Alpha-catenin Expression Research Articles

Abstract PR13: Loss of alpha-catenin expression is associated with race, aggressive disease, and chemoresistance in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive and difficult-to-treat subtype of the disease. A well-documented health disparity exists within TNBC: African American (AA) women are more likely to be diagnosed with and die from the disease. Our group previously reported homozygous deletions in the CTNNA1 gene, which encodes the protein alpha-catenin, in AA TNBC. We have undertaken a basic and translational research study to understand the mechanistic role and clinical impact of alpha-catenin loss in TNBC, particularly in AA patients. To explore the association of alpha-catenin loss with race, we first examined gene expression data available in the TCGA. We determined that race was significantly associated with alpha-catenin expression, with lower expression found in AA patients. We also found a significant over-representation of AA patients in the lowest subset of expressers. We validated this association in an independent, racially diverse cohort comprising 460 breast cancer patients. Interestingly, we observed loss of nuclear alpha-catenin specifically to be associated with race. We also found strong associations between low alpha-catenin expression and survival, tumor stage, and lymph node metastasis. While its junctional role has been well studied, little is known about nuclear alpha-catenin and its role in disease. To understand the nuclear function of alpha-catenin, we sought to identify its nuclear binding partners by performing co-immunoprecipitation followed by mass spectrometry. We found nuclear alpha-catenin to interact with ATR, a kinase critical to the DNA damage response and G2/M checkpoint. To determine whether loss of alpha-catenin affected ATR function, we developed several isogenic cell line pairs. Using CRISPR/Cas9-mediated gene editing, we generated CTNNA1 knockout BT-549, MB-MDA-436, and MDA-MB-231 cell lines. We also reintroduced CTNNA1 into MDA-MB-468 cells—a line derived from an AA woman with an endogenous deletion in CTNNA1. Using these models, we found that loss of alpha-catenin promoted greater ATR activation in response to DNA damage. This was accompanied by increased phosphorylation of downstream effector proteins, including Chk1 and RPA2. Consistent with ATR hyperactivation, we found cells lacking alpha-catenin to be more sensitive to inhibitors of ATR and the G2/M checkpoint kinases Chk1 and Wee1. Additionally, alpha-catenin loss promoted DNA damage repair, thus decreasing sensitivity to DNA-damaging chemotherapies clinically utilized for TNBC treatment. In our studies, we have identified nuclear alpha-catenin as a tumor suppressor that effects TNBC’s susceptibility to chemotherapy by playing a role in ATR-directed DNA repair. Our data suggest that loss of alpha-catenin is more common in AA patients, and is associated with aggressive disease and poor prognosis. Therefore, CTNNA1 status may be important in determining appropriate therapeutic strategies for this subset of patients, which is disproportionally affected by the disease. This abstract is also being presented as Poster B088. Citation Format: Rania Bassiouni, Sandeep Singhal, Lee D. Gibbs, Yunchi Li, Patrick Pirotte, Nasreen Vohra, Kevin Gardner, John D. Carpten. Loss of alpha-catenin expression is associated with race, aggressive disease, and chemoresistance in triple-negative breast cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr PR13.

Abstract LB-021: Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression

Abstract Majority of internal lumens in our bodies are covered with a layer of epithelial cells, whose integrity is critical for the organs to function properly. The integrity of epithelial tissues depends on intact adherens junctions (AJs), which is a multiple-components complex comprising cadherins, the transmembrane adhesion receptors, and their cytoplasmic binding proteins such as p120-catenin and b-catenin etc. Functional AJs is coupled with actin filaments through linker molecules, of which a-catenin and EPLIN are best characterized. Actin polymerization and actomyosin contraction regulated by Rho GTPases and their effectors play important role in AJs maintenance and remodeling. Aberrations, structural or functional, in AJs were associated with a number of pathological conditions, such as infection, inflammation and tumors and the like. Recent studies indicated that AJs mediated the formation of cell-in-cell structures (CICs). CICs refer to the cellular structures formed between viable cells, in which one or more cells exist inside other ones. Early records on CICs could be dated back to last century, when pathologists identified this type of unusual structures in human tumor samples10. Recent progress showed that cell-in-cell structures are rather complex than initially described, and could be classified into homotypic or heterotypic CICs based on the cells involved. Heterotypic CICs are usually formed by penetration of lymphocytes into tumor cells through processes like emperitosis. Homotypic CICs are formed between cells from same type, for example, epithelial cells inside epithelial cells. Mechanisms like entosis and homotypic cell cannibalism (HoCC) are responsible for this type of CICs formation. Once formed, CICs usually result in death of the internalized cells, which lead to the conception that CICs formation is a process of cell death. Limited researches identified extensive involvement of CICs in several important biological processes including development, immune homeostasis and tumor development and evolution etc. Recently, we and others found that formation of homotypic CICs by entosis was dependent on intact AJs and polarized actomyosin contraction. Tumor cells lacking epithelial cadherins (E and P-cadherin) failed to form CICs, moreover, re-expression of E- or P-cadherin could efficiently induce CICs in these cells, suggesting that disrupting AJs is a mechanism whereby tumor cells escape entosis-mediated CICs formation. In this work, we found that tumor cells deficient of a-catenin, a key component of functional AJs, also displayed impaired CICs formation, which could be fixed by restored expression of a -catenin. Therefore, tumor cells could escape entotic CICs formation by targeting multiple AJs components including E-/P-cadherin and a-catenin, and CICs formation by entosis may constitute a novel mechanism underlying the tumor suppressive function imposed by a -catenin. Note: This abstract was not presented at the meeting. Citation Format: Manna Wang, Xiangkai Ning, Ang Chen, Hongyan Huang, Qiang Sun, Zhaolie Chen, Li Ma, Xiaoning Wang. Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-021. doi:10.1158/1538-7445.AM2017-LB-021

Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression.

Although cell-in-cell structures (CICs) could be detected in a wide range of human tumors, homotypic CICs formed between tumor cells occur at low rate for most of them. We recently reported that tumor cells lacking expression of E- and P-cadherin were incapable of forming homotypic CICs by entosis, and re-expression of E- or P-cadherin was sufficient to induce CICs formation in these tumor cells. In this work, we found that homotypic CICs formation was impaired in some tumor cells expressing high level of E-cadherin due to loss expression of alpha-catenin (α-catenin), a molecular linker between cadherin-mediated adherens junctions and F-actin. Expression of α-catenin in these tumor cells restored cell-cell adhesion and promoted CICs formation in a ROCK kinase-dependent way. Thus, our work identified α-catenin as another molecule in addition to E- and P-cadherin that were targeted to inactivate homotypic CICs formation in human tumor cells.

Abstract 3464: CEBPD (C/EBPδ) acts as a tumor suppressor in hormone receptor positive breast cancer cells and may serve as biomarker to predict the need for adjuvant therapy.

Abstract The transcription factor CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ) is expressed in normal breast epithelial cells and down-regulated in breast cancer tissue and cell lines. In the MMTV-c-Neu mouse mammary tumor model, loss of CEBPD leads to increased tumor incidence but reduced tumor metastasis, suggesting dual functions as a tumor suppressor and tumor promoter. To investigate the role of CEBPD in human breast cancer, we analyzed tissue microarrays (TMA) by immunostaining. Analysis of 79 breast tumor samples confirmed overall downregulation of CEBPD in cancer but also revealed that CEBPD was expressed in 50% of estrogen receptor (ER) positive (+) breast tumors and associated with longer disease-specific patient survival (P=0.034, log-rank test). Furthermore, CEBPD was correlated with expression of progesterone receptor (PR; P=0.0007). Results from an independent TMA of 292 tumors from patients that were randomized to no adjuvant treatment or two years of tamoxifen (TAM) treatment confirmed that CEBPD expression was an independent good prognostic marker for ER(+) patients (progression-free survival, Multivariate Cox analysis P=0.041). Within the subgroup with high PR labeling, low or no expression of CEBPD was associated with worst prognosis if untreated and associated with significant response to TAM (P=0.034). On the other hand, patients with high CEBPD had better prognosis but did not further benefit from TAM. These data indicate that CEBPD expression is significantly associated with ER/PR(+) breast cancer and has potential as prognostic and/or predictive biomarker. To investigate the functions of CEBPD we silenced its expression in the ER/PR(+) MCF-7 breast tumor cell line. We found that CEBPD attenuates proliferation and promotes estrogen-dependence. In this cell culture model, CEBPD augmented MCF-7 cell sensitivity to TAM. Furthermore, we found that CEBPD promotes cell-cell adhesion through expression of alpha-catenin. These observations are consistent with a tumor suppressor-like function for CEBPD in ER/PR(+) cancer cells. Currently, we are using ChIP-Seq and mRNA-Seq approaches in MCF-7 cells to identify a CEBPD-dependent gene signature and test its prognostic value in breast cancer gene expression data. Our long-term goal is to contribute to the development of more precise multiparameter predictors of outcome and/or response through the combination of biological assays and analysis of gene expression signatures. Citation Format: Daniel Y. Mendoza-Villanueva, Suryun Kim, H.Raza Ali, Shikha Sharan, Tapasree R. Sarkar, Carlos Caldas, Göran Landberg, Esta Sterneck. CEBPD (C/EBPδ) acts as a tumor suppressor in hormone receptor positive breast cancer cells and may serve as biomarker to predict the need for adjuvant therapy. [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 3464. doi:10.1158/1538-7445.AM2013-3464

The expression of E-cadherin-catenin complex in patients with advanced gastric cancer: role in formation of metastasis.

