An in Vivo Screen Identifies PYGO2 As a Driver for Metastatic Prostate Cancer

Published OnlineFirst May 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3564

Cancer Molecular Cell Biology Research

An In Vivo Screen Identiﬁes PYGO2 as a Driver for Metastatic Prostate Cancer Xin Lu1,2,3, Xiaolu Pan1, Chang-Jiun Wu4, Di Zhao1, Shan Feng2, Yong Zang5, Rumi Lee1, Sunada Khadka1, Samirkumar B. Amin4, Eun-Jung Jin6, Xiaoying Shang1, Pingna Deng1,Yanting Luo2,William R. Morgenlander2, Jacqueline Weinrich2, Xuemin Lu2, Shan Jiang7, Qing Chang7, Nora M. Navone8, Patricia Troncoso9, Ronald A. DePinho1, and Y. Alan Wang1

Abstract

Advanced prostate cancer displays conspicuous chromosomal inhibits prostate cancer cell invasion in vitro and progression of instability and rampant copy number aberrations, yet the identity primary tumor and metastasis in vivo. In clinical samples, PYGO2 of functional drivers resident in many amplicons remain elusive. upregulation associated with higher Gleason score and metastasis Here, we implemented a functional genomics approach to identify to lymph nodes and bone. Silencing PYGO2 expression in patient- new oncogenes involved in prostate cancer progression. Through derived xenograft models impairs tumor progression. Finally, integrated analyses of focal amplicons in large prostate cancer PYGO2 is necessary to enhance the transcriptional activation in genomic and transcriptomic datasets as well as genes upregulated response to ligand-induced Wnt/b-catenin signaling. Together, our in metastasis, 276 putative oncogenes were enlisted into an in vivo results indicate that PYGO2 functions as a driver oncogene in the gain-of-function tumorigenesis screen. Among the top positive 1q21.3 amplicon and may serve as a potential prognostic bio- hits, we conducted an in-depth functional analysis on Pygopus marker and therapeutic target for metastatic prostate cancer. family PHD finger 2 (PYGO2), located in the amplicon at 1q21.3. Significance: Amplification/overexpression of PYGO2 may PYGO2 overexpression enhances primary tumor growth and local serve as a biomarker for prostate cancer progression and metas- invasion to draining lymph nodes. Conversely, PYGO2 depletion tasis. Cancer Res; 78(14); 3823–33. 2018 AACR.

Introduction mutation frequency compared with most solid cancer types (8), yet advanced disease is characterized by rampant genomic Prostate cancer is the most commonly diagnosed noncuta- rearrangements and somatic copy number alterations (SCNA) neous malignancy and the third leading cause of cancer mor- (3–7). SCNAs affect a larger fraction of the cancer genome than tality for men in the United States (1). Bone is the most any other type of genetic alterations in cancer (9), underscoring frequent site for distant metastasis of prostate cancer, which the potential role of SCNAs in driving the malignant nature of inflicts significant morbidity and mortality (2). Genomic pro- prostate cancer. Functional driver genes residing within recur- filing of prostate cancer (3–7) has revealed overall lower rent amplifications include key prostate cancer oncogenes such as EZH2 on 7q36.1, MYC on 8q23-24, NCOA2 on 8q13.3, and 1Department of Cancer Biology, The University of Texas MD Anderson Cancer AR on Xq12 (3). Gain-of-function screens of resident genes Center, Houston, Texas. 2Department of Biological Sciences, Center for Rare and within amplicons are a proven approach in the identification of 3 Neglected Diseases, University of Notre Dame, Notre Dame, Indiana. Tumor novel oncogenes. Microenvironment and Metastasis Program, Indiana University Melvin and Bren In this study, our screen identified PYGO2 as a putative driver of Simon Cancer Center, Indianapolis, Indiana. 4Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas. 5Depart- prostate cancer progression. PYGO2 is an essential transcription b ment of Biostatistics, Indiana University, Indianapolis, Indiana. 6Department of coactivator with -catenin/TCF complex for the Wnt signaling Biological Science, Wonkwang University, Cheonbuk, Iksan, South Korea. 7Insti- pathway in Drosophila (10). With a highly conserved plant home- tute for Applied Cancer Science, The University of Texas MD Anderson Cancer odomain (PHD) in its C-terminus, PYGO2 binds to H3K4me and 8 Center, Houston, Texas. Department of Genitourinary Medical Oncology, The activates b-catenin–dependent transcriptional regulation (11). 9 University of Texas MD Anderson Cancer Center, Houston, Texas. Department Evidence suggests that PYGO2 modulates gene transcription of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas. through both Wnt-dependent and Wnt-independent mechanisms (11). Emerging data indicate its pivotal role in multiple cancers Note: Supplementary data for this article are available at Cancer Research including glioma (12), breast cancer (13), hepatic carcinoma Online (http://cancerres.aacrjournals.org/). (14), and intestinal tumors (15). Recently, PYGO2 expression X. Lu and X. Pan contributed equally to this article. was identified as a potential risk stratification marker for PSA Corresponding Authors: Ronald A. DePinho, University of Texas MD Anderson progression in prostate cancer following radical prostatectomy Cancer Center, 1881 East Road, Unit 1906, Houston TX 77054. Phone: 832-751- (16). PYGO2 is recruited by PCGEM1, a long noncoding RNA, to 9756; Fax: 713-745-7167; E-mail: [email protected]; and Y. Alan Wang, enhance AR-bound enhancer activity (17). Nevertheless, the [email protected] functional contribution of PYGO2 to prostate cancer progression, doi: 10.1158/0008-5472.CAN-17-3564 particularly bone metastasis, is not known, prompting us to 2018 American Association for Cancer Research. explore its role in prostate cancer biology.

