TMPRSS2-ERG Controls Luminal Epithelial Lineage and Antiandrogen Sensitivity in PTEN and TP53-Mutated Prostate Cancer Alexandra M

Published OnlineFirst May 29, 2018; DOI: 10.1158/1078-0432.CCR-18-0653

Cancer Therapy: Preclinical Clinical Cancer Research TMPRSS2-ERG Controls Luminal Epithelial Lineage and Antiandrogen Sensitivity in PTEN and TP53-Mutated Prostate Cancer Alexandra M. Blee1,2, Yundong He1, Yinhui Yang1,3, Zhenqing Ye4, Yuqian Yan1, Yunqian Pan1, Tao Ma4, Joseph Dugdale1, Emily Kuehn1, Manish Kohli5, Rafael Jimenez6, Yu Chen7, Wanhai Xu3, Liguo Wang4, and Haojie Huang1,8,9

Abstract

Purpose: Deletions or mutations in PTEN and TP53 tumor xenografts, and allografted mouse tumors. Trends were eval- suppressor genes have been linked to lineage plasticity in uated in TCGA, SU2C, and Beltran 2016 published patient therapy-resistant prostate cancer. Fusion-driven overexpres- cohorts and a human tissue microarray. sion of the oncogenic transcription factor ERG is observed in Results: Transgenic ERG expression in mice blocked Pten/ approximately 50% of all prostate cancers, many of which also Trp53 alteration–induced decrease of AR expression and harbor PTEN and TP53 alterations. However, the role of ERG downstream luminal epithelial genes. ERG directly suppressed in lineage plasticity of PTEN/TP53–altered tumors is unclear. expression of cell cycle–related genes, which induced RB Understanding the collective effect of multiple mutations hypophosphorylation and repressed E2F1-mediated expres- within one tumor is essential to combat plasticity-driven sion of mesenchymal lineage regulators, thereby restricting therapy resistance. adenocarcinoma plasticity and maintaining antiandrogen sen- Experimental Design: We generated a Pten-negative/Trp53- sitivity. In ERG-negative tumors, CDK4/6 inhibition delayed mutated/ERG-overexpressing mouse model of prostate cancer tumor growth. and integrated RNA-sequencing with ERG chromatin immu- Conclusions: Our studies identify a previously undeﬁned noprecipitation-sequencing (ChIP-seq) to identify pathways function of ERG to restrict lineage plasticity and maintain regulated by ERG in the context of Pten/Trp53 alteration. We antiandrogen sensitivity in PTEN/TP53–altered prostate can- investigated ERG-dependent sensitivity to the antiandrogen cer. Our ﬁndings suggest ERG fusion as a biomarker to guide enzalutamide and cyclin-dependent kinase 4 and 6 (CDK4/6) treatment of PTEN/TP53-altered, RB1-intact prostate cancer. inhibitor palbociclib in human prostate cancer cell lines, Clin Cancer Res; 1–15. 2018 AACR.

1Department of Biochemistry and Molecular Biology, Mayo Clinic College of Introduction Medicine, Rochester, Minnesota. 2Biochemistry and Molecular Biology Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, Rochester, Castration-resistant prostate cancers respond to current anti- Minnesota. 3Department of Urology, the Fourth Hospital of Harbin Medical androgen therapies with variable levels of success (1), in part, due University, Harbin, Heilongjiang, China. 4Division of Biomedical Statistics and to extensive genetic heterogeneity (2–4). While mechanisms of Informatics, Department of Health Sciences Research, Mayo Clinic College of androgen receptor (AR) pathway restoration and compensation Medicine, Rochester, Minnesota. 5Department of Oncology, Mayo Clinic College are well documented, adenocarcinoma cell lineage plasticity and 6 of Medicine, Rochester, Minnesota. Department of Laboratory Medicine and reprogramming to AR independence represents an additional Pathology, Mayo Clinic College of Medicine, Rochester, Minnesota. 7Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, resistance mechanism (5). Interestingly, the incidence of AR- New York, New York. 8Department of Urology, Mayo Clinic College of Medicine, independent tumor progression after castration and antiandrogen Rochester, Minnesota. 9Mayo Clinic Cancer Center, Mayo Clinic College of treatment has increased since the advent of enzalutamide and Medicine, Rochester, Minnesota. abiraterone use in the clinic, highlighting that prostate cancer Note: Supplementary data for this article are available at Clinical Cancer lineage plasticity is an increasingly important barrier to overcome Research Online (http://clincancerres.aacrjournals.org/). (6). Recent studies have identified a few key molecular events RB1 A.M. Blee, Y. He, and Y. Yang contributed equally to this article. involved in AR-independent tumor progression, such as / PTEN/TP53 loss, MYCN/AURKA amplification, and altered epi- Corresponding Authors: Haojie Huang, Department of Biochemistry and Molec- genetic regulators including EZH2 (7). However, the molecular ular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-293-1712; Fax: 507-293-3071; E-mail: basis underlying prostate cancer lineage plasticity and antiandro- [email protected]; Liguo Wang, Department of Health Sciences gen resistance remains poorly understood due to extensive patient Research, Mayo Clinic College of Medicine, Rochester, MN 55905. E-mail: tumor heterogeneity and model limitations. [email protected]; and Wanhai Xu, Department of Urology, the Fourth PTEN loss frequently overlaps with TP53 mutation or loss in Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China. E-mail: drug-resistant, morphologically distinct, reprogrammed tumors [email protected] (8–11). A significant proportion of both primary and castration- doi: 10.1158/1078-0432.CCR-18-0653 resistant tumors with PTEN/TP53 alteration also have AR-depen- 2018 American Association for Cancer Research. dent, TMPRSS2 fusion–driven overexpression of the ETS family

