Prostat kanserinde androjen reseptörü varyantı ARV7'nin fonksiyonel ve yapısal karakterizasyonu

The androgen receptor (AR) is critical at all stages of prostate cancer. Interestingly, Androgen Receptor Variants (ARVs) have been shown to occur in late-stage prostate cancer patients by alternative splicing of the AR. Lacking a ligand binding domain (LBD) these constitutive active variants are intrinsically resistant to all clinically approved AR antagonists. Understanding the mechanism of ARV7 activation is critical to better treating late-stage prostate cancer patients. Upon ligand binding, the AR rapidly undergoes an allosteric modification that allows the N terminal domain and LBD to form an N-C intramolecular interaction. Following nuclear localization, the AR then forms homodimers via intermolecular interactions. Given that ARV7 lack a LBD, it is unclear if the variants undergo a similar homodimerization or they in fact heterodimerize with the ARfull-length. To clarify this, the project aims to study ARV7 interactions using both cell biology and biophysics. Utilizing a commonly observed variant (ARV7) and using a Tet-inducible ARV7 model in LNCaP cells we were able to study the impact of ARV7 on cell cycle progression and AR signaling. Surprisingly, in single cell clones we found that overexpression of ARV7 causes marked cellular senescence and the degree of senescence was dependent on the expression of ARV7. This was independent of ARfull-length and was mediated by an activation of the p53 pathway. This phenotype was also observed in several androgen responsive prostate cancer cell lines. Our results indicate that constitutive activity of ARV7 was dependent on the presence of androgen receptor and a shift from androgen receptor mediated growth to ARV7 mediated growth is not tolerated in AR dependent cells. Interestingly, we were able to identify several clones that were resistant to ARV7 expression mimicking what is clinically observed. We also studied the predicted inter- or intramolecular interaction between ARV7 and ARfull-length with live cell imaging FRET microscopy. Using custom analysis software that can track and quantify the FRET signal in living cells we observed inter- and intramolecular interactions of ARfull-length upon stimulation with its ligand in temporal manner. Using this methodology our results suggest that ARfull-length and ARV7 form a C-C interaction. The experimental design we used enabled spatiotemporal identification of the interactions and revealed different conformational changes happening throughout androgen stimulation.