Repurposing hyperpolarization‐activated cyclic nucleotide‐gated channels as a novel therapy for breast cancer

Figures

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  FIGURE 1. HCN 2 and HCN3 were overexpressed in breast cancer and associated with poor survival outcomes. A, Protein expressions of HCN2 and HCN3 were determined in breast cancer cell lines compared with non‐neoplastic breast epithelial cell line MCF‐10A by western blot. Actin was used as the loading control. Results were repeated three times and representative images are shown. B, HCN2 and HCN3 were predominantly localized to the cell membrane. MDA‐MB‐231 and MDA‐MB‐453 cells were used, and fractionation was performed to isolate different cellular fractions. Western blot was employed to determine the expression of the proteins. Lamin B1, PMCA and tubulin were used as nuclear (N), cell membranous (M) and cytoplasmic (C) markers, respectively. Results were repeated three times and representative images are shown. C, HCN2 was overexpressed in breast cancer. IHC was employed to determine the expression of HCN2 in normal (N = 38) and tumor tissues (N = 279). Mann−Whitney U test (P = 7.75 × 10−14) and chi‐square test (P = 7.57 × 10−11) were employed to determine the statistical significance. The scale bar represents 18 μm. D, HCN3 was overexpressed in breast cancer. IHC was employed to determine…

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  FIGURE 2. Suppression of HCN2 and HCN3 inhibited breast cancer cell growth in vitro. The knockdown of HCN2 could suppress the cell proliferation of TNBC cells A, MDA‐MB‐231 and B, MDA‐MB‐453 in the short term as assessed by MTT assay. Two independent shRNAs against HCN2, shHCN2.1 and shHCN2.2 were used. Results were shown as mean ± SD from three independent experiments. The knockdown of HCN3 could suppress the cell proliferation of TNBC cells C, MDA‐MB‐231 and D, MDA‐MB‐453 in the short term as assessed by MTT assay. Two independent shRNAs against HCN3, shHCN3.1 and shHCN3.2 were used. Results were shown as mean ± SD from three independent experiments. Two‐way ANOVA revealed there was a statistically significant interaction between the HCN2/3 knockdown and time (A: F = 2.71, P < .05; B: F = 4.71, P < .001; C: F = 6.00, P < .001; D: F = 7.43, P < .001). Statistical significance between shCtrl and shHCN2.1 or shHCN2.2 or between shCtrl and shHCN3.1 or shHCN3.2 at each time point was determined by Bonferroni test. *, ** and *** represent P < .05, P < .01 and P < .001 respectively. The knockdown of either HCN2 or HCN3 could suppress the cell proliferation of TNBC cells E, MDA‐MB‐231 and F,…

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  FIGURE 3. Ivabradine suppressed TNBC tumor growth in vivo. A, A comparison of tumours isolated from nude mice treated with 15 mg/kg of Ivabradine (N = 5) or water (N = 5) administered subcutaneously daily for 4 weeks. MDA‐MB‐231 was used to establish the xenografts. The graph shows the change in tumor volume with the time between Ivabradine and water‐treated groups. Results were shown as mean ± SD from five tumours. Students’ t‐test was used. ** represents P < .01. B, A comparison of tumours isolated from nude mice treated with 15 mg/Kg of Ivabradine (N = 4) or water (N = 4) administered subcutaneously daily for 4 weeks. MDA‐MB‐453 was used to establish the xenografts. The graph showed the change in tumor volume with the time between Ivabradine and water‐treated groups. Students’ t‐test was used to compare tumor sizes at the various time points between the Ivabradine and water‐treated groups. Results were shown as mean ± SD from four tumours. * and ** represent P < .05 and P < .01, respectively. C, Treatment with Ivabradine could suppress cell proliferation in tumours developed from MDA‐MB‐231 (Panel A) and MDA‐MB‐453 (Panel B). IHC was employed to determine the expression of cell…