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used within 6 months of thawing. No mycoplasma contamina- Translational Relevance tion was detected in these cell lines by testing with the Lookout Prostate cancer resistance to androgen deprivation and AR- Mycoplasma PCR Detection Kit (Sigma-Aldrich). Charcoal- targeted therapies remains a pressing clinical obstacle, partly stripped serum (CSS) was purchased from Thermo Fisher Scien- explained by lineage plasticity and transition to AR-indepen- tific-Gibco (#12676029). Enzalutamide was kindly provided by dent tumor types in response to these therapies. A compre- Medivation. LNCaP-RF cells were treated with 10 mmol/L of hensive understanding of genetic prostate tumor subtypes and enzalutamide for 72 hours unless otherwise noted. Palbociclib the unique response of each mutational subtype to AR-tar- (PD-0332991) was obtained from ApexBio. LNCaP-RF cells were geted therapies is necessary to develop new, subtype-specific treated with 1 mmol/L of palbociclib for 72 hours unless otherwise therapeutic strategies that overcome therapy-induced lineage noted. For combination treatment, LNCaP-RF cells were treated plasticity. Our results demonstrate that E-twenty-six transfor- with 10 mmol/L enzalutamide and 1 mmol/L palbociclib for mation specific (ETS)-related gene (ERG) prevents PTEN- and 72 hours. tumor protein 53 (TP53)-negative tumor cell lineage plasticity and antiandrogen resistance by blocking E2F1-mediated Cell transfection and lentivirus transduction expression of lineage switch genes. These findings also reveal For lentiviral shRNA or stable plasmid expression, HEK293T the efficacy of targeting retinoblastoma (RB)/E2F1 activity cells were transiently transfected with pTsin-HA-ERG FL, pTsin- with palbociclib in ERG-negative, PTEN/TP53-altered tumors. HA-ERG-T1-E4, pTsin-EV, pLKO-shNT, pLKO-shRB, pLKO- This study redefines the role of ERG in a specific tumor subtype shERG, pLKO-shPTEN, or pLKO-shE2F1 as indicated using Lipo- and may guide evaluation of the status of concomitant ERG fectamine 2000 (Thermo Fisher Scientific) following manufac- fusion, PTEN/TP53 alteration, and RB1 when selecting ther- turer's instructions. Virus-containing supernatant was collected apeutic strategies. 48 hours posttransfection and indicated cells were infected with virus-containing supernatant and 8 mg/mL polybrene. Selection was performed with 1.5 mg/mL puromycin. Sequences of gene- specific shRNAs are listed in Supplementary Table S1. Two shRNAs per gene were tested. transcription factor ERG (2–4). ERG alone has been shown to repress a neural gene expression signature (12) as well as partially rescue the AR pathway under PTEN loss conditions (13), but the Coimmunoprecipitation and Western blotting mechanistic role of ERG in the clinically relevant context of both Coimmunoprecipitation and subsequent Western blotting PTEN/TP53 alteration remains uncharacterized. was performed as described previously (15). The following Toaddressthesegapsinthefield, we generated a mouse antibodies were used: anti-ERG (ab92513, Abcam; CM421C, modelofprostatecancerthatencompassesPten deletion, Trp53 Biocare Medical), anti-PTEN (CST9559L, Cell Signaling Tech- mutation, and ERG overexpression. Notably, we revealed a nology), anti-p53 (sc126, Santa Cruz Biotechnology), anti-AR novel function of ERG to repress expression of a subset of cell (sc816, Santa Cruz Biotechnology), anti-NKX3.1 (NB100-1828, cycle–related genes and block RB hyperphosphorylation in Novus Biologicals), anti-RB (554136, BD Biosciences), anti- Pten/Trp53-altered, Rb1-intact tumors. As a result, ERG-positive, pRB S795 (CST9301S, Cell Signaling Technology), anti-SKP2 Pten/Trp53-altered tumors had minimal expression of E2F1 (32-3300, Life Technologies), anti-CCND1 (sc718, Santa Cruz downstream targets involved in a mesenchymal cell lineage Biotechnology), anti-CDK1 (sc54, Santa Cruz Biotechnology), switch. We extended these findings to both preclinical xenograft anti-TWIST (sc6269, Santa Cruz Biotechnology), anti-CDH1 and allograft models of tumor progression and demonstrated (610181, BD Biosciences), anti-VIM (sc73258, Santa Cruz that ERG overexpression maintained AR positivity and sensi- Biotechnology), anti-ERK2 (sc1647, Santa Cruz Biotechnology), tivity to enzalutamide. In stark contrast, ERG-negative, Pten/ anti-CDK2 (sc6248, Santa Cruz Biotechnology), anti-E2F1 Trp53–altered tumors were resistant to enzalutamide treatment (sc193, Santa Cruz Biotechnology), anti-pAKT S473 (CST4060L, and instead developed a reliance on the RB/E2F1 pathway, Cell Signaling Technology), and anti-AKT (CST9272, Cell Signal- which was effectively targeted with a CDK4/6 inhibitor, palbo- ing Technology). ciclib. This study emphasizes the importance of evaluating the individual genetic profile of tumors when designing therapeutic qRT-PCR strategies, with particular emphasis on ERG fusion, RB1,and qRT-PCR was performed as described previously (15). All fi PTEN/TP53 status. quanti cations were normalized to the level of endogenous GAPDH gene. Primers used are listed in Supplementary Table S2.

