Role of KCNMA1 gene in breast cancer invasion and metastasis to brain

Abstract

Background: The prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, large-conductance calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene that encodes for the pore-forming alpha-subunit of BKCa channels in breast cancer metastasis and invasion.

Methods: We performed Global exon array to study the expression of KCNMA1 in metastatic breast cancer to brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of BKCa channel protein in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether BKCa channel inhibition attenuates breast tumor invasion and metastasis using KCNMA1 knockdown with siRNA and biochemical inhibition with Iberiotoxin (IBTX).

Results: The Global exon array and RT-PCR showed higher KCNMA1 expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased BKCa channel activity, leading to greater invasiveness and transendothelial migration, both of which could be attenuated by blocking KCNMA1.

Conclusion: Determining the relative abundance of BKCa channel expression in breast cancer metastatic to brain and the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy.

Figure 1

Expression of KCNMA1 gene using Affymetrix Genechip Exon Array. mRNA expression of KCNMA1 was significantly enhanced in the MDA-MB-361 (brain metastatic) cancer cell line as compared to the MCF-7 (non metastatic) and MDA-MB-231 (systemic metastatic) cell lines. MCF-7 (P < 0.0002) and MDA-MB-361(*P < 0.0001), (n = 3 per cell line).

Figure 2

Analysis of KCNMA1 gene expression by RT-PCR. Semiquantitative RT-PCR analyses of human primary breast cancer (n = 6), metastatic breast cancer {n = 6, sample 1 (metastasized to ovary), 2 and 3 (metastasized to liver), 4–6 (metastasized to lymph node)}, metastasized to brain (n = 4) and breast cancer cell lines reveals differential expression of KCNMA1 gene expression (α-subunit, 250 bp). GAPDH was used as the loading control. NB: Normal breast tissue, MCF-10A (normal breast cell line), MCF-7 (non mets), 231: MDA-MB-231 (Mets) and 361: MDA-MB-361 (Brain Mets). Note: Only one of six primary breast cancer tissues showed detectable amount of KCNMA1 RNA (see arrow).

Figure 3

Localization and expression of BK_Ca channel protein. A: Immunofluorescence of breast cancer cell lines stained for BK_Ca channel (green) and nuclei with DAPI (blue). Expression of BK_Ca channel was strong in MDA-MB-231 and MDA-MB-361, weak in MCF-7 and indiscernible in MCF-10A. Stains were repeated twice. B: Flow cytometric detection of BK_Ca channel protein expression in various breast cell lines. Representative histograms from (a) MCF-10A, (b) MCF-7, (c) MDA-MB-231, and (d) MDA-MB-361 showing expression of BK_Ca channel protein. C: Immunofluorescence of anti-BK_Ca channels antibody on human breast cancer tissue. Antibody labeled in green (FITC), nuclei counterstained in blue (DAPI). (a) Very few cells show BK_Ca channels staining in the primary breast cancer tissue (n = 6, See large arrow). Note: Absence of green staining in most of the cells also indicates specificity of the antibody (See small arrows). (b) Systemic metastatic breast cancer (lymph node) shows an increased number of cells stained with BK_Ca channel antibody (n = 6; See large arrows). (c) All breast cancer tissue metastatic to the brain are strongly positive for the BK_Ca channels (n = 4). Magnification = 400×. D: Representative H&E staining of breast tumor tissue sections.

Figure 4

Differential BK_Ca channel activity measured by membrane potential in breast cancer cell lines. The BK_Ca channel activity was measured in the presence of its activator NS-004 with and without siRNA for KCNMA1 gene. Pretreatment of breast cancer cell lines with siRNA significantly (*p < 0.046; **p < 0.001) inhibited the BK_Ca channel activity following addition of NS-004. Note: Decreased drop in fluorescence (indicator of BK_Ca channel opening/activity) of siRNA + NS-004- treated cells compared to NS-004 treated cells demonstrates that BK_Ca channel activity was inhibited by addition of siRNA to cells, as fluorescence quenching is an indirect measure of K⁺ ion-mediated hyperpolarization (hyperpolarization is caused by efflux of K⁺ through K⁺ channels), shown in the Y-axis. Bars represent the mean and standard error of the mean obtained from three different experiments in triplicate (n = 9).

Figure 5

KCNMA1 expression promotes breast cancer cell invasion. A: Representative images of breast cancer cells that have migrated through the Matrigel coated transwell and adhered to the lower surface of the filters. The migrated cells were stained with crystal violet (see arrows). B: Breast cancer cells pretreated with IBTX or siRNA to silence the KCNMA1 gene demonstrated significantly (*P < 0.03; **P < 0.001) reduced BK_Ca channel mediated invasion compared to untreated breast cancer cell lines. Data are expressed as percentage of migration in the untreated controls (100%). Bars represent mean ± the standard error of the mean obtained from three different experiments (n = 3).

Figure 6

Effects of IBTX and siRNA on transendothelial migration of breast cancer cells. A: Representative images of breast cancer cells that adhered to monolayer of the human brain microvasculature endothelial cell line HCMEC/D3. The adherent breast cancer cells appear white in color against the background of HCMEC/D3. (See arrows). B: Breast cancer cells pretreated with IBTX or siRNA to silence the KCNMA1 gene demonstrated significantly (*P < 0.05; **P < 0.001) reduced BK_Ca channel mediated-TEM of breast cancer cells across HCMEC/D3 monolayer compared to untreated control. Data is presented as percentage migration of treated breast cancer cells compared with migration of untreated controls (100%); Bars represent the mean ± standard error of the mean obtained from three different experiments (n = 3).