knocking out the single Drosophila Cas protein, Dcas, identified a genetic interaction with E-cadherin. As E-cadherin is often downregulated during epithelial-mesenchymal transition (EMT) prior to metastasis, if such an activity was conserved in mammals it might partially explain how Cas proteins promote aggressive tumor behavior. We here establish that Cas proteins negatively regulate E-cadherin expression in human mammary cells. Cas proteins do not affect E-cadherin transcription, but rather, BCAR1 and NEDD9 signal through SRC to promote E-cadherin removal from the cell membrane and lysosomal degradation. We also find mammary tumors arising in MMTV-polyoma virus T-antigen mice have enhanced junctional E-cadherin in a Nedd9−/− background. Cumulatively, these results suggest a new role for Cas proteins in cell-cell adhesion signaling in cancer.

E-cadherin and β-catenin expression in sebaceous eyelid adenocarcinomas

Since the E-cadherin-catenin complex is of importance in the cancerogenesis of epithelial tumors, we examined the expression of E-cadherin and β-catenin in sebaceous adenocarcinomas of the eyelid and correlated the results with clinicopathological parameters. The study included histological specimen of 32 patients with sebaceous adenocarcinomas of the eyelid. The expression of E-cadherin, β-catenin, and Ki-67 nuclear antigen was analyzed by immunohistochemistry. The membranous expression of E-cadherin and of β-catenin was reduced in 17 (53%) patients and 18 (56%) patients, respectively, while elevated cytoplasmic levels and a nuclear accumulation of β-catenin were found in 27 (84%) and six (19%) patients, respectively. The low expression of membranous β-catenin was associated with a high degree of tumor infiltration (p = 0.02), high amount of inflammation (p = 0.01), and high degree of poor tumor differentiation (p < 0.001). In a similar manner, a low membranous E-cadherin expression in tumors was significantly associated with poor tumor differentiation (p = 0.001) and high tumor cell proliferation rate (p = 0.004). The Ki-67 proliferation index was significantly higher in the group with low membranous E-cadherin expression than in the group with high membranous E-cadherin expression (62.3 ± 20.8 versus 42.1 ± 15.3; p = 0.004). The membranous E-cadherin expression and the membranous β-catenin expression were significantly associated with each other (Fisher's exact probability test, p = 0.03). Sebaceous eyelid adenocarcinomas showed a decrease in the membranous expression of E-cadherin and of β-catenin. These changes were associated with poor tumor differentiation and an increase in the tumor infiltration and inflammation, pointing at a potential role of a low E-cadherin and low β-catenin expression for poor prognosis of sebaceous eyelid adenocarcinomas. Correspondingly, the cell proliferation index and the expression of E-cadherin were inversely correlated with each other. The findings suggest that the immunohistochemical detection of a low E-cadherin and low β-catenin expression may help in the examination and staging of sebaceous eyelid adenocarcinomas.

Comparison of genetic and epigenetic alterations at 11 tumor suppressor loci in pulmonary sclerosing hemangioma and adenocarcinoma

ABSTRACTPulmonary sclerosing hemangioma (SH) is an unusual tumor of pneumocytic origin. Morphologically, SH can mimic pulmonary adenocarcinomas. Here, the authors compared genetic and epigenetic aberrations in SH with those in pulmonary adenocarcinoma. Clinicopathologic characteristics, microsatellite alterations, and CpG island methylation were analyzed in pulmonary SHs (n = 24) and adenocarcinomas (n = 34) to compare their patterns of molecular abnormalities. SHs were also analyzed immunohistochemically to characterize the expression status of proteins involved in basic biologic processes. The clinical presentation of SH cases was generally benign. Both cell types of SH stained positive for thyroid transcription factor 1 (TTF-1), epithelial membrane antigen (EMA), β-catenin, E-cadherin, and vascular endothelial growth factor (VEGF). Allelic imbalances in D3S1283, D3S1234, D3S1300, D3S1285, TP53, D17S938, and D9S179 were less frequent in SH than in adenocarcinoma; rates of allelic imbalances in D20S170 and D21S1446 were not significantly different. In SH, CpG island methylation frequencies of p16INK4a (0.0%) and RASSF1A (12.5%) were significantly lower than those in adenocarcinoma (29.4% and 38.2%, respectively); the frequencies of HOX D9, D11, and D13 gene methylation in SH were 37.5%, 33.3%, and 33.3%, respectively. The results show that pulmonary SH and adenocarcinoma share similar genetic and epigenetic aberrations, but also exhibit significant differences, especially in tumor suppressor genes.

