Regulation of the Transcriptional Coactivator FHL2 ... fileMolecular and Cellular Pathobiology Regulation of the Transcriptional Coactivator FHL2 Licenses Activation of the Androgen

Molecular and Cellular Pathobiology

Regulation of the Transcriptional Coactivator FHL2 LicensesActivation of the Androgen Receptor in Castrate-ResistantProstate Cancer

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

AbstractIt 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 remainsa 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 andprogression to lethal castrate-resistant cancer, themolecular regulators of AR activity in CRPC remain unclear, inparticular those pathways that potentiate the function of mutant AR in CRPC. Here, we identify FHL2 as a novelcoactivator of ligand-independent AR variants that are important in CRPC.We show that the nuclear localizationof FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathwaythat shows differential activation in prostate epithelial versus prostate cancer cell lines.We further identify a novelFHL2-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 filaminfragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostatecancer. Thus, ourwork providesmechanistic insight into the enhancedAR activation,most notably of the recentlyidentified AR variants, including AR-V7 that drives CRPC progression. Furthermore, our results identify the firstdisease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. Theseresults offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other humancancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer.Cancer Res; 73(16); 5066–79. �2013 AACR.

IntroductionThe androgen receptor (AR) is activated by androgens and

recruits transcriptional coregulatory proteins to activate genesrequired for the survival and proliferation of prostate epithelialcells. Prostate cancer begins as a slowly growing localizeddisease that can be treated by surgery or radiation. Androgendeprivation therapy is used to treat locally advanced, recurrent,

or metastatic prostate cancer; however, in many cases, cas-trate-resistant prostate tumors (CRPC) develop within 2 to 3years (1). There are no curative treatments for CRPC and thecurrent survival rate is 1 to 2 years following tumor relapse (1).Themolecular events underlying the transition fromhormone-sensitive prostate cancer to CRPC are unclear; however, theresurgence of AR activity is fundamental and allows cancercells to evade androgen ablation. AR reactivationmay occur viaAR genemutations or alternate splicing, resulting in expressionof ligand-independent AR variants (2). Altered expression oractivity of AR coregulatory proteins also correlates with moreaggressive cancer and a deregulated AR transcriptional net-work (3), suggesting that a shift in the AR–coregulator rela-tionshipmay also contribute to CRPC. The challenge of currentresearch is to identify pathways involved in the transition toCRPC, notably those that potentiate ligand-independent ARfunction and the activity of mutant AR.

The role of four and a half LIM protein 2 (FHL2) in cancer isdependent upon its function in the nucleus as a coactivator orcorepressor of multiple transcription factors important in can-cer, including the AR (4, 5). LIM domains form a double-zincfinger protein structure that has a role in protein-binding. FHL2comprises four and a half LIMdomains and can bind tomultiple

Authors' Affiliations: Departments of 1Biochemistry and Molecular Biol-ogy and 2Immunology, 3Prostate and Breast Cancer Research Group,Department of Anatomy and Developmental Biology, Monash University,Clayton Victoria, Australia; 4Department of Oncology, Southern Health,East Bentleigh, Victoria, Australia; and 5Melanoma Research Laboratory,Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; and6Department of Translational Medicine and Neurogenetics, Institut deG�en�etique et de Biologie Mol�eculaire et Cellulaire (IGBMC), Illkirch, Stras-bourg, France

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author:Christina A. Mitchell, Department of Biochemistryand Molecular Biology, Monash University, Building 77, Wellington Road,Clayton, Victoria 3800, Australia. Phone: 61-3-9905-4318; Fax: 61-3-9902-9500; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-4520

�2013 American Association for Cancer Research.

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proteins that regulate transcription (6, 7). FHL2 can also shuttlebetween the cytoplasm and nucleus (8). The expression of FHL2is deregulated in many cancers; however, more recent studieshave identified significant nuclear accumulation of FHL2 inseveral cancers including lung (9), colon (10) andprostate cancer(8, 11), which is absent in benign tissue and correlates withdiseaseprogressionandpoorpatientprognosis.Therefore, FHL2nuclear localization may be important during cancer progres-sion. In fibroblasts, stimulation with lysophosphatidic acid oractivation of Rho GTPase increases FHL2 nuclear localization(8); however the mechanism promoting FHL2 nuclear accumu-lation during cancer progression is unknown.FHL2 coactivates wild-type AR (4), promotes the prolifera-

tion of prostate cancer cells (12), and is integral for prostatecancer cell survival via repression of the transcription factorFOXO1, which functions downstream from the tumor sup-pressor PTEN (7). FHL2 integrates a key regulatory networkthrough binding CBP/p300 and b-catenin, which functionsynergistically to coactivate AR (6). The FHL2 gene is alsoandrogen responsive, providing a feed-forward mechanism forrobust AR activation (12). siRNA depletion of FHL2 decreasesproliferation of androgen-independent prostate cancer cells(12), suggesting a role in ligand-independent AR activation.However, to date, all studies report FHL2 activation of AR ishormone-dependent (4, 6, 8) and a mechanism for ligand-independent AR activation is not known. FHL2 localizes to thenucleus in high-grade, localized prostate cancer, correlatingwith cancer recurrence following radical prostatectomy (8, 11).However, despite being awell-definedAR coactivator, a role forFHL2 in CPRC has not been examined.We identify a novel mechanism regulating FHL2 nuclear

