HOXC8 Inhibits Androgen Receptor Signaling in Human Prostate Cancer Cells by Inhibiting SRC-3 Recruitment to Direct Androgen Target Genes

Published OnlineFirst November 2, 2010; DOI: 10.1158/1541-7786.MCR-10-0111

Molecular Cancer Signaling and Regulation Research

Sunshine Daddario Axlund1,2, James R. Lambert1, and Steven K. Nordeen1,2

Abstract HOX (homeobox) genes encode homeodomain-containing transcription factors critical to development, differentiation, and homeostasis. Their dysregulation has been implicated in a variety of cancers. Previously, we showed that a subset of genes of the HOXC cluster is upregulated in primary prostate tumors, lymph node metastases, and malignant prostate cell lines. In the present study, we show that HOXC8 inhibits androgen receptor (AR)-mediated gene induction in LNCaP prostate cancer cells and HPr-1 AR, a nontumorigenic prostate epithelial cell line. Mechanistically, HOXC8 blocks the AR-dependent recruitment of the steroid receptor coactivators steroid receptor coactivator-3 (SRC-3), and CREB binding protein to the androgen-regulated prostate-specific antigen gene enhancer and inhibits histone acetylation of androgen-regulated genes. Inhibition of androgen induction by HOXC8 is reversed upon expression of SRC-3, a member of the SRC/p160 steroid receptor cofactor family. Coimmunoprecipitation studies show that HOXC8 expression inhibits the hormone- dependent interaction of AR and SRC-3. Finally, HOXC8 expression increases invasion in HPr-1 AR nontumorigenic cells. These data suggest a complex role for HOXC8 in prostate cancer, promoting invasiveness while inhibiting AR-mediated gene induction at androgen response element–regulated genes associated with differentiated function of the prostate. A greater understanding of HOXC8 actions in the prostate and its interactions with androgen signaling pathways may elucidate mechanisms driving the onset and progression of prostate cancer. Mol Cancer Res; 8(12); 1643–55. 2010 AACR.

Introduction certain HOX (homeobox) genes are overexpressed in primary and metastatic prostate cancer (1). These observations Androgen ablation therapy for prostate cancer exploits led us to explore a potential cross talk between HOX genes the characteristic androgen dependence of prostate epithe- and androgen receptor (AR)-mediated signaling. lium for survival and growth and is the first-line strategy for There are 39 human HOX genes, arranged in 4 chro- treatment of surgically refractory or recurrent prostate mosomal clusters designated HOXA, HOXB, HOXC, and cancer. However, despite sustained androgen ablation, HOXD. HOX genes encode a large family of homeodo- the majority of men eventually cease responding and "cas- main-containing transcription factors considered "master tration-resistant prostate cancer" (CRPC) claims the life of regulators" in embryonic development because of their key the patient. Thus, a major objective of prostate cancer involvement in body patterning, growth, and differentiation research is to better understand the mechanisms underlying in vertebrates and invertebrates (2–5). As many of these the transition from androgen-dependence to a castration- processes critical to development are also known to be resistant state. Previously, our laboratory reported that dysregulated in carcinogenesis, it is not unexpected then that aberrant HOX gene regulation has been implicated in a variety of cancers including leukemias (6–12), and cervical Authors' Affiliations: 1Department of Pathology, and 2Program in Mole- (13), colorectal (14), breast (15, 16), brain (17), renal (18), cular Biology, University of Colorado Denver, School of Medicine, Aurora, lung (19, 20), and esophageal cancers (21). Colorado In the androgen-responsive, normal mature prostate, Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). little or no expression of HOXC genes is seen, but any of a subset of these genes (HOXC4, HOXC5, HOXC6, or Corresponding Author: Steven K. Nordeen, Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, 12801 E 17th HOXC8) is upregulated in primary prostate tumors, Ave., MS 8104, Aurora, CO 80045. Phone: 303-724-4301; Fax: 303-724- lymph node metastases, and malignant prostate cell lines 3712. E-mail: [email protected] (1). HOXC8 overexpression has been correlated with doi: 10.1158/1541-7786.MCR-10-0111 loss of differentiation/higher Gleason grade in human 2010 American Association for Cancer Research. prostate cancer cell lines and tissues (22). siRNA-mediated