Materials and Methods Chromatin immunoprecipitation and qPCR Cell lines, cell culture, and drug treatment Chromatin immunoprecipitation (ChIP) was performed as LNCaP, HEK293T, VCaP, and PC-3 cells were obtained from described previously (16). DNA was pulled down with indicated the ATCC. C4-2 cells were purchased from Uro Corporation. primary antibodies (anti-ERG, ab92513; anti-E2F1, sc193) or LNCaP-RF cells were described previously (14). HEK293T cells nonspeciﬁc IgG. Primers to amplify DNA by real-time qPCR are were maintained in DMEM supplemented with 10% FBS. VCaP listed in Supplementary Table S3. cells were maintained in DMEM supplemented with 13% FBS. C4- 2, LNCaP, LNCaP-RF, and PC-3 cells were maintained in Cell proliferation assays RPMI1640 medium supplemented with 10% FBS. All cell lines LNCaP-RF, VCaP, or PC-3 cells were seeded in 96-well plates were authenticated (karyotyping, mutations in p53 and ERG (3,000 cells/wells) and treated as indicated. Cells were ﬁxed at fusions, and AR, PTEN, p53, and ERG protein expression) and indicated timepoints (day 0–5) and cell growth was measured

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using sulfohodamine B (SRB) assay (n ¼ 5) as described previ- status for Beltran cohort (metastatic tumor specimens ¼ 114) was ously (17). downloaded from Supplementary Table S5 of the original study (23). Only ERG fusions with RNA-seq or NanoString Hematoxylin and eosin staining evidence were included into the analysis. ORs were calculated in Four micron–thick sections were cut from formalin-fixed par- cBioPortal where OR > 1 indicates cooccurrence and OR < 1 affin-embedded (FFPE) tumor samples from indicated samples. indicates mutual exclusivity, followed by two-tailed Fisher exact Xylene washes were used to deparaffinize the tissue, followed by tests to determine significance of the cooccurrence or mutual graded ethanol washes to rehydrate tissue. Tissue was stained with exclusivity, as described previously (24). hematoxylin, washed, and counterstained with 1% eosin. Stained tissue was dehydrated with graded ethanol washes and a final RNA-seq and data analysis xylene wash before mounting and sealing with coverslips. Total RNA was isolated from mouse prostates by homogeni- zation of frozen tissue and purified using the RNeasy Plus Mini Kit IHC and immunofluorescent cytochemistry (Qiagen). Two-hundred micrograms of high-quality total RNA Four micron–thick sections were cut from FFPE tumor samples was used to generate the RNA sequencing library. cDNA synthesis, from indicated mice, xenografts, or human tissue microarrays. end-repair, A-base addition, and ligation of the Illumina indexed Tissue was deparaffinized with xylene and rehydrated through adapters were performed according to the TruSeq RNA Sample graded ethanol washes. Antigen retrieval and immunostaining Prep Kit v2 (Illumina). The concentration and size distribution of was performed as described previously (18, 19). Antibodies for the completed libraries was determined using an Agilent Bioa- IHC and IFC include: anti-AR (ab108341), anti-ERG (ab92513), nalyzer DNA 1000 chip and Qubit fluorometry (Invitrogen). anti-CD31 (ab28364), anti-CKAE1/3 (ab27988), anti-RB pS795 Paired-end libraries were sequenced on an Illumina HiSeq (ab85607), anti-Ki67 (ab15580), anti-pAKT S473 (CST4060L), 4000 following Illumina's standard protocol using the Illumina anti-CK8/18 (ab531826), anti-CK5 (ab52635), anti-Vimentin cBot and HiSeq 3000/4000 PE Cluster Kit. Samples were (CST5741S). Ki67 and pRB S795 staining of mouse and xenograft sequenced in biological triplicates and each sample yielded tissues was quantified by counting the number of positive cells out 60–90 million paired-end reads (2 50 nucleotide read length). of 100 cells in five random fields of view at 400 per