Oncogenic Potential of Truncated-Gli3 via the Gsk3ß/Gli3/AR-V7 Axis in Castration-Resistant Prostate Cancer

The functional activation of the androgen receptor (AR) and its interplay with the aberrant Hh/Gli cascade are pivotal in the progression of castration-resistant prostate cancer (CRPC) and resistance to AR-targeted therapies. Our study unveils a novel role of the truncated form of Gli (t-Gli3) in advancing CRPC. Investigation into Gli3 regulation revealed a Smo-independent mechanism for its activation. Despite lacking a transactivation domain, t-Gli3 relies on androgen receptor variant 7 (AR-V7) for its action. Mechanistically, Gsk3ß activation leads to the t-Gli3 generation, and inhibition of Gsk3ß supports the accumulation of full-length Gli3 through a non-canonical mechanism. Knockdown of Gsk3ß (Gsk3ß KD) reduces CRPC cell proliferation, induces apoptosis via mitochondrial fragmentation, and triggers metabolomic reprogramming. Orthotropic implantation of Gsk3ß KD cells in the mouse prostate results in tumor growth retardation compared to scramble control cells. RNA-seq analysis of Gsk3ß KD reveals upregulation of pathways associated with apoptosis, tumor suppressor pathway, and downregulation of oncogenic pathway relative to control. Furthermore, combinational use of a Gsk3ß inhibitor with anti-Smo or Gli1 significantly inhibits the growth of CRPC cells, which are resistant to individual Smo or Gli1 inhibitor targeting. Intriguingly, solely targeting Gli3 proves effective in inhibiting CRPC cell growth. Overall, our study underscores the clinical significance of Gli3, emphasizing t-Gli3, and provides novel insights into the interplay of the Gsk3ß/t-Gli3/AR-V7 axis in CRPC. Overall design: To investigate the impact of functional blockage of Gsk3ß on Hh/Gli pathway, particularly regarding the posttranslational modification of Gli-family proteins. In addition to understand Gsk3ß -mediated other regulatory signaling elements involved in oncogenesis in castration-resistant prostate cancer (CRPC). To achieve this, we have developed 22Rv1 cells with Gsk3ß knockdown using shRNA. We performed differential gene expression analysis (DEGs) and HALLMARK of Gene Set Enrichment Analysis (GSEA) of RNA seq data obtained from 22Rv1-Scramble vs. 22Rv1 Gsk3ß-shRNA cells