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knockdown of HOXC8 and its transcriptional binding in prostate cancer despite its inhibitory actions on AR- partner, PBX1, results in a decrease in androgen-indepen- mediated gene expression. dent growth of DU-145 prostate cancer cells (23). Loss of HOXC6 expression in prostate cancer cells by siRNA Materials and Methods induces apoptosis, suggesting a protective role for HOXC6 in prostate cancer cell survival (24). Our present Cell lines and culture studies show that HOXC8 overexpression promotes inva- The human prostate cell lines were obtained from the siveness in a nontumorigenic prostate epithelial model, following sources: LNCaP (40, 41), Dr. J. Horoszewicz suggesting a role for HOXC genes in cancer progression. (Roswell Park Memorial Institute, Buffalo, NY), and HPr- A link between androgen signaling and HOX gene 1AR(42),Dr.K.Yamamoto(UniversityofCalifornia expression is suggested by a report indicating that a subset San Francisco). LNCaP cells were maintained in RPMI of HOX proteins bind to the steroid receptor coactivators 1640 supplemented with 10% FBS (Hyclone), penicillin CBP (CREB binding protein) and p300, inhibiting the G (100 U/mL), and streptomycin (100 mg/mL). HPr-1 histone acetyltransferase (HAT) activity of the coactivators AR cells were maintained in keratinocyte serum-free (25). A direct interaction of HOXB13 and AR has been media (Invitrogen) supplemented with penicillin G reported (26, 27). This interaction can have positive and (100 U/mL), streptomycin (100 mg/mL), gentamicin negative effects on AR- and HOXB13-regulated transcrip- (50 mg/mL), and the manufacturer-provided supplements tion (26). In the present studies directed at understanding epidermal growth factor, and bovine pituitary extract. the consequences of the overexpression of HOXC genes in prostate, we show that the overexpression of HOXC8 HOXC8 retrovirus construction and infection inhibits androgen induction at promoters regulated by pMSCV-IRES-GFP-HOXC8 was constructed by stan- androgen response elements (ARE). Investigation into the dard subcloning techniques. HOXC8 cDNA was cloned mechanistic basis for this inhibition indicate that HOXC8 into the EcoRI site within the multiple cloning site of inhibits the loading of steroid receptor coactivator-3 pMSCV-IRES-GFP (gift from Dr. James DeGregori) (SRC-3), a transcriptional coactivator critical in AR- and confirmed by sequencing. Virus preparation and cell mediated gene transcription, at direct androgen target infection were performed as previously reported (43). genes, and that inhibition of AR-induced transcription Expressing cell lines were selected by sorting for GFP is abrogated by SRC-3 overexpression. Significantly, SRC- expression and represent many independent integrants. 3 amplification and/or overexpression have been observed in many hormone-sensitive tumors including prostate RNA isolation and PCR analysis (28), breast (29), endometrial (30), and ovarian cancers Total RNA was isolated using RNeasy Plus (Qiagen) (31), as well as many nonsteroid targeted tumors such as according to the manufacturer's instructions. One micro- pancreatic cancer (32, 33), gastric cancer (34), colorectal gram of total RNA was reverse transcribed using M-MLV carcinoma (35), and hepatocellular carcinoma (36). reverse transcriptase (Promega). Real-time reverse transcrip- SRC-3 is expressed in prostate cancer cell lines and tase-PCR (RT-PCR) for HOXC8, SRC-3, PSA (prostate- prostate tumors, and its expression is correlated with specific antigen), and 18S was performed on 1/10 (HOXC8, tumor grade and stage in prostate cancer patients (37, SRC-3, PSA) or 1/20 (18S) of the synthesized cDNA by 38). It is overexpressed in tissue samples from prostate using primer and probe sets and TaqMan Fast Universal cancer patients, and its overexpression correlates with Master Mix (Applied Biosystems). Amplification signals prostate cancer proliferation and inversely correlates with were detected with the ABI Prism 7500 Fast Sequence apoptosis (28). Knockdown of SRC-3 by siRNA results in Detection System (Applied Biosystems). Fold change in – decreased cell proliferation, delays in the G1 Stransition, expression was calculated using the comparative Ct method and increased apoptosis in prostate cancer cell lines, as well (44) following confirmation that the amplification efficien- as decreased tumor growth in nude mice (28). Other cies of the target (HOXC8, SRC-3, or PSA) and endogenous studies show that SRC-3, along with the AR, is required reference control (18S) were approximately equal. for androgen-dependent and -independent proliferation of prostate cancer cells and for xenograft tumor growth (39). Luciferase reporter assays Furthermore, AR and SRC-3 are recruited to gene pro- Cells were seeded at 3 105 cells per well in 3 mL of moters involved in cell-cycle control in an androgen- RPMI 1640 supplemented with 10% FBS in 6-well plates. independent manner (39). Taken together, these data Twenty-four hours later, cells were cotransfected with 2 mg suggest that SRC-3 is required for prostate cancer cell of probasin-, MMTV (mouse mammary tumor virus)-, or proliferation and survival, and together with the AR, plays PSA-luciferase (luc) reporter constructs (45; gift from Dr. a central role in progression to CRPC. Carlos Perez-Stable, University of Miami School of Med- The present work ties together 3 major players in prostate icine) and pCMV6c-HOXC8 expression plasmid and/or cancer, AR, SRC-3, and HOXC8, and explores the con- empty vector control pCMV6c to ensure that the same sequences of their interaction. We also provide evidence amount of DNA was transfected into each well, using implicating HOXC8 overexpression in prostate cancer pro- TransIT-LT1 Transfection Reagent (Mirus Bio). After gression and discuss the role of HOXC gene overexpression 24 hours, cells were treated with 10 nmol/L of R1881

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HOXC8 Regulates Androgen Action via SRC-3 in Prostate Cancer

(New England Nuclear) in phenol red–free RPMI (Invitro- PSA enhancer reverse primer: 50-GCCTGG ATCTGAGA- gen) supplemented with 5% dextran-coated, charcoal-trea- GAGATATCAT-30; PSA enhancer probe: 50-TGCAAG- ted FBS for 18 hours. In all experiments, the total amount GATGCCTGCTTTACAAACATCC-30) and TaqMan of plasmid transfected was equalized by adding empty Fast Universal Master Mix (Applied Biosystems). expression vector as necessary. Luminescence was normalized to total protein. For coactivator cotransfection reporter Antibodies assay (Fig. 4), the net induction (I) for each condition was HOXC8 mouse monoclonal antibody was used to detect the normalized luciferase activity with hormone minus that HOXC8 protein (Covance). For Western blot analysis, SRC- without. The average inhibition of hormone induction 3 [AIB-1 (amplified in breast cancer-1), NCoA3] protein was I I mediated by HOXC8 expression alone [1 ( HOX/ CON) detected using either mouse AIB-1 monoclonal antibody (BD 100] was 58%. Percentage control HOXC8 inhibition Transduction Laboratories) or NCoA3 N-16 goat polyclonal is the ratio of the inhibition of androgen induction in antibody (Santa Cruz Biotechnology); for ChIP and co-IP HOXC8 þ coactivator transfections to the inhibition with experiments, SRC-3 was immunoprecipitated using either HOXC8 alone. mouse AIB-1 monoclonal antibody or NCoA3 N-16 goat polyclonal antibody. For Western blot analysis, AR protein Western blot analysis was detected using either rabbit N-20 polyclonal antibody Cell extracts were separated on NuPAGE 4% to 12% Bis- (Santa Cruz Biotechnology) or mouse monoclonal antibody Tris gel (Invitrogen) and transferred to polyvinylidene difluor- (BD Biosciences). For ChIP, acetylated histone H3 or H4 was ide membrane. Following incubation with antibody, signals immunoprecipitated using the appropriate antiacetylated weredetectedbyenhancedchemiluminescence(PerkinElmer) histone antibody (Upstate Cell Signaling Solutions). For and autoradiography. Bands were quantified using Image Western blot analysis, b-actin was detected using mouse J software (NIH) and normalized to b-actin loading control. monoclonal IgG1 clone AC-15 antibody (Sigma).