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Materials and Methods viruses individually and infected low-passage LHMK cells. Stable sublines were generated by blasticidin selection, with Cell culture and patient-derived xenograft models each subline individually expanded for the in vivo screen. The LHMK cell line was a generous gift from William Hahn (Dana-Farber Cancer Institute, Boston, MA; ref. 18). LHMK and 293T (obtained from ATCC) were maintained in DMEM, 10% In vivo ORF screen FBS. Prostate cancer cell lines PC-3, LNCaP, C4, C4-2, DU145, and To evaluate the tumorigenicity of the parental cell line, LHMK 22Rv1 were obtained from ATCC and maintained in RPMI640, cells were injected subcutaneously with 106 viable cells in a 10% FBS. L cells and L Wnt-3A cells were obtained from ATCC and mixture of PBS:Matrigel (BD Biosciences) in NCr nude mice maintained in DMEM, 10% FBS (for L Wnt-3A cells, 0.4 mg/mL (Taconic), which did not form tumors 6 months postimplanta- G-418 was supplemented). ATCC provides the Human STR tion. Expecting a small fraction of the candidate genes to pro- Profiling Cell Authentication Service to authenticate these cell mote tumorigenesis, we designed a multisite subcutaneous inoc- lines. All cells were routinely verified as being free of Mycoplasma ulation method to reduce the number of mice needed for the using MycoAlert Mycoplasma Detection Kit (Lonza). Patient- screen. For each LHMK-ORF subline, 106 viable cells, resus- derived xenograft (PDX) models were previously published and pended in 50 mL mixture of PBS:Matrigel, were injected subcu- generous gifts from N.M. Navone (19, 20). The scramble control taneously into prelabeled flank positions of mice (5 sites on each shRNA and PYGO2-targeting shRNA were ordered from Sigma, side of flank, so total 10 sites/mouse). The experiment was with sequences listed in Supplementary Table S1. designed so that each subline was evaluated in 10 different mice, and each mouse received injections from 10 different sublines. Generation of LHMK sublines for screening Mice were monitored for tumor formation via caliper measure- The opening reading frame (ORF) lentiviral vectors in the ment for 8 months. We did not observe formation of more than Precision LentiORF collection were obtained from the Func- two subcutaneous tumors on any mice in the screen. All animal tional Genomics Facility at MD Anderson Cancer Center experimental protocols were approved by the Institutional Ani- (Houston,TX).In96-wellplates,wepackaged288ORFlenti- mal Care and Use Committee at MD Anderson Cancer Center.

Taylor et al. Grasso et al. Barbieri et al. TCGA 8 Oncomine transcriptome datasets Holzbeierlein et al. Focal peaks identified by GISTIC2.0 Lapointe et al. LaTulippe et al. 6,909 Amplified genes Vanaja et al. Varambally et al. Yu et al. Copy number correlated Taylor et al. Filter 1 Expression (P < 0.01) Grasso et al.

1018 Genes 25,484 Genes

Metastasis > primary in ≥3/8 Metastasis > primary in ≥6/8 Filter 2 Oncomine datasets (P < 0.05) Oncomine datasets (P < 0.05)

394 Genes 363 Genes Ding et al. 741 Genes

77 Genes

PB-Cre/Pten/p53/LSL-mTert model Precision In vivo LentiORF 288 ORFs Cross-species integrative analysis screen collection

Figure 1. Oncogenomics-informed in vivo ORF screen. Three sources of candidate prostate cancer genes are integrated: 394 genes located in focal amplicons (4 genomics datasets) and expressed in correlation with copy number gain and metastasis phenotype (3/8 transcriptomic datasets); 363 genes upregulated in metastasis (6/8 transcriptomic datasets); and 77 genes from our published cross-species prostate cancer genome analysis (36). Among the total 741 candidate genes, 288 ORFs corresponding to 276 genes were available for screening.