mouse. Base calling was performed using Illumina's RTA software (ver- Staining intensity and percentage for ERG and AR staining of sion 2.5.2). Paired-end RNA-seq reads were aligned to the mouse human tissue microarrays were graded using a set of criteria. reference genome (GRCm38/mm10) using RNA-seq spliced read Intensity was graded 0–3: 0 no staining, 1 low staining, 2 medium mapper Tophat2 (v2.0.6; ref. 25). Pre- and postalignment quality staining, and 3 strong staining. A staining index score for each controls, gene-level raw read count, and normalized read count (i. tissue biopsy was obtained by multiplying the staining intensity e., FPKM) were performed using RSeQC package (v2.3.6) with and percentage values, and used for Pearson product–moment NCBI mouse RefSeq gene model (26). Differential gene expres- correlation analysis. sion analyses were conducted using edgeR (version 3.6.8) and the built-in "TMM" (trimmed mean of M values) normalization Gene set enrichment analysis method were used (27). Differentially expressed genes were Gene set enrichment analysis (GSEA) was performed with determined on the basis of the false discovery rate (FDR) thresh- a preranked list of the target genes identified by integrated analy- old 0.01. sis of RNA-seq and ChIP-seq data against curated datasets including HALLMARK_E2F_TARGETS, HALLMARK_EPITHELIAL_ ChIP-seq data analysis MESENCHYMAL_TRANSITION, CHARAFE_BREAST_CANCER_ ERG, H3K4me1, and H3K4me3 ChIP-seq data in mouse pros- LUMINAL_VS_MESENCHYMAL_DOWN, CHARAFE_BREAST_ tate tissue was downloaded from NCBI Gene Expression Omni- CANCER_LUMINAL_VS_MESENCHYMAL_UP from the Broad bus (GEO) with accession number GSE47119 (13). To be com- Institute (20). patible with our RNA-seq analysis results, raw reads were rea- ligned to the mouse reference genome (GRCm38/mm10) using Samples from patients with prostate cancer bowtie2 (version 2.2.9; ref. 28). MACS2 (version 2.0.10) was used The advanced prostate cancer dataset was generated from to identify peaks with input samples used as background and a patients undergoing standard-of-care clinical biopsies at Mayo P value cutoff 1E5 (macs2 callpeak –bdg –SPMR -f BAM; ref. 29). Clinic (Rochester, MN). A tissue microarray was constructed from ChIP-seq tag intensity tracks (bedGraph files) were generated by the FFPE samples of metastatic prostate cancer, identified after a MACS2, and then were converted into bigWig files using UCSC search of pathologic and clinical databases of archival tissues. The "wigToBigWig" tool. The association of peaks to target genes was Mayo Clinic institutional review board approved the experimen- performed by Genomic Regions Enrichment of Annotations Tool tal protocols for retrieving pathology blocks/slides and for acces- (GREAT; ref. 30). ERG ChIP-seq data in VCaP cells (GSE14092; sing electronic medical records. The human tissue microarray ref. 31), E2F1 ChIP-seq data in PC-3 cells (GSE77448; ref. 32), and contained 157 cores (16 0.6 mm and 141 1.0 mm cores) resulting H3K4me3 ChIP-seq data in LNCaP cells (GSE43791; ref. 33) were from 53 samples (20 bone metastases and 33 nonbone metasta- downloaded from GEO. ChIP-seq analysis procedure was the ses) from 51 patients. Cores in which greater than 50% of the same as described above after mapping reads to the human tissue was lost during IHC were excluded from analysis. reference genome (GRCh37/hg19).

Meta-analysis of publicly available datasets Generation of Pten/Trp53/ERG-altered mouse model and ERG fusion and genetic alterations of PTEN and TP53 for TCGA genotyping (n ¼ 333) and SU2C (n ¼ 150) cohorts were downloaded from All animal studies were approved by the Mayo Clinic Institu- cBioPortal (http://www.cbioportal.org/; refs. 21, 22). ERG fusion tional Animal Care and Use Committee (IACUC). All mice were