The E-cadherin-catenin complex plays an important role in the process of cell adhesion. Its dysfunction is associated with a decrease in cell differentiation and with increased invasiveness and metastasis. Our aim was to evaluate the expression of E-cadherin and B-catenin in advanced gastric cancer in relation to selected clinico-pathomorphological parameters. Formalin-fixed, paraffin-embedded tissue specimens were immunohistochemically stained with monoclonal antibodies E-cadherin (NCL-E-Cad, Novocastra Laboratiries Ltd; dilution 1:50), beta-catenin (NCL-B-CAT, Novocastra Laboratories Ltd; dilution 1:100), alpha-catenin (alpha-E-caten, Santa Cruz Biotechnology; dilution 1:300) and gamma-catenin (gamma-catenin, Santa Cruz Biotechnology; dilution 1:100). The expressions of E-cadherin and alpha-, beta-, gamma-catenins in the main mass of tumor and lymph node metastasis were investigated in 91 patients with gastric cancer. No statistically significant correlation was observed between the expressions of E-cadherin, alpha-, beta-catenins and histological differentiation and between the expressions of E-cadherin, alpha-, gamma-catenins and location or depth of invasion. Moreover, the expression of alpha-, gamma-catenins in the main mass of tumor was not associated with lymph node metastasis. However, we found a relationship between the expression of beta-catenin in the main mass of tumor and lymph node metastasis and tumor location. The depth of invasion was correlated with positive expression of beta-catenin in the main mass of gastric cancer. A statistically significant association was observed between the expressions of E-cadherin and beta-catenin in the main mass of tumor and lymph node involvement. The expression of alpha-catenin in the main mass of tumor was also associated with histological differentiation and Lauren's classification. Statistical analysis showed an association between the expression of E-cadherin and postoperative survival time. No significant correlation was found between the expression of alpha-, beta-, gamma-catenins and survival time. Our results may suggest that the E-cadherin-catenin complex is the factor indicative of metastasis and disease progression in gastric cancer. Also the expression of E-cadherin may play a role as a prognostic factor.

Association between lymphangiogenesis-/micrometastasis- and adhesion-related molecules in resected stage I NSCLC

Background The purpose of this study was to clarify the role and clinical significance of lymphangiogenesis/micrometastases and adhesion molecules in resected stage I non-small cell lung cancer (NSCLC). Methods Immunohistochemical (IHC) staining was used to analyze the protein expression of vascular endothelial growth factor-C (VEGF-C), VEGF, E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin in paraffin-embedded tumor samples from 117 well-characterized stage I NSCLC patients and to compare the protein expression, clinical variables and survival outcome. As a micrometastatic parameter in lymph nodes (LNs), cytokeratin (CK) staining was performed. Results The positive expression of VEGF-C and VEGF were detected in 54 (48.7%) and 86 (73.5%), respectively. We identified micrometastatic tumor cells in pathological N0 LNs in 34 (29.1%) of 117 patients. E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin were identified in 70 (59.8%), 41 (35.0%), 83 (70.9%), and 61 (52.1%) specimens, respectively. The VEGF-C expression was found more frequently in squamous cell carcinoma (SQ) and in the tumors with negative expression of beta-catenin than counter features. The VEGF expression was found more frequently in the tumors with a negative expression of E-cadherin. Micrometastasis was found more frequently in a pathological T2 status and in the tumors with a negative expression of alpha-catenin. Beta-catenin and gamma-catenin expressions were found less and more frequently in SQ, respectively. A univariate and multivariate survival analysis demonstrated that old age, pathological T2 status, and micrometastasis were independently associated with an increased risk of poor survival in the patients who underwent a surgical resection of stage I NSCLC. Conclusions Complicated relationships exist between lymphangiogenesis/micrometastases and adhesion molecules with a specific histology. The detection of lymph nodal micrometastasis by CK may therefore be a useful marker for predicting a poor prognosis in patients who undergo a complete resection of stage I NSCLC.

Alpha-Catenin, ARHGAP21 and β-Catenin Are Abnormally Expressed in Bone Marrow Cells From Patients with Myelodysplatic Syndromes and Are a Possible Target for Decitabine Treatment.

