Abstract
Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca2+ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na+. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.
Highlights
Each year around 1.3 million men worldwide are diagnosed with prostate cancer.[1]
Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca2+ activated monovalent cation channel that contributes to the pathophysiology of several diseases
To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na+
Summary
Each year around 1.3 million men worldwide are diagnosed with prostate cancer.[1] After lung cancer, prostate cancer is the most frequently diagnosed malignancy in men, and it is the fifth leading cause of cancer-related death in men.[1] Androgen deprivation therapy (ADT) is commonly used as initial treatment of prostate cancer This therapy can be initially efficient, most cases develop resistance to ADT and transform into castrationresistant prostate cancer (CRPC). The two most commonly used inhibitors for studies of TRPM4 to date are flufenamic acid and 9-phenanthrol Both have been reported to have low potency[22,23] and a long list of offtargets.[24] new selective inhibitors are required to accurately and efficiently study the role of TRPM4 and its ion conductivity in different disease model systems. We investigated the underlying mechanism with rescue cells expressing TRPM4 or a dominant negative construct, which is unable to conduct Na+ (TRPM4-D984A)
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