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In Vivo Screen Identiﬁes PYGO2 as a Prostate Cancer Gene

A LentiORF Lentivirus library 96-well LHMK SubQ n Infection ( = 10)

Blasticidin One stable Selection subline/ORF

B C

LHMK-RFP-IRES-turboGFPnuc Figure 2. In vivo ORF screen identiﬁed genes KRAS promoting prostate tumorigenesis. A, Procedure for lentivirus packaging, ORF FGFR1 stable overexpression in LHMK and in β vivo tumorigenesis screen. B, Images of -Actin LHMK overexpressing the control vector RFP-IRES-turboGFPnuc. Scale bar, 50 D mm. C, ORF-driven KRAS or FGFR1 150 overexpression in LHMK, conﬁrmed with TROAP Western blot analysis. D, Top hits with Note: RFP (neg. ctrl.) 0% 20% or higher incidence rate from the 120 in vivo screen. PYGO2 RPS20 90 ZKSCAN5 EZH2 TAF6 TOMM40L 60 MTBP

Earliest onset day MOS KRAS (pos. ctrl.) 30 CCNE2 FGFR1 (pos. ctrl.) 0 0 20 40 60 80 100 Tumor incidence %

Tissue specimens, histology, and Western blot analysis BioScience) for 3 days. For soft agar assay, DMEM with 1% A prostate cancer tissue microarray with 80 cases and Gleason FBS, 0.6% LE Agarose (Lonza) was used as base layer while cells grade information was purchased (PR803b, US Biomax). Archived were seeded in 2 104 cells/mL in DMEM with 1% FBS, 0.3% prostate cancer FFPE specimens of adjacent normal, primary tumor, SeaPlaque Agarose as top layer (Lonza). After incubation at and metastasis (total n ¼ 49) were requested from MD Anderson 37C for 3 weeks, the colonies were stained by crystal violet and Cancer Center Prostate Cancer SPORE program (Specialized Pro- quantified. grams of Research Excellence) under approved IRB protocol at MD Anderson Cancer Center. For all clinical samples, written informed Migration and invasion assay consent was obtained from the patients. The studies were conduct- Cells were first starved in DMEM with 1% FBS overnight and ed in accordance with recognized ethical guidelines (Declaration then seeded in serum-free DMEM at 5 105 cells/200 mL to the of Helsinki, CIOMS, Belmont Report, U.S. Common Rule). chamber inserts (BD Falcon) for migration or BioCoat Matrigel Hematoxylin and eosin stain, IHC, and Western blot analysis were Invasion Chamber (BD Falcon) for invasion. DMEM with 10% performed as described previously (21). Primary antibodies used FBS were placed at the bottom as chemoattractant. Migrated or include PYGO2 (HPA023689, Sigma, for IHC; GTX119726, invaded cells on the membrane were stained with crystal violet for GeneTex, for Western blot analysis), KRAS (sc-30, Santa Cruz quantification. Biotechnology), FGFR1, b-catenin, c-Myc, Met, H3K4me2, H3K4me3, H3 (9740, 8480, 5605, 8198, 9725, 9751, and 4499, Quantitative RT-PCR Cell Signaling Technology), and b-actin (A2228, Sigma). RNA was isolated by RNeasy Kit (Qiagen) and reverse tran- scribed using SuperScript III cDNA Synthesis Kit (Life Technol- Cell proliferation and soft agar assay ogies). Quantitative PCR was performed using SYBR-GreenER For two-dimensional (2D) proliferation, cells were seeded to Kit (Life Technologies). Primers are listed in Supplementary 24-well plates with confluence tracked by IncuCyte (Essen Table S1.

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Figure 3. Candidate prostate cancer genes promote soft agar colony formation, migration, and invasion. A, Normalized confluence curves on 2D culture for selected top hit genes showing modest change of cell proliferation. B, Significant increase of colony formation on soft agar by selected top hit genes compared with RFP control. C, Significant increase of cell migration by selected top hit genes compared with RFP control. D, Significant increase of cell invasion by selected top hit genes compared with RFP control. Representative images of invaded cells are shown.

Functional validation using animal models , P < 0.001; #, P > 0.05). To display PYGO2 expression from Experimental bone metastasis assay using intracardiac injection four Oncomine transcriptomic datasets containing primary and noninvasive imaging was performed as reported previously and metastatic prostate cancer samples (3, 4, 27, 28), log2 (22). PDX models were passaged in the flank of C.B-17 SCID median-centered ratio of PYGO2 probe data was drawn as a (Taconic) mice as reported previously (19, 20). The tumors were box plot with whiskers displaying 10–90 percentile using measured by caliper and treated by intratumoral injection of 10 mg GraphPad Prism. siRNA targeting PYGO2 (Sigma-Aldrich, SASI_HS01_00059018, or 1:1 ratio of SASI_Hs01_00059021 and SASI_Hs02_00363399) or control siRNA (Sigma-Aldrich, SIC001) twice a week, using Results MaxSuppressor In Vivo RNA-LANCEr II (Bioo Scientific) follow- In vivo ORF screen identified putative genes involved in prostate ing the manufacturer's protocol and our recent report (23). cancer progression To enlist genes with putative function in promoting prostate Luciferase reporter assay cancer progression, we performed an integrated oncogenomic TCF/LEF reporter plasmids, M50 Super 8x TOPFlash and M51 analysis to enrich for cancer-relevant genes and cull passenger Super 8x FOPFlash (TOPFlash mutant), were gifts from Randall genes. First, genes with focal copy number gains were identified Moon (University of Washington, Seattle, WA; Addgene plasmid using GISTIC2 (29) from 4 prostate cancer genomic datasets: # 12456, 12457; ref. 24). Activation of Wnt/b-catenin signaling Taylor et al., Grasso et al., Barbieri et al., and The Cancer Genome was achieved by using conditioned medium from Wnt3A-secret- Atlas (TCGA; Fig. 1; refs. 3–6). This analysis resulted in 6,909 ing L cells and control L cells (25). PC3 sublines were transfected genes, which were further selected based on two filters: genes with with Lipofectamine LTX Reagent (Life Technologies) following copy number correlated expression in at least 1 of the 4 datasets the manufacturer's protocol, and the reporter assay was per- (P < 0.01) and genes with higher expression in metastasis com- formed as described previously (26). pared with primary tumor (P < 0.05) in at least 3 of 8 Oncomine transcriptomic datasets (3, 4, 27, 30–34). The gene expression Statistical analysis data for these 8 datasets were directly queried from Oncomine Unless otherwise indicated, data represent mean SD, with (35). After applying the filters, 394 genes remained (Fig. 1). Student t test assuming two-tailed distributions used to calculate Second, to enrich for genes potentially contributing to metastasis, statistical significance between groups. P < 0.05 was considered 363 genes upregulated in metastasis compared with primary statistically significant (annotation: , P < 0.05; , P < 0.01; tumor were identified in at least 6 of 8 Oncomine datasets. Third,