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housed in standard conditions with a 12-hour light/12-hour dark cohort analyzed in Fig. 5 was downloaded from Supplementary cycle and access to food and water ad libitum. The indicated groups Table S5 of the original study (23). The ERG, H3K4me1, and of mice were generated by crossing the following mice: Probasin H3K4me3 ChIP-seq datasets analyzed in Fig. 3 were accessed from (Pb)-driven Cre4 recombinase transgenic mice, acquired from the the NCBI Gene Expression Omnibus (GEO) with accession num- National Cancer Institute (NCI) Mouse Repository and originally ber GSE47119 (13), the ERG and H3K4me3 ChIP-seq datasets generated in the laboratory of Dr. Pradip Roy-Burnam at Univer- analyzed in Fig. 4 were accessed from the NCBI GEO with the sity of Southern California (Los Angeles, CA; ref. 34); transgenic accession numbers GSE14092 (31) and GSE43791 (33), and ERG mice purchased from the Jackson Laboratory (010929), the E2F1 and H3K4me3 ChIP-seq datasets analyzed in Supple- originally generated in the laboratory of Dr. Valeri Vasioukhin mentary Fig. S5 were accessed from the NCBI GEO with the at Fred Hutchinson Cancer Research Center (Seattle, WA; ref. 35); accession numbers GSE77448 (32) and GSE43791 (https:// Pten loxp/loxp conditional mice, acquired from Jackson Labora- www.ncbi.nlm.nih.gov/geo/; ref. 33). The HALLMARK_E2F, tory (004597) and originally generated in the laboratory of Dr. HALLMARK_EPITHELIAL_TO_MESENCHYMAL, and CHARA- Hong Wu at University of California (Los Angeles, CA; ref. 36); FE_BREAST_CANCER datasets for GSEA in Fig. 3 were accessed Trp53 loxp/loxp conditional mice, acquired from the NCI Mouse from the Broad Institute (http://software.broadinstitute.org/gsea/ Repository and originally generated in the laboratory of Dr. Tyler index.jsp; ref. 20). The RNA-seq data generated from mouse Jacks at Massachusetts Institute of Technology (Cambridge, MA; prostate tissues in Fig. 3 is accessible from the NCBI GEO with ref. 37); and Trp53 loxp-STOP-loxp-R172H conditional mice, the accession number GSE103871. acquired from the NCI Mouse Repository and originally generated in the laboratory of Dr. Tyler Jacks at Massachusetts Institute of Statistical analysis Technology (Cambridge, MA; ref. 37). PCR genotyping primers All data are shown as mean values SE for experiments are listed in Supplementary Table S4. performed with at least three replicates. Differences between two groups were analyzed using paired Student t tests unless otherwise Generation and treatment of prostate cancer cell line xenografts noted. P values < 0.05 were considered significant. and mouse-derived allografts All animal studies were approved by the Mayo Clinic IACUC. All mice were housed in standard conditions with a 12-hour light/ Results 12-hour dark cycle and access to food and water ad libitum. NOD- Generation and characterization of a clinically relevant Pten/ SCID IL2 receptor g-null (NSG) mice were generated in house and Trp53/ERG triple-mutant mouse model at 6 weeks of age, were randomly divided into different experi- By mining the whole-exome sequencing data from TCGA mental treatment groups as indicated (six mice per group). For patients with primary prostate cancer (PRPC; N ¼ 333; ref. 3), prostate cancer cell line xenografts, 5 106 LNCaP-RF cells per we revealed significant cooccurrence (P ¼ 1.11 10 6,OR¼ 3.01; injection were suspended in 0.1 mL of 50% PBS and 50% Corning 95% CI ¼ 1.89–4.84) of PTEN/TP53 deletions or mutations with Matrigel Matrix and implanted by subcutaneous injection into the ERG gene fusions, one of the most frequent genetic alterations in left flank of each NSG mouse (one implantation per mouse) using prostate cancer (ref. 38; Fig. 1A and B). In contrast, while a similar a 16 gauge needle. LNCaP-RF cells were tested and ensured to be trend was observed in the SU2C metastatic castration-resistant mycoplasma-free prior to injection using the Lookout Mycoplas- prostate cancer (mCRPC) patients (N ¼ 150; ref. 4), the correla- ma PCR Detection Kit purchased from Sigma-Aldrich, and were tion (P ¼ 0.043, OR ¼ 2.04; 95% confidence interval ¼ 0.98– stably expressing either pTsin-EV or pTsin-HA-ERG-T1-E4. For 4.33) was much weaker than that in TCGA PRPC patients (Fig. 1A mouse-derived allografts, three ARlow/KRTlow DMT prostate and B). Given that AR is more commonly expressed in PRPC tumors and three ARhigh/KRThigh TMT prostate tumors were compared with mCRPC, especially neuroendocrine CRPC (NEPC; homogenized and 200 mL of tissue per NSG mouse was implanted refs. 23, 39), these data suggest that ERG fusions are prone to by subcutaneous injection into the left flank of each NSG mouse cooperate with PTEN and TP53 gene alterations in the pathogen- (one implantation per mouse) using a 16 gauge needle. Once the esis of AR-positive prostate cancer. It is important to note that in implanted cells grew to reach a size of 100 mm3 measured the mCRPC SU2C cohort, only 3.6% of samples displayed neu- externally with calipers (approximately 4–5 weeks posttransplan- roendocrine (ARlow/KRTlow) features (4), which may partly tation), drug treatment began. Mice were treated with vehicle (100 explain the apparent under-representation of AR loss samples in mL sodium lactate), enzalutamide (30 mg/kg/day), palbociclib the SU2C dataset (Fig. 1A). To genetically test this hypothesis in (100 mg/kg/day), or combination by oral gavage, once daily five vivo, we generated four cohorts of mice recapitulating the genetic days per week for three weeks. Mouse weight and tumor size was alterations most frequently occurring in human prostate cancers measured every three days by measuring tumor length (L) and (such as R175H in TP53; refs. 3, 4, 40; Supplementary Fig. S1): (i) width (W) using a caliper, and tumor volume (TV) was calculated "wild-type" (Cre-negative littermates); (ii) ERG transgenic alone, with the following formula: TV ¼ (L W2)/2. Posttreatment, with Met33 N-terminally truncated ERG driven by the AR-depen- xenografted tissue was harvested and collected for subsequent dent probasin (Pb) promoter (hereafter termed Pb-ERG); (iii) study. prostate-specific Pten deletion and Trp53 deletion and mutation (Ptenpc / ;Trp53pcR172H/ , hereafter termed double mutant or Data availability DMT) where Trp53 R172H is the mouse equivalent to human The datasets generated and/or analyzed during the current TP53 R175H; and (iv) Ptenpc / ;Trp53pcR172H/ ;Pb-ERG (hereafter study are available in the following repositories. The Cancer termed triple mutant or TMT; Supplementary Fig. S2A). We Genome Atlas (TCGA) and Stand Up To Cancer (SU2C) datasets generated these four groups of mice by using Pb-driven Cre analyzed in Fig. 1 and Supplementary Fig. S1 were accessed from recombinase (Pb-Cre4; ref. 34), Pb-ERG (35), Ptenloxp/loxp (36), cBioPortal (http://www.cbioportal.org/; refs. 21, 22). The Beltran and Trp53loxp-stop-loxp-R172H/loxp (37) as breeders. For comparison,