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  FIGURE 4. RNA‐seq analysis revealed transcriptome changes following inhibition of HCN. A, Ivabradine exerted distinctively different effects on TNBC cells compared with non‐cancerous breast cells. MDA‐MB‐231, MDA‐MB‐453 and MCF‐10A were used. The cells were either treated with 100 μM of Ivabradine or water for 72 h. RNA sequencing was performed to determine the gene expression profile. Gene expression levels were determined by transcript per millions (TPM). The differential expressions of genes in each of the cell lines between 72 h Ivabradine‐treated and water control were determined. The results from MDA‐MB‐231 and MDA‐MB‐453 were grouped as TNBC. The mean value of the differential expression level of each of the genes from MDA‐MB‐231 and MDA‐MB‐453 was calculated. A heatmap was plotted to show the distinctive effect of Ivabradine on differential gene expression profiles between normal breast and TNBC cells. B, Venn diagram was plotted to show the number of genes commonly and upregulated explicitly in MDA‐MB‐231 and MDA‐MB‐453 compared with MCF‐10A. A total of 177 genes were found. C, Gene ontology enrichment analysis was performed to identify the biological processes that were…

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  FIGURE 5. Inhibition of HCN channels reduced the level of intracellular Ca2+ and triggered ER‐stress. A, Ivabradine depleted intracellular Ca2+ levels in breast cancer cell lines but not in normal epithelial cell line MCF‐10A. A total of 4 μM of Fura‐2 was used to stain intracellular calcium ions for 30 min. Live cell imaging was performed to capture the image (Panel A). Infinite F200 microplate reader was employed to record the fluorescent signal from 10 000 cells (Panel B). Results were shown as mean ± SD from three independent experiments. Students’ t‐test was employed to determine statistical significance between H2O and Ivabradine‐treated groups. *** represents P < .001. The scale bar represents 5 μm. B, HCN inhibition by Ivabradine significantly induced ER‐stress response as measured by ATF4 luciferase reporter activities. Renilla luciferase was used as the internal control. A total of 10 μM of Ivabradine was used to treat the cells for 72 h. Results were shown as mean ± SD from three independent experiments. Students’ t‐test was employed to determine statistical significance between H2O and Ivabradine‐treated groups. *** represents P < .001. C, Ivabradine could enhance the protein…

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  FIGURE 6. Knockdown of HNC2‐ and HCN3‐induced ER‐stress and apoptosis in breast cancer cells. Knockdown of HCN2 and HCN3 in A, MDA‐MB‐231 and B, MDA‐MB‐453 could enhance the activity of ATF4. The cells were treated with 50 pmol of siHCN2, siHCN3, or non‐targeting siRNA (siCtrl) for 72 h. ER‐stress response was determined by ATF4 luciferase reporter assay. Results were shown as mean ± SD from three independent experiments. Students’ t‐test was employed to determine statistical significance between two groups. * and *** represents P < .05 and P < .001, respectively. C, Knockdown of HCN2 and HCN3 promoted cytochrome c release to cytoplasm. The cells were treated with 50 pmol of siHCN2, siHCN3 and non‐targeting siRNA (siCtrl) for 72 h. Mitochondria Isolation Kit was performed to isolate cytoplasmic fraction which was free of mitochondria. Western blot was employed to determine the expression of cytochrome c in the fraction. Tubulin served as loading control and cytoplasmic marker. GAPDH served as mitochondrial marker. Representative image shown from three independent experiments. Knockdown of HCN2 and HCN3 enhanced caspase activity in D, MDA‐MB‐231 and E, MDA‐MB‐453 but not in F, MCF‐10A. The…

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  FIGURE 7. The synergistic effect of Ivabradine with paclitaxel. The curve showed the effect of paclitaxel on ER‐stress in A, MDA‐MB‐231 and B, MDA‐MB‐453. The curve showed the effect of Ivabradine on ER stress in C, MDA‐MB‐231 and D, MDA‐MB‐453. The effect of the addition of 100, 250 and 500 nM of Ivabradine on ER‐stress in E, MDA‐MB‐231 and F, MDA‐MB‐453 treated with paclitaxel. ER‐stress was determined using luciferase reporter with ATF4 response element. The assay was performed 24 h post drug treatment. Results were shown as mean ± SD from six independent experiments. G, A table showed the EC50 of paclitaxel with or without Ivabradine. Combination Index (CI) was calculated. The dose‐dependent effect of paclitaxel on caspase activities in H, MDA‐MB‐231 and I, MDA‐MB‐453. The dose‐dependent effect of Ivabradine on caspase activities in J, MDA‐MB‐231 and K, MDA‐MB‐453. Results were shown as mean ± SD from six independent experiments. One‐way ANOVA was used. Bonferroni was used as post‐test of one‐way ANOVA to determine the statistical significance between the control (0 nM) and the treatment groups. *** represents P < .001. L, Cotreatment of Ivabradine and paclitaxel could enhance…