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In Vivo Screen Identiﬁes PYGO2 as a Prostate Cancer Gene

A 2.7% (74, Taylor et al.) B Diploid 5.5% (55, Baca et al.) Gain or amplification 7.7% (13, Grasso et al.) Shallow deletion 100 8.7% (333, TCGA) 53.6% (28, Beltran et al.) TCGA Dataset 80 76.9% (13, Kumar et al.) (treatment-naïve, primary tumors) 60 33.3% (15, Taylor et al.) 40 35.4% (48, Grasso et al.) Gleason score 3+3 3+4 4+3 ≥8 45.9% (148, Robinson et al.) N 65 102 78 88 20 54.4% (79, Beltran et al.) 2 PYGO2 Gain/amp % ***, P = 0.0004 (χ test) 0 67.6% (136, Kumar et al.) Primary Metastasis C PYGO2 Diploid D PYGO2 Gain or amplification Grasso et al. Taylor et al. Tamura et al. Lapointe et al. P = 1.05E–10 P = 0.006 P = 0.008 P = 0.018

2 3 2 100 100 2 2 1 1 ratio ratio

ratio 1 2 2 ratio 2 50 50 2 1 Log 0 Log Log Log 0 free survival 0 0 Percent disease 0 0 -1 0 50 100 150 200 Percent biochemical 0 1,000 2,000 recurrence free survival Time (months) Time (days) *, P = 0.0146 (Log-rank test) **, P = 0.0040 (Log-rank test)

Intensity-0 Intensity-1 E F PYGO2 (0) PYGO2 (1) PYGO2 (0) PYGO2 (1) PYGO2 (2) PYGO2 (3) PYGO2 (2) PYGO2 (3) 10 15 8 12 6 9 4 Intensity-2 Intensity-3 6 2 Patient cases

Patient cases 3 0 0 6 and 7 8 and 9 10 Gleason score sum Normal Primary LN met Bone met **, P = 0.0094 (χ2 test) ***, P < 0.0001 (χ2 test)

Figure 4. PYGO2 is ampliﬁed in prostate cancer and correlates with higher Gleason score and metastasis. A, Frequency of PYGO2 copy number gain and ampliﬁcation in a variety of prostate cancer genomics datasets categorized by disease site and treatment. B, Fraction of PYGO2 copy number status in different Gleason score categories in the TCGA dataset. C, Correlation of PYGO2 copy number status with disease-free survival (n ¼ 329) or biochemical recurrence (n ¼ 281) in the TCGA dataset. D, PYGO2 mRNA expression level in primary tumor and metastasis in four prostate cancer studies with data compiled from Oncomine. E, In the TMA, PYGO2 expression as measured by IHC and plotted against Gleason grade categories. F, In an archived prostate cancer clinical cohort from MD Anderson Cancer Center, PYGO2 expression was plotted against categories as normal prostate, primary prostate tumor, lymph node (LN) metastases, and bone metastases.

77 amplified genes were identified from integrated analysis of our igenic capability when inoculated orthotopically or subcutane- previous telomerase reactivation prostate cancer mouse model ously in nude mice (18), providing a suitable system to identify and human prostate cancer genomics (36). From these diverse putative oncogenes through a gain-of-function approach. ORF- datasets and experimental systems, a total of 741 putative metas- encoded lentivirus was packaged in 96-well plates and used to tasis-promoting genes were identified (Supplementary Table S2), transduce LHMK cells, followed by blasticidin selection, to estab- among which 288 ORFs (corresponding to 276 unique genes) lish 288 individual ORF-expressing sublines (Fig. 2A). Overex- were available at the time of experimentation from the Precision pression of red fluorescent protein (RFP) in the same LentiORF LentiORF Collection for lentiviral overexpression and ORF screen- backbone was used as the negative control (Fig. 2B). ORFs ing (Supplementary Table S3). encoding KRAS and FGFR1 were used as positive controls (Fig. We employed LHMK cells for the in vivo screen, which were 2C), the choice of which was justified given the prostate cancer– derived from primary human prostate epithelial cells after immor- promoting role of RAS/MAPK (3, 37) or FGF/FGFR1 signaling, talization with SV40 LT and hTERT followed by transformation respectively (38). ORF-driven overexpression was validated for a with MYC and PI3K (18). LHMK cells exhibit very limited tumor- number of randomly selected genes using quantitative RT-PCR

