TRPM8 indicates poor prognosis in colorectal cancer patients and its pharmacological targeting reduces tumour growth in mice by inhibiting Wnt/β-catenin signalling

Abstract

Background and purpose: Transient receptor potential melastatin type-8 (TRPM8) is a cold-sensitive cation channel protein belonging to the TRP superfamily of ion channels. Here, we reveal the molecular mechanism of TRPM8 and its clinical relevance in colorectal cancer (CRC).

Experimental approach: TRPM8 expression and its correlation with the survival rate of CRC patients was analysed. To identify the key pathways and genes related to TRPM8 high expression, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted in CRC patients. TRPM8 functional role was assessed by using Trpm8^-/- mice in models of sporadic and colitis-associated colon cancer. TRPM8 pharmacological targeting by WS12 was evaluated in murine models of CRC.

Key results: TRPM8 is overexpressed in colon primary tumours and in CD326⁺ tumour cell fraction. TRPM8 high expression was related to lower survival rate of CRC patients, Wnt-Frizzled signalling hyperactivation and adenomatous polyposis coli down-regulation. In sporadic and colitis-associated models of colon cancer, either absence or pharmacological desensitization of TRPM8 reduced tumour development via inhibition of the oncogenic Wnt/β-catenin signalling. TRPM8 pharmacological blockade reduced tumour growth in CRC xenograft mice by reducing the transcription of Wnt signalling regulators and the activation of β-catenin and its target oncogenes such as C-Myc and Cyclin D1.

Conclusion and implications: Human data provide valuable insights to propose TRPM8 as a prognostic marker with a negative predictive value for CRC patient survival. Animal experiments demonstrate TRPM8 involvement in colon cancer pathophysiology and its potential as a drug target for CRC.

Keywords: TRPM8; Wnt/β-catenin; colon cancer; pharmacology; transient receptor potential channels.

FIGURE 1

Transient receptor potential melastatin type‐8 (TRPM8) channel expression in patients with colorectal cancer (CRC). (a, b) Kaplan–Meier survival analysis of CRC patients with low TRPM8 expression (green) and high TRPM8 expression (red) was used to analyse the (a) progression‐free survival probability and (b) disease‐specific survival probability. P values were determined using the log‐rank test, and the exact values are reported at the left bottom of the figures. (c, d) mRNA expression of TRPM8 was analysed by reverse transcription quantitative polymerase chain reaction (RT–qPCR) and calculated by using the 2^−ΔΔCt formula (n = 34 different biological samples) in normal tissues (collected at least 10 cm from the tumour lesion) and primary tumours removed from patients with CRC diagnosis. Error bars represent ±SEM. P values were determined using paired t test. *P < 0.05 versus normal. (e) Representative immunoblots of TRPM8 protein levels in human CRC specimens (primary tumours) and the surrounding nontumorous tissues (normal). Lower panel shows densitometric analysis of western blot analysis. Results show mean ± SEM of eight different biological samples. P value were determined using paired t tests. *P < 0.05 versus normal. (f, g) Representative flow cytometric analysis showing the enrichment of CD326⁺ human tumour cells before (f) and after (g) cells purification from primary specimens. CD31 was used to exclude the infiltration of leukocytes and endothelial cells. (h) mRNA expression of TRPM8 was analysed by RT–qPCR and calculated by using the 2^−ΔΔCt formula (n = 6 independent experiments) in immortalized human colonic epithelial cells (HCEC) and primary tumour cells isolated from the bulk tumour by magnetic separation. Error bars represent ±SEM. P values were determined using unpaired t tests. *P < 0.05 versus HCEC.