Expression of hypoxia‐associated proteins in sporadic medullary thyroid cancer is associated with desmoplastic stroma reaction and lymph node metastasis and may indicate somatic mutations in the VHL gene

Medullary thyroid carcinomas (MTCs) are mostly aggressive but slowly growing malignant tumours that metastasize early to loco-regional lymph nodes. Desmoplastic stroma reaction is a strong risk factor associated with lymph node metastases. We evaluated immunohistochemically the expression of two hypoxia-associated proteins, carbonic anhydrase IX (CAIX) and hypoxia-induced factor 1α (HIF1α), and ki-67, intercellular matrix adhesion molecule E-cadherin and the stroma remodelling marker tenascin C in a series of 100 sporadic MTCs and corresponding lymph node metastases, if present. Moderate to strong expression of CAIX was seen in 53 cases, and of HIF1α in 51 cases, showing a strong correlation (p < 0.001; Spearman's coefficient of correlation, 0.59). Expression correlated with the degree of desmoplasia (pCAIX = 0.001 and pHIF1α = 0.001), with tenascin C expression (pCAIX = 0.001,pHIF1α = 0.038), with the ki-67 proliferation index (pCAIX = 0.001, pHIF1α = 0.001) and with the presence of lymph node metastases (pCAIX < 0.001 and pHIF1α = 0.007). The absence of membranous E-cadherin staining was significantly associated with the grade of desmoplasia, tenascin expression and lymph node metastases(p ≤ 0.05) but not with ki67 proliferation index or expression of hypoxia-associated factors. Expression of hypoxia-associated proteins was in most cases identical between primary tumours and lymph node metastases.Two cases showed strong uniform expression of CAIX and HIF1α in the primary tumour as well as in the lymph node metastases, and sequencing revealed mutations in the coding regions of the Von-Hippel–Lindau gene (VHL ).Our findings suggest that despite of the fact that MTCs have only slowly growth, tumour hypoxia plays an important role in the development of loco-regional metastases. Since traditional cytotoxic chemotherapy has only little effect on MTCs, targeting hypoxia-associated and -regulated proteins might be of benefit for patients.

The Calcium-Sensing Receptor-Dependent Regulation of Cell–Cell Adhesion and Keratinocyte Differentiation Requires Rho and Filamin A

Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

Abstract 120: Over-expression of microRNA-221 causes neoplastic cell transformation through induction of the epithelial-mesenchymal transition in non-tumorigenic liver cells

Abstract MicroRNAs (miRNAs) are negative regulators of gene expression with immense significance in cancer progression. In cancer cells, aberrant expression of miRNAs has been associated with every aspect of tumor biology, including tumor progression, invasiveness, metastasis, and acquisition of resistance of malignant cells to chemotherapeutic agents. MicroRNA miR-221 is frequently up-regulated in several major human cancers, including liver cancer. More importantly, up-regulated miR-221 is capable of enhancing liver carcinogenesis; it is not clear, however, if over-expression of any given miRNA, including miR-221, is sufficient to initiate neoplastic cell transformation. The goal of our study was to determine the tumorigenic potential of the miR-221 on normal non-transformed liver cells. Non-tumorigenic TRL-1215 rat liver cells were transfected with 20 nM of either scrambled RNA oligonucleotide (control) or pre-miR-221. At 72 hours post-transfection, adherent cells were harvested, re-seeded, and transfection was repeated, a process that was repeated for 10 months. After 1, 2, 4, and 10 months, control and miR-221-transfected cells were collected for subsequent analyses. Ectopic transfection of miR-221 for 1, 2, and 4 months resulted in significant down-regulation of the experimentally confirmed targets of the miR-221, including p27, Ddit4, and Esr1 genes and a number of newly predicted target genes. Previous reports have linked the oncogenic properties of miR-221 in liver carcinogenesis to down-regulation of critical cancer-related genes p27, Ddit4, and Esr1; however, despite a profound and early inhibition of these target genes, we did not find any evidence of neoplastic cell transformation until 10 months of pre-miRNA-221 transfection. At that time, we detected substantial morphological changes in the miR-221-transfected cells and the accumulation of neoplastic phenotypic features (i.e., soft agar colony formation and increase in cell invasiveness). These changes were accompanied by a significant decrease in the levels of plasma membrane E-cadherin. Array-based RT-PCR analysis demonstrated changes in the expression of a number of extracellular matrix and adhesion genes, including more than a 50 fold up-regulation of the mesenchymal cell marker N-cadherin (Cdh2) gene. In conclusion, our results demonstrate that the up-regulation of miR-221 not only enhances liver tumorigenesis, but also initiates the neoplastic cell transformation of the normal liver cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 120. doi:10.1158/1538-7445.AM2011-120