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LHMK B A * LNCaP **

) 1.5 3 1,200

PYGO2 800 PYGO2 1.0

β-Actin 400 β-Actin 0.5

0 Tumor weight (g) Tumor volume (mm RFP PYGO2 0.0 GFP PYGO2

C LNCaP-PYGO2-IRES-GFP Primary Draining tumor lymph node incidence metastasis LN LN GFP 10/10 0/10

PYGO2 10/10 3/10 1o tumor 1o tumor

Figure 5. PYGO2 overexpression promotes prostate cancer tumor growth and invasion to draining lymph nodes. A, PYGO2 overexpression in LHMK cells significantly increased subcutaneous tumor growth in mice (n ¼ 4). B, PYGO2 overexpression in LNCaP significantly increased subcutaneous tumor growth in mice (n ¼ 10). C, Summary of tumor incidence by LNCaP sublines and fluorescence imaging showing the invasion of LNCaP-PYGO2-IRES-GFP from subcutaneous tumor to local draining lymph node. In A and B, , P < 0.05; , P < 0.01, Mann–Whitney test.

(Supplementary Fig. S1A), which all showed various levels of ized) was accompanied by their effect on increased migration and overexpression of the putative targets. In the screen, the 288 invasion (Fig. 3C and D). Together, the robustness of PYGO2 in sublines and RFP control subline were inoculated into mice the in vivo ORF screen coupled with strong effect in the 3D colony subcutaneously (n ¼ 10 for each ORF). Mice were monitored for assay (second only to KRAS; Fig. 3B) prompted further functional tumor development for 8 months. Although no tumor growth investigation of this putative prostate cancer–promoting gene. was detected for the RFP control (total 30 sites were tested), the Overexpression of BOP1 (block of proliferation 1) led to strongest positive controls KRAS and FGFR1 generated 100% and 30% enhancement of migration and invasion (Fig. 3C and D). Located incidence of tumors, respectively (Supplementary Table S4). at 8q24.3, BOP1 is close to MYC at 8q24.21. These two genes tend Importantly, 38 genes were identified as positive hits based on to be coamplified in the broad amplification peak at 8q24 a 10% to 50% tumor incidence rate (Supplementary Table S4), (Supplementary Fig. S1D), which is commonly attributed to the among which 10 genes produced more than 2 tumors out of the oncogenic function of MYC. Therefore, we reasoned that the 10 tested sites (Fig. 2D; Supplementary Fig. S1B). The top 10 hits amplification of BOP1 might be, at least partly, a passenger effect include EZH2, known to be frequently upregulated in advanced from MYC amplification, which would make a study on BOP1 less prostate cancer and to promote metastasis, and CCNE2, which is significant in terms of finding independent biomarker and/or overexpressed in metastatic prostate cancer and critical for cell- therapeutic target for prostate cancer. TOMM40L overexpression cycle G1–S transition (39). Notably, the presence of known led to higher tumor incidence rate and shorter onset day than prostate cancer–promoting genes among the top hits suggests PYGO2 (Fig. 2D). The function of TOMM40L was not studied the possibility that the other genes may represent bona fide before. The commercially available reagents for TOMM40L are oncogenes involved in prostate cancer progression. To rule out limited, making it difficult to perform clinical characterization of the possibility that the negative hits were merely due to failure of its expression and related functional studies. LentiORF-driven gene overexpression, we randomly selected 26 negative hits from the lenti-ORF infected cells and showed that 23 PYGO2 expression is correlated with higher Gleason score and of 26 genes were upregulated more than 2-fold (Supplementary bone metastasis Fig. S1C). PYGO2 resides on cytoband 1q21.3, a region amplified in Reasoning that in vitro assays could complement the in vivo advanced prostate cancer (3, 40, 41) but containing no known result to illuminate biological effects, we performed proliferation, definitive prostate cancer oncogenes. When surveyed through migration, and invasion assays for the sublines of the top hits. prostate cancer databases in cBioPortal, the status of PYGO2 copy Although meager differences were observed in the 2D growth number was retrieved from 7 studies (3, 4, 6, 7, 42–44) and curve assay (Fig. 3A), soft-agar assay showed that sublines over- showed higher gain or amplification in primary castration-resis- expressing genes like KRAS, PYGO2, MOS, CCNE2, and MTBP tant prostate cancer (CRPC; 53.6%–76.9%) or metastatic CRPC could form significantly more colonies than RFP control (Fig. 3B). (33.3–67.7%) compared with treatment-na€ve primary prostate The gain of colony formation potential by genes such as PYGO2, cancer (2.7%–8.7%; Fig. 4A). In the TCGA dataset, PYGO2 gain/ MOS, and MTBP (with functions in prostate cancer uncharacter- amplification is associated with higher Gleason score in

