Regulation of the Transcriptional Coactivator FHL2 Licenses Activation of the Androgen Receptor in Castrate-Resistant Prostate Cancer

Published OnlineFirst June 25, 2013; DOI: 10.1158/0008-5472.CAN-12-4520

Cancer Molecular and Cellular Pathobiology Research

Meagan J. McGrath1, Lauren C. Binge1,2, Absorn Sriratana1, Hong Wang3, Paul A. Robinson1, David Pook3,4, Clare G. Fedele1,5, Susan Brown1, Jennifer M. Dyson1, Denny L. Cottle1, Belinda S. Cowling1,6, Birunthi Niranjan3, Gail P. Risbridger3, and Christina A. Mitchell1

Abstract 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. Although the 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 that 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 show that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathway that 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. Cancer Res; 73(16); 1–14. 2013 AACR.

Introduction or metastatic prostate cancer; however, in many cases, cas- The androgen receptor (AR) is activated by androgens and trate-resistant prostate tumors (CRPC) develop within 2 to 3 recruits transcriptional coregulatory proteins to activate genes years (1). There are no curative treatments for CRPC and the required for the survival and proliferation of prostate epithelial current survival rate is 1 to 2 years following tumor relapse (1). cells. Prostate cancer begins as a slowly growing localized The molecular events underlying the transition from hormone- disease that can be treated by surgery or radiation. Androgen sensitive prostate cancer to CRPC are unclear; however, the deprivation therapy is used to treat locally advanced, recurrent, resurgence of AR activity is fundamental and allows cancer cells to evade androgen ablation. AR reactivation may occur via AR gene mutations or alternate splicing, resulting in expression Authors' Afﬁliations: Departments of 1Biochemistry and Molecular Biol- ogy and 2Immunology, 3Prostate and Breast Cancer Research Group, of ligand-independent AR variants (2). Altered expression or Department of Anatomy and Developmental Biology, Monash University, activity of AR coregulatory proteins also correlates with more 4 Clayton Victoria, Australia; Department of Oncology, Southern Health, aggressive cancer and a deregulated AR transcriptional net- East Bentleigh, Victoria, Australia; and 5Melanoma Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; and work (3), suggesting that a shift in the AR–coregulator rela- 6Department of Translational Medicine and Neurogenetics, Institut de tionship may also contribute to CRPC. The challenge of current Gen etique et de Biologie Moleculaire et Cellulaire (IGBMC), Illkirch, Stras- bourg, France research is to identify pathways involved in the transition to CRPC, notably those that potentiate ligand-independent AR Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). function and the activity of mutant AR. The role of four and a half LIM protein 2 (FHL2) in cancer is Corresponding Author: Christina A. Mitchell, Department of Biochemistry and Molecular Biology, Monash University, Building 77, Wellington Road, dependent upon its function in the nucleus as a coactivator or Clayton, Victoria 3800, Australia. Phone: 61-3-9905-4318; Fax: 61-3-9902- corepressor of multiple transcription factors important in can- 9500; E-mail: [email protected] cer, including the AR (4, 5). LIM domains form a double-zinc doi: 10.1158/0008-5472.CAN-12-4520 ﬁnger protein structure that has a role in protein-binding. FHL2 2013 American Association for Cancer Research. comprises four and a half LIM domains and can bind to multiple

