prostate cancer activation of the androgen receptor in castrate-resistant Regulation of the transcriptional coactivator FHL2 licenses

It is now clear that progression from localized prostate cancer to incurable castrate-resistant prostate cancer (CRPC) is driven by continued androgen-receptor (AR) signaling independently of androgen. Thus, there remains a strong rationale to suppress AR activity as the single most important therapeutic goal in CRPC treatment. While expression of ligand-independent AR splice variants confers resistance to AR-targeted therapy, and progression to lethal castrate-resistant cancer, the molecular regulators of AR activity in CRPC remain unclear, in particular those pathways which potentiate the function of mutant AR in CRPC. Here we identify FHL2 as a novel coactivator of ligand-independent AR variants that are important in CRPC. We demonstrate that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain-cleavage of the cytoskeletal protein filamin, a pathway which shows differential activation in prostate epithelial versus prostate cancer cell lines. We further identify a novel FHL2-AR-filamin transcription complex, revealing how deregulation of this axis promotes the constitutive, ligand-independent activation of AR variants which are present in CRPC. Critically, the calpain-cleaved filamin fragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostate cancer. Thus, our work provides mechanistic insight into the enhanced AR activation, most notably of the recently identified AR variants including AR-V7 that drives CRPC progression. Furthermore, our results identify the first disease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. These results offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other human cancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer. LNCaP and DU145 prostate cancer cells were co-transfected using lipofectamine 2000 (Invitrogen); 1 μ g HA-FHL2 or HA-vector; Flag-vector, Flag-AR full-length or trAR; His-vector or His-Ar-V7; 2 μ g Myc-Filamin(R16-23; 90kDa filamin fragment) or Myc-vector; 20 ng phRL-TK (Renilla) and 2 μ g of ARE3-luciferase or PSA-luciferase. For androgen-depletion versus androgen-stimulation cells were cultured in phenol-red free RPMI media 10 endogenous FHL2, filamin and m-calpain was demonstrated (SuppFig2G). Furthermore, expression of a calpain-resitant GFP-filamin ( Δ 1762-1764) mutant (23), in M2 FIL- cells inhibited the increase in transactivation activity observed for Gal-FHL2 following ionomycin-stimulated calpain activation (SuppFig2H). Therefore calpain-cleavage of filamin drives FHL2 nuclear localization. androgen (DHT) stimulation. In the absence of calpain inhibition HA-FHL2 coactivated AR-transcription of both p(ARE 3 )- and PSA-reporters to a greater extent in prostate cancer cells, relative to the PNT1a cells. Treatment of LNCaP and DU145 cells with calpeptin reduced FHL2 coactivation of AR to basal levels observed in PNT1a cells. Calpain-dependent activation of endogenous AR-target genes by FHL2 was examined using qRT-PCR. In LNCaP cells, expression of HA-FHL2 increased PSA (Fig2G), hK2 and TMPRSS2 (SuppFig4A and 4C) mRNA levels relative to HA-vector control transfected cells, which was abrogated by calpain inhibition. Collectively, these studies demonstrate that the enhanced susceptibility of filamin to calpain cleavage exclusively in prostate cancer cells results in enhanced FHL2 nuclear localization and AR activation. Filamin phosphorylation at ser2152 protects against calpain cleavage (25). The Ca 2+/ calmodulin-dependent protein phosphatase calcineurin, dephosphorylates filamin to promote calpain cleavage (26) and calcineurin expression is increased in prostate cancer (27). We show here that filamin dephosphorylation by calcineurin in prostate cancer cell lines was responsible for the enhanced susceptibility of filamin to calpain cleavage, a pathway which initiates FHL2 nuclear localization and increased AR activation (SuppFig5). regulates nuclear cancer Co-immunostaining luminal basal (CK-5) epithelial cell FHL2 expression luminal and basal cells, filamin in basal indicating FHL2 and filamin co-expression occurs in basal cells of benign prostate. The nuclear localization of filamin in benign prostate and localized prostate cancers In contrast, we observed similar cytoplasmic filamin staining in benign prostate using both N- and C-terminal specific antibodies indicating that filamin remains intact in the cytoplasm. filamin further studies are required to examine the complex interplay between FHL2, filamin and the in CRPC. Regulation of nuclear FHL2 by calpain-cleavage of filamin may important Our mechanism for FHL2 nuclear accumulation during cancer progression is likely to have implications for other cancers. FHL2 nuclear localization is increased in human colorectal cancer, and during tumor development in the Apc mouse model, where FHL2 suppression inhibits tumor initiation Transcriptional targets for FHL2 in colon cancer include β -catenin (46) and the oncogenic EpICD transcription complex (47). Calpain expression and activity is increased in human colorectal cancer and in colorectal polyps, where it may be an early tumorigenesis event (48) and we predict this may increase filamin cleavage and promote nuclear FHL2. Filamin is a potential diagnostic biomarker for colorectal cancer as it is shed by tumors into patient feces Although this latter study did not examine the presence of the 90kDa filamin fragment generated by calpain-cleavage, of note was the observation that filamin appeared at a lower molecular weight than predicted. Future studies will be directed towards examining calpain-dependent regulation of FHL2 in other cancers.