localization and transactivation function, through interactionwith the actin crosslinking protein filamin (13). In the cyto-plasm, filamin promotes actin fiber formation and is impli-cated in cell motility and prostate cancer invasiveness (14). Inthe nucleus, a truncated filamin fragment directly corepressesthe AR (15). Nuclear filamin is also required to maintain theandrogen-dependent growth of prostate cancer cells and sen-sitizes CRPC cells to androgen-deprivation therapy by a mech-anism that is largely unknown (16, 17). Therapeutic targeting ofnuclearfilamin is a recently suggested treatment for CPRC (17).We show that the nuclear localization of FHL2 and subsequentAR activation is driven by calpain cleavage of filamin, apathway that shows differential activation in prostate epithe-lial versus prostate cancer cells. Critically, deregulation of theFHL2-AR–filamin complex facilitates ligand-independent acti-vation of AR variants present in CRPC, hence providing mech-anistic insight into the enhanced AR activation that drivesCRPC progression. Moreover, this is the first study to identify adisease-specific mechanism for the enhanced FHL2 nuclearlocalization, which is associated with the progression of anincreasing number of different cancers.

Materials and MethodsCell linesCOS1, LNCaP, DU145, and 22Rv1 cells were purchased from

American Type Culture Collection (ATCC) and PNT1a werepurchased from SigmaAldrich (European Collection of Animal

Cell Cultures; ECACC), and all cultured in RMPI-1640 contain-ing 10% fetal calf serum and 2 mmol/L L-glutamine. ATCC andECACC test the authenticity of these cell lines using shorttandem repeat analyses. PNT1a and 22Rv1 cells were usedimmediately following receipt. Bulk frozen stocks of LNCaPand DU145 cells were prepared immediately following receiptand used within 3 months following resuscitation; during thisperiod, cell lines were authenticated by AR immunostaining,morphologic inspection, and tested negative for myocoplasmaby PCR (November 2012). M2FIL� and A7FILþ cell lines werefrom T. Stossel (Brigham and Women's Hospital, HarvardMedical School, Boston, MA), cultured as previously described(20), and routinely validated by immunoblotting lysates with afilamin antibody (Fig. 1B). The authenticity of noncancer COS1cells (cultured in Dulbecco's Modified Eagle Medium 10%Newborn Calf Serum and 2 mmol/L L-glutamine) was notvalidated.

Luciferase assaysLuciferase assays were conducted by plating 5� 104 cells per

12-well plate. For Gal-FHL2 assays, cells were cotransfectedwith 500 ng pG5E1b-luciferase, 20 ng phRL-TK (Renilla), 50 ngof pCMXGal-vector or pCMXGal-FHL2, and 500 ng of GFP-vector, GFP-filamin (wild-type), GFP-filamin (D1,762–1,764) orHA-vector, HA-calpastatin. COS1, M2FIL�, and A7FILþ cellswere transfected using Lipofectamine (Invitrogen) and 24hours posttransfection were serum starved or serum starvedand then treated with lysophosphatidic acid, ionomycin, orcalcimycin. For AR transcriptional assays PNT1a, LNCaP, andDU145 prostate cancer cells were cotransfected using Lipo-fectamine 2000 (Invitrogen); 1 mg HA-FHL2 or HA-vector; flag-vector, flag-AR full-length, or trAR; His-vector or His-Ar-V7;2 mg Myc-filamin full-length or (R 16–23; 90 kDa filamin frag-ment) or Myc-vector; 20 ng phRL-TK (Renilla), and 2 mg ofARE3-luciferase or prostate-specific antigen (PSA)-luciferase.For androgen-depletion versus androgen-stimulation cellswere cultured in phenol-red–free RPMI media containing10% charcoal-stripped serum (Gibco; Invitrogen) � treatmentwith 10 nmol/L dihydrotestosterone (DHT) for 24 hours.Enzalutaminde (MDV3100; Selleck Chemicals) was used at10 mmol/L for 24 hours (18). Cells were lysed in passive lysisbuffer and luciferase activity measured using the Dual Lucif-erase Reporter Kit (Promega). All luciferase data were nor-malized for transfection efficiency by correcting for constitu-tive Renilla luciferase activity and is presented relative to therelevant control cells.