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proteins that regulate transcription (6, 7). FHL2 can also shuttle Cell Cultures; ECACC), and all cultured in RMPI-1640 contain- between the cytoplasm and nucleus (8). The expression of FHL2 ing 10% fetal calf serum and 2 mmol/L L-glutamine. ATCC and is deregulated in many cancers; however, more recent studies ECACC test the authenticity of these cell lines using short have identified significant nuclear accumulation of FHL2 in tandem repeat analyses. PNT1a and 22Rv1 cells were used several cancers includinglung(9), colon (10)and prostate cancer immediately following receipt. Bulk frozen stocks of LNCaP (8, 11), which is absent in benign tissue and correlates with and DU145 cells were prepared immediately following receipt diseaseprogressionandpoorpatientprognosis.Therefore,FHL2 and used within 3 months following resuscitation; during this nuclear localization may be important during cancer progres- period, cell lines were authenticated by AR immunostaining, sion. In fibroblasts, stimulation with lysophosphatidic acid or morphologic inspection, and tested negative for myocoplasma activation of Rho GTPase increases FHL2 nuclear localization by PCR (November 2012). M2FIL and A7FILþ cell lines were (8); however the mechanism promoting FHL2 nuclear accumu- from T. Stossel (Brigham and Women's Hospital, Harvard lation during cancer progression is unknown. Medical School, Boston, MA), cultured as previously described FHL2 coactivates wild-type AR (4), promotes the prolifera- (20), and routinely validated by immunoblotting lysates with a tion of prostate cancer cells (12), and is integral for prostate filamin antibody (Fig. 1B). The authenticity of noncancer COS1 cancer cell survival via repression of the transcription factor cells (cultured in Dulbecco's Modified Eagle Medium 10% FOXO1, which functions downstream from the tumor sup- Newborn Calf Serum and 2 mmol/L L-glutamine) was not pressor PTEN (7). FHL2 integrates a key regulatory network validated. through binding CBP/p300 and b-catenin, which function synergistically to coactivate AR (6). The FHL2 gene is also Luciferase assays androgen responsive, providing a feed-forward mechanism for Luciferase assays were conducted by plating 5 104 cells per robust AR activation (12). siRNA depletion of FHL2 decreases 12-well plate. For Gal-FHL2 assays, cells were cotransfected proliferation of androgen-independent prostate cancer cells with 500 ng pG5E1b-luciferase, 20 ng phRL-TK (Renilla), 50 ng (12), suggesting a role in ligand-independent AR activation. of pCMXGal-vector or pCMXGal-FHL2, and 500 ng of GFP- However, to date, all studies report FHL2 activation of AR is vector, GFP-filamin (wild-type), GFP-filamin (D1,762–1,764) or hormone-dependent (4, 6, 8) and a mechanism for ligand- HA-vector, HA-calpastatin. COS1, M2FIL, and A7FILþ cells independent AR activation is not known. FHL2 localizes to the were transfected using Lipofectamine (Invitrogen) and 24 nucleus in high-grade, localized prostate cancer, correlating hours posttransfection were serum starved or serum starved with cancer recurrence following radical prostatectomy (8, 11). and then treated with lysophosphatidic acid, ionomycin, or However, despite being a well-defined AR coactivator, a role for calcimycin. For AR transcriptional assays PNT1a, LNCaP, and FHL2 in CPRC has not been examined. DU145 prostate cancer cells were cotransfected using Lipo- We identify a novel mechanism regulating FHL2 nuclear fectamine 2000 (Invitrogen); 1 mg HA-FHL2 or HA-vector; flag- localization and transactivation function, through interaction vector, flag-AR full-length, or trAR; His-vector or His-Ar-V7; with the actin crosslinking protein filamin (13). In the cyto- 2 mg Myc-filamin full-length or (R 16–23; 90 kDa filamin frag- plasm, filamin promotes actin fiber formation and is impli- ment) or Myc-vector; 20 ng phRL-TK (Renilla), and 2 mgof cated in cell motility and prostate cancer invasiveness (14). In ARE3-luciferase or prostate-specific antigen (PSA)-luciferase. the nucleus, a truncated filamin fragment directly corepresses For androgen-depletion versus androgen-stimulation cells the AR (15). Nuclear filamin is also required to maintain the were cultured in phenol-red–free RPMI media containing androgen-dependent growth of prostate cancer cells and sen- 10% charcoal-stripped serum (Gibco; Invitrogen) treatment sitizes CRPC cells to androgen-deprivation therapy by a mech- with 10 nmol/L dihydrotestosterone (DHT) for 24 hours. anism that is largely unknown (16, 17). Therapeutic targeting of Enzalutaminde (MDV3100; Selleck Chemicals) was used at nuclear filamin is a recently suggested treatment for CPRC (17). 10 mmol/L for 24 hours (18). Cells were lysed in passive lysis We show that the nuclear localization of FHL2 and subsequent buffer and luciferase activity measured using the Dual Lucif- AR activation is driven by calpain cleavage of filamin, a erase Reporter Kit (Promega). All luciferase data were nor- pathway that shows differential activation in prostate epithe- malized for transfection efficiency by correcting for constitu- lial versus prostate cancer cells. Critically, deregulation of the tive Renilla luciferase activity and is presented relative to the FHL2-AR–filamin complex facilitates ligand-independent acti- relevant control cells. vation of AR variants present in CRPC, hence providing mechanistic insight into the enhanced AR activation that drives Prostate sections CRPC progression. Moreover, this is the first study to identify a Sections of benign prostate tissue and localized prostate disease-specific mechanism for the enhanced FHL2 nuclear cancer were provided by the Australian Prostate Cancer Bio- localization, which is associated with the progression of an Resource (Cabrini Human Research Ethics Committee 03-14- increasing number of different cancers. 04-08; Monash University, Clayton, Victoria, Australia; Human Research Ethics Committee 2004/145MC). CRPC samples were Materials and Methods provided by Dr. David Pook, Department of Oncology, South- Cell lines ern Health, East Bentleigh, Victoria, Australia (10247A). Anti- COS1, LNCaP, DU145, and 22Rv1 cells were purchased from gen retrieval was conducted as previously described (19) and American Type Culture Collection (ATCC) and PNT1a were incubated with primary antibodies overnight at 4C; FHL2 purchased from Sigma Aldrich (European Collection of Animal (1:50, rabbit), filamin (1:1,000, N- or C-terminal), AR-V7 (1:500),

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Figure 1. FHL2 localizes constitutively to the nucleus in filamin-deficient cells. A, FHL2 comprises four and a half LIM domains. LIM domains 3 and 4 were used as "bait" in yeast two- hybrid analysis (top). Filamin domain structure comprising N- terminal actin-binding domain followed by 24 Ig-like repeats (bottom). Two hinge regions are cleaved by calpain (arrows). Using yeast two-hybrid analysis, we identified FHL2 binds repeat 23 of filamin C. B, filamin and FHL2 expression in M2FIL versus A7FILþ cells. b-Tubulin was used as a loading control. C, A7FILþ versus M2FIL cells stained for FHL2 and either filamin, focal adhesions (paxillin), or phalloidin (actin). Scale bar, 100 mm; 25 mm in high magnification images. D, A7FILþ and M2FIL cells were serum starved or lysophosphatidic acid stimulated before staining for FHL2 and nuclei (propidium iodide). Scale bar, 100 mm.