Coimmunoprecipitation Glutathione-S-transferase pull-down assay LNCaP cells (7 106) were seeded on 15-cm dishes in HOXC8 cDNA was cloned into pGEX-4T1 (GE phenol red–free RPMI 1640 supplemented with 5% dex- Healthcare) and purified using the glutathione-S-transferase tran-coated, charcoal-treated FBS and grown to near con- (GST) Purification Kit (Clontech) according to the man- fluency. Cells were treated with 10 nmol/L of R1881 or ufacturer's instructions. To assess interaction between GST- ethanol control for 24 hours. Coimmunoprecipitation (co- HOXC8 and SRC-3, baculovirus-expressed, Flag-tagged IP) was performed as previously described by Gnanapraga- SRC-3 protein was used. To assess interaction between sam et al. (38), with an additional step added to preclear the GST-HOXC8 and AR, cell extract from a confluent 15-cm lysate with 100 mL of protein A agarose. Immune complexes dish of LNCaP cells treated for 24 hours with 10 nmol/L of were collected by centrifugation, washed, and analyzed by R1881 was used. Cells were harvested in cold PBS and lysed Western blot as described. in 1 mL of GST lysis buffer. GST pull-down assay was performed as previously described (47). Chromatin immunoprecipitation Cells were grown to near confluency in 15-cm dishes in Invasion phenol red–free RPMI (Invitrogen) supplemented with 5% Invasion was assayed using Cell Biolabs Cytoselect 96- dextran-coated, charcoal-treated FBS for 3 to 5 days. For well Cell Migration and Invasion Assay (Cell Biolabs, Inc.) histone acetylation analyses, cells were transfected with 2 according to manufacturer's directions. LNCaP or HPr-1 mg/mL of MMTV-luc and 200 ng/mL of pCMV6c- AR cells were starved for 18 hours and then 5 104 cells in HOXC8 or pCMV6c-Empty vector 18 hours before hor- serum-free RPMI (LNCaP) or keratinocyte serum-free mone treatment. Cells were treated with 10 nmol/L of medium without supplements (HPr-1 AR) were added to R1881 or vehicle control for 30 [SRC-3 and CBP chro- the upper invasion chamber. Cells were incubated for 48 matin immunoprecipitation (ChIP)] or 60 minutes (histone hours at 37C, and cell invasion was assessed. Invasion acetylation ChIP). ChIP analysis was performed as pre- values are reported as mean RFU (relative fluorescence viously described by Lambert et al. (46). DNA was purified units) of quadruplicate samples. using the QIAprep Spin Miniprep Kit (Qiagen) and eluted in water. Control experiments using serial dilutions of input Results DNA were performed to affirm that the PCRs were within the linear range of amplification cycles. For histone acet- HOXC8 inhibits AR-mediated transcription ylation ChIP experiments, PCR for MMTV-luc was per- It has been previously shown that HOX proteins bind to formed as previously described (46), and samples were the steroid receptor coactivators CBP and p300, inhibiting analyzed by agarose gel electrophoresis. For PSA ChIP the HAT activity of the coactivators (25). We therefore asked experiments, real-time PCR for the PSA enhancer region whether overexpression of HOXC8 might inhibit AR- was performed on 1/10 (PSA enhancer) or 1/20 (18s) of the mediated transcription via inhibition of CBP/p300. These samples, using primer and probe set (PSA enhancer forward experiments were performed in LNCaP cells, as they express primer: 50- ACACCTTTTTTTTTCTGGATTGTTG-30; functional AR and very low endogenous HOXC8 mRNA

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A C 8 Figure 1. HOXC8 overexpression specifically inhibits AR-mediated 7 gene induction in LNCaP prostate 6 cancer cells cells. A and B, HOXC8 5 LNCaP cells were transfected 4 with a probasin-lucreporter 3 vector and increasing amounts of β

-actin Luc activity 2 HOXC8 expression vector.

0 20 100 500 ng pCMV- (LU/ng of protein) 1 Total transfected plasmid HOXC8 0 was held constant by balancing pRSV-luc pRSV-luc + with empty expression vector. 1 μg HOXC8 Cells were treated with 10 nmol/L B D of R1881 for 18 hours. A, Western blot for HOXC8 (60s exposure) 50 and actin (1s exposure). B, lucifer- 40 EtOH ase activity normalized to total R1881 40 30 protein. LNCaP cells were transfected with (C) pRSV-luc or (D) 20 30 pCMV-bgal along with the HOXC8 10 20 expression vector or the empty Luc activity Luc activity vector control. Luciferase data (LU/ng of protein) 0 (LU/ng of protein) 10 shown are from a single experi- 0 20 100 500 ment and are representative of 3 ng pCMV-HOXC8 0 (B) or 2 (C and D) separate experi- β β pCMV- gal pCMV- gal + ments. Values represent mean 1 μg of HOXC8 SD (B, n ¼ 4; C and D, n ¼ 3).

