SAT633 The RNA Helicase UPF1 Mediates AR- And SRF-dependent Transcription In Prostate Cancer
Authors: Chauhan Gaurav , Venkadakrishnan Varadha Balaji , Dahiya Ujjwal , Ghanem Yara , Srinivasan Gideon Jebaraj , Willard Belinda , Hu Qiang , Cortes Eduardo , Liu Song , Heemers Hannelore 
Published: October 2023

Disclosure: G. Chauhan: None. V. Venkadakrishnan: None. U. Dahiya: None. Y. Ghanem: None. G. Srinivasan: None. B. Willard: None. Q. Hu: None. E. Cortes: None. S. Liu: None. H. Heemers: None.

The ligand-activated androgen receptor (AR) is a transcription factor that drives prostate cancer (CaP). Blocking androgen-activation of AR via androgen deprivation therapy (ADT) is the default treatment for metastatic CaP. Despite initial remission, ADT invariably fails and CaP progresses to castration-recurrent CaP (CRPC), which still relies on aberrantly activated AR. Alternative approaches are needed to inhibit AR action in CaP that has failed ADT. Our lab has been exploring the therapeutic potential of a non-canonical AR signaling mechanism wherein AR stimulates another transcription factor, Serum Response Factor (SRF). AR-SRF action correlates with CaP progression and is enriched in CRPC. Inhibiting SRF-dependent AR action may be an effective treatment strategy following failure of ADT but remains poorly understood. Previously, we determined that the Rho effector Protein Kinase N1 (PKN1) transduces signaling from AR to SRF. This signaling was mediated by PKN1 and SRF interaction at AR-SRF target genes, whose transcription relied on PKN1’s kinase moiety. In Co-IP, ChIP and qRT-PCR assays, kinase-dead PKN1 prevented SRF-PKN1 interaction, recruitment of PKN1 to SRF target genes and AR-SRF target gene expression. Here, we performed biotin-based Turbo-ID-mediated proximity labeling coupled with mass spectrometry (PLA-MS) to elucidate SRF-PKN1 complex function. We isolated 26 significant and mostly novel PKN1 interactors with preferential roles in transcription, RNA binding and DNA repair. The majority was not previously linked to AR or SRF action or CaP progression. PLA-MS studies which we combined with SRF IP, androgen treatment or nuclear localization independently returned several of these PKN1 interactors. A prominent hit was the RNA helicase UPF1, involved in mRNA surveillance and nonsense-mediated mRNA decay (NMD). Co-IP assays confirmed that both SRF and PKN1 interacted with UPF1 and showed wild-type PKN1 increased while kinase-dead PKN1 decreased UPF1-SRF interactions. Moreover, silencing of UPF1 decreased CRPC cell viability, consistent with effects of SRF and PKN1 loss. UPF1 protein sequence contained 3 putative PKN1 consensus phosphorylation motifs, 2 of which were confirmed in in vitro kinase assays. RNA-Seq studies combined with pathway analyses revealed considerable overlap between AR-dependent UPF1- and PKN1-dependent gene signatures and cell functions. Confirming clinical relevance of the UPF1-SRF-PKN1 interactions, we found overlap in gene expression profiles of CaP specimens that showed high PKN1, high SRF and high UPF1 expression, which was enriched in CRPCs and associated with functions in cell proliferation and cell division. Taken together, our work isolated UPF1, which is druggable, as an entirely novel regulator of clinically relevant AR-SRF-PKN1 action in CaP growth, and suggest a previously unrecognized role for NMD in CaP progression.

Presentation: Saturday, June 17, 2023