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HRP conjugated antibodies with DAB detection (Dako) and hematoxylin nuclei counterstaining. For immunofluorescence, sections were incubated with Alexa Fluor-conjugated second- ary antibodies (1:500), with nuclear TO-PRO–3 iodide. A Fisher exact test determined the statistical significance of FHL2 and filamin nuclear staining in CRPC. For other detailed methods, refer to Supplementary Methods. Results FHL2 localizes to the nucleus in filamin-deficient cells To identify new FHL2-binding partners, we used LIM domains 3 and 4 as bait in a yeast two-hybrid screen of a human skeletal muscle cDNA library and identified several interacting clones encoding the Ig-like repeat 23 of filamin C (amino acids 2,500–2,558; Fig. 1A). The 3 filamin isoforms, A, B, and C share significant amino acid homology in repeats 22–24, to which FHL2 binds (Supplementary Fig. S1; ref. 13). Filamin A is expressed in prostate cancer (16) and was used for all subsequent studies. In vitro binding studies confirmed a direct interaction between FHL2 and filamin (Supplementary Fig. S2A). Coimmunoprecipitation of HA-FHL2 and truncated Myc-filamin (repeats 22–24) was shown, also delineating the FHL2-binding site to this C-terminal region of filamin (Sup- plementary Fig. S2B). FHL2 localization was examined in filamin-deficient fi (M2FIL ) cells compared with the lamin-replete A7FILþ sub- line (20), which both express FHL2 (Fig. 1B). Cells were imaged using a confocal laser scanning microscope and a single optical section taken at the ventral cell surface for visualization of FHL2 staining at the cytoskeleton. In this focal plane the nucleus is not visible. In A7FILþ cells, FHL2 localized to the cytoskeleton, colocalizing with both filamin and actin, and was also present at focal adhesions, colocalizing with paxillin (Fig. 1C, a–i). In M2FIL cells, FHL2 localized only to focal adhesions, consistent with its association with integrins (21), but not actin stress fibers, which were absent due to loss of filamin actin crosslinking (Fig. 1C, m–t; ref. 20). The nuclear localization and transactivation activity of FHL2 was examined in M2FIL versus A7FILþ cells following stimulation with lysophosphatidic acid, which stimulates FHL2 nuclear localization (8). Confocal imaging of A7FILþ and M2FIL cells as a Z-stack was used to compare FHL2 nuclear and cytoplasmic staining. In unstimulated A7FILþ cells FHL2 was at the cytoskeleton, but translocated to the nucleus following lysophosphatidic acid Figure 1. Continued ( ) E, nuclear and cytoplasmic subcellular fractions stimulation and in contrast was constitutively nuclear in prepared from unstimulated A7FILþ and M2FIL cells and immunoblotted for FHL2. GAPDH and lamin A/C control cytoplasm or nuclear proteins M2FIL cells (Fig. 1D). Subcellular fractionation in unstimu- were used. Ponceau Red staining of Western blot membranes (loading lated cells revealed FHL2 was enriched in the nuclear control). F, the nuclear:cytoplasmic ratio of FHL2 immunostaining was fraction in M2FIL cells, with relatively less FHL2 present quantified in unstimulated A7 þ and M2 (serum starved) or following FIL FIL in the nucleus of A7 þ cells (Fig. 1E). Quantification of lysophosphatidic acid stimulation by the measuring fluorescence FIL the nuclear:cytoplasmic ratio for FHL2 immunostaining intensity. G, A7FILþ and M2FIL cells were cotransfected with a Gal- luciferase reporter and Gal-FHL2 (or Gal-vector, not shown). Cells were (representative images Fig. 1D) further revealed a constitu- lysophosphatidic acid stimulated and luciferase activity measured. Data tively higher ratio in M2 cells than A7 þ cells under FIL FIL represent mean from n ¼ 6 SEM; , P ¼ 0.01. nonstimulated conditions (Fig. 1F). A Gal-FHL2 fusion protein that transactivates a Gal-luciferase reporter is used to cytokeratin (CK)-5 (1:200), CK-8 (1:200), and normal rabbit quantify FHL2 transactivation activity in the nucleus (4, 8). m 0 or mouse immunoglobulin (Ig)G (5 g/mL). 3,3 -diamino- In unstimulated A7FILþ cells, Gal-FHL2 exhibited low trans- benzidine (DAB) staining was conducted using Envisionþ activation of the Gal-luciferase reporter, which increased

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Figure 2. Calpain cleavage of filamin induces FHL2 nuclear localization and AR coactivation. A, lysates from prostate epithelial (PNT1a) and prostate cancer (LNCaP and DU145) cell lines (þ/) calpain inhibition, immunoblotted for filamin (C-term), FHL2, and b-tubulin (loading control). B, coimmunoprecipitation of endogenous FHL2 and filamin in prostate cancer cell lines. NonI, nonimmune control. C, coimmunoprecipitation in COS1 cells coexpressing Myc- FilaminR16–23, (90 kDa filamin fragment) and either HA-vector control or HA-FHL2. D, cells calpain inhibition using calpeptin were costained for FHL2 and filamin (C-terminal) and nuclear stain TO-PRO-3 iodide (Topro). www.aacrjournals.org Cancer Res; 73(16) August 15, 2013 OF5