Prostate sectionsSections of benign prostate tissue and localized prostate

cancer were provided by the Australian Prostate Cancer Bio-Resource (Cabrini Human Research Ethics Committee 03-14-04-08; Monash University, Clayton, Victoria, Australia; HumanResearch Ethics Committee 2004/145MC). CRPC samples wereprovided by Dr. David Pook, Department of Oncology, South-ern Health, East Bentleigh, Victoria, Australia (10247A). Anti-gen retrieval was conducted as previously described (19) andincubated with primary antibodies overnight at 4�C; FHL2(1:50, rabbit), filamin (1:1,000, N- or C-terminal), AR-V7 (1:500),

Regulation of Nuclear FHL2 in Cancer by Filamin Cleavage

www.aacrjournals.org Cancer Res; 73(16) August 15, 2013 5067




Figure 1. FHL2 localizesconstitutively to the nucleus infilamin-deficient cells. A, FHL2comprises four and a half LIMdomains. LIM domains 3 and 4were used as "bait" in yeast two-hybrid analysis (top). Filamindomain structure comprising N-terminal actin-binding domainfollowed by 24 Ig-like repeats(bottom). Two hinge regions arecleaved by calpain (arrows). Usingyeast two-hybrid analysis, weidentified FHL2 binds repeat 23 offilamin C. B, filamin and FHL2expression in M2FIL� versus A7FILþcells. b-Tubulin was used as aloading control. C, A7FILþ versusM2FIL� cells stained for FHL2 andeither filamin, focal adhesions(paxillin), or phalloidin (actin). Scalebar, 100 mm; 25 mm in highmagnification images. D, A7FILþand M2FIL� cells were serumstarved or lysophosphatidic acidstimulated before staining for FHL2and nuclei (propidium iodide).Scale bar, 100 mm.

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Cancer Res; 73(16) August 15, 2013 Cancer Research5068




cytokeratin (CK)-5 (1:200), CK-8 (1:200), and normal rabbitor mouse immunoglobulin (Ig)G (5 mg/mL). 3,30-diamino-benzidine (DAB) staining was conducted using Envisionþ

HRP� conjugated antibodies with DAB detection (Dako) andhematoxylin nuclei counterstaining. For immunofluorescence,sections were incubated with Alexa Fluor-conjugated second-ary antibodies (1:500), with nuclear TO-PRO–3 iodide. A Fisherexact test determined the statistical significance of FHL2 andfilamin nuclear staining in CRPC.

For other detailed methods, refer to SupplementaryMethods.

ResultsFHL2 localizes to the nucleus in filamin-deficient cells

To identify new FHL2-binding partners, we used LIMdomains 3 and 4 as bait in a yeast two-hybrid screen of ahuman skeletal muscle cDNA library and identified severalinteracting 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 Ais expressed in prostate cancer (16) and was used for allsubsequent studies. In vitro binding studies confirmed a directinteraction between FHL2 and filamin (SupplementaryFig. S2A). Coimmunoprecipitation of HA-FHL2 and truncatedMyc-filamin (repeats 22–24) was shown, also delineating theFHL2-binding site to this C-terminal region of filamin (Sup-plementary Fig. S2B).

FHL2 localization was examined in filamin-deficient(M2FIL�) cells compared with the filamin-replete A7FILþ sub-line (20), which both express FHL2 (Fig. 1B). Cells were imagedusing a confocal laser scanningmicroscope and a single opticalsection taken at the ventral cell surface for visualization ofFHL2 staining at the cytoskeleton. In this focal plane thenucleus is not visible. In A7FILþ cells, FHL2 localized to thecytoskeleton, colocalizing with both filamin and actin, and wasalso present at focal adhesions, colocalizing with paxillin (Fig.1C, a–i). InM2FIL� cells, FHL2 localized only to focal adhesions,consistent with its associationwith integrins (21), but not actinstress fibers, which were absent due to loss of filamin actincrosslinking (Fig. 1C, m–t; ref. 20). The nuclear localization andtransactivation activity of FHL2 was examined in M2FIL�versus A7FILþ cells following stimulation with lysophosphati-dic acid, which stimulates FHL2 nuclear localization (8).Confocal imaging of A7FILþ and M2FIL� cells as a Z-stack wasused to compare FHL2 nuclear and cytoplasmic staining. Inunstimulated A7FILþ cells FHL2 was at the cytoskeleton, buttranslocated to the nucleus following lysophosphatidic acidstimulation and in contrast was constitutively nuclear inM2FIL� cells (Fig. 1D). Subcellular fractionation in unstimu-lated cells revealed FHL2 was enriched in the nuclearfraction in M2FIL� cells, with relatively less FHL2 presentin the nucleus of A7FILþ cells (Fig. 1E). Quantification ofthe nuclear:cytoplasmic ratio for FHL2 immunostaining(representative images Fig. 1D) further revealed a constitu-tively higher ratio in M2FIL� cells than A7FILþ cells undernonstimulated conditions (Fig. 1F). A Gal-FHL2 fusion pro-tein that transactivates a Gal-luciferase reporter is used toquantify FHL2 transactivation activity in the nucleus (4, 8).In unstimulated A7FILþ cells, Gal-FHL2 exhibited low trans-activation of the Gal-luciferase reporter, which increased

Figure 1. (Continued ) E, nuclear and cytoplasmic subcellular fractionsprepared from unstimulated A7FILþ and M2FIL� cells and immunoblottedfor FHL2. GAPDH and lamin A/C control cytoplasm or nuclear proteinswere used. Ponceau Red staining of Western blot membranes (loadingcontrol). F, the nuclear:cytoplasmic ratio of FHL2 immunostaining wasquantified in unstimulated A7FILþ andM2FIL� (serum starved) or followinglysophosphatidic acid stimulation by the measuring fluorescenceintensity. G, A7FILþ and M2FIL� cells were cotransfected with a Gal-luciferase reporter and Gal-FHL2 (or Gal-vector, not shown). Cells werelysophosphatidic acid stimulated and luciferase activity measured. Datarepresent mean from n ¼ 6 � SEM; �, P ¼ 0.01.