Abstract 1778Poster Board I-804 IntroductionMyelodysplastic syndrome (MDS) encloses a group of clonal hematopoietic disorders clinically and morphologically characterized by ineffective hematopoiesis. The gene encoding alpha-catenin (CTNNA1) is expressed at a much lower level in leukemia-initiating stem cells from individuals with MDS del(5q). Thus, loss of alpha-catenin tumor suppressor expression in hematopoietic cells may provide a growth advantage that collaborates MDS pathogenesis. ARHGAP21, a negative regulator of RhoGTPase signaling pathways, is a partner of alpha-catenin that controls its recruitment to the adherens junctions. ARHGAP21 is upregulated during myeloid differentiation, and could be involved in the malignant process of hematopoietic cells. In addition, alpha-catenin is a target for decitabine (DAC) treatment, a demethylating agent with potent antitumorigenic properties against MDS. The aim of this work was to evaluate the expression of alpha-catenin, ARHGAP21 and beta-catenin (gene CTNNB1) in bone marrow cells from MDS patients with or without del(5q) and to analyze CTNNA1, ARHGAP21 and CTNNB1 expression after DAC treatment. PATIENTS AND METHODScells were isolated from bone marrow of 6 MDS patients, including 5 refractory anemia (RA), being two with del(5q), and 1 refractory anemia with excess blasts (RAEB), based on the French-American-British classification, and 4 control subjects (normal hematopoietic tissues were obtained from healthy donors). The study was approved by the National Ethical Committee Board and bone marrow samples were collected at the Hematology and Hemotherapy Center, University of Campinas, after all participants provided informed written consent. Alpha-catenin, ARHGAP21 and beta-catenin localization in CD34+ cells was obtained using confocal microscopic analysis. ARHGAP21 localization was also analyzed in HS-5 stromal cells that were submitted to a CTNNA1 RNA interference (RNAi) approach. Leukemia cells lines (HL-60 and P-39) and bone marrow mononuclear cells obtained from 7 MDS patients, 5 RA and 2 refractory anemia with ringed sideroblasts (RARS), were treated with DAC for 72 hours; then mRNA expression of CTNNA1, ARHGAP21 and CTNNB1 was analyzed by Real-time PCR (normalized by GAPDH and beta-actin). RESULTSalpha-catenin, ARHGAP21 and beta-catenin are preferentially localized in the nucleus of CD34+ cells from MDS patients in contrast to the preferential cytoplasm and membrane localization in healthy donors and in MDS patients with del(5q). In del(5q) patients and healthy donors, ARHGAP21 and alpha-catenin co-localizated in the cell membrane. ARHGAP21 was abnormally expressed in cells with decreased CTNNA1 expression: in HS-5 stromal cells, ARHGAP21 was localized at the cytoplasm (mainly in the perinuclear region) and at the nucleus, in contrast, ARHGAP21 was poorly detectable in the nucleus of CTNNA1-RNAi treated cells. DAC treatment of MDS cells and leukemia cell lines induced CTNNA1, ARHGAP21, and CTNNB1 expression in a dose-dependent way. In HL60 and P39 cells, ARHGAP21 relocate to the cell membrane after DAC treatment. CONCLUSIONThe abnormal localization of alpha-catenin, ARHGAP21 and beta-catenin in MDS may compromise the reorganization of actin dynamics at sites of cell–cell contact that stabilizes cadherin-mediated cell–cell adhesion; moreover, these results also suggest a deficient recruitment of alpha-catenin to the cell membrane and an aberrant signaling in the Wnt pathway. In addition, ARHGAP21, alpha-catenin and beta-catenin are a target for DAC treatment in MDS. Supported by: FAPESP.Keywords: alpha-catenin, ARHGAP21, beta-catenin, myelodysplastic syndrome, Rho-GAP, decitabine DisclosuresNo relevant conflicts of interest to declare.

Loss of Coxsackie and adenovirus receptor downregulates α-catenin expression

Background:The Coxsackie and adenovirus receptor (CAR) has been shown to inhibit cancer cell proliferation, migration, and invasion. The underlying mechanisms, however, are poorly understood.Methods:The differential gene expression in the human colon cancer cell line DLD1 on RNAi-mediated functional CAR knockdown was analysed using oligo-array technology. Expression of α-catenin was determined by quantitative RT-PCR and western blotting. Proliferation, migration, and invasion after CAR knockdown were assessed by in vitro assays, and cell morphology in a three-dimensional context was evaluated using matrigel.Results:Oligo-array technology identified α-catenin as the strongest downregulated gene after CAR knockdown. Western blotting and quantitative RT-PCR confirmed a reduced α-catenin expression after CAR knockdown in DLD1 cells and in the rat intestinal cell line IEC-6. Functionally, both cell lines showed a marked increase in proliferation, migration, and invasion on CAR knockdown. In matrigel, both cell lines formed amorphous cell clusters in contrast to well-organised three-dimensional structures of CAR-expressing vector controls. Ectopic ‘re’-expression of α-catenin in DLD1 and IEC-6 CAR knockdown cells reversed these functional and morphological effects.ConclusionThese data suggest that an interaction of CAR and α-catenin mediates the impact of CAR on cell proliferation, migration, invasion, and morphology.