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Figure 2. (Continued) E, FHL2 localization in PTN1a cells expressing Myc-vector or Myc- FilaminR16–23. TO-PRO-3 iodide (nuclei). Scale bar, 100 mm. F, cells were cotransfected with ﬂag-AR, HA-FHL2, and p(ARE3)-luciferase and left untreated or pretreated with calpeptin before stimulation with androgen (10 nmol/L DHT). For all luciferase assays, data represent the mean from n ¼ 5–8 SEM; , P ¼ 0.01. G, expression of the AR-target gene PSA was examined by qRT-PCR in LNCaP cells transfected with HA-FHL2 (or HA-vector) calpain inhibition using calpeptin. Data represent the mean from n ¼ 4 SEM; , P ¼ 0.05.

following lysophosphatidic acid stimulation, but constitu- also abrogated by calpain inhibition using calpeptin (Sup- tively activated transcription in M2FIL cells irrespective of plementary Fig. S2E) or the calpain-inhibitory protein cal- lysophosphatidic acid treatment(Fig.1G).Therefore,inthe pastatin(SupplementaryFig.S2F;ref.22).Therefore,calpain presence of intact filamin, FHL2 is sequestered at the activation induces FHL2 nuclear localization and transacti- cytoskeleton and in the absence of filamin, FHL2 localizes vation activity. To address whether these processes are to the nucleus and activates transcription. filamin-dependent, complex formation between endogenous FHL2, filamin, and m-calpain was shown (Supplementary Calpain cleavage of filamin induces FHL2 nuclear Fig.S2G).Furthermore,expressionofacalpain-resitantGFP- fi D – localization lamin ( 1,762 1,764) mutant (23), in M2FIL cells inhibited þ Filamin is a substrate for Ca2 -dependent calpain pro- the increase in transactivation activity observed for Gal- teases, with 2 cleavage sites; within hinge I, between repeats FHL2 following ionomycin-stimulated calpain activation 15 and 16, and within hinge II between repeats 23 and (Supplementary Fig. S2H). Therefore, calpain cleavage of 24 (Fig. 1A; ref. 16). Calpain cleavage of filamin generates filamin drives FHL2 nuclear localization. 2 fragments; N-terminal 170 kDa (repeats 1–15) and a C-terminal 90 kDa fragment (repeats 16–23; Fig. 1A). The Calpain cleavage of filamin is increased in prostate effect of filamin-cleavage on FHL2 nuclear localization and cancer cells, resulting in nuclear FHL2 accumulation and transactivation function was examined. Calpain activation increased AR coactivation þ was induced in COS1 cells by ionomycin (Ca2 ionophore) Calpain expression and activity are increased in prostate treatment, resulting in filamin cleavage (Supplementary Fig. cancer (24); therefore, the affect of calpain cleavage of S2C) and FHL2 nuclear localization (Supplementary Fig. filamin on FHL2 nuclear localization and AR coactivation S2D) and both events were reduced by calpain inhibition was assessed in prostate cancer cell lines. Filamin cleavage using calpeptin. Calpain activation also increased transac- in a prostate epithelial cell line (PNT1a) versus prostate tivation of a Gal-luciferase reporter by Gal-FHL2, an effect cancer cell lines (LNCaP and DU145) was compared by

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immunoblotting using a C-terminal antibody that recog- noprecipitation of the 90 kDa filamin fragment (Myc-fila- nizes the full-length filamin (280 kDa) and the C-terminal minR16–23) with HA-FHL2 (Fig. 2C). fragment generated following calpain cleavage (90 kDa; Intact filamin localizes to the cytoskeleton; however, fol- ref. 16). A 90 kDa filamin fragment was present in prostate lowing calpain cleavage, the 90 kDa filamin fragment translo- cancer cell lines (LNCaP and DU145) and was partially cates to the nucleus and the 170 kDa fragment is retained in the reduced following calpain inhibition using calpeptin (100 cytoplasm (16). In PNT1a cells, filamin was not cleaved and mmol/L for 24 hours), whereas PNT1a cells expressed only both FHL2 and filamin (C-terminal antibody) colocalized at the full-length filamin (Fig. 2A). Higher doses of calpain inhibitor cytoskeleton (Fig. 2D). In LNCaP and DU145 prostate cancer or longer incubation resulted in cell death and could not be cell lines, which exhibited significant filamin-cleavage, nuclear used to further reduce filamin cleavage. Filamin cleavage in costaining of FHL2 and filamin was observed, which was LNCaP and DU145 cells was confirmed by immunoblotting reduced by calpain inhibition using calpeptin. Ectopic expres- with an N-terminal filamin antibody, which recognizes the sion of the 90 kDa filamin fragment (Myc-FilaminR16–23)in 170 kDa cleaved fragment (Supplementary Fig. S3A). Coim- PNT1a cells induced cytoskeleton to nuclear translocation of munoprecipitation revealed endogenous FHL2 bound only FHL2 (Fig. 2E), evidence that the 90 kDa filamin fragment full-length filamin in PNT1a cells and to both full-length promotes nuclear FHL2. FHL2 coactivation of the AR was filamin and the 90 kDa fragment in LNCaP and DU145 cells examined under conditions of calpain inhibition, using 2 fi (Fig. 2B). The latter interaction was con rmed by coimmu- AR-regulated reporters p(ARE3)-luciferase (Fig. 2F) and PSA-