FIGURE 2

Loss of Trpm8 reduces azoxymethane (AOM)‐induced sporadic colon tumours. (a) Schematic representation of AOM protocol and timeline. Mice were injected with 10 mg·kg⁻¹ of AOM at the indicated time. (b) Representative colonoscopy images of wild‐type (WT) and Trpm8 ^−/− mice carried out on the day of killing. All mice were subjected to endoscopy (n = 8). (c) Number of tumours per mouse in WT and Trpm8 ^−/− mouse colon treated with vehicle (black) or AOM (red) (n = 8 mice). Error bars represent ±SEM. P value was determined using two‐way ANOVA followed by Sidak's multiple comparisons test. *P < 0.05 Trpm8 ^−/− AOM versus vehicle, *P < 0.05 Trpm8 ^−/− AOM versus WT AOM and *P < 0.05 WT AOM versus vehicle. (d) Kaplan–Meier survival analysis of WT and Trpm8 ^−/− mice treated with AOM. P values were determined using the Mantel–Cox test. n.s. = non‐significant and *P < 0.05 versus Day 0. (e) Representative confocal images of β‐catenin (green) and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue) immunostaining in WT and Trpm8 ^−/− tumours collected at Day 91. Scale bars = 50 μm. Analyses were carried out from three mice for each experimental group.

FIGURE 3

Loss of Trpm8 reduces azoxymethane (AOM)/dextran sodium sulfate (DSS)‐induced colitis‐associated colon cancer. (a) Schematic representation of AOM/DSS protocol and timeline. Mice were injected intraperitoneally with 12.5 mg·kg⁻¹ AOM on the first day of the experiment. DSS was administered in drinking water at the indicated doses at the indicated time. (b) Representative colonoscopy images taken at the end of the carcinogenic protocol (Day 63). All mice were subjected to endoscopy (n = 6). (c) Number of tumours per mouse counted microscopically in wild‐type and Trpm8 ^−/− mice, treated with vehicle (black) or AOM/DSS (red); n = 6 mice for wild‐type vehicle and wild‐type and Trpm8 ^−/− AOM; n = 5 mice for Trpm8 ^−/− vehicle. Error bars represent ±SEM. P values were determined using two‐way ANOVA followed by Sidak's multiple comparisons test. *P < 0.05 Trpm8 ^−/− AOM/DSS versus vehicle, *P < 0.05 Trpm8 ^−/− AOM/DSS versus wild‐type AOM/DSS and *P < 0.05 wild‐type AOM/DSS versus vehicle. (d) Tumour grade of wild‐type (black) and Trpm8 ^−/− (red) mouse tumours, evaluated by colonoscopy (tumours counted in n = 6 mice). Error bars represent ±SEM. P value was determined using two‐way ANOVA followed by Sidak's multiple comparisons test. *P < 0.05 Grade 1 Trpm8 ^−/− versus wild‐type mice. (e) Analysis of the inflammatory parameters by the murine endoscopic index of colitis severity (MEICS) in wild‐type and Trpm8 ^−/− mice treated with vehicle (black) or AOM/DSS (red); n = 6 mice for wild‐type vehicle and wild‐type and Trpm8 ^−/− AOM; n = 5 mice for Trpm8 ^−/− vehicle. Error bars represent ±SEM. P values were determined using two‐way ANOVA followed by Sidak's multiple comparisons test. n.s. = non‐significant Trpm8 ^−/− AOM/DSS versus wild‐type AOM/DSS and *P < 0.05 wild‐type and Trpm8 ^−/− AOM/DSS versus vehicle. (f, g) Analysis of the macroscopic inflammatory parameters colon weight/colon length ratio (f) and spleen weight (g) in wild‐type and Trpm8 ^−/− mice treated with vehicle (black) or AOM/DSS (red); n = 6 mice for wild‐type vehicle and wild‐type and Trpm8 ^−/− AOM; n = 5 mice for Trpm8 ^−/− vehicle. Error bars represent ±SEM. P values were determined using two‐way ANOVA followed by Sidak's multiple comparisons test. n.s. = non‐significant and *P < 0.05 (f) wild‐type and Trpm8 ^−/− AOM/DSS versus vehicle.

