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, like AR-V7, may be major drivers of CRPC. Our lab recently identified a novel mechanism of AR regulation via the transmembrane protein TM4SF3 (Transmembrane 4 superfamily 3), which exhibits a physical interaction, nuclear co-localization, 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 de-ubiquitination of both proteins, showing that mutual stabilization results from de-ubiquitination. 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.

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 concentration 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 4,900/min to a peak number of ∼198,000 followed by a loss of actin at 5,400/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 two-fold 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.

Pkd2 is the fission yeast homolog 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 the pkd2 mutant cells. Pkd2 was translated and inserted into the lipid bilayer of giant unilamellar vesicles using a cell-free expression system. The reconstituted Pkd2 permeated calcium when the membrane was stretched via hypo-osmotic shock. In vivo, inactivation of Pkd2 through a temperature-sensitive mutation pkd2-B42 reduced the average intracellular calcium level by 34%. Compared to the wild type, the hypomorphic mutation pkd2-81KD reduced the amplitude of hypo-osmotic 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.

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.

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 concentration 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 4,900/min to a peak number of ~198,000 followed by a loss of actin at 5,400/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 two-fold 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.