Trpm8 Expression in Human and Mouse Castration Resistant Prostate Adenocarcinoma Paves the Way for the Preclinical Development of TRPM8-Based Targeted Therapies

Abstract

Metastatic prostate cancer (mPCa) is one of the leading causes of cancer-related mortality in both the US and Europe. Androgen deprivation is the first-line therapy for mPCa; however, resistance to therapy inevitably occurs and the disease progresses to the castration resistant stage, which is uncurable. A definition of novel targeted therapies is necessary for the establishment of innovative and more effective protocols of personalized oncology. We employed genetically engineered mouse models of PCa and human samples to characterize the expression of the TRPM8 cation channel in both hormone naïve and castration resistant tumors. We show that Trpm8 expression marks both indolent (Pten-null) and aggressive (Pten/Trp53 double-null and TRAMP) mouse prostate adenocarcinomas. Importantly, both mouse and human castration-resistant PCa preserve TRPM8 protein expression. Finally, we tested the effect of TRPM8 agonist D-3263 administration in combination with enzalutamide or docetaxel on the viability of aggressive mouse PCa cell lines. Our data demonstrate that D-3263 substantially enhances the pro-apoptotic activity of enzalutamide and docetaxel in TRAMP-C1 e TRAMP-C2 PCa cell lines. To conclude, this study provides the basis for pre-clinical in vivo testing of TRPM8 targeting as a novel strategy to implement the efficacy of standard-of-care treatments for advanced PCa.

Validation of six commercial antibodies for detection of heterologous and endogenous TRPM8 ion channel expression

“…However, using their recommended dilutions, only three of them yielded satisfactory results for our standards. Interestingly, Alomone antibody has been widely used for WB [38][39][40] but, in our conditions, it did not work properly. However, in many of these articles, the antibody dilution was lower than recommended and the reported TRPM8 bands were dubious and/or no full membrane/molecular weight markers were shown, making it difficult to rule out the presence of unspecific bands or crossdetection.…”

“…In our hands, Alomone, ECM2 and ECM3 did not have enough sensitivity and/or specificity to detect endogenous TRPM8, at least with the conventional conditions we used. There is abundant literature using Alomone antibody for detection of endogenous TRPM8 by ICC and/or IHC in different tissues [39,44–48]. However, in many of the published figures, the staining pattern was not consistent with the known restrict-ed expression of TRPM8 [31,49].…”

Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

“…However, using their recommended dilutions, only three of them yielded satisfactory results for our standards. Interestingly, the Alomone antibody has been widely used for WB [38][39][40], but in our conditions, it did not work properly. However, in many of these articles, the antibody dilution was lower than recommended, and the reported TRPM8 bands were dubious and/or no full membrane/molecular weight markers were shown, making it difficult to rule out the presence of unspecific bands or cross-detection.…”

“…In our hands, Alomone, ECM2 and ECM3 did not have enough sensitivity and/or specificity to detect endogenous TRPM8, at least with the conventional conditions we used. There is abundant literature using Alomone antibody for the detection of endogenous TRPM8 by ICC and/or IHC in different tissues [39,[44][45][46][47][48]. However, in many of the published figures, the staining pattern was not consistent with the known restricted expression of TRPM8 [31,49].…”

Therapeutic potential of TRPM8 channels in cancer treatment

“…Following that, knocking down TRPM8 channels in osteosarcoma and prostate cancer cell lines increased apoptosis induced by epirubicin, suggesting an enhancement in chemosensitivity ( Wang et al, 2013 ; Liu T. et al, 2018 ). In aggressive prostate cancer cells was demonstrated that the agonist D-3263 significantly improves the pro-apoptotic effect of enzalutamide and docetaxel, antiandrogen and antineoplastic, respectively, used in the treatment of prostate cancer ( Genovesi et al, 2022 ).…”