1) KCNMA1 gene and protein expression in breast cancer patients
We used gene expression data (RNA-seq) from 981 breast cancer samples (TCGA Cell 2015 [23] and TCGA Provisional). In all five subtypes BK channel KCNMA1 gene expression levels are dramatically upregulated, comparison to normal breast cells (FPKM ~ 0.7) (Figure 1). TNBC patients are represented by red dots.
Using transcriptomics and a targeted proteomics approach, the gene-specific correlation of mRNA levels and protein copy number has been well established in human cells and tissues [24]. Previous studies have demonstrated that BK channel alpha subunit protein (main subunit forming the pore of the channel) is abundantly expressed in MDA-MB-231 cells, weakly expressed in MCF7, and nearly undetectable in normal breast epithelial cells MCF10A [15]. Therefore, we set out to investigate the protein expression of BK channels in TNBC patients’ tissues.
Figure 2 shows a representative figure for protein expression of BK channel alpha subunit in primary TNBC tissue using an antibody that targets an epitope in the 1st extracellular loop of transmembrane domains 1 and 2 (corresponding to amino acid residues 199-213 of rat KCNMA1 (Alomone Labs). MDA-MB-231 (MDA231 in the figure) was used as a positive control. Mouse brain (MB) known to express BK channels [25, 26] was used as an additional positive control (stronger signals in a more sensitive fluorescence image of Western blot is provided in Supplemental Figure 1). In addition to the glycosylated channel protein (around 200kD), there exist smaller fragments recognized by the antibody that are likely the proteolyzed C-terminals of the channel protein reported in previous studies [27]. The interpretation of the results was confirmed by incubation of the antigen (2B) that showed disappearance of the signals in 2A. After total expression signals being normalized to b-actin, BK channel protein expression levels are nearly 14-fold higher in primary TNBC than in normal human breast tissue (Normal: 0.345±0.177; TNBC: 4.793±1.074, n=4-6, p<0.001) (2C). Figure 2D shows that BK channel proteins are also abundantly expressed in different types of TNBC cells (SUM159, HCC1143), but barely detectable in MCF10A normal breast cells.
To confirm the increased protein expression levels of BK channels in TNBC patients, we performed IHC experiments in seven TNBC tissue and three normal breast tissue samples. We used a BK channel antibody that had been successfully applied in identifying KCNMA1 channel protein expression in the Human Protein Atlas (Sigma-Aldrich, HPA054648). Supplemental Figure 2 shows a normal breast tissue (A) and a TNBC tissue (B). The averaged percentage of protein expression area is shown in (C). BK channel protein levels were increased by ~ 9-fold in TNBC than in normal breast tissue (TNBC = 3.56±1.33, n=7; Normal = 0.41±0.10, n=3; p<0.01).
Figure 1 raised several questions: 1) Why are depolarized TNBC cells overexpressing a hyperpolarizing BK channel? 2) Are these overexpressed BK channels activated (open)? These questions led us to the hypothesis that these overexpressed BK channels are not activated. If this hypothesis is correct, then opening these channels can be exploited as a novel strategy for targeted therapy in treatment of TNBC.
For ion channels whose activity is dependent on Em, gene/protein expression levels are not intrinsically correlated with channel activity. At the resting Em of the cell, ion channels are active when they are open, inactive when they are closed. We therefore set up to investigate biophysical properties of BK channels in TNBC cells.
2) Voltage-dependent activation of BK channels in TNBC cells
Previously, we showed that the resting Em, which is within the physiological voltage range, in MDA-MB-231 cells is depolarized compared to normal mammary epithelial cells (HMEC) (Em_MDA-MB-231: about -40mV, Em_HMEC: about-67mV) [12]. To investigate whether BK channels in MDA-MB-231 are open at -40mV, we studied biophysical properties of BK channels in MDA-MB-231 cells using whole-cell and perforated patch clamp techniques.
Figure 3A shows the typical BK channel currents activated by the depolarizing pulse protocol (below 3A). The currents were confirmed to be generated from BK channels by iberiotoxin (IbTX, 100nM) (known as a potent specific blocker of BK channel (with IC50 of 250pM) since it does not affect other ion channels [28]). Figure 3B shows the average voltage-dependent activation curve of BK channel in eight (8) MDA-MB-231 cells. At -40mV (resting Em in MDA-MB-231), only 1% of BK channels are open.