Figure 3. FHL2 and filamin localize to the nucleus in CRPC and not benign prostate tissue or localized prostate cancer. A, sections of human benign prostate or localized prostate cancer were stained for FHL2, filamin (C-terminal; nuclear fragment repeats 16–23), or filamin (N-terminal; cytoplasmic fragment repeats 1–15). In control studies, benign prostate tissue was stained with nonimmune rabbit or mouse IgG. B, serial sections of human CRPC from 2 representative patients stained for FHL2 and filamin. Boxed regions indicate areas shown in high magnification, Scale bars, 50 mm. Open arrows, basal epithelial cells; closed arrows,luminal epithelial cells; arrowheads, filamin stromal staining. PCa, prostate cancer.

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fi Table 1. Frequency of nuclear localization of arrow) and basal (open arrow) epithelial cells and lamin was restricted to the cytoplasm of basal cells (Fig. 3A). FHL2, filamin, and AR-V7 in human prostate Coimmunostaining with luminal (CK-8) and basal (CK-5) samples epithelial cell markers confirmed FHL2 expression in luminal and basal cells and filamin only in basal cells (Supple- Localized mentary Fig. S6), indicating FHL2 and filamin coexpression prostate occurs in basal cells of benign prostate. The nuclear local- Benign cancer CRPC ization of filamininbenignprostateandlocalizedprostate FHL2 0/8 0/8 6/8a cancers has been reported (14). In contrast, we observed Filamin 0/8 0/8 6/8a similar cytoplasmic filamin staining in benign prostate using AR-V7 —— 7/8 both N- and C-terminal–specific antibodies (Fig. 3A), indicating that filamin remains intact in the cytoplasm. Similar aP ¼ 0.001. overlapping cytoplasmic N- and C-terminal filamin antibody staining was observed in the PNT1a prostate epithelial cells luciferase (Supplementary Fig. S4A). Cells were left untreated (Supplementary Fig. S3B), where filamin was not cleaved or treated with the calpain inhibitor calpeptin, followed by (Fig.2AandSupplementaryFig.S3A).Ourdataareconsis- androgen (DHT) stimulation. In the absence of calpain inhi- tent with the absence of filamin cleavage in benign prostate, bition, HA-FHL2 coactivated AR transcription of both p(ARE3)- correlating with absent nuclear FHL2. and PSA reporters to a greater extent in prostate cancer cells, In low-grade localized prostate cancers, FHL2 was restricted relative to the PNT1a cells. Treatment of LNCaP and DU145 to the cytoplasm and filamin was not detected in cancer cells cells with calpeptin reduced FHL2 coactivation of AR to basal (Fig. 3A). Loss of basal cells is a hallmark of neoplastic foci in levels observed in PNT1a cells. Calpain-dependent activation localized prostate cancer (29); therefore, the absence of filamin of endogenous AR-target genes by FHL2 was examined using in localized cancer is consistent with filamin expression exclu- quantitative real-time PCR (qRT-PCR). In LNCaP cells, expres- sively in CK5þ8 basal cells in benign prostate. Notably, the sion of HA-FHL2 increased PSA (Fig. 2G), hK2, and TMPRSS2 majority of CRPC samples examined (75%) showed strong (Supplementary Fig. S4B and S4C) mRNA levels relative to HA- nuclear FHL2 staining and a reemergence of filamin staining vector control transfected cells, which was abrogated by in the nucleus of cancer cells (Fig. 3B; Table 1). A correlation calpain inhibition. Collectively, these studies show that the was also observed between the CRPCs, which exhibited both enhanced susceptibility of filamin to calpain cleavage exclu- nuclear FHL2 and filamin (Table 2), suggesting an association sively in prostate cancer cells results in enhanced FHL2 nuclear between generation of the nuclear filamin fragment by calpain localization and AR activation. Filamin phosphorylation at cleavage and FHL2 nuclear localization in CRPC. þ Ser2152 protects against calpain cleavage (25). The Ca2 /cal- modulin-dependent protein phosphatase calcineurin depho- Ligand-independent activation of the AR by FHL2 sphorylates filamin to promote calpain cleavage (26) and CRPC is driven by ligand-independent AR activation and calcineurin expression is increased in prostate cancer (27). is associated with high expression of AR variants, which are We show here that filamin dephosphorylation by calcineurin in absent or expressed at low levels in benign prostate or hormone- prostate cancer cell lines was responsible for the enhanced na€ve prostate cancer (30). We examined whether FHL2 pro- susceptibility of filamin to calpain cleavage, a pathway that motes ligand-independent coactivation of 2 AR variants, trAR initiates FHL2 nuclear localization and increases AR activation (31) and AR-V7 (AR3; refs. 30, 32), which have truncation of the (Supplementary Fig. S5). ligand-binding domain (LBD) and promote androgen-independent growth. Interestingly, trAR is generated by calpain cleavage FHL2 and filamin localize to the nucleus in castrate- of the AR (31). HA-FHL2 binds both trAR and full-length AR (Fig. resistant prostate cancer 4A). In DU145 cells, FHL2 coactivated full-length AR under We investigated whether calpain cleavage of filamin reg- androgen-stimulated conditions and, significantly, FHL2 coac- ulates FHL2 nuclear localization during prostate cancer tivation of trAR was ligand independent (Fig. 4B). The nuclear 90 progression. Benign prostatic epithelium is composed of kDa C-terminal filamin fragment, which we show binds FHL2, multiple cell types including basal and luminal cells, defined also represses AR activity (33). Therefore, we determined wheth- by expression of specific CK proteins (28). In benign prostate er the FHL2-filamin complex coregulates AR activity. In contrast tissue, FHL2 localized to the cytoplasm of luminal (closed to FHL2, the 90 kDa filamin fragment (Myc-filaminR16–23)bound