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A B C Lung met – shControl shPYGO2 #860 Lung met + 100% 400 shPYGO2 #861 ) 3 ** 80% shControl 300 60% *** PYGO2 200 sh#860 *** 40% *** 100 20% β-Actin *** % of Cohort * *** ** sh#861

Tumor volume (mm Tumor 0 0% PC3 0 10 20 30 40 50 shCtrl sh#860 sh#861 Days after injection

D E F shPYGO2 shPYGO2 X-ray H&E shControl #860 #861 shControl shPYGO2 #860 500 shPYGO2 #861 T

250 Day 0

** 0 ** Normalized BLI signal 0 10 20 30 40 50 Days after injection T Day 42 shPYGO2 #861 shControl

Figure 6. PYGO2 silencing reduces primary tumorigenicity and metastatic potential of PC3 cells. A, PYGO2 knockdown by two independent shRNA clones in PC3 shown by Western blot analysis. B, Significant decrease of subcutaneous tumor size by PYGO2 knockdown in PC3 (n ¼ 25 for each group). Data represent mean SEM. C, Incidence of spontaneous lung metastasis from subcutaneous tumors formed by PC3 sublines (n ¼ 15 for each group). , P < 0.05; , P < 0.01, Fisher exact test. D and E, Weakened bone colonization ability by PYGO2 knockdown in PC3-TR cells, shown by both bioluminescence signals (D, normalized to day 0) and representative images (E, n ¼ 7 for each group). Data, mean SEM. F, Osteolysis in the long bones induced by PC3-TR sublines, shown by X-ray radiographs and hematoxylin and eosin staining. Scale bar, 500 mm. treatment-na€ve primary prostate cancer (Fig. 4B), as well as larger tumors as compared with RFP controls (Fig. 5A; Sup- shorter disease-free survival and shorter biochemical recurrence- plementary Fig. S2B). To test the protumor function of PYGO2 free survival (Fig. 4C). Copy number–correlated expression of in a different prostate cancer cell line, we overexpressed PYGO2 is evident across several datasets (Supplementary PYGO2 in LNCaP, which also has a low endogenous level of Fig. S2A). Regarding metastasis, PYGO2 is significantly upregu- PYGO2 (Fig. 5B). Compared with GFP control, PYGO2 over- lated at the transcriptional level in metastatic prostate cancer expression led to significant increase of subcutaneous tumor compared with primary tumors (Fig. 4D). At the protein level, weight (Fig. 5B). Based on the IRES-GFP cassette in the over- þ tissue microarray (TMA) analysis showed that, although PYGO2 expression vector, we identified GFP tumor cells in draining expression was not detectable in normal prostate, stronger lymph nodes in 3 of 10 mice inoculated with LNCaP-PYGO2 PYGO2 expression was correlated with higher Gleason score cells (Fig. 5C; Supplementary Fig. S2C). Thus, PYGO2 over- (Fig. 4E). Furthermore, from an archived clinical prostate cancer expression promotes both primary tumor growth and regional sample cohort at MD Anderson Cancer Center, which includes lymph node invasion. normal, primary tumors, lymph node metastases, and bone metastases, IHC analysis showed that PYGO2 expression was PYGO2 depletion inhibits prostate cancer metastasis and PDX highly upregulated in metastases (Fig. 4F). The clinical expression tumor growth analysis, in addition to the in vivo functional screen and in vitro To determine whether PYGO2 is required for prostate cancer functional validation, strongly supports a direct role of PYGO2 in progression, we used two independent PYGO2 shRNA vectors to promoting prostate cancer progression. deplete PYGO2 levels in the aggressive prostate cancer cell line PC3 (Fig. 6A; ref. 45). PYGO2 knockdown resulted in a modest PYGO2 overexpression promotes prostate tumor growth and decrease in cell proliferation in vitro (Supplementary Fig. S3A) but invasion to lymph nodes significant reduction of cell invasion (Supplementary Fig. S3B). To determine whether PYGO2 upregulation enhances pro- When inoculated subcutaneously in mice, PYGO2 knockdown state cancer progression, we first retested the LHMK sublines cells showed reduced tumorigenic potential (Fig. 6B). To evaluate expressing RFP or PYGO2 by subcutaneous inoculation in whether PYGO2 knockdown affects spontaneous metastasis of NSG mice. The LHMK-PYGO2 subline formed significantly PC3 to lung, we removed the subcutaneous tumors at day 50

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A B C PDX: MDA-PCa-180 400 MDA-PCa-180 ) 3 300 Start Tx

PDX Model 118b 180 118b 180 200 siControl siPYGO2 siControl ++–– 100

Tumor volume (mm Tumor ** siPYGO2 –++– 0 0 25 30 35 40 Days after inoculation PDX: MDA-PCa-118b PYGO2 MDA-PCa-118b 200 ) 3

β-Actin 150 Start Tx siControl 100 siPYGO2

50 ** Tumor volume (mm Tumor 0 0 10 20 30 40 Days after inoculation

D E 123 1,500 WNT3a medium - TOP Flash β-Catenin *** L medium - TOP Flash * c-Myc 1,000 WNT3a medium - FOP Flash L medium - FOP Flash Met 500 1. PC3-shControl H3K4me3 2. PC3-shPYGO2 #860 H3K4me2 3. PC3-shPYGO2 #861 Luciferase signal (AU) 0 H3 shControl β-Actin shPYGO2 #860shPYGO2 #861