Tumor suppressor function of BCSC‐1 in nasopharyngeal carcinoma

BCSC-1 is dramatically upregulated in CNE-2L2 human nasopharyngeal carcinoma cells with reduced malignancy (AS cells) and is proposed to be a candidate tumor suppressor gene. We therefore examined the effect of BCSC-1 expression on malignant behaviors of CNE-2L2 cells. Growth in vitro and tumorigenesis in nude mice of wild-type CNE-2L2 cells (W cells) were inhibited by ectopic BCSC-1, and those of AS cells were promoted by BCSC-1 suppression. The tumor suppressor function of BCSC-1 was further confirmed by a study showing that intratumor BCSC-1 injection caused growth suppression of the tumor from W cells inoculated in nude mice. Immunohistochemistry exhibited marked reduction of BCSC-1 expression in 11 of 39 human nasopharyngeal carcinoma specimens. Because BCSC-1 expression was as rich as that in normal cells in the rest of the carcinoma specimens and was poor in CNE-2L2 cells, HNE-1 human nasopharyngeal carcinoma cells with rich BCSC-1 expression were used as a control in the study. No effect of BCSC-1 transfection on growth of the cells was observed. The data suggest that BCSC-1 suppression might play roles in tumorigenesis of some nasopharyngeal carcinomas and that BCSC-1 might be a potential gene therapy target in nasopharyngeal carcinomas with poor BCSC-1 expression. Enhanced aggregation of cells together with increased E-cadherin and alpha-catenin expression and reduced Wnt signaling might be involved in the mechanisms of tumor suppressor function of BCSC-1.

Loss of α-Catenin Decreases the Strength of Single E-cadherin Bonds between Human Cancer Cells

The progression of several human cancers correlates with the loss of cytoplasmic protein alpha-catenin from E-cadherin-rich intercellular junctions and loss of adhesion. However, the potential role of alpha-catenin in directly modulating the adhesive function of individual E-cadherin molecules in human cancer is unknown. Here we use single-molecule force spectroscopy to probe the tensile strength, unstressed bond lifetime, and interaction energy between E-cadherins expressed on the surface of live human parental breast cancer cells lacking alpha-catenin and these cells where alpha-catenin is re-expressed. We find that the tensile strength and the lifetime of single E-cadherin/E-cadherin bonds between parental cells are significantly lower over a wide range of loading rates. Statistical analysis of the force displacement spectra reveals that single cadherin bonds between cancer cells feature an exceedingly low energy barrier against tensile forces and low molecular stiffness. Disassembly of filamentous actin using latrunculin B has no significant effect on the strength of single intercellular E-cadherin bonds. The absence of alpha-catenin causes a dominant negative effect on both global cell-cell adhesion and single E-cadherin bond strength. These results suggest that the loss of alpha-catenin alone drastically reduces the adhesive force between individual cadherin pairs on adjoining cells, explain the global loss of cell adhesion in human breast cancer cells, and show that the forced expression of alpha-catenin in cancer cells can restore both higher intercellular avidity and intercellular E-cadherin bond strength.

Up-regulation of a-catenin is associated with increased lymph node involvement in colorectal cancer

To investigate the changing pattern of alpha-catenin expression and its relationship to clinical and pathological features of colorectal cancer (CRC) patients. Archival tumor samples were analyzed using immunohistochemistry (IHC) for alpha-catenin in 91 patients with advanced CRC. The values of alpha-catenin membrane index (MI) and cytoplasmic index (CI) were significantly related to the depth of tumor invasion (P = 0.027, P = 0.020, respectively), high indices being associated with increased depth of the primary tumor invasion (T3 and T4). Similarly, patients with high alpha-catenin expression had a significantly increased risk of lymph node metastasis (32/39 vs 37/52 for MI and 37/45 vs 32/46 for CI) (P = 0.001, P = 0.0001, respectively, for LNN status). An altered expression (i.e., cytoplasmic pattern) was also related (P = 0.047) to the response to chemotherapy; patients with low CI were more responsive (CR: 7/46) than patients with high CI values (CR: 0/45). There was a marginal effect on survival in patients time with metastases (SWM) (P = 0.087); patients with low CI showing slightly longer SWM, but no such effect on disease free survival (DFS) or disease specific survival (DSS). As to co-expression with another member of the adhesion complex (beta-catenin), high alpha-catenin/beta-catenin MI index was of marginal significance in predicting longer DSS (P = 0.063, log-rank). The results implicate that high alpha-catenin expression is intimately involved in the key regulatory mechanisms leading to invasive phenotype, lymph node metastases, and progressive disease in CRC.