3) BK channel opener hyperpolarizes Em in MDA-MB-231 cells
Opening large conductance (>200pS) [29] BK channels causes loss of intracellular K+ ions to the outside of the cell, leading to membrane hyperpolarization. In MDA-MB-231 cells, we used a potent selective BK channel opener, BMS-191011 (1mM) [30] to study how opening BK channels may affect cell Em. We found that BMS-191011 (1mM) hyperpolarized Em by 15mV (Em_con = -30.71±8.20mV, Em_BMS = -45.86±8.95mV, n=8; p<0.01) within 15min of application (Figure 3C). IbTX (100nM) did not induce significant change in Em (Em_con = -30.71±8.20mV, Em_IbTX = -30.86±6.64mV, n=8; p>0.05), but completely reversed hyperpolarization induced by BMS-191011 (Em_con = -30.71±8.20mV, Em_BMS+IbTX = -28.43±7.30mV, n=8; p>0.05) (Figure 3C). The results provided additional evidence that the majority of BK channels are closed at Em in MDA-MB-231 cells.
4) BK channel opener induced death of human TNBC cells
To test whether hyperpolarization by opening BK channels can induce death of TNBC cells, we studied effects of BK channel opener in TNBC cell lines. Treatment of BMS - 191011 at 20mM for two days did not affect normal breast MCF10A cells (Supplemental Fig.3, left panels), but dramatically induced cell death of MDA-MB-231 cells (Supplemental Fig.3, middle panels). BMS-191011 halted growth of MCF-7 cells but induced death of much fewer cells compared to MDA-MB-231 (Supplemental Fig.3, right panels). Figure 4A shows the percentages of dead/dying cells are 0.92 ± 0.29% (n=5) for MCF10A, 14.76±1.94% (n=5) for MCF-7, and 63.82±6.21% (n=5) for MDA-MB-231, p<0.0001 (One-way ANOVA).
To ensure that BMS-191011 induced cell death is via opening BK channels, we used another specific BK channel opener, NS11021, which has a different chemical structure [31]. Figure 4B shows time - and concentration – dependent effects of NS11021 on the growth of MDA-MB-231. At day 5, most cells treated with 20mM NS11021 were dead (Supplemental Fig.4). For the same day, NS11021 induced cell death at different concentration is statistically significant compared to untreated group (p<0.0001, n=6).
To test whether BK channel opener mediated hyperpolarization-induced cell death is independent of TNBC subtypes, we studied effects of BMS-191011 on additional TNBC cell lines, SUM159 (Basal A, like MDA-MB-231) and HCC1143 (Basal B) [32]. BMS-191011 inhibited cell growth of SUM159 (4C) (and Supplemental Fig.5) and HCC1143 (4D) (and Supplemental Fig.6) cells in a similar way compared to that in MDA-MB-231. Additional controls were performed to rule out the potential side effects of DMSO on the cell growth of TNBC. Supplemental Figure 7 shows an example for 0.5% DMSO (maximal volume used in drug treatment) that has no effect in cell death of HCC1143. We also tested that DMSO had no effects in cell growth of MDA-MB-231 and SUM159 cell lines.
Additionally, we tested whether a mutated BK channel that is permanently open (A313D), leading to hyperpolarization [33] may induce MDA-MB-231 cell death. Supplemental Figure 8 shows that after 2 days of transfection, 59.4±13.7% (n=5) of MDA-MB-231 cells expressing A313D died, in comparison to 17.2±5.3% (n=5) of death in cells expressing wild-type (WT) channels or 18.8±7.4% of death in cells expressing on GFP plasmid (n=5, p<0.003). After 4 days of transfection, all MDA-MB-231 cells expressing A313D were dead, whereas most cells expressing WT channels or GFP alone were alive.