F Localized PCa CRPC

shControl

High PYGO2 expression High PYGO2 expression

Low PYGO2 expression Low PYGO2 expression

Figure 7. Intratumoral infusion of PYGO2 siRNA blocks prostate tumor growth in PDX models. A, Strong nuclear expression of PYGO2 in two PDX models of CRPC, detected by IHC. Scale bar, 100 mm. B, Reduction of PYGO2 level in two PDX models by PYGO2-targeting siRNA, detected by Western blot analysis. C, PYGO2-targeting siRNA (Sigma-Aldrich, SASI_HS01_00059018) impaired PDX tumor growth. Arrows, start day for intratumoral siRNA infusion. D, Luciferase assay measuring effect of PYGO2 knockdown in PC3 on the response to Wnt-3A–mediated TOPFlash reporter activity. E, Effect of PYGO2 knockdown in PC3 on the expression of indicated proteins, detected by Western blot analysis. F, GSEA analysis for transcriptomic samples. Samples from Grasso et al. (4), dichotomized by normalized expression level of the PYGO2 probe A_23_P411953. Kyoto Encyclopedia of Genes and Genomes Wnt pathway with 138 genes was the gene set for the analysis. Localized prostate cancer (PCa) and CRPC samples were analyzed separately, with false discovery rate Q-value being 0.051 and 0.070, respectively. Both false discovery rate Q-values are <0.25, the recommended false discovery rate cut-off value by the GSEA User Guide.

postinoculation and assessed metastasis formation in the lung 2 As bone is the most frequent site of distant metastasis of months later by gross inspection and histology. Although 60% of prostate cancer, we performed intracardiac injection to compare mice previously inoculated with the PC3-shControl subline the bone colonization capability of shControl and shPYGO2 developed spontaneous lung metastasis nodules, less than 20% sublines of PC3 after labeling PC3 with a triple reporter (TR) of mice inoculated with the shPYGO sublines of PC3 developed containing ﬁreﬂy luciferase, GFP, and thymidine kinase (46). lung metastasis nodules (Fig. 6C; Supplementary Fig. S3C). Noninvasive bioluminescence imaging revealed that PYGO2 Expression of PYGO2 in PC3 cells remains pronounced in lung knockdown impaired the ability of PC3-TR cells to colonize the metastasis (Supplementary Fig. S3D). bone and form osteolytic lesions (Fig. 6D–F). PYGO2 is also

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In Vivo Screen Identiﬁes PYGO2 as a Prostate Cancer Gene

expressed by a few other prostate cancer cell lines, including cancer, including the metastatic process and development of 22Rv1, C4, and C4-2 (Supplementary Fig. S3E). CRPC (48). Therefore, the implication of PYGO2 in Wnt pathway As PDX models more closely resemble the clinical disease, we has significant clinical relevance. Future studies to investigate the examined the effect of targeting PYGO2 in two PDX models: molecular mechanism of PYGO2 in prostate cancer progression MDA-PCa-180 (derived from primary CRPC; ref. 19) and MDA- will provide new opportunities to target lethal prostate cancer. We PCa-118b (derived from bone metastatic CRPC; ref. 20). We envision at least two potential approaches to target PYGO2. First, first performed IHC for PYGO2 and detected high PYGO2 the PHD finger in PYGO2 is responsible for binding to di- and expression in both models (Fig. 7A). Through intratumoral trimethylated lysine 4 of histone H3 (H3K4me2/3). Therefore, infusion of siRNA (either scramble control or PYGO2-target- small-molecule inhibitors blocking the PHD finger (49) may ing),wewereabletosignificantly attenuate PYGO2 protein serve as useful agents for PYGO2-overexpressed lethal prostate level (Fig. 7B). In both models, PYGO2-targeting siRNA treat- cancer. Second, siRNAs that effectively downregulate PYGO2 in ment inhibited subcutaneous PDX tumor growth (Fig. 7C). For vivo may provide another avenue. siRNA or shRNA as therapeutics MDA-PCa-180, we also demonstrated the antitumor effect by is being actively developed, although challenges remain in the an independent siRNA mixture (Supplementary Fig. S3F). delivery of these agents. That said, recent progress using exosomes Spontaneous metastasis to lung or bone from the PDX tumors to deliver siRNA or shRNA in vivo (50) marks a new direction for was not detected based on histologic evaluation, and metastasis moving this idea forward. was not reported to occur in these two models previously (19, 20). These results support PYGO2 as a therapeutic target for Disclosure of Potential Conflicts of Interest prostate cancer. No potential conflicts of interest were disclosed. To explore the function of PYGO2 as a coactivator of the b Wnt/ -catenin pathway in the context of prostate cancer, we Authors' Contributions compared the ability of PC3-shControl and PC3-shPYGO2 b Conception and design: X. Lu, X. Pan, R.A. DePinho, Y.A. Wang sublines to activate the Wnt/ -catenin reporter TOPFlash Development of methodology: X. Lu, X. Pan, X. Shang, Y.A. Wang (24) under conditioned medium from L Wnt-3A cells (25). As Acquisition of data (provided animals, acquired and managed patients, control, FOPFlash and conditioned medium from L cells were provided facilities, etc.): X. Lu, X. Pan, D. Zhao, S. Feng, R. Lee, S. Khadka, used. Interestingly, PYGO2 knockdown significantly reduced E.-J. Jin, X. Shang, P. Deng, Y. Luo, W. R. Morgenlander, Q. Chang, N.M. Navone, the Wnt-3A–induced TOPFlash activity (Fig. 7D). At the protein P. Troncoso, Y.A. Wang Analysis and interpretation of data (e.g., statistical analysis, biostatistics, level, PYGO2 knockdown moderately decreased the expression b b computational analysis): X. Lu, X. Pan, C.-J. Wu, D. Zhao, S. Feng, Y. Zang, of -catenin and Wnt/ -catenin targets c-Myc and Met (Fig. 7E). S.B. Amin, X. Lu, R.A. DePinho, Y.A. Wang PYGO2 knockdown has little effect on H3K4me2 and Writing, review, and/or revision of the manuscript: X. Lu, X. Pan, P. Troncoso, H3K4me3 levels (Fig. 7E). Our results on the connection of R.A. DePinho, Y.A. Wang PYGO2 with Wnt signaling were supported by the gene set Administrative, technical, or material support (i.e., reporting or organizing enrichment analysis (GSEA) showing that Wnt pathway is data, constructing databases): X. Lu, X. Pan, D. Zhao, P. Deng, J. Weinrich, S. Jiang, Y.A. Wang enriched in both localized prostate cancer and CRPC samples Study supervision: X. Lu, R.A. DePinho, Y.A. Wang with high PYGO2 expression phenotype (Fig. 7F). Other (provided pathologic expertise): P. Troncoso

