Evaluating RSPO2 activation and its association with outcomes and potential role as a driver in double-negative prostate cancer (DNPC).
Authors: Deacon Aiden , Gustafson Ava , Boytim Ella , Moline David , Kairies Elin , Kellen Sam , Makovec Allison , Anike Alexis , Ludwig Megan , Drake Justin , Antonarakis Emmanuel S. , Hwang Justin 
Published: February 2025

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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.