5) BK channel opener induced apoptosis and caspase-3 activation in TNBC
To understand the mechanism that mediates hyperpolarization - induced death in TNBC cells, we studied apoptosis, a well-studied programmed cell death mechanism. We performed time-lapse imaging experiment demonstrating that low concentration (1mM) of BMS-191011 induced rapid cell shrinkage, a distinguished event unique to apoptosis [34], within 20-60min (Supplemental Fig.9).
We also studied effect of BK channel opener in caspase activation, an established mechanism and a strong indicator of apoptosis [35]. We first used a fluorescent caspase 3/7 green dye to test whether BK channel opener may induce caspase activation in MDA-MB-231. Figure 5 shows that after 3 days of 10mM BMS-191011 treatment (5A), strong fluorescent signals (5B) were detected in MDA-MB-231. Furthermore, Figure 5C shows the presence of pro-caspase-3 protein expression in three TNBC cell lines, MDA-MB-231, HCC1143, and SUM159. BMS-191011 (20mM) treatment for two days induced cleaved caspase-3 expression in MDA-MB-231 (5D). These results suggested that BK channel opener can induce caspase-3 activation in MDA-MB-231 cells. Thus, hyperpolarization can induce activation of caspase-3 and apoptosis in TNBC cells.
6) BK channel opener prevented migration of MDA-MB-231
Majority of breast cancer patients die due to tumor metastasis and one critical step of metastasis is migration [36]. If BMS-191011 effectively induced cell death, it may prevent migration of MDA-MB-231 cells. Supplemental Figure 10 shows a typical scratch assay (or “wound heal”) imaging experiment [37]. While control (untreated) MDA-MB-231 cells can recover from the scratch within 4 hours (upper), 100nM BMS-191011 prevented migration of MDA-MB-231 cells within 4 hours (lower). Similar results were obtained in an additional four experiments.
7) BK channel opener induced arrest in cell cycle G2 phase in MDA-MB-231
Figure 6 shows the effect of opening BK channels in MDA-MB-231 cell cycle using flow cytometry. In the absence of BMS-191011 treatment, cell distribution in G1/S/G2 phases are approximately equal (A), BMS-191011 treatment after 24h resulted in arrest in S/G2 phases associated with a decrease distribution in G1 phase (B). After 48h, cells are all arrested in G2 phase (C). As a control, we showed that DMSO (1%) had no effects on cell cycle in MDA-MB-231 cells after 24h treatment (Supplemental Fig.11). Similar results were repeated in an additional three experiments.
8) BK channel opener induced slower growth of MDA-MB-231 xenograft in NSG mice
To test the inhibitory effects of BK channel opener on TNBC tumor in vivo, we generated MDA-MB-231 xenograft in female NSG mice at 4-week old age [21]. After a sizable tumor was formed (typically after 1-2 weeks of cell injection), BMS-191011 at 100mg/kg (or 1-2mg/mouse) was directly injected into the tumor for better control of the dose and the potential loss of the drug due to rapid metabolism in mice. The drug was given twice a week in the treat group. For control group, saline was given twice a week. To avoid large variation in tumor sizes due to heterogeneity of breast cancer, we selected pairs of tumors (one as a control – injected PBS only, the other treated – injected drug in PBS) that had similar tumor sizes, and performed ultrasound imaging for four weeks to monitor effect of BK channel opener during the growth of tumors.
Figure 7 shows a representative example for three pairs of control and treated tumors. The injection of the drug began at week5 when three pairs (C1/T1, C2/T2, and C3/T3) had similar tumor sizes, the treated tumor (T) grew significantly slower than the control tumor (C) each week (7A). In week 8, averaging data showed a 33% reduction of tumor volume in drug-treated group (T= 710±105, n=8) compared to the control group (C=1056±106, n=8) (p<0.05) (7B).
9) BK channel opener and cardiac function in vivo
Cardiac toxicity is a major concern in anti-cancer drugs [38]. Supplemental Figure 12 shows the echocardiograph results for MDA-MB-231 xenograft treated mice with high dose (0.1mg/kg) of BMS-191011 compared to control (PBS treated) mice (n=3). There are no significant differences (p>0.05) between the two groups in cardiac function including heart rate, ejection fraction, left ventricular mass, and cardiac output.