Evaluating RSPO2 activation and its association with outcomes and potential role as a driver in double-negative prostate cancer (DNPC).

236 Background: Several aggressive tumor variants have emerged since the broad use of 2nd generation androgen therapies for metastatic prostate cancers (mPCs). Close to 20% of mPCs are double negative prostate cancers (DNPC) that lack androgen receptor (AR) and do not exhibit histopathological features of neuroendocrine prostate cancer (NEPC). Our study focused on the R-spondin family of genes ( RSPO1/2/3/4 ), which are secreted proteins classified as WNT regulators. In mPCs, we examined their genomic alterations, impact on clinical outcomes, interaction with CTNNB1 (master regulator of WNT signaling), or other genes associated with DNPC and epithelial-mesenchymal transition (EMT). Methods: Alterations in RSPOs , CTNNB1 , and APC were examined with respect to survival using data from the TCGA Pan Cancer Atlas (n=10967) and a non-redundant set of prostate tumors from cBioPortal (n=2510). Kaplan-Meier and log-rank tests were used to evaluate survival. Alphafold2 was used to model the protein structures in silico . Differential gene expression, Algorithm for Linking Activity Networks (ALAN), and Gene Set Enrichment Analysis (GSEA) were used to model gene expression and determine enriched pathways. Proliferative effects of RSPO2 and CTNNB1 were analyzed in AR+ (LNCaP) and AR- (PC3) cell lines. Results: Pan cancer RSPO2 amplification rates (5%) were greater than CTNNB1 mutations (4%) but less than APC loss (8%). In mPC, RSPO2 amplifications (22%) were greater than CTNNB1 mutations or APC loss (9%, 8%). RSPO1/3/4 alterations were of low prevalence. Pan cancer, RSPO2 and CTNNB1 alterations were associated with poor disease-free survival (HR=1.578, 1.448, 95%CI=1.17-2.13, 1.06-1.964, p<0.001 respectively), whereas APC loss or RSPO1/3/4 alterations were not prognostic. In primary prostate cancer, RSPO2 amplifications were associated with worse survival (HR=1.63, 95%CI=1.05-2.54, p<0.05). In silico protein structure modeling indicated stark differences between RSPO2 and RSPO1/3/4 . Using RNA-seq data from 208 mPCs, our gene-gene interaction analysis and GSEA indicated that RSPO2 expression was robustly associated with DNPC driver genes FGFR1/2 , the Hallmark EMT pathway, and EMT transcription factors ZEB1 , SNAI1/2 , TWIST1/2 , while being anti-correlated with expression of AR, AR co-factors ( FOXA1 , HOXB13 ), and Hallmark androgen signaling. Unlike RSPO2 , CTNNB1 was associated with Hallmark EMT and Androgen response. Finally, RSPO2 overexpression promoted proliferation in the two PC cell lines regardless of AR expression. Conclusions: RSPO2 has unique features from RSPO1/3/4 in which amplifications are associated with worse outcomes in prostate and other cancers. Relative to CTNNB1 , RSPO2 exhibits unique signaling properties that imply RSPO2 has oncogenic functions other than WNT signaling, which may explain how it promotes growth of cell lines without AR expression.