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, coimmunoprecipitationof 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 andfilamin (C-terminal) and nuclear stain TO-PRO-3 iodide (Topro).

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following lysophosphatidic acid stimulation, but constitu-tively activated transcription in M2FIL� cells irrespective oflysophosphatidic acid treatment (Fig. 1G). Therefore, in thepresence of intact filamin, FHL2 is sequestered at thecytoskeleton and in the absence of filamin, FHL2 localizesto the nucleus and activates transcription.

Calpain cleavage of filamin induces FHL2 nuclearlocalizationFilamin is a substrate for Ca2þ-dependent calpain pro-

teases, with 2 cleavage sites; within hinge I, between repeats15 and 16, and within hinge II between repeats 23 and24 (Fig. 1A; ref. 16). Calpain cleavage of filamin generates2 fragments; N-terminal 170 kDa (repeats 1–15) and aC-terminal 90 kDa fragment (repeats 16–23; Fig. 1A). Theeffect of filamin-cleavage on FHL2 nuclear localization andtransactivation function was examined. Calpain activationwas induced in COS1 cells by ionomycin (Ca2þ ionophore)treatment, resulting in filamin cleavage (Supplementary Fig.S2C) and FHL2 nuclear localization (Supplementary Fig.S2D) and both events were reduced by calpain inhibitionusing calpeptin. Calpain activation also increased transac-tivation of a Gal-luciferase reporter by Gal-FHL2, an effect

also abrogated by calpain inhibition using calpeptin (Sup-plementary Fig. S2E) or the calpain-inhibitory protein cal-pastatin (Supplementary Fig. S2F; ref. 22). Therefore, calpainactivation induces FHL2 nuclear localization and transacti-vation activity. To address whether these processes arefilamin-dependent, complex formation between endogenousFHL2, filamin, and m-calpain was shown (SupplementaryFig. S2G). Furthermore, expression of a calpain-resitant GFP-filamin (D1,762–1,764) mutant (23), in M2FIL� cells inhibitedthe increase in transactivation activity observed for Gal-FHL2 following ionomycin-stimulated calpain activation(Supplementary Fig. S2H). Therefore, calpain cleavage offilamin drives FHL2 nuclear localization.

Calpain cleavage of filamin is increased in prostatecancer cells, resulting innuclear FHL2accumulationandincreased AR coactivation

Calpain expression and activity are increased in prostatecancer (24); therefore, the affect of calpain cleavage offilamin on FHL2 nuclear localization and AR coactivationwas assessed in prostate cancer cell lines. Filamin cleavagein a prostate epithelial cell line (PNT1a) versus prostatecancer cell lines (LNCaP and DU145) was compared by

Figure 2. (Continued ) E, FHL2localization in PTN1a cellsexpressing Myc-vector or Myc-FilaminR16–23. TO-PRO-3 iodide(nuclei). Scale bar, 100 mm. F, cellswere cotransfected with flag-AR,HA-FHL2, and p(ARE3)-luciferaseand left untreated or pretreatedwith calpeptin before stimulationwith androgen (10 nmol/L DHT).For all luciferase assays, datarepresent the mean from n¼ 5–8�SEM; �, P ¼ 0.01. G, expression ofthe AR-target gene PSA wasexamined by qRT-PCR in LNCaPcells transfected with HA-FHL2(or HA-vector) � calpain inhibitionusing calpeptin. Data represent themean from n ¼ 4 � SEM;�, P ¼ 0.05.






immunoblotting using a C-terminal antibody that recog-nizes the full-length filamin (280 kDa) and the C-terminalfragment generated following calpain cleavage (90 kDa;ref. 16). A 90 kDa filamin fragment was present in prostatecancer cell lines (LNCaP and DU145) and was partiallyreduced following calpain inhibition using calpeptin (100mmol/L for 24 hours), whereas PNT1a cells expressed onlyfull-length filamin (Fig. 2A). Higher doses of calpain inhibitoror longer incubation resulted in cell death and could not beused to further reduce filamin cleavage. Filamin cleavage inLNCaP and DU145 cells was confirmed by immunoblottingwith an N-terminal filamin antibody, which recognizes the170 kDa cleaved fragment (Supplementary Fig. S3A). Coim-munoprecipitation revealed endogenous FHL2 bound onlyfull-length filamin in PNT1a cells and to both full-lengthfilamin and the 90 kDa fragment in LNCaP and DU145 cells(Fig. 2B). The latter interaction was confirmed by coimmu-

noprecipitation of the 90 kDa filamin fragment (Myc-fila-minR16–23) with HA-FHL2 (Fig. 2C).