(Supplementary Fig. S1A) and protein levels Fig. S3A) and protein (Supplementary Fig. S3B) expres- (Supplementary Fig. S1B). A progressive increase in the sion levels are increased in LNCaP-HOXC8 compared expression of HOXC8 (Fig. 1A) decreased androgen- with the negative control LNCaP-Empty cell line. dependent activity of a probasin-luc reporter vector Although many prostate cancer cell lines exhibit low levels (Fig. 1B). This cannot be ascribed to a general squelching of HOXC8, the expression of HOXC8 in LNCaP- or inhibitory effect of HOXC8 overexpression, as expres- HOXC8 cells is comparable to the levels seen in the sion of neither pCMV-bgal (Fig. 1C) nor pRSV-luc prostate cancer cell line, PC-3, that endogenously (Fig. 1D) is affected. expresses HOXC8 (Supplementary Fig. S1). These Many HOX proteins share overlapping function. Our HOXC8- and empty vector–transduced LNCaP stable laboratory previously observed the overexpression of cell lines were transiently transfected with the andro- HOXC genes, including HOXC6, in primary tumors, gen-responsive MMTV-luc or PSA-luc reporter plasmids. metastases, and in prostate cancer cell lines, including The HOXC8 expressing lines exhibit diminished AR- LNCaP (1). We asked whether further overexpression of mediated promoter activity of both reporters (Fig. 2A HOXC6 in LNCaP cells also inhibits androgen-mediated and B). gene induction. Similar to the results of overexpressing To determine whether this effect was specific to prostate HOXC8, HOXC6 expression inhibits androgen-induced cancer cells, or to LNCaP cells in particular, we performed transcription in a dose-dependent manner (Supplementary similar experiments in the immortalized, nontumorigenic Fig. S2A). HOXB13, important in normal prostate func- prostate epithelial cell line HPr-1 AR, which was transduced tion as well as in prostate cancer, exhibited a similar with a retrovirus to stably overexpress HOXC8 or empty inhibitory effect (Supplementary Fig. S2B). These data vector control. Real-time RT-PCR and Western blot ana- support the notion that, among the subset of HOX proteins lyses confirm that HOXC8 mRNA (Supplementary tested, there may exist functional redundancy with respect Fig. S3C) and protein (Supplementary Fig. S3D) expression to inhibition of AR-mediated signaling. The availability of levels are increased in HPr-1 AR-HOXC8 cells compared an adequately specific commercial antibody for the detec- with the negative control HPr-1 AR-Empty cells. Andro- tion of HOXC8 and the null background in LNCaP gen-induced activity directed by the MMTV-luc (Fig. 2C) prompted us to focus further studies on HOXC8. and PSA-luc (Fig. 2D) reporter plasmids was diminished in To validate the transient transfection reporter assays and HPr-1 AR cells stably overexpressing HOXC8. These data to investigate additional activities of HOXC8, an LNCaP indicate that the inhibitory effect of HOXC8 on androgen- cell line stably overexpressing HOXC8 was constructed by mediated induction of different direct androgen target retroviral transduction. HOXC8 mRNA (Supplementary promoters is observed in both tumorigenic and nontumori-

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HOXC8 Regulates Androgen Action via SRC-3 in Prostate Cancer

A C

MMTV-luc EtOH MMTV-luc 30 R1881 18 25 16 14 20 * 12 10 * 15 8 10 6 Luc activity

Luc activity 4 5 2 (LU/ng of protein) (LU/ng of protein) 0 0 HPr-1 AR-Empty HPr-1 AR-HOXC8 LNCaP-Empty LNCaP-HOXC8

B D PSA-luc 35 PSA-luc 18 16 30 14 35 12 * 20 * 10 15 8

Luc activity 10

Luc activity 6

4 (LU/ng of protein) 5 (LU/ng of protein) 2 0 0 HPr-1 AR-Empty HPr-1 AR-HOXC8 LNCaP-Empty LNCaP-HOXC8

Figure 2. AR-mediated transcription is inhibited in prostate cells stably overexpressing HOXC8. Retroviral transduction was employed to stably overexpress HOXC8 in prostate cancer cells (LNCaP; A and B) and nontumorigenic prostate (HPr-1 AR; C and D) cell lines. Paired control cell lines were created by transduction of the empty vector. HOXC8-expressing and control cell lines were transfected with MMTV-luc (A and C) or PSA-luc (B and D) reporter and treated with 10 nmol/L of R1881 for 18 hours. Luciferase activity is normalized to total protein. Data shown are from a single experiment and representative of 3 independent experiments. Values represent mean SD (n ¼ 4; *, P < 0.05). genic prostate cell lines and when HOXC8 is transiently or Overexpression of SRC-3, but not CBP, reverses the stably overexpressed. HOXC8-induced inhibition of AR-mediated We also tested whether androgen induction of an endo- transcription genous gene was inhibited by HOXC8 expression. We The report of Shen et al. (25) that HOX genes interact compared the androgen induction of the endogenous with the transcriptional coactivator CBP and inhibit its PSA gene in LNCaP-HOXC8 and LNCaP-Empty as well HAT activity suggests a possible mechanistic basis for the as in HPr-1 AR-HOXC8 and HPr-1 AR-Empty cells. In HOXC8-mediated inhibition of androgen-induced gene both comparisons, the androgen induction of the gene was expression. If suppression of CBP coactivator function were reduced in the HOXC8-expressing line (Fig. 3). responsible for the inhibition of AR action, we reasoned that overexpression of CBP might overcome the inhibition by HOXC8. We overexpressed CBP and other cofactors HOXC8 inhibition of AR-mediated transcription is not that are major coregulators of steroid signaling, the CBP due to downregulation of the AR relative p300 and the 3 members of the p160 steroid To rule out the possibility that HOXC8 inhibits AR- receptor coactivator family of coregulators SRC-1, SRC-2, mediated transcription by downregulating AR expression and SRC-3. levels, real-time RT-PCR and Western blot analyses of AR LNCaP prostate cancer cells were cotransfected with the mRNA and protein levels was performed in LNCaP- probasin-luc reporter, 100 ng of HOXC8 expression vector HOXC8 and compared with that of the empty vector (or empty vector control) and 100 or 500 ng of the control cell line. LNCaP-HOXC8 and LNCaP- indicated coactivator expression vector (or empty vector Empty cells express nearly identical AR mRNA (Supple- control). Western blot analysis of coactivator protein expres- mentary Fig. S4A) and protein (Supplementary Fig. S4B) sion levels following transfection is shown in Supplementary levels, indicating that the inhibition of AR-mediated tran- Figure S5. The results of the coactivator coexpression scription by HOXC8 cannot be attributed to downregula- experiment are presented in Figure 4. The average inhibition of AR expression levels. AR protein levels are also tion of the hormone induction by HOXC8 from duplicate unchanged in HPr-1 AR-HOXC8 cells when compared experiments is 58% without added coactivators. This is the with the empty vector control cell line (data not shown). control inhibition (Fig. 4, leftmost bar), and the inhibition

