Bevan, C. L. | bioRxiv Science

bioRxiv2021

A suite of activity-based probes to dissect the KLK activome in drug-resistant prostate cancer

Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network - the KLK activome - with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors with unprecedented potency and selectivity enabling simultaneous orthogonal analysis of KLK2, KLK3 and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP) we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.

biochemistry↗

bioRxiv2020

Genome-wide analyses of transcription factors and co-regulators across seven cohorts identified reduced PPARGC1A expression as a driver of prostate cancer progression

Defining altered transcription factor (TF) and coregulators that are oncogenic drivers remains a challenge, in part because of the multitude of TFs and coregulators. We addressed this challenge by using bootstrap approaches to test how expression, copy number alterations or mutation of TFs (n = 2662), coactivators (COA; n= 766); corepressor (COR; n = 599); mixed function coregulators (MIXED; n = 511) varied across seven prostate cancer (PCa) cohorts (three of localized and four advanced disease). COAS, CORS, MIXED and TFs all displayed significant down-regulated expression (q.value < 0.1) and correlated with protein expression ({rho} 0.4 to 0.55). Stringent expression filtering identified commonly altered TFs and coregulators including well-established (e.g. ERG) and underexplored (e.g. PPARGC1A, encodes PGC1) in localized PCa. Reduced PPARGC1A expression significantly associated with worse disease-free survival in two cohorts of localized PCa. Stable PGC1 knockdown in LNCaP cells increased growth rates and invasiveness and RNA-Seq revealed a profound basal impact on gene expression (~2300 genes; FDR < 0.05, logFC > 1.5), but only modestly impacted PPAR{gamma} responses. GSEA analyses of the PGC1 transcriptome revealed that it significantly altered the AR-dependent transcriptome, and was enriched for epigenetic modifiers. PGC1-dependent genes were overlapped with PGC1-ChIP-Seq genes and significantly associated in TCGA with higher grade tumors and worse disease-free survival. Together these data demonstrate an approach to identify cancer-driver coregulators in cancer and that PGC1 expression is clinically significant yet underexplored coregulator in aggressive early stage PCa.

cancer biology↗