Intact filamin localizes to the cytoskeleton; however, fol-lowing calpain cleavage, the 90 kDa filamin fragment translo-cates to the nucleus and the 170 kDa fragment is retained in thecytoplasm (16). In PNT1a cells, filamin was not cleaved andboth FHL2 andfilamin (C-terminal antibody) colocalized at thecytoskeleton (Fig. 2D). In LNCaP and DU145 prostate cancercell lines, which exhibited significant filamin-cleavage, nuclearcostaining of FHL2 and filamin was observed, which wasreduced by calpain inhibition using calpeptin. Ectopic expres-sion of the 90 kDa filamin fragment (Myc-FilaminR16–23) inPNT1a cells induced cytoskeleton to nuclear translocation ofFHL2 (Fig. 2E), evidence that the 90 kDa filamin fragmentpromotes nuclear FHL2. FHL2 coactivation of the AR wasexamined under conditions of calpain inhibition, using 2AR-regulated reporters p(ARE3)-luciferase (Fig. 2F) and PSA-

Figure 3. FHL2 and filamin localizeto the nucleus in CRPC and notbenign prostate tissue or localizedprostate cancer. A, sections ofhuman benign prostate or localizedprostate cancer were stained forFHL2, filamin (C-terminal; nuclearfragment repeats 16–23), or filamin(N-terminal; cytoplasmic fragmentrepeats 1–15). In control studies,benign prostate tissue was stainedwith nonimmune rabbit or mouseIgG. B, serial sections of humanCRPC from 2 representativepatients stained for FHL2 andfilamin. Boxed regions indicateareas shown in high magnification,Scale bars, 50 mm. Open arrows,basal epithelial cells; closedarrows,luminal epithelial cells;arrowheads, filamin stromalstaining. PCa, prostate cancer.

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luciferase (Supplementary Fig. S4A). Cells were left untreatedor treated with the calpain inhibitor calpeptin, followed byandrogen (DHT) stimulation. In the absence of calpain inhi-bition, HA-FHL2 coactivated AR transcription of both p(ARE3)-and PSA reporters to a greater extent in prostate cancer cells,relative to the PNT1a cells. Treatment of LNCaP and DU145cells with calpeptin reduced FHL2 coactivation of AR to basallevels observed in PNT1a cells. Calpain-dependent activationof endogenous AR-target genes by FHL2 was examined usingquantitative real-time PCR (qRT-PCR). In LNCaP cells, expres-sion of HA-FHL2 increased PSA (Fig. 2G), hK2, and TMPRSS2(Supplementary Fig. S4B and S4C) mRNA levels relative to HA-vector control transfected cells, which was abrogated bycalpain inhibition. Collectively, these studies show that theenhanced susceptibility of filamin to calpain cleavage exclu-sively in prostate cancer cells results in enhanced FHL2nuclearlocalization and AR activation. Filamin phosphorylation atSer2152 protects against calpain cleavage (25). The Ca2þ/cal-modulin-dependent protein phosphatase calcineurin depho-sphorylates filamin to promote calpain cleavage (26) andcalcineurin expression is increased in prostate cancer (27).We showhere thatfilamin dephosphorylation by calcineurin inprostate cancer cell lines was responsible for the enhancedsusceptibility of filamin to calpain cleavage, a pathway thatinitiates FHL2 nuclear localization and increases AR activation(Supplementary Fig. S5).

FHL2 and filamin localize to the nucleus in castrate-resistant prostate cancerWe investigated whether calpain cleavage of filamin reg-

ulates FHL2 nuclear localization during prostate cancerprogression. Benign prostatic epithelium is composed ofmultiple cell types including basal and luminal cells, definedby expression of specific CK proteins (28). In benign prostatetissue, FHL2 localized to the cytoplasm of luminal (closed

arrow) and basal (open arrow) epithelial cells and filaminwas restricted to the cytoplasm of basal cells (Fig. 3A).Coimmunostaining with luminal (CK-8) and basal (CK-5)epithelial cell markers confirmed FHL2 expression in lumi-nal and basal cells and filamin only in basal cells (Supple-mentary Fig. S6), indicating FHL2 and filamin coexpressionoccurs in basal cells of benign prostate. The nuclear local-ization of filamin in benign prostate and localized prostatecancers has been reported (14). In contrast, we observedsimilar cytoplasmic filamin staining in benign prostate usingboth N- and C-terminal–specific antibodies (Fig. 3A), indi-cating that filamin remains intact in the cytoplasm. Similaroverlapping cytoplasmic N- and C-terminal filamin antibodystaining was observed in the PNT1a prostate epithelial cells(Supplementary Fig. S3B), where filamin was not cleaved(Fig. 2A and Supplementary Fig. S3A). Our data are consis-tent with the absence of filamin cleavage in benign prostate,correlating with absent nuclear FHL2.

In low-grade localized prostate cancers, FHL2was restrictedto the cytoplasm and filamin was not detected in cancer cells(Fig. 3A). Loss of basal cells is a hallmark of neoplastic foci inlocalized prostate cancer (29); therefore, the absence of filaminin localized cancer is consistent with filamin expression exclu-sively in CK5þ8� basal cells in benign prostate. Notably, themajority of CRPC samples examined (75%) showed strongnuclear FHL2 staining and a reemergence of filamin stainingin the nucleus of cancer cells (Fig. 3B; Table 1). A correlationwas also observed between the CRPCs, which exhibited bothnuclear FHL2 and filamin (Table 2), suggesting an associationbetween generation of the nuclear filamin fragment by calpaincleavage and FHL2 nuclear localization in CRPC.