FIGURE 4

Kyoto Encyclopedia of Genes and Genomes (KEGG) Wnt signalling pathway. (a) Overlay of the differentially expressed genes (DEGs) between high and low Trpm8 expression groups on the KEGG pathway. The overexpressed and underexpressed genes are represented in red and green, respectively. (b) Gene set enrichment analysis (GSEA) enrichment plot. In green, the enrichment profile; in black on the X‐axis, the hits. Colour scale on the X‐axis goes from high (red) to low (blue) expression of genes in the high Trpm8 group. (c) Cnetplot showing the linkages between genes and biological concepts. The overexpressed and underexpressed genes are represented in red and green, respectively. (d, e) Expression of Wnt2 (d) and Wnt11 (e) by RNA sequencing analysis on the colon of wild‐type (blue) and Trpm8 ^−/− (orange) mice, treated with vehicle or azoxymethane (AOM) (n = 6 mice). P values were determined using multiple Student's t test. *P < 0.05 wild‐type vehicle versus Trpm8 ^−/− vehicle.

FIGURE 5

Pharmacological modulation of Trpm8 reduces tumour growth in azoxymethane (AOM) and xenograft models of colon cancer by impairing the Wnt/β‐catenin pathway. (a) Schematic representation of the AOM protocol and timeline. Mice were injected with 10 mg·kg⁻¹ of AOM at the indicated time. WS12 (10 mg·kg⁻¹) was administered intraperitoneally every other day. (b) Number of tumours per mouse counted on wild‐type (blue) and Trpm8 ^−/− (orange) mouse colon, treated with vehicle (n = 8 mice), AOM (n = 8 mice) or AOM + WS12 (n = 6 wild‐type mice and n = 3 Trpm8 ^−/− mice, three dead mice values were excluded, and this experimental group was not subjected to statistical analysis). P values were determined using two‐way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05 Trpm8 ^−/− AOM versus vehicle, *P < 0.05 wild‐type AOM + WS12 versus AOM and *P < 0.05 wild‐type AOM versus vehicle. (c) Upper panel: Schematic representation of the xenograft protocol. WS12 (10 mg·kg⁻¹) was administered intraperitoneally every day. Lower panel: Pictures of athymic mice bearing xenograft tumours on the day of killing. Tumors are indicated with black (vehicle) and blue (WS12) circles (d) Analysis of tumour volume of mice receiving vehicle (black) or WS12 (blue) in an 8‐day time course. Tumour size was measured every day and the volume was calculated. Each dot represents the mean ± SEM of seven mice. P values were determined using the Tukey–Kramer test; *P < 0.05 versus vehicle. (e, f) Analysis of tumour volume (e) and weight (f) of the explanted tumours from mice treated with vehicle (black) or WS12 (blue), at the end of the experiment (n = 7). P values were determined using unpaired Student's t tests; *P < 0.05 versus vehicle. (g) β‐Catenin mRNA expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT–qPCR) and calculated by using the 2^−ΔΔCt formula in xenografted tumours explanted from mice treated with vehicle (black) or WS12 (blue) (n = 5 different biological samples). P values were determined using unpaired Student's t tests; *P < 0.05 versus vehicle. (h) Representative immunoblots of β‐catenin protein levels in xenografted tumours explanted from mice receiving vehicle (black) or WS12 (blue). Lower panel shows densitometric analysis of western blot analysis. Results show mean ± SEM of seven different biological samples for each experimental group (i.e., vehicle and WS12) (n = 1 outlier has been removed from WS12 group with ROUT test). P values were determined using unpaired Student's t tests; *P < 0.05 versus vehicle. (i) Wnt signalling pathway‐associated genes (Axin 2 [axis inhibition protein 2], Sox‐9 [SRY‐box transcription factor 9], Cyclin D1, C‐Myc, CD44, MMP7 [matrix metalloproteinase 7], MMP2 [matrix metalloproteinase 2], OLFM4 [olfactomedin protein 4] and SMAD4) mRNA expression was evaluated by RT–qPCR and calculated by using the 2^−ΔΔCt formula in xenografted tumours explanted from mice treated with vehicle (black) or WS12 (blue) (n = 5 different biological samples). P values were determined using multiple Student's t tests; *P < 0.05 versus vehicle.