Insulin‐like growth factor‐I receptor, E‐cadherin and αv integrin form a dynamic complex under the control of α‐catenin

Dynamic crosstalk between cell adhesion molecules, extracellular matrix and soluble informative factors is essential for cancer cell migration and invasion. Here, we investigated the mechanisms by which the E-cadherin/catenin complex and alpha v integrin can modulate insulin-like growth factor-I (IGF-I)-induced cell migration. Human colon mucosa, human colon cancer cell lines, HT29-D4 and HCT-8 derivatives that differ in their expression of alpha-catenin, were used as models. Interactions between E-cadherin, alpha v integrin and IGF-I receptor (IGF-IR) were analyzed by coimmunoprecipitation and immunolocalization experiments. The impact of these interactions on cell mobility was determined by haptotaxis assays. We report that alpha v integrin, E-cadherin and IGF-IR form a ternary complex in both cultured cancer cells and human normal colonic mucosa. alpha-Catenin regulates the scaffolding of this complex. IGF-IR ligation by IGF-I induces the disruption of the complex and the relocalization of alpha v integrin from cell-cell contacts to focal contact sites. This perturbation is correlated with the observed increase in cell migration. These results suggest that regulation of the alpha v integrin/E-cadherin/IGF-IR scaffolding is essential for the modulation of cell mobility. Its alteration could be of major importance to sustain alterations in cell adhesion that occur during cancer cell invasion and metastasis.

The prognostic value of E‐cadherin, α‐, β‐ and γ‐catenin in bladder cancer patients who underwent radical cystectomy

To determine the value of the loss of expression of E-cadherin and cadherin associated molecules as useful markers for both prognosis and chemosensitivity in bladder cancer patients who have undergone radical cystectomy. In 55 paraffin embedded specimens of radical cystectomy at our hospital from 1982 to 2000, the expression of E-cadherin, alpha-, beta- and gamma-catenin was examined by immunohistochemical staining. To evaluate the prognostic significance of these molecules, Kaplan-Meier survival curves were constructed and a statistical analysis was calculated by a log-rank test. A multivariate test (tumor stage, tumor grade, lymph node metastasis, configuration, the expression of E-cadherin, alpha-, beta- and gamma-catenin) was performed to detect prognostic markers. Normal expression was found in 33 cases (60.0%) for E-cadherin, 29 (52.7%) for alpha-catenin, 31 cases (56.4%) for beta-catenin, and 31 cases (56.4%) for gamma-catenin. The expression patterns for E-cadherin, alpha-, beta- and gamma-catenin were significantly correlated with each other (P < 0.01). Survival analysis showed a significant difference between normal and aberrant expression in each staining. A multivariate analysis revealed that the expression of alpha- catenin was an independent prognostic factor (P = 0.0191). In 23 patients that received adjuvant chemotherapy, there was a significant difference in survival between the normal and aberrant expression of alpha-catenin, but not other molecules. Alpha-catenin may not only be a good prognostic marker, but also one of key molecules that determine the chemosensitivities in patients with invasive bladder cancer.

The prognostic value of E‐cadherin and the cadherin‐associated molecules α‐, β‐, γ‐catenin and p120ctn in prostate cancer specific survival: A long‐term follow‐up study

To determine the value of loss of expression of E-cadherin and cadherin associated molecules as prognostic markers for prostate cancer patients in a long-term follow-up study. Sixty-five prostate cancer specimens, obtained from patients with different stages of prostate cancer who underwent a radical prostatectomy or TUR-P between 1987 and 1991, were used for immunohistochemical analysis of the expression pattern of E-cadherin, alpha-, beta-, gamma-catenin and p120(ctn). Clinical records of these patients were studied for follow-up data and the prognostic value of expression of these adhesion molecules was determined by Kaplan-Meier survival analyses and multivariable proportional hazard regression analysis. Normal staining patterns were found in 36 cases (55.4%) for E-cadherin, 37 cases (56.9%) for alpha-catenin, 40 cases (61.5%) for beta-catenin, 25 cases (38.5%) for gamma-catenin, and 40 cases (61.5%) for p120(ctn). Overall, a strong correlation was found between the expression of E-cadherin and other cadherin-associated molecules. The 5-year survival rates for each staining were as follows: E-cadherin (normal 79.2%, aberrant 26.8%), alpha-catenin (normal 79.2%, aberrant 26.8%), beta-catenin (normal 73.1%, aberrant 27.3%), gamma-catenin (normal 86.4%, aberrant 37.1%), and p120(ctn) (normal 72.8%, aberrant 30.0%). There was a significant difference in survival between normal and aberrant expression in each staining (log rank P < 0.0001). The proportional hazard regression model including tumor stage and Gleason score revealed alpha-catenin expression as the best prognostic marker for patients with prostate cancer. Our data revealed a strong correlation between E-cadherin expression and other cadherin-associated molecules. Among these markers, alpha-catenin seems the best prognostic marker for prostate cancer specific survival. Larger studies are needed to confirm this result.

