CACNA1D overexpression and voltage-gated calcium channels in prostate cancer during androgen deprivation

Figures

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  Figure 1. Profiling CACNA1D and CaV1.3 expression in clinical PCa datasets and tissue microarrays. (A) Human CACNA1D transcript expression was determined by RNA-seq analysis from 7932 TCGA samples from 17 different cancer tissues. Data was obtained from The Human Protein Atlas (www.proteinatlas.org/) ENSG00000157388-CACNA1D/pathology (v21.1.proteinatlas.org), is presented as box-and-whisker plots (median, 10–90 percentile) and was analysed with Kruskal–Wallis, Dunn’s multiple comparison test p < 0.001 (***). (B) Scatter plot showing CACNA1D expression in primary (n = 131) PCa and metastatic PCa (n = 19) compared to adjacent benign prostate tissue (n = 29). Data was derived from GEO dataset, GSE21032 (GPL 5188) conducted by Memorial Sloan-Kettering Cancer Centre (MSKCC). Data is expressed as median and inter-quartile range and was analysed using Kruskal–Wallis, Dunn’s multiple comparison test, p < 0.001 (***). C. CACNA1D expression was compared by Gleason grade (6, 7 and 8–9) between primary and metastatic PCa samples. Data was derived from GEO dataset, GSE21032 (GPL 5188, MSKCC) and analysed using Kruskal–Wallis, Dunn’s multiple comparison test. Data is expressed as median and…

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  Figure 2. CaV1.3 expression profiling using in vitro PCa cell lines. (A) qPCR was performed to assess CACNA1D transcript expression in normal and PCa cell lines. RPLPO was used as an endogenous reference control. Fold changes were determined by normalisation to RPLPO and RWPE-1 transcript levels. Data was analysed using 1-way ANOVA (Dunnett’s) where p < 0.01 (**), p < 0.001 (***) comparing PCa cells to RWPE-1 (N indicated above each bar). (B) Protein expression of CaV1.3 and androgen receptor, AR was analysed in the panel of cell lines in Western blots with GAPDH as a loading control (N = 3). Uncropped original blots are displayed in Supplementary Fig. S4. (C) Immunofluorescence micrographs of CaV1.3 (green) and nuclear staining by DAPI (blue) for each cell line (N = 3). White boxes in the top row of micrographs are shown at higher magnification in the bottom row. (D) Summary data (mean, SEM) of proliferation across LNCaP, C4-2 and C4-2B treated with nifedipine (0–100 µM) for 72 h (N = 3) showing concentration-dependent decrease in proliferation.

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  Figure 3. Changes in CACNA1D and CaV1.3 expression during recapitulation of hormone therapy. (A) CACNA1D transcript expression in CSFBS-cultured and ENZ-treated LNCaP. Transcription expression was normalised to control/vehicle CACNA1D expression and RPLPO housekeeping gene (N indicated above the bars, ‘d’ denotes days). Data was analysed with Kruskal–Wallis test (Dunn’s multiple comparison), p < 0.05 (*) or p < 0.001 (***). (B) Western blotting micrographs showing CaV1.3 and NSE expression during CSFBS-culturing and ENZ treatment (4–14 days, N = 3). GAPDH was used as a loading control. Uncropped original blots are displayed in Supplementary Figs. S5 and S6. (C) Representative light micrographs showing neurite-like extensions in CSFBS-cultured LNCaP compared to untreated controls at 10 days. Scale bar (white line) = 100 µm. (D) Average neurite length (µm) in time controls and CSFBS-cultured LNCaP, and during ENZ were compared. Data was analysed using unpaired t-tests with Welch’s correction where p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (N indicated within bars). (E) Nifedipine had no effect on neurite length (µm) in 10 day and 14 day CSFBS-cultured LNCaP. Data was analysed using 1-way…

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  Figure 4. CaV1.3-mediated Ca2+ signalling emerges in LNCaP during CSFBS culturing. (A) Pseudocolour micrographs of Cal-520 AM-loaded LNCaP (cultured in media containing normal FBS) during live-cell recordings. Pseudocolour scale indicating increased fluorescence intensity from blue–green–yellow–red, indicating increased intracellular [Ca2+]. Fluorescence micrographs in normal Hanks' (0 s) and during perfusion with high K+ Hanks containing Bay K 8644 (10 µM, denoted by solid horizontal bar) (100 s) are shown. Five cells (1–5, white arrows) were selected and ΔF/F0 measured then plotted on a fluorescence intensity-time graph (coloured lines, lower panel) (N = 5 recordings). (B) Pseudocolour micrographs showing CSFBS-cultured LNCaP (10 days) pre- (0 s) and during exposure (100 s) to high K+/Bay K 8644. Fluorescence intensity of 5 selected cells demonstrating Ca2+-transients in 4 of the 5 cells (lower panel) (N = 3 recordings). (C) Mean percentage of responding cells per recording is shown at several time points during CSFBS along with matched time controls. Data analysed with 1-way ANOVA with Šídák's multiple comparison test, p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) (N = 3). (D)…

