MDM2 regulates the stability of AR, AR-V7, and TM4SF3 proteins in prostate cancer.

Androgen receptor (AR) and its constitutively active splice variant, AR Variant 7 (AR-V7), regulate genes essential for the development and progression of prostate cancer. Degradation of AR and AR-V7 by the ubiquitination proteasomal pathway is important for the regulation of both their protein stability. Our published results demonstrate that the interaction of TM4SF3 with either AR or AR-V7 leads to mutual stabilization due to a reduction in their ubiquitination and proteasomal degradation. These results led us to search for a common E3 ligase for AR, AR-V7, and TM4SF3. Depletion by siRNA of several E3 ligases identified MDM2 as the common E3 ligase. MDM2 inhibition by siRNA depletion or using a pharmacological inhibitor (MDM2i) of its E3 ligase activity led to elevated levels of endogenous AR, AR-V7, and TM4SF3 in prostate cancer cells. MDM2 knockdown in PC-3 cells, which do not express AR, also increased TM4SF3, demonstrating that MDM2 affects the TM4SF3 protein independent of AR. We further demonstrate that MDM2i treatment reduced the ubiquitination of AR and TM4SF3, suggesting that MDM2 can induce the ubiquitination of these proteins. Increased AR and AR-V7 protein levels induced by MDM2i treatment resulted in the expected increased expression of AR-regulated genes and enhanced proliferation and migration of both LNCaP and Enzalutamide-resistant CWR-22Rv1 prostate cancer cells. Thus, our study expands the known roles of MDM2 in prostate cancer to include its potential involvement in the important mutual stabilization that TM4SF3 exhibits when interacting with either AR or AR-V7.

Peptides disrupting TM4SF3 interaction with AR or AR-V7 block prostate cancer cell proliferation.

Androgen receptor (AR) plays a vital role in the development and progression of prostate cancer from the primary stage to the usually lethal stage known as castration-resistant prostate cancer (CRPC). Constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain are partially responsible for the abnormal activation of AR and may be involved in resistance to AR-targeting drugs occurring in CRPC. There is increasing consensus on the potential of drugs targeting protein-protein interactions. Our lab has recently identified transmembrane 4 superfamily 3 (TM4SF3) as a critical interacting partner for AR and AR-V7 and mapped the minimal interaction regions. Thus, we hypothesized that these interaction domains can be used to design peptides that can disrupt the AR/TM4SF3 interaction and kill prostate cancer cells. Peptides TA1 and AT1 were designed based on the TM3SF3 or AR interaction domain, respectively. TA1 or AT1 was able to decrease AR/TM4SF3 protein interaction and protein stability. Peptide TA1 reduced the recruitment of AR and TM4SF3 to promoters of androgen-regulated genes and subsequent activation of these AR target genes. Peptides TA1 and AT1 were strongly cytotoxic to prostate cancer cells that express AR and/or AR-V7. Peptide TA1 inhibited the growth and induced apoptosis of both enzalutamide-sensitive and importantly enzalutamide-resistant prostate cancer cells. TA1 also blocked the migration and malignant transformation of prostate cancer cells. Our data clearly demonstrate that using peptides to target the important interaction AR has with TM4SF3 provides a novel method to kill enzalutamide-resistant prostate cancer cells that can potentially lead to new more effective therapy for CRPC.

The Transmembrane Protein TM4SF3 Interacts With AR and AR-V7 and is Recruited to AR Target Genes.

Prostate cancer transitions from an early treatable form to the lethal castration-resistant prostate cancer (CRPC). Androgen receptor (AR) and constitutively active AR splice variants, such as AR-V7, may be major drivers of CRPC. Our laboratory recently identified a novel mechanism of AR regulation via the transmembrane protein transmembrane 4 superfamily 3 (TM4SF3), which exhibits a physical interaction, nuclear colocalization, and mutual stabilization with AR. Here, we have mapped the interaction domains within AR and TM4SF3 and discovered that TM4SF3 also physically interacts with AR-V7, regulating its protein stability and the viability of CRPC cells expressing AR-V7. Ubiquitination of TM4SF3 and AR-V7 was detected for the first time and TM4SF3 interaction with either AR or AR-V7 resulted in mutual deubiquitination of both proteins, showing that mutual stabilization results from deubiquitination. Interestingly, nuclear TM4SF3 was co-recruited to the promoters of AR- and AR-V7-regulated genes and required for their expression, showing that TM4SF3 interaction is critical for their transcriptional functions. The results collectively show the multiple critical regulatory functions of TM4SF3 on AR or AR-V7 in prostate cancer cells.

Dual-Acting Peptides Target EZH2 and AR: A New Paradigm for Effective Treatment of Castration-Resistant Prostate Cancer.

Prostate cancer starts as a treatable hormone-dependent disease, but often ends in a drug-resistant form called castration-resistant prostate cancer (CRPC). Despite the development of the antiandrogens enzalutamide and abiraterone for CRPC, which target the androgen receptor (AR), drug resistance usually develops within 6 months and metastatic CRPC (mCRPC) leads to lethality. EZH2, found with SUZ12, EED, and RbAP48 in Polycomb repressive complex 2 (PRC2), has emerged as an alternative target for the treatment of deadly mCRPC. Unfortunately, drugs targeting EZH2 have shown limited efficacy in mCRPC. To address these failures, we have developed novel, dual-acting peptide inhibitors of PRC2 that uniquely target the SUZ12 protein component, resulting in the inhibition of both PRC2 canonical and noncanonical functions in prostate cancer. These peptides were found to inhibit not only the EZH2 methylation activity, but also block its positive effect on AR gene expression in prostate cancer cells. Since the peptide effect on AR levels is transcriptional, the inhibitory peptides can block the expression of both full-length AR and its splicing variants including AR-V7, which plays a significant role in the development of drug resistance. This dual-mode action provides the peptides with the capability to kill enzalutamide-resistant CRPC cells. These peptides are also more cytotoxic to prostate cancer cells than the combination of enzalutamide and an EZH2 inhibitory drug, which was recently suggested to be an effective treatment of mCRPC disease. Our data show that such a dual-acting therapeutic approach can be more effective than the existing front-line drug therapies for treating deadly mCRPC.

Rehabilitation of Severely Attrited Teeth with Hobo Twin Stage Technique: A Case Report.

Restoration of excessively worn dentition is a challenging treatment procedures. It requires efficient diagnosis and treatment plan. Hobo's techniques and Pankey Mann Schuyler's philosophy are widely used and documented for full mouth rehabilitation. We have reported the case of a 56-year- old male patient who presented with the severely worn dentition and had difficulty in chewing. To rehabilitate this case Hobo's twin stage technique had been adopted as it is based on scientific data and mathematical analysis for both disocclusion and anterior guidance thus reducing chair side time. Keywords: attrited; hobo; occlusion; rehabilitation.