Table 2. CRPC samples exhibiting nuclear localization of FHL2, ﬁlamin, and AR-V7

CRPC1 CRPC2 CRPC3 CRPC4 CRPC5 CRPC6 CRPC7 CRPC8 FHL2 þþþþþþ Filamin þþþþþþ AR-V7 þþþþþþþ

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Figure 4. FHL2 competes with ﬁlamin for binding to wild-type AR, but not a truncated AR (trAR). A, coimmunoprecipitation in COS1 cells coexpressing HA-FHL2 (or HA-vector) and either FLAG-tagged full-length AR or trAR. B, DU145 cells were cotransfected with combinations of HA-vector or HA-FHL2; FLAG-vector, FLAG-AR (full-length) or FLAG-trAR, and the AR-responsive reporter p(ARE3)-luciferase. Cells were maintained under androgen-depleted conditions ( Androgen) or stimulated with DHT (þ Androgen) and luciferase activity measured. C, coimmunoprecipitation in COS1 cells coexpressing Myc-FilaminR16–23 (or Myc-vector) and either FLAG-tagged full-length AR or trAR. D, competitive-binding assay; cells were cotransfected as indicated and also coexpressed Myc-vector or Myc-ﬁlaminR16–23. Lysates were immunoprecipitated (IP) and immunoblotted (WB) as indicated.

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Figure 4. (Continued) E and F, filamin represses FHL2 coactivation of wild-type AR (E), but not trAR (F); DU145 cells were cotransfected as indicated and cells also coexpressed Myc-vector or Myc-FilaminR16–23. FHL2 coactivation of full-length AR and trAR was measured under androgen-dependent and androgen-independent conditions. For all luciferase assays, data represent the mean from n ¼ 8 SEM; , P ¼ 0.05. G, AR coregulation by the FHL2-filamin complex. The 90 kDa filamin fragment competes with FHL2 for binding to full-length AR and also represses ligand-dependent AR coactivation by FHL2. ARE, androgen-response element; FLNa, filamin A (a); FHL2, but not the 90 kDa filamin fragment, binds to AR variants trAR and AR-V7, such that coactivation of truncated AR variants by FHL2 is constitutive (b).

full-length AR, but not trAR (Fig. 4C). Competitive-binding lutamide acts on the full-length AR by directly binding the LBD studies revealed Myc-filamin16–23 disrupted HA-FHL2 binding (18) and expression of AR variants including AR-V7, which lack to full-length AR, but not trAR (Fig. 4D). Furthermore, FHL2 the LBD, may confer resistance to this therapy (36, 37). coactivation of full-length AR (Fig. 4E), but not trAR (Fig. 4F), Enzalutamide does not directly inhibit the transcriptional was repressed by the 90 kDa filamin fragment. Therefore, FHL2 activity of AR-V7 (36) and AR-V7–expressing prostate cancer and filamin compete for binding to full-length AR, but not trAR. cells are resistant to enzalutamide (37). FHL2 activation of full- Moreover, FHL2 coactivation of full-length AR is repressed by length AR, trAR, and AR-V7 following enzalutamide treatment filamin, but the ligand-independent activation of trAR by FHL2 was examined in DU145 cells that do not express endogenous is constitutive (Fig. 4G). AR. AR-V7 (Fig. 5F) and trAR (Supplementary Fig. S7A) Similar results were obtained using the AR-V7 variant, which coactivation of the ARE3-luciferase reporter under andro- is generated by alternate splicing of the AR gene (30, 32) and has gen-depleted conditions was not affected by enzalutamide generated significant interest as a pathologically-relevant AR treatment and was also increased by FHL2 irrespective of variant expressed in CRPC (30, 32, 34, 35). AR-V7 bound to enzalutamide treatment. In contrast, activation of the ARE3- fi fi FHL2, but not lamin (Myc- laminR16–23; Fig. 5A) and endog- luciferase reporter by full-length AR was reduced by enzalu- enous FHL2, and AR-V7 bind in the 22Rv1 prostate cancer tamide (Supplementary Fig. S7B). Surprisingly, coexpression of cell line (Fig. 5B). Ligand-independent coactivation of AR-V7 FHL2 was sufficient to restore the transcriptional activity of by FHL2 was constitutive and not repressed by the 90 kDa full-length AR in the presence of this antiandrogen (Supple- filamin fragment (Fig. 5C and D). AR-V7 is frequently expressed mentary Fig. S7B). Therefore, FHL2 can not only sustain 20-fold higher in CPRC than hormone-naive prostate cancer enhanced activation of AR variants lacking the LBD irrespec- (30) and correlates with poor survival (34). In serial sections of tive of enzalutamide treatment, but can also reverse the human CRPC, FHL2 and AR-V7 colocalized in the nucleus of inhibitory affects of enzalutamide on full-length AR. cancer cells (Fig. 5E) and 75% of CRPCs exhibited both nuclear FHL2 and AR-V7 (Table 1, Table 2). Therefore, FHL2 nuclear Discussion localization in CRPC may promote constitutive, ligand-inde- We report a novel pathway for AR activation in prostate pendent activation of this clinically important AR variant. cancer. In a prostate epithelial cell line FHL2, nuclear locali- The antiandrogen enzalutamide promotes tumor regression zation and AR coactivation are tightly regulated, whereby, the in a mouse xenograft model of CRPC (18) and has progressed absence of filamin cleavage results in FHL2 sequestration at rapidly toward approval for treating metastatic CRPC. Enza- the cytoskeleton and low AR activity. Conversely, in prostate