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  Figure 5. Voltage-gated inward currents in LNCaP emerge after CSFBS culturing. (A) Representative recordings from untreated LNCaP in Ba2+/TEA Hanks, and detection of inward currents in divalent-free solution. (B) Summary current–voltage plot of mean current from 6 cells (N = 2 cultures) in BaLNCaP exhibited high K/TEA and divalent free conditions. (C) Summary current–voltage plot of divalent-free currents in untreated LNCaP (n = 8, N = 2 cultures, overlapping with n = 6 data from panel B) and CSFBS-LNCaP up to 25 days treatment (n = 11, N = 4). Two-way ANOVA, Bonferroni post-hoc tests showed significant increase in mean current at − 50, − 40, − 30 and − 20 mV (*p < 0.05, **p < 0.01; ***p < 0.001). (D) Representative currents in divalent-free from an LNCaP cultured in CSFBS or 25 days in the presence of nifedipine and upon further addition of mibefradil. (E) Summary current–voltage plot for the effect of nifedipine (1 μM) alone and combined with (0.3 μM mibefradil) in CSFBS treated cells (n = 6; N = 4) where mibefradil significantly reduced current amplitude. (F) In CSFBS treated cells (14 days), nifedipine-sensitive currents were less frequently encountered and the effect of nifedipine was…

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  Figure 6. CaV-mediated Ca2+ signalling in LNCaP after Enzalutamide treatment. (A) LNCaP exhibited high K+/Bay K8644-Ca2+ -transients after ENZ for 4 days or 10 days; Kruskal–Wallis with Dunn’s multiple comparison test, p < 0.01 (**) (N = 3). (B) The percentage of cells, treated with ENZ for 10 days, responding to the stimulus was significantly reduced in the presence of nifedipine (1 µM, unpaired t-test, p < 0.01 (**) (N = 3). Note, data for 10 day ENZ in the absence of nifedipine is replotted from panel (A). (C) Summary current–voltage plot of divalent-free currents in untreated, ENZ-LNCaP 4 days and 14 days treatment (n = 8, N = 2 cultures; n = 11, N = 4 cultures; n = 5, N = 2 cultures respectively). Two-way ANOVA, Bonferroni post-hoc tests showed significant increase in mean current between untreated vs 4 days ENZ, or untreated vs 14 days ENZ (*p < 0.05, **p < 0.01; ***p < 0.001). (D) Summary current–voltage plot for ENZ-LNCaP (4 days treatment) (n = 9 cells, N = 4 cultures) currents recorded in divalent-free before and after nifedipine (1 μM). (E) Representative recordings from ENZ-LNCap (14 days) in divalent-free and in nifedipine. (F) Summary current–voltage graph showing currents in…

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  Figure 7. CaV-mediated Ca2+ signalling in T2E+ VCaP. (A) Representative currents from VCaP in Ba2+ /TEA Hanks and in divalent-free solution. (B) A portion of the inward current was Ni2+-sensitive; the residual current–voltage plot was right-shifted by 20 mV (n = 7 cells, N = 5 cultures). (C) Summary current–voltage plot for VCaP currents (n = 9 cells, N = 7 cultures) recorded in Ba2+/TEA Hanks and in divalent-free solution. (D) Representative recordings from 5 VCaP perfused with Hanks. Oscillating cells (green and red) were typical of 2.9 ± 1.2% (mean ± SEM) VCaP within each field of view (N = 5 recordings). (E) VCaP were challenged with high K+/Bay K 8644 (10 µM) as indicated with a horizontal bar, and Ca2+ transients could frequently be recorded. Five ROI are plotted on a fluorescence-time graph (typical of N = 3 recordings). (F) ENZ significantly increased the percentage of high K+/Bay K 8644-responsive VCaP per recording compared with vehicle controls (1-way ANOVA with Šídák's multiple comparison test, p < 0.01 (**) or p < 0.001 (***), N = 4). (G) The average amplitude (ΔF/F0) of high K+/Bay K 8644-induced Ca2+events were similar in the absence and presence of ENZ or nifedipine.