BRD7 suppresses the growth of Nasopharyngeal Carcinoma cells (HNE1) through negatively regulating β-catenin and ERK pathways

BRD7 is a novel gene which involved NPC in our lab. Our previous studies showed that BRD7 was expressed at high level in normal nasopharyngeal epithelial tissues, but at low level in nasopharyngeal carcinoma biopsies and cell lines. In these papers, we found that ectopic expression of BRD7 can decrease cell proliferation and capability to form colonies in soft agar. FCM (Flow cytometry) assay indicated that the cell cycle progression from G1 to S phase was inhibited and the expression of cyclinD1 was significantly decreased after being transfected with BRD7 in HNE1 cells (NPC cells). To further investigate the molecular mechanism of BRD7 suppression of NPC cells growth, the cDNA microarray was performed to detect difference in gene expression profile induced by BRD7. The results indicated that 21 genes expression were changed after being transfected with BRD7 and the differentially expressed gene including alpha-catenin, cyclinD1, E2F3 was confirmed by western-blot. Next, we found that even though no obvious changes of the total expression of beta-catenin were observed, the accumulation of beta-catenin in nucleus was blocked. In addition, it was found that the expression of beta-catenin was up-regulated in the complex composed of beta-catenin and alpha-catenin in HNE1 cells induction of BRD7. So, we concluded that over-expression of BRD7 increased the expression of alpha-catenin which "hold" beta-catenin in the complex and inhibited its accumulating in nucleus. At last, we demonstrated the c-jun, p-MEK, and p-ERK1/2 expression were down-regulated, and the Ap-1 promoter activity was inactive after being transfected with BRD7. We also found that over-expression of BRD7 can inactivate the c-jun and p-ERK1/2 after being treated with EGF in HNE1 cells. These results indicated that BRD7 played a negative role in ERK1/2 pathway. Taken together, our present results provide new insights for BRD7 function to inhibit NPC cells growth through negative regulating beta-catenin and ERK1/2 pathways.

Discovery of epigenetically silenced genes in acute myeloid leukemias

The demethylating 5-aza-2'deoxycytidine (DAC) and the histone deacetylase inhibitor (HDACi) suberoyl anilide bishydroxamide (SAHA) possess potent antitumorigenic properties in myeloid disorders. However, the transcriptome alterations mediated by these drugs are poorly understood. We analyzed the transcriptional effects of DAC and SAHA in the AML cell line KG-1. Microarray analyses revealed 76 genes expressed in normal CD34+ cells, absent in KG-1 cells but whose expression was induced after drug treatment. A total of 39 of these genes harbored CpG islands in their promoters. We examined the expression level of these genes in 120 AML patient samples representing diverse karyotpyes. Gas2l1, tfIIs, ehd3, enolase 2, mx1, dral, astml and pxdn were diminished across all AML karyotypes examined. Ehd3 was methylated in 63% of AML patients examined. This methylation was lost upon complete remission, and not observed in normal CD34+ cells. CD34+ cells expressed ehd3 at approximately 10-fold higher levels than AML samples. Another highlighted gene, alpha-catenin, is located at q31 of chromosome 5. Analyses of 29 5q- AML/myelodysplastic syndrome (MDS) samples revealed marked decreases in expression of alpha-catenin, compared to non-5q- MDS samples (6.6+/-9-fold). However, no methylation was detected, suggesting indirect effects of these drugs on the expression of alpha-catenin.