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cancer cell lines, calpain cleavage of filamin drives nuclear Over- and underexpression of FHL2 is reported in prostate FHL2 and enhanced AR activation. In addition, FHL2 locali- cancer (8, 11, 38), we revealed FHL2 nuclear localization by the zation and hence AR activation in prostate cancer cells is mechanism identified here may also be important. In tumors, driven both by increased calpain activity and the susceptibility which had progressed to CRPC, nuclear localization of FHL2 and of filamin-to-calpain cleavage following dephosphorylation by calpain-cleaved filamin was consistently observed in the same calcineurin. patient biopsies supporting our contention that calpain

Figure 5. FHL2 coactivates an AR variant; AR-V7 present in CRPC. A, coimmunoprecipitation in COS1 cells coexpressing HA-FHL2 (or HA-vector) and His-AR- V7. B, coimmunoprecipitation of endogenous FHL2 and AR-V7 from 22Rv1 cells. NonI, nonimmune sera. C, DU145 cells were cotransfected with HA-vector or HA-FHL2, His-vector or His-AR-V7, and p(ARE3)-luciferase as indicated. Cells were maintained under androgen-depleted conditions and luciferase activity measured. D, luciferase assays were repeated as above in cells coexpressing Myc-vector or Myc-FilaminR16–23. E, serial sections from 2 representative CRPCs showing nuclear colocalization of FHL2 and AR-V7. Boxed regions indicate areas shown in high magniﬁcation. Scale bar, 50 mm for all images.

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Figure 5. (Continued) F, ARE3-luciferase assays were repeated in DU145 cells expressing AR-V7 HA-FHL2 expression as above, enzalutamide treatment. For all luciferase assays, data represent the mean from n ¼ 4–8 SEM; , P ¼ 0.05. G, differential regulation of FHL2 nuclear localization and AR coactivation during prostate cancer progression. In benign prostate tissue, the absence of ﬁlamin cleavage results in FHL2 cytoplasmic sequestration and low AR coactivation. In CRPC, ﬁlamin cleavage to the 90 kDa fragment activates FHL2 nuclear accumulation and FHL2 promotes the constitutive ligand-independent coactivation of the AR variant AR-V7.

mediates filamin and FHL2 nuclear association. Critically, FHL2 the prototype AR, including genes, which regulate cell-cycle and filamin were not detected in the nucleus of benign prostate progression (32, 35). Despite its importance in CRPC, the tissue or low-grade localized prostate cancer (Fig. 5G). The regulation of AR-V7 is largely uncharacterized, the identifica- reemergence of filamin staining in CRPC cells (absent in local- tion of which may lead to novel therapeutic targets for incur- ized cancer) is interesting given the prediction that cancer stem able CPRC. Vav3, a RhoGTPase guanine nucleotide exchange cells, which reinitiate CRPC tumor growth following androgen- factor, which like FHL2 enhances AR-V7 coactivation, is critical deprivation therapy, reside in the CK5þ8 basal cell compart- for CRPC growth and survival (41). AR-V7 is also regulated by ment (28) and are shown here to coexpress FHL2 and filamin. the phosphoinositide 3-kinase/Akt/FOXO1 signaling pathway, Calpain expression and activity are higher under androgen- whereby FOXO1 inhibits AR-V7 activity (42). Interestingly, depleted conditions (39), suggesting that androgen ablation FHL2 represses FOXO1 activity in prostate cancer cells (7). therapy creates an environment that is not only highly selective The absence of filamin binding to C-terminally truncated for expression of ligand-independent variants including AR-V7 AR variants is consistent with a requirement for the AR–LBD (40), but is also conducive for promoting nuclear FHL2. There- for filamin association (33) and indicates filamin repressive fore, of note are our data showing that FHL2 activates AR-V7 and function is limited to the prototype AR. Differential regula- both proteins colocalize in the nucleus of CRPC. tion of full-length AR versus truncated AR by the FHL2– filamin complex is an important finding given the prevalence The FHL2-filamin complex is a novel AR regulatory of AR mutations in CRPC (10%–30%), many of which result mechanism that may be deregulated in CRPC in AR mutants that lack all or part of the LBD (2). Given that We show that the nuclear 90 kDa filamin fragment competes inhibition of calpain activity blocks filamin cleavage, FHL2 with FHL2 for binding full-length AR and represses ligand- nuclear localization, and AR coactivation, we speculate that dependent AR coactivation by FHL2. This is supported by the calpain inhibitors may provide a novel avenue for CRPC findings that Ig-repeats 22–24 of filamin bind AR (33), a region treatment. We have further shown FHL2 coactivation of AR- that overlaps with the FHL2-binding site (Ig repeat 23) iden- V7 and trAR is resistant to enzalutamide. Expression of tified here. Therefore, we identify filamin as an important FHL2 also restored full-length AR transcriptional activity negative AR regulator via modulation of the AR-coactivator in enzalutamide-treated prostate cancer cells. Therefore, we relationship, whereby the activation of the prototype AR is predict that FHL2 may reduce the efficacy of enzalutamide mediated by a balance between the coactivator FHL2 and the treatment of CRPC by maintaining AR function and calpain corepressor filamin (Fig. 4G, a). We showed how loss in this inhibitors may provide an adjunct to enzalutamide therapy. balance due to the absence of filamin repression results in Targeting calpain activity is suggested for several cancers to constitutive activation of pathologic AR variants notably AR- limit the development, metastases, and neovascularization V7, by FHL2 (Fig. 4G-b). AR-V7 is a major AR variant present in of primary tumors (43). Calpain inhibition also decreases the CRPC that activates a transcriptional program distinct from androgen-independent proliferation of Rv1 cells (31) and

