Therapeutic potential of TRPM8 antagonists in prostate cancer

Abstract

Transient receptor potential melastatin-8 (TRPM8) represents an emerging target in prostate cancer, although its mechanism of action remains unclear. Here, we have characterized and investigated the effects of TRPM8 modulators in prostate cancer aggressiveness disclosing the molecular mechanism underlying their biological activity. Patch-clamp and calcium fluorometric assays were used to characterize the synthesized compounds. Androgen-stimulated prostate cancer-derived cells were challenged with the compounds and the DNA synthesis was investigated in a preliminary screening. The most effective compounds were then employed to inhibit the pro-metastatic behavior of in various PC-derived cells, at different degree of malignancy. The effect of the compounds was then assayed in prostate cancer cell-derived 3D model and the molecular targets of selected compounds were lastly identified using transcriptional and non-transcriptional reporter assays. TRPM8 antagonists inhibit the androgen-dependent prostate cancer cell proliferation, migration and invasiveness. They are highly effective in reverting the androgen-induced increase in prostate cancer cell spheroid size. The compounds also revert the proliferation of castrate-resistant prostate cancer cells, provided they express the androgen receptor. In contrast, no effects were recorded in prostate cancer cells devoid of the receptor. Selected antagonists interfere in non-genomic androgen action and abolish the androgen-induced androgen receptor/TRPM8 complex assembly as well as the increase in intracellular calcium levels in prostate cancer cells. Our results shed light in the processes controlling prostate cancer progression and make the transient receptor potential melastatin-8 as a 'druggable' target in the androgen receptor-expressing prostate cancers.

Figure 1

Effect of TRPM8 modulators on DNA synthesis elicited by androgens in PC-derived LNCaP cells. LNCaP cells were used. In (a), lysate protein from cells unstimulated (–) or stimulated for 24 h with 10 nM R1881 were analyzed by WB technique, using the antibodies against the indicated proteins. WB are representative of three different experiments and densitometric analysis of data is reported in Supplementary Information. In (b–f) quiescent LNCaP cells on coverslips were left untreated or treated for 18 h with 10 nM R1881, in the absence or presence of decreasing (1 μM, 100 nM, 10 nM or 1 nM) of the indicated TRPM8 modulators. Cells were pulsed in vivo with 100 μM BrdU and its incorporation into newly synthesized DNA was analyzed by IF, as described in “Methods”. BrdU incorporation was expressed as % of total cells. Means and standard errors (SEMs) are shown. n, represents the number of experiments. *p < 0.05 for the indicated experimental points vs. the corresponding untreated control.

Figure 2

TRPM8 antagonists, 4 and 6, specifically affect the DNA synthesis and cell cycle progression elicited by androgens in LNCaP cells. LNCaP cells were employed. When indicated in Figure, the compounds 4 and 6 were used at 1 μM. Panel (a) shows representative images captured from one experiment presented in Fig. 1 (panels d,e). Fluorescence (red) in the lower section is from reactivity with the anti-BrdU antibody. Hoechst 33,258 staining (blue) is shown in the upper section. Bar, 10 μM. In (b), quiescent cells on coverslips were left untreated or treated for 18 h with 1 nM R1881 in the absence or presence of the indicated compounds. Cells were pulsed in vivo with 100 μM BrdU and its incorporation into DNA was analyzed and expressed as in Fig. 1. In (c–f), quiescent cells were left untreated or treated with 10 nM R1881 (c–d) or 1 nM R1881 (e–f) for 15 h, in the absence or presence of the indicated compounds. Lysate proteins were prepared and analyzed by WB technique, using the antibodies against the indicated proteins. WB are representative of three different experiments and the corresponding densitometric analysis is reported in Supplementary Information. In (g), quiescent cells on coverslips were left untreated or treated for 18 h with 10 nM estradiol (E₂), in the absence or presence of the indicated compounds. Cells were pulsed in vivo with 100 μM BrdU and its incorporation into DNA was analyzed and expressed as in Fig. 1. In (b,g), means and standard errors (SEMs) are shown. n, represents the number of experiments. * p < 0.05 for the indicated experimental points vs. the corresponding untreated control.

Figure 3

TRPM8 antagonists, 4 and 6, affect the DNA synthesis in various PC-derived cells. In (a), lysate protein from C4-2B, 22Rv1 and DU-CaP were analyzed by WB technique, using the antibodies against the indicated proteins. WB are representative of three different experiments and densitometric analysis of data is reported in Supplementary Information. In (b), cycling C4-2B cells on coverslips were left un-challenged or challenged for 18 h with increasing (100 nM, 1 μM) concentrations of TRPM8 modulators 4 and 6 (left panel) or enzalutamide (10 μM; right panel). In c and d, quiescent 22Rv1 (c) and DU-CaP (d) cells on coverslips were left untreated or treated for 18 h with 10 nM R1881, in the absence or presence of compounds 4 and 6 (at 1 μM) or enzalutamide (at 10 μM). In (b–d), cells were pulsed in vivo with 100 μM BrdU. Its incorporation into newly synthesized DNA was analyzed by IF and expressed as % of total cells. Means and standard errors (SEMs) are shown. n, represents the number of experiments. *p < 0.05 for the indicated experimental points vs. the corresponding untreated control.

