Breast cancer metastatic potential: Correlation with increased heterotypic gap junctional intercellular communication between breast cancer cells and osteoblastic cells

Abstract

Key words: adhesion; breast cancer; connexin; cadherin; gap junctional intercellular communication; metastasisGap junctions are intercellular channels that facilitate the movement of small (Ͻ1 kDa) ions and signaling molecules between cells. 1 A breakdown in GJIC has been correlated with tumorigenesis. 2 Previous studies suggested that the breakdown in homotypic communication may contribute to breast cancer metastatic potential. 3 The breast metastasis suppressor gene BRMS1 suppresses metastasis in human breast carcinomas. 4 Transfection of BRMS1 cDNA into the metastatic breast cancer cell line MDA-MB-435 (435) reduces their metastatic potential and restores homotypic GJIC, supporting the concept that metastasis-suppressed cells are capable of significant homotypic GJIC as well as the correlation between breakdown in homotypic GJIC and metastasis. 3 The purpose of the current study was 3-fold: (i) to determine whether breast carcinoma cells can form gap junctions with bone cells; (ii) to ascertain whether heterotypic GJIC might be relevant to cancer metastasis; and (iii) to explore whether BRMS1 expression might regulate heterotypic GJIC. Since breast carcinomas so commonly colonize bone, we explored heterotypic GJIC (i.e., between 435 cells and hFOB1.19 cells). We examined 435 cells because they metastasize to bone when injected intracardially in mice. 5 BRMS1-transfected 435 cells were used to study the effect of metastasis suppression on heterotypic GJIC with hFOB cells since BRMS1 transfectants are genotypically related to 435 cells. Osteoblasts were used for heterotypic studies because osteoblastic bone lining cells cover all bone surfaces 6 and tumor cells might have to adhere to and migrate through a layer of bone lining cells in addition to a layer of endothelial cells to reach the marrow cavity. 7 MATERIAL AND METHODS Cell linesHTERT-HME1 (HTERT) cells were maintained in specially formulated medium (both cells and medium from Clontech, Lexington, KY). 8 MCF-7 cells were purchased from the ATCC (Rockville, MD) 9 and maintained in EMEM supplemented with 10% FBS (Hyclone, Logan, UT) and 1 mg/ml bovine insulin. 435 cells 10 were maintained in DMEM/F-12 medium (GIBCO Life Technologies, Rockville, MD) supplemented with 5% FBS. 435/ BRMS1 cells (clones 3 and 6) are 435 cells transfected with BRMS1 cDNA 4 and were maintained in the 435 cell medium supplemented with geneticin (500 g/ml). The hFOB1.19 cell line 11 was maintained in DMEM/F-12 medium supplemented with 10% FBS and 1% penicillin-streptomycin. In previous studies, 435/BRMS1 cells were subcultured by an EDTA method while parental 435 cells were maintained by trypsinization. 12 Therefore, Abbreviations: BRMS1, breast metastasis suppressor gene; Cx32, connexin 32; DiI, 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethyl indocarbocyanine perchlorate (DiIC18); E-cadherin, epithelial cadherin; ECL, enhanced chemiluminescence; GJIC, gap junctional intercellular communication; hFOB, human fetal osteoblast; HRP, horseradish peroxidase; MAb, monoclonal antibody; N-cadherin,...

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