Ligand-independent activation of the AR by FHL2CRPC is driven by ligand-independent AR activation and

is associated with high expression of AR variants, which areabsent or expressed at low levels in benignprostate or hormone-na€�ve prostate cancer (30). We examined whether FHL2 pro-motes ligand-independent coactivation of 2 AR variants, trAR(31) and AR-V7 (AR3; refs. 30, 32), which have truncation of theligand-binding domain (LBD) and promote androgen-indepen-dent growth. Interestingly, trAR is generated by calpain cleavageof the AR (31). HA-FHL2 binds both trAR and full-length AR (Fig.4A). In DU145 cells, FHL2 coactivated full-length AR underandrogen-stimulated conditions and, significantly, FHL2 coac-tivationof trARwas ligand independent (Fig. 4B). The nuclear 90kDa C-terminal filamin fragment, which we show binds FHL2,also repressesAR activity (33). Therefore, we determinedwheth-er the FHL2-filamin complex coregulates AR activity. In contrastto 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 þ þ � þ þ � þ þFilamin þ þ � þ þ � þ þAR-V7 þ þ þ þ þ � þ þ

Table 1. Frequency of nuclear localization ofFHL2, filamin, and AR-V7 in human prostatesamples

Benign

Localizedprostatecancer CRPC

FHL2 0/8 0/8 6/8a

Filamin 0/8 0/8 6/8a

AR-V7 — — 7/8

aP ¼ 0.001.






Figure 4. FHL2 competes with filamin for binding to wild-type AR, but not a truncated AR (trAR). A, coimmunoprecipitation in COS1 cells coexpressingHA-FHL2 (or HA-vector) and either FLAG-tagged full-length AR or trAR. B, DU145 cells were cotransfected with combinations of HA-vector orHA-FHL2; FLAG-vector, FLAG-AR (full-length) or FLAG-trAR, and the AR-responsive reporter p(ARE3)-luciferase. Cells were maintained underandrogen-depleted conditions (� Androgen) or stimulated with DHT (þ Androgen) and luciferase activity measured. C, coimmunoprecipitation in COS1cells coexpressing Myc-FilaminR16–23 (or Myc-vector) and either FLAG-tagged full-length AR or trAR. D, competitive-binding assay; cells werecotransfected as indicated and also coexpressed Myc-vector or Myc-filaminR16–23. Lysates were immunoprecipitated (IP) and immunoblotted (WB) asindicated.

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full-length AR, but not trAR (Fig. 4C). Competitive-bindingstudies revealed Myc-filamin16–23 disrupted HA-FHL2 bindingto full-length AR, but not trAR (Fig. 4D). Furthermore, FHL2coactivation of full-length AR (Fig. 4E), but not trAR (Fig. 4F),was repressed by the 90 kDa filamin fragment. Therefore, FHL2and filamin compete for binding to full-length AR, but not trAR.Moreover, FHL2 coactivation of full-length AR is repressed byfilamin, but the ligand-independent activation of trAR by FHL2is constitutive (Fig. 4G).Similar results were obtained using the AR-V7 variant, which

is generated by alternate splicing of theAR gene (30, 32) andhasgenerated significant interest as a pathologically-relevant ARvariant expressed in CRPC (30, 32, 34, 35). AR-V7 bound toFHL2, but not filamin (Myc-filaminR16–23; Fig. 5A) and endog-enous FHL2, and AR-V7 bind in the 22Rv1 prostate cancercell line (Fig. 5B). Ligand-independent coactivation of AR-V7by FHL2 was constitutive and not repressed by the 90 kDafilamin fragment (Fig. 5C andD). AR-V7 is frequently expressed20-fold higher in CPRC than hormone-naive prostate cancer(30) and correlates with poor survival (34). In serial sections ofhuman CRPC, FHL2 and AR-V7 colocalized in the nucleus ofcancer cells (Fig. 5E) and 75% of CRPCs exhibited both nuclearFHL2 and AR-V7 (Table 1, Table 2). Therefore, FHL2 nuclearlocalization in CRPC may promote constitutive, ligand-inde-pendent activation of this clinically important AR variant.The antiandrogen enzalutamide promotes tumor regression

in a mouse xenograft model of CRPC (18) and has progressedrapidly toward approval for treating metastatic CRPC. Enza-

lutamide acts on the full-length AR by directly binding the LBD(18) and expression of AR variants including AR-V7, which lackthe LBD, may confer resistance to this therapy (36, 37).Enzalutamide does not directly inhibit the transcriptionalactivity of AR-V7 (36) and AR-V7–expressing prostate cancercells are resistant to enzalutamide (37). FHL2 activation of full-length AR, trAR, and AR-V7 following enzalutamide treatmentwas examined in DU145 cells that do not express endogenousAR. AR-V7 (Fig. 5F) and trAR (Supplementary Fig. S7A)coactivation of the ARE3-luciferase reporter under andro-gen-depleted conditions was not affected by enzalutamidetreatment and was also increased by FHL2 irrespective ofenzalutamide treatment. In contrast, activation of the ARE3-luciferase reporter by full-length AR was reduced by enzalu-tamide (Supplementary Fig. S7B). Surprisingly, coexpression ofFHL2 was sufficient to restore the transcriptional activity offull-length AR in the presence of this antiandrogen (Supple-mentary Fig. S7B). Therefore, FHL2 can not only sustainenhanced activation of AR variants lacking the LBD irrespec-tive of enzalutamide treatment, but can also reverse theinhibitory affects of enzalutamide on full-length AR.