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A 200 3 180 160 2.5 140 120 2 100 80 1.5 60

1 Percent control HOXC8 inhibition 20 Fold induction PSA 0.5 0 * ng of plasmid: 100 500 100 500 100 500 100 500 100 500 Vector CBP p300 SRC-1 SRC-2 SRC-3 0 control LNCaP- LNCaP- Empty HOXC8 Figure 4. SRC-3 overexpression reverses the HOXC8-induced inhibition of AR-mediated transcription. LNCaP cells were cotransfected with a probasin-luc reporter, 100 ng of HOXC8 expression vector or empty B vector control and 100 or 500 ng of the indicated coactivator expression 5 vector. Total transfected plasmid was held constant by balancing with empty expression vector. Cells were treated with 10 nmol/L of R1881 4.5 for 18 hours. The inhibition of the hormone induction by HOXC8 was 4 58% without added coactivators. This is the control inhibition (leftmost bar), and the inhibition imposed by HOXC8 when coactivators 3.5 are expressed is compared with this standard. Data shown are the 3 average from 2 independent experiments in which each condition was tested in duplicate. Values represent mean range. 2.5 2 ** 1.5 Fold induction PSA from left). These data suggest that SRC-3, rather than CBP, 1 may be the critical target for HOXC8 inhibition of AR- 0.5 mediated signaling. This result is of particular interest, as will 0 be discussed, and directed subsequent efforts toward elucidat- ingtheinterplaybetweenHOXC8andSRC-3inthecontextof HPr-1AR- HPr-1AR- Empty HOXC8 AR signaling in prostate cells. SRC-3 expression in LNCaP prostate cancer cell lines Figure 3. Induction of the endogenous PSA gene is inhibited when In consideration of the finding that overexpression of HOXC8 is stably overexpressed. A, LNCaP and B, HPr-1 AR cells SRC-3, but not CBP, abrogates the HOXC8-mediated stably expressing HOXC8 and paired control cells were serum starved for 48 hours and then treated with 10 nmol/L of R1881 for 6 hours. inhibition of AR transcriptional activity, we asked whether PSA mRNA levels were analyzed by real-time RT-PCR analysis. SRC-3 mRNA or protein levels are altered with HOXC8 Data shown are from a single experiment and representative of overexpression. SRC-3 mRNA and protein levels were 3 separate experiments. Values represent mean SD (n ¼ 3; compared in LNCaP-Empty and LNCaP-HOXC8 prostate ** *, P < 0.01, , P < 0.05). cancer cell lines by real-time RT-PCR and Western blot analysis. LNCaP-Empty and LNCaP-HOXC8 cells express imposed by HOXC8 when coactivators are expressed is very similar SRC-3 mRNA (Supplementary Fig. S6A) and compared with this standard. Contrary to our expectation, protein (Supplementary Fig. S6B) levels. Therefore, overexpression of CBP only modestly counters the HOXC8-mediated changes in overall SRC-3 expression HOXC8-mediated inhibition of the hormone induction levels are not a contributing factor in the HOXC8 inhibi- (Fig. 4, second and third bars from the left). Both p300 and tion of AR-mediated signaling. SRC-1 enhance the HOXC8-mediated inhibition of AR- mediated transcription somewhat (Fig. 4, from left, bars 4– HOXC8 overexpression inhibits the hormone- 7), increasing the inhibition from 58% to about 70% to dependent interaction of AR and SRC-3 80%. Overexpression of SRC-2 weakly diminishes the Gnanapragasam et al. (38) showed that SRC-3 binds AR HOXC8 inhibition (Fig. 4, from left, bars 8–9). In contrast in a ligand-dependent manner in LNCaP prostate cancer to the other coactivators, overexpression of SRC-3 reverses cells and that this interaction is important in enhancing the HOXC8-mediated inhibition. At the highest level of transcriptional activity in prostate cancer cells. We, there- expression,SRC-3completelyrestoresthehormoneinduction fore, tested whether overexpression of HOXC8 might alter despite the overexpression of HOXC8 (Fig. 4, last 2 bars the physical interaction between AR and SRC-3.