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Figure 1. ERG tempers PTEN/TP53 alteration–induced loss of ARhigh luminal epithelial cells. A, Oncoprint image with percentage of ERG, PTEN, TP53, and AR genetic alterations in 333 primary prostate cancer patient samples (top, TCGA cohort; ref. 3) and 150 advanced mCRPC patient samples (bottom, SU2C cohort; ref. 4). B, Contingency tables used by Fisher exact test (two-tailed) to examine association between ERG fusion and PTEN/TP53 alterations in primary TCGA (left) and mCRPC SU2C (right) cohorts. C, Histologic characterization of mouse prostate tissue from 16–20 weeks of age. Wild-type n ¼ 8, Pb-ERG n ¼ 9, DMT n ¼ 10, TMT n ¼ 12. Top, hematoxylin and eosin (H&E) staining. Subsequent rows, IHC for AR, ERG, CD31, Pan-KRT, KRT8/18, KRT5, and Vimentin. CD31 as an endothelial cell marker to distinguish between endogenous endothelial versus transgenic ERG. Asterisk in Vimentin IHC tissue indicates a stromal compartment that is distinct from the Vimentinlow adenocarcinoma.

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we also generated prostate-specific Pten deletion (Ptenpc / ), Pten mice (Fig. 2A; Supplementary Fig. S3A and S3B), a phenomenon and Trp53 double deletion (Ptenpc / ;Trp53pc / ) (hereafter reminiscent of prostate-specific Pten/Rb1 double KO tumors (8). termed double knockout or DKO) mice, as well as prostate- At present, the exact cause-and-effect of altered cellular localiza- specific Pten and Trp53 double deletion plus ERG transgenic tion of phosphorylated AKT remains to be elucidated. Previous (Ptenpc / ;Trp53pc / ;Pb-ERG) mice (Supplementary Fig. S2A– study has shown that in the presence of PTEN loss, ERG partially S2C). Pten/Trp53 DKO mice have been shown to develop plastic, rescues AR function (13). However, further analyses showed that dedifferentiated tumors (8–10, 41) and served as controls for ARhigh/KRThigh tumors in TMT mice had lower levels of phos- comparison purposes with the Trp53-mutant lines, which repre- phorylated RB (pRB S795) and lower expression of a subset of cell sent an unstudied portion of patients with PTEN deletion/TP53 cycle–promoting genes compared with ARlow/KRTlow tumors in mutation. DMT and TMT mice (Fig. 2C and D, see Fig. 3 below). Further- At the age of 8–10 weeks, 100% of both Ptenpc / ;Trp53pc / more, expression of cell lineage regulators commonly associated (DKO) and Ptenpc / ;Trp53pcR172H/ (DMT) mice developed with epithelial-to-mesenchymal transition (EMT) and neuroen- well-differentiated adenocarcinomas with high expression of AR docrine cell lineage was also much lower in ARhigh/KRThigh TMT proteins (ARhigh; Supplementary Fig. S2B). In contrast, AR expres- tumors than that in ARlow/KRTlow tumors (Fig. 1C; see Fig. 3 sion was dramatically reduced (ARlow) in prostate tumors in below). These data argue that ERG-induced preservation of the approximately 90% of DKO and DMT mice at the age of 16– late-stage ARhigh/KRThigh phenotype was not solely mediated by 20 weeks (Fig. 1C; Supplementary Fig. S2B). Consistent with the restored AR activity in the PTEN loss context, but may require reduced AR expression, the level of pan-keratin (pan-KRT) in additional drivers. tumors, used as an indicator of epithelial cells as opposed to Loss of RB function has been implicated in development of mesenchymal cells, was also markedly reduced (KRTlow) in both plastic, antiandrogen-resistant prostate tumors in Pten and Trp53- DKO and DMT mice at the age of 16–20 weeks compared with deficient mice (8, 10). In agreement with functional loss of RB as mice 8–10 weeks younger (Supplementary Fig. S2B). In addition, reflected by increased RB phosphorylation (Fig. 2C and D), cell DMT tumor cells from mice at the age of 16–20 weeks were also proliferation as indicated by Ki67 staining was much higher in negative for both luminal epithelial cell marker KRT8/18 and ARlow/KRTlow dedifferentiated tumors in both DMT and TMT basal epithelial cell marker KRT5, but positive for vimentin (VIM), mice compared with that in ARhigh/KRThigh tumors in TMT mice a mesenchymal cell marker (Fig. 1C). These results