Acknowledgments Discussion The project was supported by R01 CA084628 (to R.A. DePinho), In summary, through functional screen and analysis of P01CA117969 (to R.A. DePinho), MDACC Knowledge Gap Award (to Y.A. recurrently amplified genes in prostate cancer, we identified Wang), MDACC Prostate Cancer Moon Shot Award (to R.A. DePinho and Y.A. PYGO2 as a prostate cancer–promoting gene capable of driving Wang), and Department of Defense Prostate Cancer Research Program Idea Development Award W81XWH-14-1-0576 (to X. Lu). X. Lu is also supported by disease progression and metastasis. Another candidate prostate the Indiana Clinical and Translational Sciences Institute (CTSI) KL2 Young cancer gene located on 1q21.3, CREB3L4 (a.k.a. AIbZIP, an Investigator Award, which is funded in part by grants KL2TR001106 and androgen-regulated gene), has been reported as highly UL1TR001108 (A. Shekhar, principal investigator) from the National Institutes expressed in prostate cancer (47). However, CREB3L4 is distinct of Health, National Center for Advancing Translational Sciences, Clinical and in that its expression is neither correlated with copy number Translational Sciences Award. S. Feng is supported by Walther Cancer Foun- gain (3) nor upregulated in metastatic prostate cancer when we dation Advancing Basic Cancer grants. Cancer Center Support Grant P > P30CA016672 (MD Anderson Cancer Center) provided facility-based experi- surveyed the 8 Oncomine datasets ( 0.5 for all datasets). In mental support. Data analysis assistance is partly supported from the Collab- fact, CREB3L4 was not among the 60 of 178 genes located in orative Core for Cancer Bioinformatics, shared by Indiana University Simon 1q21.2-q22 with transcript levels correlated with copy number Cancer Center (grant P30CA082709) and Purdue University Center for Cancer gain (3). From the 60 genes in 1q21.2-q22, 13 genes passed our Research (grant P30CA023168), supported by the Walther Cancer Foundation. gene selection filters and 6 genes (ENSA, LYSMD1, RPRD2, The authors thank Dr. Denise J. Spring for manuscript proofreading, and all the FLAD1, KRTCAP2,andPYGO2) were screened with available members of the DePinho laboratory and the Lu laboratory for a variety of technical support and helpful suggestions. lentiviral ORFs. Only PYGO2 emerged as a functional hit in our tumor models. Our results indicate that PYGO2 promotes primary tumor growth, lymph node invasion, and bone metas- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked tasis. Together, we conclude that PYGO2 is a key driver gene of advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate 1q21.3 that is targeted for increased expression via copy num- this fact. ber gain in prostate cancer. Hyperactivated Wnt signaling pathway has been increasingly Received November 22, 2017; revised March 27, 2018; accepted May 10, identified to play important roles in promoting advanced prostate 2018; published first May 16, 2018.

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www.aacrjournals.org Cancer Res; 78(14) July 15, 2018 3833

An In Vivo Screen Identifies PYGO2 as a Driver for Metastatic Prostate Cancer

Xin Lu, Xiaolu Pan, Chang-Jiun Wu, et al.

Cancer Res 2018;78:3823-3833. Published OnlineFirst May 16, 2018.

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