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been previously shown that in the presence of hormone, AR A predominately occupies the distal enhancer region, not the LNCaP-Empty LNCaP-HOXC8 proximal promoter region, of the PSA gene in LNCaP cells EtOH R1881 EtOH R1881 (48–50). In LNCaP-HOXC8 cells, the occupancy by SRC- IP: SRC-3 3 at the PSA enhancer region is greatly inhibited when WB: AR compared with that observed in the LNCaP-Empty control cell line (Fig. 6A). The SRCs interact directly with steroid Input: receptors bound to target DNA elements and serve as a SRC-3 platform to recruit additional coregulators including the HAT coactivator CBP (51, 52). Recruitment of these AR factors by SRC/p160 proteins is critical for steroid receptor–mediated local chromatin remodeling and assembly of transcriptional machinery around target gene promoters B 10 9 (53, 54). We, therefore, also assessed recruitment of CBP 8 and found its androgen-dependent recruitment to be dimin- 7 ished in LNCaP-HOXC8, consistent with the idea that the 6 inhibition of SRC-3 recruitment in turn diminishes CBP 5 recruitment (Fig 6B). 4 A potential explanation for reduced recruitment of SRC- 3 3 is that HOXC8 inhibits recruitment of AR to target AR/SRC-3 input 2 response elements. However, we observe no significant 1 change in either the basal or hormone-induced occupancy 0 R1881: –+ – + of AR at the PSA enhancer region in LNCaP-HOXC8 cells when compared with that of the LNCaP-Empty control cell LNCaP-Empty LNCaP-HOXC8 line (Fig. 6C). This suggests that HOXC8 imposes its inhibitory effects on AR signaling independent of AR Figure 5. The hormone-dependent interaction of AR/SRC-3 is inhibited binding to target genes by inhibiting further recruitment in LNCaP-HOXC8 prostate cancer cells. A, LNCaP-Empty and of cofactors, including SRC-3 and CBP, to AR target genes. LNCaP-HOXC8 cells were cultured in 10 nmol/L of R1881 or ethanol The SRC-3 loading experiments show that basal as well as for 24 hours. SRC-3 was immunoprecipitated and the association with AR hormone-induced levels of SRC-3 loading are diminished was assessed by Western blot analysis by using an antibody directed against AR. AR protein levels were similar in all samples as shown in by HOXC8 expression. SRC-3 is a coactivator for many the input (bottom). B, quantitation of AR bound to SRC-3 (data from A, factors acting at many promoters, and HOXC8 may be top, normalized to SRC-3 input, middle). Data shown are from a inhibiting SRC-3 at many or all of these. This suggestion single experiment and representative of 3 separate experiments. implies that HOXC8 would impose pleiotropic actions beyond that of androgen-regulated genes through its inhibitory effect on SRC-3. Co-IP experiments were performed to examine binding Both CBP and SRC-3 possess intrinsic HAT activity (51, interactions between SRC-3 and AR, using LNCaP cells 55, 56). Coactivator-dependent chromatin modification is a stably overexpressing HOXC8 and LNCaP control cells. critical step in hormone receptor–mediated induction of LNCaP-Empty cells exhibit an increase in the interaction of gene expression. We predicted that the HOXC8-mediated SRC-3 and AR upon hormone treatment (Fig. 5A, top left). inhibition of coactivator recruitment would be reflected in In contrast, LNCaP-HOXC8 cells exhibit diminished inter- reduced acetylation of histones at target promoters. As can action of SRC-3 and AR following treatment with R1881 be seen in Figure 7A, androgen-induced acetylation of (Fig. 5A, top-right bands), showing that HOXC8 expres- histones H3 and H4 at the MMTV promoter is indeed sion disrupts the ligand-enhanced interaction between suppressed by HOXC8. Quantitation of these data normal- SRC-3 and AR. This is not due to a decrease in SRC-3 ized to input is shown in Figure 7B. or AR protein levels (Fig. 5A, middle and bottom, respectively). Although a slight decrease is observed in SRC-3 HOXC8 does not directly interact with SRC-3, AR, protein levels after treatment with androgen (Fig. 5A, or CBP middle), this decrease is seen in both the LNCaP-Empty To further assess the mechanism of HOXC8-mediated and LNCaP-HOXC8 cell lines. Quantitation of these data inhibition of AR signaling, experiments were conducted to normalized to input is presented in Figure 5B. test for direct interactions between HOXC8 and SRC-3. In vitro GST pull-down assays were performed using HOXC8 expression inhibits recruitment of SRC-3 and the fusion protein GST-HOXC8 as bait along with Flag- CBP and acetylation of histones tagged SRC-3 purified from baculovirus expression vector– ChIP analyses were performed to assess whether the infected insect cells. No interaction was detected between recruitment of SRC-3 to the endogenous AR target gene GST-HOXC8 and purified SRC-3 above the minimal PSA is also inhibited by HOXC8 overexpresssion. It has levels observed with the GST control (Supplementary

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A IP: α ac-H3 α ac-H4 Input No Ab A 2.5

R1881: – + + – + + – + + – + + 2 HOXC8: –– + ––+––+––+

1.5 B 7 6

1 5 SRC-3 loading 4 0.5 3

0 Acetylationl/input 2 R1881: –+ –+ 1 LNCaP-Empty LNCaP-HOXC8 0 α ac-H3 α ac-H4 R1881: ––++++ B 2.5 HOXC8: ––+ ––+

2 Figure 7. HOXC8 expression inhibits histone acetylation at an androgen- responsive gene promoter in prostate cancer cells. A, MMTV-luc 1.5 and 100 ng of HOXC8 or empty control expression vectors were transiently transfected into LNCaP cells. Cells were treated with 10 nmol/L R1881 as indicated and ChIP was performed to assess acetylation of 1 histones H4 and H3 at the MMTV hormone-responsive promoter.

CBP loading CBP B, quantitation of IP samples normalized to input is indicated. Data 0.5 shown are from a single experiment and representative of 3 separate experiments. 0 R1881: –+–+ Fig. S7B) or extracts from untreated cells (data not shown). We also examined binding between HOXC8 and AR, SRC- LNCaP-Empty LNCaP-HOXC8 3, or CBP by co-IP by using LNCaP cellular extracts, but no binding interactions were detected either in the presence or C 14 absence of hormone (data not shown). All in vitro binding 12 assays have limitations; therefore, the possibility of interactions between HOXC8 and SRC-3 or AR in the context 10 of the cell cannot be completely discounted. 8 HOXC8 effects on in vitro surrogate markers of 6 tumorigenesis and progression AR loading Overexpression of HOXC genes is associated with 4 prostate cancer (1) and, in the case of HOXC8, increased 2 Gleason grade (22). Recent studies have implicated HOXC8 in the regulation of cell cycle and proliferation 0 (57–60), apoptosis (58, 61), cell differentiation (62–67), R1881: –+ –+ cell adhesion (58, 61, 68), cytoskeleton/motility/migra- LNCaP-Empty LNCaP-HOXC8 tion (61), and tumorigenesis (58, 60, 61, 68, 69). On the basis of these reports and the present lack of understanding as to HOXC8 function in prostate carcinogenesis, its Figure 6. Recruitment of SRC-3 and CBP to the endogenous effects on in vitro surrogate markers of prostate cell PSA enhancer is inhibited in LNCaP-HOXC8 cells. ChIP assays tumorigenicity were examined. were performed to analyze the occupancy of SRC-3 (A), CBP (B), and AR (C) at the endogenous PSA enhancer region. Cells were treated Malignant transformation is associated with certain phe- with R1881 or ethanol for 60 minutes. Occupancy was normalized to notypic changes including dysregulation of cell cycle and that of empty vector control cells treated with ethanol. Data shown cell death pathways, loss of contact inhibition and ancho- are from a single experiment and representative of 3 separate experiments. rage independence, and increased motility and invasiveness. n ¼ Values represent mean SD ( 3). We observed no dramatic changes in anchorage-independent growth, apoptosis, or migration in conjunction with Fig. S7A). Interaction between HOXC8 and AR was also HOXC8 overexpression in prostate cancer cells or non- examined, but no binding above background was detected tumorigenic prostate cell lines. A mild inhibition of growth between GST-HOXC8 and AR by using extracts from of LNCaP and HPr-1 AR cells was seen with an accom- hormone-treated LNCaP as a source of AR (Supplementary panying increase in G1 phase of the cell cycle (data not