suggest that (Fig. 2E and F). It is worth noting that proliferation in ARhigh/ tumors in DKO and DMT mice at the age of 16–20 weeks KRThigh tumors in TMT mice was still much higher than that in transitioned to minimal luminal epithelial phenotypes and were malignant and nonmalignant prostate tissues in Pten KO alone less differentiated compared with tumors in mice at younger ages, and ERG transgenic alone mice, respectively (Fig. 2E and F), as indicated by the comparatively weak but detectable pan-keratin reinforcing the concept that ERG is an oncogenic protein that and AR levels. These data provide support to the previous obser- promotes prostate tumorigenesis by cooperating with other vation that loss of Pten and Trp53 induces lineage plasticity in lesions. Nevertheless, these data suggest that ERG may regulate mouse prostate cancer (8–10, 41). the cell cycle and subsequently, RB activity. In striking contrast, at the same age (16–20 weeks), approximately 50% of Ptenpc / ;Trp53pcR172H/ ;Pb-ERG (TMT) mice ERG downregulates a subset of cell cycle–promoting genes in developed well-differentiated ARhigh/KRThigh adenocarcinomas Pten/Trp53–altered mouse prostate tumors while the other 50% developed ARlow/KRTlow tumors reminiscent To define the molecular mechanisms by which ERG mod- of those in DMT mice (Fig. 1C). Notably, ARlow/KRTlow tumors in ulates prostate cancer cell lineage plasticity, we performed RNA TMT mice lacked transgenic ERG expression in the majority of sequencing (RNA-seq) analysis in three ARlow/KRTlow tumors tumor cells, but as expected, endogenous ERG was highly from DMT mice and three ARhigh/KRThigh tumors in TMT mice. expressed in CD31-positive endothelial cells of blood vessels We selected DMT ARlow/KRTlow tissues rather than TMT ARlow/ (Fig. 1C). Importantly, lack of epithelial expression of transgenic KRTlow tissues for this analysis to ensure no possible contam- ERG correlated with decreased expression of AR proteins in these ination from any tumor cells that may have low levels of ERG Pten/Trp53–altered tumors (Fig. 1C; Supplementary Fig. S2C). expression. Although we did not observe strongly positive ERG- This observation is supported by the previous report that ERG expressing tumor cells by IHC in the TMT ARlow/KRTlow tissues knockdown decreases the AR-positive luminal cell population in (Fig.1C),thepresenceofthePb-ERG transgene in these mice TMPRSS2-ERG–expressing VCaP prostate cancer cells (12). would not allow us to eliminate that possibility. RNA-seq data Together, these findings reveal that PTEN/TP53 alteration induces for one DMT tumor was excluded from further analysis due to loss of the ARhigh luminal epithelial cell lineage in prostate cancer its poor correlation with the other two biological replicates and this phenomenon is disrupted in the presence of ERG (Supplementary Fig. S4A). Differential gene expression analy- expression. ses revealed 1,281 and 1,598 genes that were significantly Compared with Pten wild-type prostate tissues ("wild-type" down- and upregulated by ERG, respectively, in ARhigh/KRThigh and Pb-ERG genotypes), Pten-null PIN lesions in Ptenpc / mice or TMT prostate tumors in comparison with ARlow/KRTlow DMT tumors in DMT and TMT mice had increased, but comparable tumors (Fig. 3A; Supplementary Fig. S4B). After integrating levels of phosphorylated AKT (pAKT S473; Fig. 2A and B; Sup- RNA-seq data with ERG ChIP-coupled sequencing (ChIP-seq) plementary Fig. S3A), reinforcing the concept that PTEN loss is a data obtained from prostate tumors in Rosa26 TMPRSS2-ERG key driver of initial tumorigenesis in these models (42–44). mice (13), we found 76% (972 of 1,281) of ERG-downregu- Intriguingly, plasma membrane expression of pAKT S473 was lated genes and 82% (1,314 out of 1,598) of ERG-upregulated detected in the luminal epithelial cells of ARhigh/KRThigh tumors in genes contained ERG ChIP-seq peaks in their promoter and/or TMT mice, whereas no typical plasma membrane staining of pAKT enhancer regions (Fig. 3A), suggesting that they are putative S473 was detected in ARlow/KRTlow tumor cells in DMT and TMT ERG target genes.

