Molecular interaction and functional coupling between type 3 inositol 1,4,5-trisphosphate receptor and BKCa channel stimulate breast cancer cell proliferation

作者：Mound Abdallah; Rodat Despoix Lise; Bougarn Salim; Ouadid Ahidouch Halima; Matifat Fabrice^*

来源：European Journal of Cancer, 2013, 49(17): 3738-3751.

DOI：10.1016/j.ejca.2013.07.013

• Breast cancer
• Proliferation
• Type 3 inositol 1,4,5-trisphosphate receptor voltage- and Ca2+-dependent K+ channels
• BKCa channel
• Ca2+

Background: The implication of ion channels and inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ signalling (IICS) in the carcinogenesis processes, including deregulation of cell proliferation, migration and invasion, is increasingly studied. Studies from our laboratory have shown that type 3 IP3 receptor (IP(3)R3) and voltage-and Ca2+-dependent K+ channels BKCa channels are involved in human breast cancer cell proliferation. In this context, we investigated the probable interaction between these two proteins (IP(3)R3 and BKCa channel) in normal and in breast cancer cells. %26lt;br%26gt;Methods: MCF-7 and MCF-10A cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay in the presence or absence of adenosine triphosphate (ATP). Furthermore, cell-cycle analysis was carried out and cell cycle protein expression was examined by Western blotting. Immunocytochemistry and co-immunoprecipitation assays were used to check co-localisation of BKCa and IP(3)R3 and their molecular interaction. Finally, whole cell patch-clamp and Ca2+ imaging were performed to assess the functional interaction. %26lt;br%26gt;Results: Our results are in favour of a functional and a molecular coupling between IP(3)R3 and BKCa channel that is involved in MCF-7 proliferation. Indeed, ATP increased MCF-7 cell proliferation and this effect was impaired when the expression of BKCa and/or IP(3)R3 has been reduced by specific small interfering RNAs (siRNAs). Flow cytometry experiments showed that both siRNAs led to cell cycle arrest in the G0/G1 phase and these results were confirmed by the analysis of cell cycle protein expression. Specifically, BKCa and IP(3)R3 silencing decreased both cyclin-D1 and cyclin-dependent kinase 4 (CDK4) expression levels. Furthermore, ATP elicited a phospholipase C (PLC)-dependent elevation of internal Ca2+ that triggered in turn an iberiotoxin (IbTx)- and a tetra-ethyl-ammonium (TEA)-sensitive membrane hyperpolarisation that was strongly reduced in the cells with silenced IP(3)R3 or BKCa. In the same way, intracellular application of Ins(2,4,5)P-3 triggered an IbTx-sensitive membrane hyperpolarisation. Moreover, intracellular Ca2+ chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N%26apos;,N%26apos;-tetraacetic acid (BAPTA) prevented ATP-induced BKCa activation. BKCa and IP(3)R3 also co-immunoprecipitated and this interaction seemed to occur in cholesterol-enriched microdomains. Conversely, in the normal breast cell line MCF-10A, neither ATP application nor BKCa silencing affected cell proliferation. Furthermore, IP(3)R3 and BKCa did not co-immunoprecipitate, suggesting the absence of a molecular coupling between BKCa and IP(3)R3 in the MCF-10A normal cell line. %26lt;br%26gt;Conclusion: Altogether, our results suggest a molecular and functional link between BKCa channel and IP(3)R3 in cancer cells. Our findings led us to propose this coupling between BKCa and IP(3)R3 as an important mechanism for tumour cell proliferation.

• 出版日期2013-11