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Pathologically stabilized GLI3 promotes androgen-independent growth of SPOP mutant prostate cancer cells through reactivation of an AR signaling axis

ABSTRACT: Although androgen deprivation therapy triggers a rapid response in men with metastatic prostate cancer, most patients eventually progress to lethal castration resistant prostate cancer (CRPC) characterized by androgen receptor (AR)-dependent tumor growth despite castrate levels of circulating androgens. Current treatment options for CRPC remain limited and non-curative, rendering improved outcomes dependent on deeper mechanistic insight and new therapeutic targets. In this RNA sequencing study we profiled the transcriptomes of SPOP WT and mutant LNCaP cells before and after androgen deprivation by RNA-sequencing. Using standard criteria (minimum 2-fold change), we identified 981 genes that were differentially expressed in SPOP mutant vs SPOP WT LNCaP cells grown in the absence of androgens . Strikingly, a significant fraction (595 or ~60%) of these genes overlapped with genes (3510 in number) differentially expressed as a function of androgen in WT LNCaP cells, indicating partial restoration of AR signaling in SPOP mutant cells following androgen withdrawal. To determine the contribution of GLI3 to this process, we performed RNA-sequencing following GLI3 knockdown in androgen-deprived SPOP mutant LNCaP cells. Among the 595 androgen-responsive genes in LNCaP cells whose hormone-independent expression is regulated by SPOP 187 (or ~31%) are also regulated by GLI3, thus revealing a prominent role for GLI3 in restoration of AR signaling in SPOP mutant cells following androgen withdrawal. Enrichment analysis using Gene Ontology Biological Processes revealed these 187 GLI3-regulated genes to be prominently linked with “chromosome segregation”, “DNA replication”, “transcriptional regulation of in G1/S transition”, “regulation of mitotic cell cycle”, and “chromatin silencing” among others, suggesting possible molecular genetic bases for androgen independent growth of SPOP mutant cells. Taken together, these findings indicate that SPOP mutant prostate cancer cells acquire robust androgen-independent growth through reestablishment of an AR signaling axis that involves functional crosstalk with the SHH/GLI3 pathway.

ORGANISM(S): Homo sapiens

PROVIDER: GSE134682 | GEO | 2022/07/12

REPOSITORIES: GEO

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Project description:Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (~25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (~23%), five events involved transcripts derived from known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of the alternative TSC2 mRNA isoform was directly regulated by androgens. Furthermore, by chromatin immunoprecipitation, we observed recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa. Total 8 samples were analysed. 4 control (LNCaP cells grown in RPMI-1640 media with charcoal-stripped 10% FBS devoid of steroids) and 4 treatment (LNCaP cells grown in media devoid of steroid, and subsequently treated with 10nM R1881 synthetic androgen analogue for 24 hours).

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Pathologically stabilized GLI3 promotes androgen-independent growth of SPOP mutant prostate cancer cells through reactivation of an AR signaling axis

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