Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells,with Limited Impact on Cancer Hallmark Functions

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.     

Inactivation of uPA and its receptor uPAR by 3,3′‐diindolylmethane (DIM) leads to the inhibition of prostate cancer cell growth and migration

3,3′‐Diindolylmethane (DIM) has been studied for its putative anti‐cancer properties, especially against prostate cancer; however, its exact mechanism of action remains unclear. We recently provided preliminary data suggesting down‐regulation of uPA during B‐DIM (a clinically active DIM)‐induced inhibition of invasion and angiogenesis in prostate cancer cells. Since the expression and activation of uPA plays important role in tumorigenicity, and high endogenous levels of uPA and uPAR are found in advanced metastatic cancers, we investigated their role in B‐DIM‐mediated inhibition of prostate cancer cell growth and motility. Using PC3 cells, we found that B‐DIM treatment as well as the silencing of uPA and uPAR by siRNAs led to the inhibition of cell growth and motility. Conversely, over‐expression of uPA/uPAR in LNCaP and C4‐2B cells resulted in increased cell growth and motility, which was effectively inhibited by B‐DIM. Moreover, we found that uPA as well as uPAR induced the production of VEGF and MMP‐9, and that the down‐regulation of uPA/uPAR by siRNAs or B‐DIM treatment resulted in the inhibition of VEGF and MMP‐9 secretion which could be responsible for the observed inhibition of cell migration. Interestingly, silencing of uPA/uPAR led to decreased sensitivity to B‐DIM indicating important role of uPA/uPAR in B‐DIM‐mediated regulation of prostate cancer cell growth and migration. Our data suggest that chemopreventive and/or therapeutic activity of B‐DIM is in part due to down‐regulation of uPA–uPAR leading to reduced production of VEGF/MMP‐9 which ultimately leads to the inhibition of cell growth and migration of aggressive prostate cancer cells. J. Cell. Biochem. 107: 516–527, 2009. © 2009 Wiley‐Liss, Inc.     

Prohibitin is a cholesterol‐sensitive regulator of cell cycle transit

Cholesterol is essential in establishing most functional animal cell membranes; cells cannot grow or proliferate in the absence of sufficient cholesterol. Consequently, almost every cell, tissue, and animal tightly regulates cholesterol homeostasis, including complex mechanisms of synthesis, transport, uptake, and disposition of cholesterol molecules. We hypothesize that cellular recognition of cholesterol insufficiency causes cell cycle arrest in order to avoid a catastrophic failure in membrane synthesis. Here, we demonstrate using unbiased proteomics and standard biochemistry that cholesterol insufficiency causes upregulation of prohibitin, an inhibitor of cell cycle progression, through activation of a cholesterol‐responsive promoter element. We also demonstrate that prohibitin protects cells from apoptosis caused by cholesterol insufficiency. This is the first study tying cholesterol homeostasis to a specific cell cycle regulator that inhibits apoptosis. J. Cell. Biochem. 111: 1367–1374, 2010. © 2010 Wiley‐Liss, Inc.     

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

TRAF6 promotes TGFβ-induced invasion and cell-cycle regulation via Lys63-linked polyubiquitination of Lys178 in TGFβ type I receptor

Transforming growth factor β (TGFβ) can act either as a tumor promoter or a tumor suppressor in a context-dependent manner. High levels of TGFβ are found in prostate cancer tissues and correlate with poor patient prognosis. We recently identified a novel TGFβ-regulated signaling cascade in which TGFβ type I receptor (TβRI) is activated by the E3 ligase TNF-receptor-associated factor 6 (TRAF6) via the Lys63-linked polyubiquitination of TβRI. TRAF6 also contributes to activation of TNF-α-converting enzyme and presenilin-1, resulting in the proteolytic cleavage of TβRI and releasing the intracellular domain of TβRI, which is translocated to the nucleus to promote tumor invasiveness. In this report, we provide evidence that Lys178 of TβRI is polyubiquitinated by TRAF6. Moreover, our data suggest that TRAF6-mediated Lys63-linked ubiquitination of the TβRI intracellular domain is a prerequisite for TGFβ regulation of mRNA for cyclin D1 (CCND1), expression, as well as for the regulation of other genes controlling the cell cycle, differentiation, and invasiveness of prostate cancer cells.     

Inhibition of Janus Kinase 1 synergizes docetaxel sensitivity in prostate cancer cells

Prostate cancer (PCa) is the second most common malignancy and is the fifth leading cause of cancer mortality among men globally. Docetaxel-based therapy remains the first-line treatment for metastatic castration-resistant prostate cancer. However, dose-limiting toxicity including neutropenia, myelosuppression and neurotoxicity is the major reason for docetaxel dose reductions and fewer cycles administered, despite a recent study showing a clear survival benefit with increased total number of docetaxel cycles in PCa patients. Although previous studies have attempted to improve the efficacy and reduce docetaxel toxicity through drug combination, no drug has yet demonstrated improved overall survival in clinical trial, highlighting the challenges of improving the activity of docetaxel monotherapy in PCa. Herein, we identified 15 lethality hits for which inhibition could enhance docetaxel sensitivity in PCa cells via a high-throughput kinome-wide loss-of-function screen. Further drug-gene interactions analyses identified Janus kinase 1 (JAK1) as a viable druggable target with existing experimental inhibitors and FDA-approved drugs. We demonstrated that depletion of endogenous JAK1 enhanced docetaxel-induced apoptosis in PCa cells. Furthermore, inhibition of JAK1/2 by baricitinib and ruxolitinib synergizes docetaxel sensitivity in both androgen receptor (AR)–negative DU145 and PC3 cells, but not in the AR-positive LNCaP cells. In contrast, no synergistic effects were observed in cells treated with JAK2-specific inhibitor, fedratinib, suggesting that the synergistic effects are mainly mediated through JAK1 inhibition. In conclusion, the combination therapy with JAK1 inhibitors and docetaxel could be a useful therapeutic strategy in the treatment of prostate cancers.