DiscussionWe report a novel pathway for AR activation in prostate

cancer. In a prostate epithelial cell line FHL2, nuclear locali-zation and AR coactivation are tightly regulated, whereby, theabsence of filamin cleavage results in FHL2 sequestration atthe cytoskeleton and low AR activity. Conversely, in prostate

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 andcells also coexpressed Myc-vector or Myc-FilaminR16–23. FHL2 coactivation of full-length AR and trAR was measured under androgen-dependentand androgen-independent conditions. For all luciferase assays, data represent the mean from n ¼ 8 � SEM; �, P ¼ 0.05. G, AR coregulation by theFHL2-filamin complex. The 90 kDa filamin fragment competeswith FHL2 for binding to full-length ARandalso represses ligand-dependent ARcoactivation byFHL2. 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 thatcoactivation of truncated AR variants by FHL2 is constitutive (b).






cancer cell lines, calpain cleavage of filamin drives nuclearFHL2 and enhanced AR activation. In addition, FHL2 locali-zation and hence AR activation in prostate cancer cells isdriven both by increased calpain activity and the susceptibilityof filamin-to-calpain cleavage following dephosphorylation bycalcineurin.

Over- and underexpression of FHL2 is reported in prostatecancer (8, 11, 38), we revealed FHL2 nuclear localization by themechanism identified here may also be important. In tumors,whichhadprogressed toCRPC, nuclear localizationof FHL2andcalpain-cleaved filamin was consistently observed in the samepatient biopsies supporting our contention that calpain

Figure5. FHL2coactivates anARvariant; AR-V7present inCRPC.A, coimmunoprecipitation inCOS1cells coexpressingHA-FHL2 (orHA-vector) andHis-AR-V7. B, coimmunoprecipitation of endogenous FHL2 and AR-V7 from 22Rv1 cells. NonI, nonimmune sera. C, DU145 cells were cotransfected withHA-vector or HA-FHL2, His-vector or His-AR-V7, and p(ARE3)-luciferase as indicated. Cells were maintained under androgen-depleted conditions andluciferase activity measured. D, luciferase assays were repeated as above in cells coexpressing Myc-vector or Myc-FilaminR16–23. E, serial sectionsfrom 2 representative CRPCs showing nuclear colocalization of FHL2 and AR-V7. Boxed regions indicate areas shown in high magnification. Scalebar, 50 mm for all images.

McGrath et al.





mediates filamin and FHL2 nuclear association. Critically, FHL2and filamin were not detected in the nucleus of benign prostatetissue or low-grade localized prostate cancer (Fig. 5G). Thereemergence of filamin staining in CRPC cells (absent in local-ized cancer) is interesting given the prediction that cancer stemcells, which reinitiate CRPC tumor growth following androgen-deprivation therapy, reside in the CK5þ8� basal cell compart-ment (28) and are shown here to coexpress FHL2 and filamin.Calpain expression and activity are higher under androgen-depleted conditions (39), suggesting that androgen ablationtherapy creates an environment that is not only highly selectivefor expression of ligand-independent variants including AR-V7(40), but is also conducive for promoting nuclear FHL2. There-fore, ofnote areourdata showing thatFHL2activatesAR-V7andboth proteins colocalize in the nucleus of CRPC.

The FHL2-filamin complex is a novel AR regulatorymechanism that may be deregulated in CRPCWe show that the nuclear 90 kDa filamin fragment competes

with FHL2 for binding full-length AR and represses ligand-dependent AR coactivation by FHL2. This is supported by thefindings that Ig-repeats 22–24 of filamin bind AR (33), a regionthat overlaps with the FHL2-binding site (Ig repeat 23) iden-tified here. Therefore, we identify filamin as an importantnegative AR regulator via modulation of the AR-coactivatorrelationship, whereby the activation of the prototype AR ismediated by a balance between the coactivator FHL2 and thecorepressor filamin (Fig. 4G, a). We showed how loss in thisbalance due to the absence of filamin repression results inconstitutive activation of pathologic AR variants notably AR-V7, by FHL2 (Fig. 4G-b). AR-V7 is a major AR variant present inCRPC that activates a transcriptional program distinct from

the prototype AR, including genes, which regulate cell-cycleprogression (32, 35). Despite its importance in CRPC, theregulation of AR-V7 is largely uncharacterized, the identifica-tion of which may lead to novel therapeutic targets for incur-able CPRC. Vav3, a RhoGTPase guanine nucleotide exchangefactor, which like FHL2 enhances AR-V7 coactivation, is criticalfor CRPC growth and survival (41). AR-V7 is also regulated bythe phosphoinositide 3-kinase/Akt/FOXO1 signaling pathway,whereby FOXO1 inhibits AR-V7 activity (42). Interestingly,FHL2 represses FOXO1 activity in prostate cancer cells (7).

