An Alternative Splice Variant of KCNMA1 Drives Breast Cancer Metastasis and Invasion.
Authors: D. Khaitan, U. Sankpal, N. Ningaraj
Journal: Cancer Research (2009)

Abstract Background: We have recently shown that KCNMA1, which encodes the pore forming α-subunit of large-conductance calcium-activated voltage-sensitive potassium (BKCa) channel, plays a highly critical role in breast tumor cells metastatic to brain and somewhat less critical role in primary breast cancer cell metastasis. The resulting functional heterogeneity of BKCa channels is attributed generally to alternative splicing of KCNMA1 that generate BKCa channel isoform with altered channel properties. We have identified a KCNMA1 splice variant (KCNMA1vE22) expressed in breast cancer cell line (MDA-MB-361) metastatic to brain with a deletion of 108 bp in exon 22 between the S9 and S10 protein subunit (C-terminus). In this study we investigated the biological function of KCNMA1vE22 in vitro and in vivo.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 KCNMA1vE22 was identified from A-172 tumor cells and cloned into mammalian expression vector (pcDNA6). MCF-7 cells (non-metastatic, null type for KCNMA1vE22) and HEK cells (null type for KCNMA1 and KCNMA1vE22) were transfected with pcDNA6/KCNMA1vE22 and stable clones selected. The expression of KCNMA1 and KCNMA1vE22 was confirmed using RT-PCR, western blot and qPCR. The biological role of the splice variant was studied using parental and transfected MCF-7 cells by cell proliferation, matrigel invasion, transendothelial migration and membrane potential assays in vitro and tumorogenecity using s.c xenograft mouse model.Results: Based on the preliminary screening of breast tumor samples, KCNMA1vE22 was undetectable in primary as well as systemic metastatic breast tumor samples. 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 KCNMA1vE22 showed a eight-fold increase in invasion while a three-fold increase in TEM was observed compared to parental MCF-7 cells. A marked increase in the proliferation rate of KCNAM1vE22-transfected cells compared to MCF-7 alone was observed, suggesting that KCNAM1vE22 has a profound effect on breast cancer cell proliferation. The in vitro results were validated by subcutaneous mouse model experiment. We found statistically significant increase in mean tumor volume in mice with KCNMA1vE22 transfected compared with parental MCF-7 cells.Conclusions: Our results clearly show that KCNMA1vE22 promotes breast cancer cell invasion, and possibly metastasis to brain. Perhaps the discovery and validation of brain specific metastasis-associated KCNMA1 alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. The variant KCNMA1vE22 that we have discovered potentially may fill the gap to serve as a new generation of biomarker of breast cancer metastasis to brain. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6166.