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Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells, with Limited Impact on Cancer Hallmark
Anna Borgström1, Barbara Hauert1, Sven Kappel1
1Institute of Biochemistry and Molecular Medicine, National Center of Competence in Research NCCR TransCure, University of Bern, Bern, Switzerland.
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.
Insights
Transient receptor potential melastatin 4 (TRPM4) channels impact prostate cancer progression. TRPM4 knockout cells showed reduced viability and migration, but blocking TRPM4 ion conductivity alone was insufficient to halt cancer hallmarks.
Area of Science:
- Molecular Biology
- Cancer Research
- Ion Channel Physiology
Background:
- Transient receptor potential melastatin 4 (TRPM4) is a calcium-activated monovalent cation channel implicated in various disease pathologies.
- TRPM4's role in prostate cancer progression and its potential as a therapeutic target remain incompletely understood.
Purpose of the Study:
- To investigate the function of TRPM4 in prostate cancer by generating and analyzing TRPM4 knockout cells.
- To assess the impact of TRPM4 on cancer hallmarks including proliferation, viability, migration, and adhesion.
- To evaluate the efficacy of novel TRPM4 inhibitors and the role of TRPM4 ion conductivity in prostate cancer.
Main Methods:
- Generation of CRISPR/Cas9 TRPM4 knockout DU145 prostate cancer cells.
- Analysis of cancer hallmark functions: proliferation, viability, migration, and adhesion assays.
- Prostate cancer patient tissue analysis for TRPM4 protein expression.
- Whole-cell patch clamp electrophysiology to test TRPM4 inhibitors.
- Rescue experiments using TRPM4 wild-type and dominant-negative mutant transfections.
Main Results:
- TRPM4 knockout cells exhibited significantly lower proliferation, viability, and migration compared to parental cells.
- TRPM4 protein expression positively correlated with local and metastatic progression in prostate cancer patient tissues.
- While novel inhibitors partially blocked TRPM4 currents, they lacked specificity in cellular assays.
- Functional TRPM4, but not its pore mutant, partially rescued cell viability, while both rescued cell cycle distribution, indicating a complex role for ion conductivity.
Conclusions:
- TRPM4 significantly contributes to prostate cancer cell viability, migration, cell cycle regulation, and adhesion.
- TRPM4 protein expression is associated with advanced prostate cancer.
- Targeting TRPM4 ion conductivity alone is insufficient to fully inhibit its pro-cancer functions in prostate cancer cells.
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