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shown). None of these assays suggested a critical role of HOXC8 in prostate cancer tumorigenesis or progression. A 300 HOXC8 expression increases invasiveness in HPr-1 AR prostate epithelial cells but not in LNCaP prostate 250 cancer cells Invasive capacity is the single most important trait that 200 distinguishes benign from malignant lesions. Invasion assays in vitro require cells to migrate through an extracellular matrix or basement membrane extract barrier by first enzy- 150 matically degrading the matrix barrier in order to become

established in a new location. HOXC8 has been implicated Invasion (RFU) 100 as a regulator of cadherin-11 and osteopontin (58, 61). Cadherin-11 has been shown to produce significant changes 50 in the invasive capacity of cancer cells (70), and cumulative evidence suggests that osteopontin functions in the regulation of tumor metastasis and invasion (71, 72). 0 The ability of LNCaP prostate cancer and HPr-1 AR LNCaP-Empty LNCaP-HOXC8 nontumorigenic prostate epithelial cells overexpressing HOXC8 to invade through a coated membrane was assessed. B 100 LNCaP-HOXC8 cells invade to a similar degree as the 90 LNCaP-Empty control cell line (Fig. 8A). HPr-1 AR-Empty cells invade to a level that is less than 20% as that of LNCaP 80 cells, but in contrast to paired LNCaP cell lines, invasion by 70 HPr-1 AR-HOXC8 cells is nearly double that of HPr-1 AR- 60 Emptycontrolcells(Fig.8B).ThesedatasuggestthatHOXC8 expression may direct protumorigenic actions in prostate 50 carcinogenesis by promoting increased invasive capacity. 40

Discussion Invasion (RFU) 30 20 Inappropriate expression of HOX genes is associated with a number of cancers (6–10, 14–20). In particular, any of the 10 4 genes of the HOXC cluster may be overexpressed in 0 prostate cancer (1). The present study suggests a complex HPr-1 AR-Empty HPr-1 AR-HOXC8 role for HOXC8 in prostate cancer, promoting invasiveness while inhibiting AR-mediated gene induction at ARE- regulated genes associated with differentiated function of Figure 8. HOXC8 expression increases invasiveness in nontumorigenic the prostate. HOXC8 overexpression may well affect the HPr-1 AR cells but not in LNCaP prostate cancer cells. Invasion by LNCaP prostate cancer cells (A) and HPr-1 AR cells (B) overexpressing participation of SRC-3 at promoters in addition to ARE- HOXC8 when compared with empty vector control cells. Invasion driven promoters. SRC-3 serves as a cofactor for many was measured at 48 hours. Data shown in A and B are pooled from transcription factors, and a general inhibition of its activity 2 separate experiments. Values represent mean SD (n ¼ 4; *, P < 0.01). by HOXC8 would be expected to have pleiotropic dis- ruptive effects on cellular homeostasis. overexpression in prostate cancer seems to be especially critical in AR-mediated gene induction. Among the p160 Mechanism of HOXC8-dependent inhibition of family of coactivators, SRC-3 is the preferred coactivator for androgen-mediated gene expression hormone-activated AR. SRC-3 is selectively phosphorylated Although a report that HOX genes inhibit the HAT when cells are treated with androgen (74), its binding affinity activity of CBP (25) suggested that HOXC8 might inhibit for AR is 10- to 100-fold stronger than that of SRC-1 or AR-meditated gene induction through this mechanism, we SRC-2 (75), and it activates AR more potently than other find evidence that it is SRC-3, rather than CBP, p300, SRC- SRCs in the presence of 5a-dihydrotestosterone (DHT; ref. 1, or SRC-2, that seems to be the key target of HOXC8. 75). Thus, SRC-3 represents an attractive target for HOXC8 SRC-3 is an intriguing candidate because like HOX genes, it inhibition of AR function. is overexpressed in many cancers. Indeed, one of its many names (NCoA-3/AIB-1/pCIP/ACTR/RAC3/TRAM-1), Inhibition of androgen action—a conundrum? AIB-1 (amplified in breast cancer-1), came from its identi- Because prostate growth and maintenance are androgen fication as 1 of 3 gene amplifications frequently observed in dependent, then why doesn't HOXC8 expression serve a human breast carcinomas (73). The involvement of SRC-3 tumor suppressor function rather than being associated with