Figure 4

Effect of TRPM8 modulators on migration and invasion induced by androgens in LNCaP cells. Quiescent LNCaP cells were used. In (a), cells were wounded and left unstimulated or stimulated with 10 nM R1881 for 20 h, in the absence or presence of the indicated compounds (at 1 μM). In (b), wounded cells were left unstimulated or stimulated with 10 nM R1881 for 20 h, in the absence or presence of the indicated concentrations of TRPM8 modulators. In (a), phase-contrast images are representative of three different experiments, each in duplicate. In (b), the wound area was calculated using Leica Suite Software. Data are presented as % of residual wound width. The standard deviations were < 0,05 for each experimental condition. LNCaP, 22Rv1 and DU-CaP cells were used for migration (left panels in c–e) or invasion (right panels in c–e) assays. The cells were left unstimulated or stimulated with 10 nM R1881, in the absence or presence of the indicated compounds (at 1 μM). Migrating or invading cells were detected and counted as reported in “Methods”. Results from three different experiments were collected and expressed as fold increase. Means and SEMs are shown. n represents the number of experiments. *p < 0.05 for the indicated experimental points vs. the corresponding untreated control.

Figure 5

TRPM8 antagonists, 4 and 6, inhibit the androgen-induced increase in LNCaP cell spheroid size. LNCaP cells were employed in 3D cultures and 3 days after the cell’s embedding in Matrigel, representative images were acquired as described in “Methods”. 3D cultures were left untreated or treated with 10 nM R1881, in the absence or presence of the indicated compounds (at 1 μM) for 15 days. Shown are phase-contrast images, representative of 3 different experiments and captured at 15th day. Scale bar, 100 μm. In (b), the spheroid size was calculated as described in “Methods” and expressed as fold increase in the relative spheroid size. Means and SEMs from three different experiments are shown. * p < 0.05 for the indicated experimental points vs. the corresponding untreated control.

Figure 6

TRPM8 antagonists inhibit the rapid androgen action by disrupting the ligand-induced AR/TRPM8 complex assembly in LNCaP cells. In (a–e), quiescent LNCaP were used. When indicated the compounds 4 and 6 were used at 1 μM. Enzalutamide (enz) was added at 10 μM. In (a), cells were left untreated or treated with increasing R1881 concentration (0,1, 1 or 10 nM), in the absence or presence of the indicated compounds for 48 h. Conditioned media were collected and PSA was assayed as described in “Methods”. Means and SEMs from three different experiments are shown. *p < 0.05 for the indicated experimental points versus the corresponding untreated control. In (b–e), cells were unstimulated or stimulated with 10 nM R1881, in the absence or presence of the indicated compounds for 10 min. Lysate proteins were analyzed by WB technique using the antibodies against the indicated proteins. P-FAK stands for Tyr 397-P-FAK; P-paxillin stands for Tyr 118-P-paxillin; P-RSK stands for P-Ser 380 RSK; P-ERK stands for phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204); P-Src stands for P-Tyr 416 Src and P-Akt stands for P-Ser 473 Akt. The filters were stripped and re-probed using anti tubulin antibody, as loading control. In the upper panel in (d) and left section in (e), lysate proteins were analyzed by WB using the antibodies against AR or TRPM8. Lysate proteins were immunoprecipitated using the anti-AR antibody (anti-AR, lower panel in (d) and right section in (e) or control IgG (anti-IgG; middle panel in (d) and middle section in e). Proteins in immune-complexes were revealed by WB using the antibodies against the indicated proteins. Results in (b–e) are representative of 3 different experiments and the densitometric analysis is reported in Supplementary Information.

Figure 7

TRPM8 antagonists inhibit the androgen-induced increase of intracellular Ca²⁺ levels in LNCaP cells. In (a), LNCaP cells were made quiescent and loaded with 1 µM 4-Fluo AM, as described in “Methods”. Cells were left untreated or treated for 240 s with 10 nM R1881, in the absence or presence of the indicated compounds (at 1 μM). Different fields were analyzed by fluorescence (upper images) or phase-contrast (lower images) microscopy, as described in “Methods”. Representative images were acquired using a DFC 450C camera (Leica). Bar, 10 μM. In (b), the mechanism of TRPM8 antagonist action in PC cells is shown. Androgens rapidly induce association of AR with TRPM8 in androgen-sensitive PC cells. Once assembled, this complex leads to intracellular calcium increase and promotes proliferation as well as pro-metastatic phenotype of PC cells. By perturbing the androgen-challenged AR/TRPM8 complex assembly, TRPM8 antagonists (compounds 4 and 6) lower the intracellular calcium levels and impair the proliferation, invasion and growth in 3D of PC cells.