The absence of filamin binding to C-terminally truncatedAR variants is consistent with a requirement for the AR–LBDfor filamin association (33) and indicates filamin repressivefunction is limited to the prototype AR. Differential regula-tion of full-length AR versus truncated AR by the FHL2–filamin complex is an important finding given the prevalenceof AR mutations in CRPC (10%–30%), many of which resultin AR mutants that lack all or part of the LBD (2). Given thatinhibition of calpain activity blocks filamin cleavage, FHL2nuclear localization, and AR coactivation, we speculate thatcalpain inhibitors may provide a novel avenue for CRPCtreatment. We have further shown FHL2 coactivation of AR-V7 and trAR is resistant to enzalutamide. Expression ofFHL2 also restored full-length AR transcriptional activityin enzalutamide-treated prostate cancer cells. Therefore, wepredict that FHL2 may reduce the efficacy of enzalutamidetreatment of CRPC by maintaining AR function and calpaininhibitors may provide an adjunct to enzalutamide therapy.Targeting calpain activity is suggested for several cancers tolimit the development, metastases, and neovascularizationof primary tumors (43). Calpain inhibition also decreases theandrogen-independent proliferation of Rv1 cells (31) and

Figure 5. (Continued ) F, ARE3-luciferase assayswere repeated inDU145 cells expressingAR-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 ARcoactivation during prostate cancer progression. In benign prostate tissue, the absence of filamin cleavage results in FHL2 cytoplasmic sequestrationand low AR coactivation. In CRPC, filamin cleavage to the 90 kDa fragment activates FHL2 nuclear accumulation and FHL2 promotes the constitutiveligand-independent coactivation of the AR variant AR-V7.






prostate cancer invasiveness (44). In contrast, a recentreport has suggested that enhancing generation of thenuclear filamin fragment using genistein-combined polysac-charide may be a potential CRPC treatment (17). This comesfrom observations that the 90 kDa filamin fragment main-tains sensitivity of CRPC cells to androgen deprivation (16,17). Our current results, however, clearly indicate thatfurther studies are required to examine the complex inter-play between FHL2, filamin, and the AR in CRPC.

Regulation of nuclear FHL2 by calpain cleavage offilamin may be important in other cancers

Our mechanism for FHL2 nuclear accumulation duringcancer progression is likely to have implications for othercancers. FHL2 nuclear localization is increased in humancolorectal cancer and during tumor development in the Apcmouse model, where FHL2 suppression inhibits tumor initi-ation (10). Transcriptional targets for FHL2 in colon cancerinclude b-catenin (45) and the oncogenic EpICD transcriptioncomplex (46). Calpain expression and activity is increased inhuman colorectal cancer and in colorectal polyps, where itmaybe an early tumorigenesis event (47) and we predict this mayincrease filamin cleavage and promote nuclear FHL2. Filaminis a potential diagnostic biomarker for colorectal cancer as it isshed by tumors into patient feces (48). Although this latterstudy did not examine the presence of the 90 kDa filaminfragment generated by calpain cleavage, of note was theobservation that filamin appeared at a lower molecular weightthan predicted. Future studies will be directed towards exam-ining calpain-dependent regulation of FHL2 in other cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception anddesign:M.J.McGrath, P.A. Robinson, S. Brown,G.P. Risbridger,C.A. MitchellDevelopment of methodology: M.J. McGrath, P.A. Robinson, C.G. Fedele,S. Brown, C.A. MitchellAcquisition of data (provided animals, acquired and managed patients,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. RisbridgerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.J. McGrath, L.C. Binge, A. Sriratana, C.G. Fedele,C.A. MitchellWriting, review, and/or revision of the manuscript: M.J. McGrath, L.C.Binge, P.A. Robinson, D. Pook, J.M. Dyson, D.L. Cottle, B. Niranjan, G.P. Risbridger,C.A. MitchellAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. SriratanaStudy supervision: M.J. McGrath, S. Brown, B. Niranjan, C.A. Mitchell

AcknowledgmentsThe authors thank the Associate Professors John Pedersen and Mark Fryden-

berg (Departments of Anatomy and Cell Biology, and Surgery, Monash Univer-sity) for supplying the human tissues and also Dr. Melissa Papargiris (AustralianProstate Cancer Collaboration) for assisting with the acquisition of patientsamples.

Grant SupportThe Australian Prostate Cancer BioResource is supported by the National

Health and Medical Research Council of Australia Enabling Grant (No. 614296)and by a research infrastructure grant from the Prostate Cancer FoundationAustralia.

Received December 18, 2012; revised May 30, 2013; accepted June 13, 2013;published OnlineFirst June 25, 2013.

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2013;73:5066-5079. Published OnlineFirst June 25, 2013.Cancer Res Meagan J. McGrath, Lauren C. Binge, Absorn Sriratana, et al. Cancer

ProstateActivation of the Androgen Receptor in Castrate-Resistant Regulation of the Transcriptional Coactivator FHL2 Licenses

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