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

Abdallah Mound, Lise Rodat-Despoix, Salim Bougarn, Halima Ouadid-Ahidouch, Fabrice Matifat

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

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 (IP3R3) 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 3R3 and BKCa channel) in normal and in breast cancer cells. 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 IP3R3 and their molecular interaction. Finally, whole cell patch-clamp and Ca2+ imaging were performed to assess the functional interaction. Results: Our results are in favour of a functional and a molecular coupling between IP 3R3 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 IP3R3 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 IP3R3 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 IP3R3 or BKCa. In the same way, intracellular application of Ins(2,4,5)P3 triggered an IbTx-sensitive membrane hyperpolarisation. Moreover, intracellular Ca 2+ chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid (BAPTA) prevented ATP-induced BKCa activation. BKCa and IP3R3 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 3R3 and BKCa did not co-immunoprecipitate, suggesting the absence of a molecular coupling between BKCa and IP3R3 in the MCF-10A normal cell line. Conclusion: Altogether, our results suggest a molecular and functional link between BKCa channel and IP 3R3 in cancer cells. Our findings led us to propose this coupling between BKCa and IP3R3 as an important mechanism for tumour cell proliferation.

Original languageEnglish
Pages (from-to)3738-3751
Number of pages14
JournalEuropean Journal of Cancer
Volume49
Issue number17
DOIs
Publication statusPublished - 1 Nov 2013
Externally publishedYes

Fingerprint

Inositol 1,4,5-Trisphosphate Receptors
Adenosine Triphosphate
Cell Proliferation
Breast Neoplasms
Cell Cycle Proteins
Small Interfering RNA
Cyclin-Dependent Kinase 4
Cell Line
Cell Cycle Resting Phase
Ethane
Inositol 1,4,5-Trisphosphate
Membranes
Cyclin D1
MCF-7 Cells
G1 Phase
Type C Phospholipases
Cell Cycle Checkpoints
Ion Channels
Ammonium Compounds
Immunoprecipitation

Keywords

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

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Molecular interaction and functional coupling between type 3 inositol 1,4,5-trisphosphate receptor and BKCa channel stimulate breast cancer cell proliferation. / Mound, Abdallah; Rodat-Despoix, Lise; Bougarn, Salim; Ouadid-Ahidouch, Halima; Matifat, Fabrice.

In: European Journal of Cancer, Vol. 49, No. 17, 01.11.2013, p. 3738-3751.

Research output: Contribution to journalArticle

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T1 - Molecular interaction and functional coupling between type 3 inositol 1,4,5-trisphosphate receptor and BKCa channel stimulate breast cancer cell proliferation

AU - Mound, Abdallah

AU - Rodat-Despoix, Lise

AU - Bougarn, Salim

AU - Ouadid-Ahidouch, Halima

AU - Matifat, Fabrice

PY - 2013/11/1

Y1 - 2013/11/1

N2 - 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 (IP3R3) 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 3R3 and BKCa channel) in normal and in breast cancer cells. 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 IP3R3 and their molecular interaction. Finally, whole cell patch-clamp and Ca2+ imaging were performed to assess the functional interaction. Results: Our results are in favour of a functional and a molecular coupling between IP 3R3 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 IP3R3 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 IP3R3 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 IP3R3 or BKCa. In the same way, intracellular application of Ins(2,4,5)P3 triggered an IbTx-sensitive membrane hyperpolarisation. Moreover, intracellular Ca 2+ chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid (BAPTA) prevented ATP-induced BKCa activation. BKCa and IP3R3 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 3R3 and BKCa did not co-immunoprecipitate, suggesting the absence of a molecular coupling between BKCa and IP3R3 in the MCF-10A normal cell line. Conclusion: Altogether, our results suggest a molecular and functional link between BKCa channel and IP 3R3 in cancer cells. Our findings led us to propose this coupling between BKCa and IP3R3 as an important mechanism for tumour cell proliferation.

AB - 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 (IP3R3) 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 3R3 and BKCa channel) in normal and in breast cancer cells. 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 IP3R3 and their molecular interaction. Finally, whole cell patch-clamp and Ca2+ imaging were performed to assess the functional interaction. Results: Our results are in favour of a functional and a molecular coupling between IP 3R3 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 IP3R3 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 IP3R3 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 IP3R3 or BKCa. In the same way, intracellular application of Ins(2,4,5)P3 triggered an IbTx-sensitive membrane hyperpolarisation. Moreover, intracellular Ca 2+ chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid (BAPTA) prevented ATP-induced BKCa activation. BKCa and IP3R3 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 3R3 and BKCa did not co-immunoprecipitate, suggesting the absence of a molecular coupling between BKCa and IP3R3 in the MCF-10A normal cell line. Conclusion: Altogether, our results suggest a molecular and functional link between BKCa channel and IP 3R3 in cancer cells. Our findings led us to propose this coupling between BKCa and IP3R3 as an important mechanism for tumour cell proliferation.

KW - BK channel

KW - Breast cancer

KW - Ca

KW - Proliferation

KW - Type 3 inositol 1,4,5-trisphosphate receptor

KW - voltage- and Ca-dependent K channels

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