α‐catenin is a significant prognostic factor than E‐cadherin in esophageal squamous cell carcinoma

The purpose of the present study was to analyze clinicopathologic variables in esophageal squamous cell carcinoma (ESCC) according to expression of E-cadherin and alpha-catenin which play an important role in cell adhesion. We immunohistochemically examined E-cadherin and alpha-catenin in 205 patients with ESCC. The expression results were classified into two groups: preserved expression (+) and reduced expression (-). The incidence of E-cadherin (-) and alpha-catenin (-) was 52% and 54%, respectively and significantly related each other. For both E-cadherin and alpha-catenin, reduced expression was significantly related to tumor depth, nodal metastasis, stage, recurrence, and prognosis. In the E-cadherin (+) group, the alpha-catenin (+) and alpha-catenin (-) patients differed significantly in tumor depth, nodal metastasis, stage, hematogenous and lymphatic recurrences (P < 0.001, <0.001, <0.001, <0.001 and =0.007, respectively). According to coexpression of E-cadherin and alpha-catenin, the prognosis was best in patients with E-cadherin (+) and alpha-catenin (+), and worst in patients with E-cadherin (-) and alpha-catenin (-). Multivariate analysis revealed that alpha-catenin expression was an independent prognostic factor. The examination of expression of E-cadherin and especially alpha-catenin is useful for predicting lymph node metastasis and clinical outcome of ESCC.

Varicocele-Induced Testicular Dysfunction May Be Associated with Disruption of Blood-Testis Barrier

The objective of this study was to examine E-cadherin and alpha-catenin expression at the junctions between adjacent Sertoli cells in testicular specimens from patients with varicocele in order to determine the presence of a possible link between blood-testis barrier and pathophysiology of varicocele. A total of 51 testicular biopsies were obtained from 28 infertile men with unilateral or bilateral varicocele. Twenty-three patients had bilateral and 5 had unilateral varicocele, Grade I varicocele was detected in 30 (59%), grade II in 15 (29%) and grade III in 6 (12%) patients. Abnormal expression of E-cadherin and alpha-catenin at the junctions between adjacent Sertoli cells was demonstrated in 100% and 90% of the patients with varicocele, respectively. In those with grade I-III varicocele, the mean E-cadherin and alpha-catenin expression were 7.6 +/- 11.4 and 39 +/- 36; 7.6 +/- 0.0 and 49 +/- 30; 8.3 +/- 9.3 and 58 +/- 33, respectively, but the difference was not significant. Reduced E-cadherin and alpha-catenin expression at the junctions between adjacent Sertoli cells may be associated with disruption of blood-testis barrier in varicocele.

Expression and Subcellular Localization of E-Cadherin, .ALPHA.-Catenin, and .BETA.-Catenin in 8 Feline Mammary Tumor Cell Lines

Protein expression and subcellular localization of E-cadherin, alpha-catenin, and beta-catenin in 8 feline mammary tumor cell lines were examined by western blot analysis and fluorescence immunocytochemistry. A low E-cadherin expression was observed in FNN-m cells. Furthermore, compared to other cell lines, two E-cadherin bands existed in FMC-p1 cells and were localized in the perinuclear region; distinct radial lines were observed in the cytoplasm. A low alpha-catenin expression was observed in FON-m cells, but there were no apparent abnormalities in its localization. In contrast, similar levels of beta-catenin expression and cytoplasmic localization were observed in all cell lines.

Expression of adhesion proteins E-cadherin, α-catenin, β-catenin and γ-catenin is different in T1 and T2 breast tumours

Breast cancer is the most common malignancy in women. Although an increasing number of patients with breast cancer are being cured by surgery, a considerable number of patients suffer relapse in the form of metastases after surgery. E-cadherin and catenins have documented roles in breast cancer progression. Mammography is supposed to decrease breast cancer mortality by detecting tumours while they are small and before they have reached a clinically detectable stage. In the present study, we wanted to evaluate whether there are differences in expression patterns of adhesion proteins, shown to be crucial in the metastatic process, between small tumours detected by mammography and clinically detected large tumours. Expression of E-cadherin, alpha-catenin, beta-catenin and gamma-catenin was analysed using immunohistochemistry methods in 86 invasive breast carcinomas detected by mammography and compared with 90 clinically palpable invasive breast carcinomas. In the group of tumours detected by mammography (86 samples), reduced expression of E-cadherin was observed in 12 (14%) samples. Reduced expression of alpha-catenin was observed in four (4.6%) samples, and three (3.5%) samples showed reduced expression of beta-catenin. All samples showed strong expression of gamma-catenin. When expression patterns of these proteins were evaluated in 90 clinically detected tumours, we observed reduced expression of E-cadherin in 58 (64.4%) samples, 12 (13.3%) samples showed reduced expression of alpha-catenin, while nine (10%) samples showed reduced expression of beta-catenin. Strong expression of gamma-catenin was detected in all tumours also in this group.Statistical analyses revealed a highly significant difference in expression of E-cadherin (p<0.001). However, no statistically significant differences were observed in expression of alpha-catenin (p = 0.081) and beta-catenin (p = 0.092) between the two groups of tumours. Results indicate that T1 breast tumours harbour less alterations in E-cadherin-catenin complexes and therefore are probably less likely to disseminate, and patients probably have a better prognosis than if tumours are diagnosed as T2.