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prostate cancer invasiveness (44). In contrast, a recent Disclosure of Potential Conflicts of Interest report has suggested that enhancing generation of the No potential conflicts of interest were disclosed. nuclear filamin fragment using genistein-combined polysac- charide may be a potential CRPC treatment (17). This comes Authors' Contributions from observations that the 90 kDa filamin fragment main- Conception and design: M.J. McGrath, P.A. Robinson, S. Brown, G.P. Risbridger, tains sensitivity of CRPC cells to androgen deprivation (16, C.A. Mitchell Development of methodology: M.J. McGrath, P.A. Robinson, C.G. Fedele, 17). Our current results, however, clearly indicate that S. Brown, C.A. Mitchell further studies are required to examine the complex inter- Acquisition of data (provided animals, acquired and managed patients, play between FHL2, filamin, and the AR in CRPC. provided facilities, etc.): M.J. McGrath, L.C. Binge, A. Sriratana, H. Wang, D. Pook, J.M. Dyson, D.L. Cottle, B.S. Cowling, B. Niranjan, G.P. Risbridger Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Regulation of nuclear FHL2 by calpain cleavage of computational analysis): M.J. McGrath, L.C. Binge, A. Sriratana, C.G. Fedele, fi C.A. Mitchell lamin may be important in other cancers Writing, review, and/or revision of the manuscript: M.J. McGrath, L.C. Our mechanism for FHL2 nuclear accumulation during Binge, P.A. Robinson, D. Pook, J.M. Dyson, D.L. Cottle, B. Niranjan, G.P. Risbridger, cancer progression is likely to have implications for other C.A. Mitchell Administrative, technical, or material support (i.e., reporting or orga- cancers. FHL2 nuclear localization is increased in human nizing data, constructing databases): A. Sriratana colorectal cancer and during tumor development in the Apc Study supervision: M.J. McGrath, S. Brown, B. Niranjan, C.A. Mitchell mouse model, where FHL2 suppression inhibits tumor initi- ation (10). Transcriptional targets for FHL2 in colon cancer Acknowledgments b include -catenin (45) and the oncogenic EpICD transcription The authors thank the Associate Professors John Pedersen and Mark Fryden- complex (46). Calpain expression and activity is increased in berg (Departments of Anatomy and Cell Biology, and Surgery, Monash Univer- sity) for supplying the human tissues and also Dr. Melissa Papargiris (Australian human colorectal cancer and in colorectal polyps, where it may Prostate Cancer Collaboration) for assisting with the acquisition of patient be an early tumorigenesis event (47) and we predict this may samples. increase filamin cleavage and promote nuclear FHL2. Filamin is a potential diagnostic biomarker for colorectal cancer as it is Grant Support shed by tumors into patient feces (48). Although this latter The Australian Prostate Cancer BioResource is supported by the National study did not examine the presence of the 90 kDa filamin Health and Medical Research Council of Australia Enabling Grant (No. 614296) fragment generated by calpain cleavage, of note was the and by a research infrastructure grant from the Prostate Cancer Foundation observation that filamin appeared at a lower molecular weight Australia. than predicted. Future studies will be directed towards exam- Received December 18, 2012; revised May 30, 2013; accepted June 13, 2013; ining calpain-dependent regulation of FHL2 in other cancers. published OnlineFirst June 25, 2013.

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Regulation of the Transcriptional Coactivator FHL2 Licenses Activation of the Androgen Receptor in Castrate-Resistant Prostate Cancer

Meagan J. McGrath, Lauren C. Binge, Absorn Sriratana, et al.

Cancer Res Published OnlineFirst June 25, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-4520

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