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prostate cancer and/or worse prognosis? One possibility is increased invasion by HOXC8 expression, thereby predis- that any inhibition of androgen signaling by HOXC gene posing the tumor to survive in the face of a subsequent overexpression is sufficiently countermanded by concomi- androgen withdrawal through androgen deprivation therapy tant overexpression of SRC-3 to maintain androgen-driven (ADT). This model could account for the relatively rapid growth of prostate cancer. Another possibility is that max- failure of ADT by suggesting that an adaptation to dimin- imal AR activity is not favorable to growth promotion in ished androgen signaling has already occurred before andro- prostate cancer. LNCaP cells, widely used as a model of gen ablation by ADT is imposed. androgen-dependent prostate cancer growth, exhibit a There is precedence for the notion that dysregulated biphasic response to androgens. Maximal growth promo- HOXC expression may be an early event in carcinogenesis. K tion is seen at levels of androgen not only below the d for Oct-4, a homeobox gene critical in the genesis of testicular the receptor, but also below those considered to be normal germ cell tumors, is expressed in precursor lesions (76, 77), physiologic levels of androgens (Daddario Axlund and whereas homeobox genes NKX3.1 and CDX2 are down- Nordeen, unpublished results). Thus, to the extent that regulated at the earliest stages of carcinogenesis of the LNCaP growth responses reflect prostate cancer in vivo, prostate and colon, respectively (78, 79). partial suppression of AR activity at physiologic androgen levels may effectively shift the growth response to a more HOXC gene expression and prostate cancer growth-promoting place on the dose–response curve. Additional studies are required to distinguish between the Alternatively, HOXC genes may be acting differentially different explanations to reconcile the apparent paradox that on critical genes in the prostate. PSA is a gene representing a HOXC8 overexpression is associated with prostate cancer differentiated function of the prostate. Perhaps HOXC yet can inhibit androgen-dependent signaling. Our studies expression inhibits genes associated with prostate differen- reveal that HOXC8 promotes invasiveness in prostate tiation but has no effect, or a stimulatory effect, on genes epithelial cells but does not further promote invasiveness involved in tumorigenesis. Our data showing that HOXC8 in tumorigenic prostate cancer cells, consistent with an early expression inhibits AR-mediated induction of the PSA gene, role of HOXC genes in promoting prostate cancer progres- coupled with the finding that HOXC8 expression increases sion and suggesting that HOX genes play an important role invasiveness in prostate epithelial cells, and recent work on in the disruption of cellular homeostasis leading to cancer. HOXB13 (26) suggest this may in fact be the case. The findings of this work are summarized in the model Norris et al. (26) have recently shown that HOXB13 presented in Figure 9 depicting the inhibition of direct AR regulates AR target gene activity in both positive and negative target genes by HOXC8 through its inhibition of SRC-3 manners. HOXB13 interacts with AR and inhibits the recruitment. The model also suggests that effects of HOXC8 transcription of genes that contain an ARE, whereas, in acting as a transcription factor in its own right along with its complex together, AR-HOXB13 confers androgen respon- transcription partner PBX may be mediating increased siveness to genes whose promoters contain a HOXB13 invasion and other activities promoting tumorigenesis. Pre- response element. Furthermore, they show that in the case liminary microarray studies implicate HOXC8 in the reg- of genes that contain a HOXB13 element adjacent to an ARE, ulation of various genes involved in matrix degradation HOXB13 and AR synergistically enhance the transcription of including MMP1, MMP2, TIMP1, PLAU, and SERPINE these genes (26). These observations support the notion that 1 (data not shown). Other studies identifying putative HOX proteins may play a dynamic role in the normal transcriptional targets of HOXC8 have implicated it in prostatic development and tumorigenesis through both the the regulation of cell cycle (57–60), apoptosis (58, 61), cell repression and activation of AR-mediated signaling. Like adhesion (58, 61, 68), migration (61), and tumorigenesis HOXC8 or HOXC6, we see that HOXB13 inhibits AR- (58, 60, 61, 68, 69). Confirmation of these and other mediated transcription at ARE-driven promoters. Although it HOXC8 targets implicated in cancer may prove extremely is plausible that HOXB13 and HOXC8 act in a similar valuable and help achieve a greater understanding of the role fashion to inhibit direct AR target genes, we have been unable of HOXC8 in prostate tumorigenesis. to detect an interaction between HOXC8 and AR in contrast An estimated 91% of men with newly diagnosed prostate to what has been previously reported for HOXB13 and AR cancer are likely to have stage 1 or stage 2 disease, in which (27). To assess whether HOXC8 modulates AR function in case the 5-year relative survival is nearly 100% (80). With both positive and negative manners via direct or indirect such a favorable prognosis, many question the benefit of interaction with AR, SRC-3, or other coregulators important exposing men with early-stage and low- or moderate-grade in AR signaling, further investigations will be required. prostate cancer to radical surgery or radiation therapy (81). A final mechanism, which is not mutually exclusive, to It is, therefore, critical to be able to identify men whose explain a role of HOXC overexpression in prostate cancer in histologic early-stage prostate cancer will progress and who the face of a suppressive activity on androgen action invokes would, therefore, benefit from aggressive therapy (82, 83). the concept that HOXC overexpression is critical to dis- Hence, there is a great need for reliable prostate cancer ruption of cellular homeostasis at an early stage of prostate markers that can predict cancers requiring aggressive ther- tumorigenesis. The developing tumor is forced to adapt to apy, thereby enabling better informed treatment options. diminished androgen signaling that accompanies other Given the correlation of HOXC8 with increased Gleason protumorigenic actions of HOXC expression, including grade (22) and data herein showing that HOXC8 increases

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HOXC8 Regulates Androgen Action via SRC-3 in Prostate Cancer

Figure 9. Model of HOXC8 action in human prostate cells. HOXC8 acts as a transcription factor to control gene expression in conjunction with its partner PBX (left) possibly to mediate increased invasiveness and other protumorigenic actions of HOXC8, which simultaneously inhibits the androgen-dependent recruitment of SRC-3 and subsequent recruitment of CBP to ARE-regulated genes (right), resulting in decreased transcription of direct AR target genes.

invasiveness of nontumorigenic prostate cells, further ana- Grant Support lysis of the consequences of HOXC gene expression and its use as a prognostic marker is warranted. This work was supported by Prostate Cancer Research Program Predoctoral Fellowship W81XWH-06-1-0066, Department of Defense (S. Daddario Axlund) and, in part, by NIH NCI CA84269 (S. K. Nordeen). Disclosure of Potential Conflicts of Interest The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance No potential conflicts of interest were disclosed. with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments

The authors thank Drs. Andrew Bradford and Heide Ford for critical reading of Received 03/24/2010; revised 09/27/2010; accepted 10/22/2010; the manuscript. published OnlineFirst 11/02/2010.

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www.aacrjournals.org Mol Cancer Res; 8(12) December 2010 1655

HOXC8 Inhibits Androgen Receptor Signaling in Human Prostate Cancer Cells by Inhibiting SRC-3 Recruitment to Direct Androgen Target Genes

Sunshine Daddario Axlund, James R. Lambert and Steven K. Nordeen

Mol Cancer Res 2010;8:1643-1655. Published OnlineFirst November 2, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-10-0111

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