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 cytoplasmic tail of the mechanosensitive channel Pkd2 regulates its internalization and clustering in eisosomes.

Polycystins are a family of conserved ion channels, mutations of which lead to one of the most common human genetic disorders, namely, autosomal dominant polycystic kidney disease. Schizosacchromyces pombe possesses an essential polycystin homologue, Pkd2, which directs Ca2+ influx on the cell surface in response to membrane tension, but its structure remains unsolved. Here, we analyzed the structure-function relationship of Pkd2 based on its AlphaFold-predicted structure. Pkd2 consists of three domains, the extracellular lipid-binding domain (LBD), nine-helix transmembrane domain (TMD) and C-terminal cytoplasmic domain (CCD). Our genetic and microscopy data revealed that LBD and TMD are essential for targeting Pkd2 to the plasma membrane from the endoplasmic reticulum. In comparison, CCD ensures the polarized distribution of Pkd2 by promoting its internalization and preventing its clustering in the eisosome, a caveolae-like membrane compartment. The domains of Pkd2 and their functions are conserved in other fission yeast species. We conclude that both extracellular and cytoplasmic domains of Pkd2 are crucial for its intracellular trafficking and function. We propose that mechanosensitive channels can be desensitized through either internalization or clustering in low-tension membrane compartments.

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.

Membrane stretching activates calcium permeability of a putative channel Pkd2 during fission yeast cytokinesis.

Pkd2 is the fission yeast homologue of polycystins. This putative ion channel localizes to the plasma membrane. It is required for the expansion of cell volume during interphase growth and cytokinesis, the last step of cell division. However, the channel activity of Pkd2 remains untested. Here, we examined the calcium permeability and mechanosensitivity of Pkd2 through in vitro reconstitution and calcium imaging of pkd2 mutant cells. Pkd2 was translated and inserted into the lipid bilayers of giant unilamellar vesicles using a cell-free expression system. The reconstituted Pkd2 permeated calcium when the membrane was stretched via hypoosmotic shock. In vivo, inactivation of Pkd2 through a temperature-sensitive mutation pkd2-B42 reduced the average intracellular calcium level by 34%. Compared with the wild type, the hypomorphic mutation pkd2-81KD reduced the amplitude of hypoosmotic shock-triggered calcium spikes by 59%. During cytokinesis, mutations of pkd2 reduced the calcium spikes, accompanying cell separation and the ensuing membrane stretching, by 60%. We concluded that fission yeast polycystin Pkd2 allows calcium influx when activated by membrane stretching, representing a likely mechanosensitive channel that contributes to the cytokinetic calcium spikes.

Counting actin in contractile rings reveals novel contributions of cofilin and type II myosins to fission yeast cytokinesis.

Cytokinesis by animals, fungi, and amoebas depends on actomyosin contractile rings, which are stabilized by continuous turnover of actin filaments. Remarkably little is known about the amount of polymerized actin in contractile rings, so we used low concentrations of GFP-Lifeact to count total polymerized actin molecules in the contractile rings of live fission yeast cells. Contractile rings of wild-type cells accumulated polymerized actin molecules at 4900/min to a peak number of ∼198,000 followed by a loss of actin at 5400/min throughout ring constriction. In adf1-M3 mutant cells with cofilin that severs actin filaments poorly, contractile rings accumulated polymerized actin at twice the normal rate and eventually had almost twofold more actin along with a proportional increase in type II myosins Myo2, Myp2, and formin Cdc12. Although 30% of adf1-M3 mutant cells failed to constrict their rings fully, the rest lost actin from the rings at the wild-type rates. Mutations of type II myosins Myo2 and Myp2 reduced contractile ring actin filaments by half and slowed the rate of actin loss from the rings.

Androgen up-regulation of Twist1 gene expression is mediated by ETV1.

Twist1, a basic helix-loop-helix transcription factor that regulates a number of genes involved in epithelial-to-mesenchymal transition (EMT), is upregulated in prostate cancer. Androgen regulation of Twist1 has been reported in a previous study. However, the mechanism of androgen regulation of the Twist1 gene is not understood because the Twist1 promoter lacks androgen receptor (AR)-responsive elements. Previous studies have shown that the Twist1 promoter has putative binding sites for PEA3 subfamily of ETS transcription factors. Our lab has previously identified Ets Variant 1 (ETV1), a member of the PEA3 subfamily, as a novel androgen-regulated gene that is involved in prostate cancer cell invasion through unknown mechanism. In view of these data, we hypothesized that androgen-activated AR upregulates Twist1 gene expression via ETV1. Our data confirmed the published work that androgen positively regulates Twist1 gene expression and further showed that this positive effect was directed at the Twist1 promoter. The positive effect of androgen on Twist1 gene expression was abrogated upon disruption of AR expression by siRNA or of AR activity by Casodex. More importantly, our data show that disruption of ETV1 leads to significant decrease in both androgen-mediated upregulation as well as basal level of Twist1, which we are able to rescue upon re-expression of ETV1. Indeed, we are able to show that ETV1 mediates the androgen upregulation of Twist1 by acting on the proximal region of Twist1 promoter. Additionally, our data show that Twist1 regulates prostate cancer cell invasion and EMT, providing a possible mechanism by which ETV1 mediates prostate cancer cell invasion. In conclusion, in this study we report Twist1 as an indirect target of AR and androgen regulation through ETV1.

Peptide B targets soluble guanylyl cyclase α1 and kills prostate cancer cells.

Among androgen-regulated genes, soluble guanylyl cyclase α1 (sGCα1) is significant in promoting the survival and growth of prostate cancer cells and does so independent of nitric oxide (NO) signaling. Peptides were designed targeting sGCα1 to block its pro-cancer functions and one peptide is discussed here. Peptide B-8R killed both androgen-dependent and androgen-independent prostate cancer cells that expressed sGCα1, but not cells that do not express this gene. Peptide B-8R induced apoptosis of prostate cancer cells. Importantly, Peptide B-8R does not affect nor its cytotoxicity depend on NO signaling, despite the fact that it associates with sGCα1, which dimerizes with sGCß1 to form the sGC enzyme. Just as with a previously studied Peptide A-8R, Peptide B-8R induced elevated levels of reactive oxygen species (ROS) in prostate cancer cells, but using a ROS-sequestering agent showed that ROS was not responsible the cytotoxic activity of Peptide B-8R. Interestingly, Peptide B-8R induced elevated levels of p53 and phosphorylated p38, but neither of these changes is the cause of the peptide's cytotoxicity. Additional drugs were used to alter levels of iron levels in cells and these studies showed that Peptide B-8R activity does not depend on Ferroptosis. Thus, future work will be directed at defining the mechanism of cytotoxic action of Peptide B-8R against prostate cancer cells.