Role of <i>KCNMA1</i> in Breast Cancer Metastasis and Angiogenesis.
Authors: N. Ningaraj, D. Khaitan, U. Sankpal
Journal: Cancer Research (2009)

Abstract Background: Despite great advances in understanding the etiology and molecular biology of breast cacncers, the treatment options for patients affected with this disease remain severly limited. The KCNMA1, which encodes the pore forming α-subunit of large-conductance calcium-activated voltage-sensitive potassium (BKCa) channel, may play highly critical roles in breast tumor cells metastasis and angiogenesis within the brain. We sought to investigate the above roles of KCNMA1 using breast tumor models.Methods: We validated the results from the breast tumor cell lines data with human normal breast tissue, primary, non-metastatic and metastatic breast tumors by RT-PCR. The KCNMA1 was cloned into mammalian expression vector (pcDNA6). MCF-7 cells (non-metastatic, KCNMA1 low expressing) and HEK cells (null type for KCNMA1) were transfected with pcDNA6/KCNMA1 and stable clones selected. The expression of KCNMA1 was confirmed using RT-PCR, western blot and qPCR. The biological role of KCNMA1 was studied using parental and transfected MCF-7 cells by cell proliferation, matrigel invasion, transendothelial migration and membrane potential assays in vitro and tumorogenecity and angiogenesis using subcutaneous xenograft mouse model. To study the role of KCNMA1 in angiogenesis, human brain microvascular endothelial cells (HCMEC/D3) were co cultured with breast tumor cells and VEGF levels determined by ELISA.Results: The KCNMA1 was overexpressed in metastatic breast tumor samples compared to primary breast tumor. Stably transfected HEK and MCF-7 cells showed 2.5 to 3-fold increase in KCNMA1 mRNA expression, which corroborated with the increased protein levels and BKCa channel activity. In addition MCF-7 cells expressing KCNMA1 showed a five-fold increase in invasion while a two-fold increase in TEM was observed compared to parental MCF-7 cells. A marked increase in the proliferation rate of KCNMA1-transfected cells compared to MCF-7 alone was observed, suggesting that KCNMA1 has a profound effect on breast cancer cell proliferation. A two-fold increase in VEGF secretion in KCNMA1-overexpressing MCF-7 cells was observed. The results also indicated that the conditioned media from MCF-7 cells increased BKCa channel expression in HCMEC/D3 cells by nearly 1.5-fold as compared to HCMEC/D3 cells cultured without the conditioned media. The proliferation of HCMEC/D3 cells was increased by three-folds in the presence of VEGF or conditioned media from MCF-7 overexpressing KCNMA1. The proliferation was attenuated by BKCa channels inhibitor (Iberiotoxin). The in vitro results were validated using subcutaneous mouse model experiment. We found statistically significant increase in mean tumor volume in mice with KCNMA1-transfected MCF-7 cells compared with parental cells.Conclusions: Our results clearly show that KCNMA1 promotes breast cancer cell invasion, and possibly metastasis. Taken together these experiments showed a critical role of KCNMA1 expression on VEGF secretion by MCF-7 cells and endothelial cell proliferation. These studies will contribute to a fundamental understanding of the role of KCNMA1 in metastatic breast cancer biology and angiogenesis. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6165.