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Figure 2. ERG prevents Pten/Trp53 alteration–induced proliferation and loss of membrane-localized phosphorylated AKT in mouse prostate tumors. A, IHC for pAKT S473 in mouse prostate tissues from 16–20 weeks of age. Wild-type n ¼ 8, Pb-ERG n ¼ 9, DMT n ¼ 10, TMT n ¼ 12, Ptenpc/ n ¼ 8. B, Protein levels of pAKT S473, total AKT, and AR in mouse prostate tissues at 16–20 weeks of age. Both blots for each protein of interest were exposed and developed on the same piece of film. ERK2 as a loading control. Band intensity was quantified and normalized to ERK2 for each lane. Asterisk ¼ outlier samples with significantly low levels of total protein. C, IHC for pRB S795 in mouse prostate tissues from 16–20 weeks of age as described in A. D, Quantification of pRB S795 staining as shown in C. E, IHC for Ki67 in mouse prostate tissues from 16–20 weeks of age as described in A. F, Quantification of Ki67 IHC as shown in E.

Gene ontology (GO) analysis of the 972 ERG-downregulated breast cancer cell type as compared with the mesenchymal-like genes demonstrated a significant enrichment of genes that breast cancer cell type, while ERG-upregulated genes were regulate the cell cycle (Fig. 3B), in agreement with our finding significantly overlapped with genes upregulated in the luminal that ARhigh/KRThigh tumors in TMT mice display decreased RB breast cancer cell type (ref. 45; Fig. 3C). Further analysis of phosphorylation and cell proliferation in comparison with RNA-seq profiles between ARlow/KRTlow tumors in DMT mice ARlow/KRTlow tumorsinDMTandTMTmice(Fig.2C–F). GSEA and ARhigh/KRThigh tumors in TMT mice revealed that ERG revealed that ERG-downregulated genes significantly overlap expression resulted in drastic upregulation of AR pathway genes with hallmark E2F targets and EMT genes (Fig. 3C). Additional (e.g., Ar and Nkx3.1) and luminal epithelial lineage genes (e.g., comparison demonstrated that ERG-downregulated genes also Cdh1 and Krt8), and robust downregulation of cell-cycle genes correlated with luminal epithelial-to-mesenchymal changes in (e.g., Ccnd1 and Cdk1) and nonluminal epithelial (mesenchy- other cancer types. For examples, these genes were also signif- mal and neuroendocrine) lineage-regulatory genes (ref. 11; e.g., icantly overlapped with genes downregulated in the luminal Twist1 and Sox11;Fig.3D).

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ERG-downregulated genes are exemplified in Fig. 3E and were and VIM (Fig. 4A), similar to ARhigh/KRThigh tumors in TMT mice further confirmed by reverse transcription–coupled quantitative (Fig. 1C). Consistent with ARlow/KRTlow DMT or DKO tumors, PCR (qRT-PCR; Fig. 3F). qRT-PCR analysis of key cell-cycle and PTEN- and ERG-negative CRPC cell lines PC-3 and LNCaP-RF, EMT genes and Western blot analysis of AR proteins further which lack or express very low levels of functional p53, respec- confirmed that ARlow/KRTlow tumors in DMT and TMT mice tively, displayed little to no expression of AR, but increased shared similar molecular traits (Fig. 2B; Supplementary Fig. hyperphosphorylated RB and augmented expression of cell- S4C). It is important to note that although TMT ARlow/KRTlow cycle–driven proteins and mesenchymal-specific proteins (Fig. tissues were not analyzed by RNA-seq, the trends in gene expres- 4A). Overexpression of full-length or fusion (T1-E4) ERG in sion observed in DMT ARlow/KRTlow tissues were seemingly LNCaP-RF and PC-3 cells partially reversed these trends in a conserved in the TMT ARlow/KRTlow tissues (Fig. 2B; Supplemen- dose-dependent manner (Fig. 4B; Supplementary Fig. S5B) and tary Fig. S4C). ERG ChIP-seq data clearly showed ERG-binding decreased cell proliferation (Fig. 4C), as detected in ERG-positive, peaks in the promoter region of cell-cycle genes such as Ccnd1, and Pten/Trp53-mutated mouse prostate tumors (Fig. 2E and F). Cdk1, but not cell lineage–regulatory genes such as Twist1 and Conversely, concomitant knockdown of endogenous Sox11 (Fig. 3E), suggesting that cell-cycle–related genes are likely TMPRSS2-ERG and PTEN in TP53-mutated VCaP cells, mimicking direct targets of ERG while Twist1 and Sox11 are not. These data the situation in ARlow/KRTlow tumors in DMT or TMT mice, suggest that in the context of Pten/Trp53 alteration, ERG tran- resulted in increased expression of cell-cycle–related proteins, scriptionally downregulates a subset of key cell-cycle–promoting hyperphosphorylation of RB, upregulation of nonepithelial cell genes and maintains AR signaling. markers TWIST1 and VIM, and decreased expression of AR and the VCaP cells harbor intact PTEN, one allele loss of TP53 and a epithelial cell markers CDH1 and NKX3.1 (Supplementary Fig. gain-of-function mutation R248W, which is a hotspot mutation S5A and S5C). in CRPC (40) (Supplementary Fig. S1). ERG is also overexpressed Previous study has indicated that hypophosphorylated RB can in this cell line due to TMPRSS2-ERG fusion (TMPRSS2 exon 1 be recruited by AR to repress cell-cycle genes (46). Coimmuno- fused with ERG exon 4 or termed T1-E4 ERG). Importantly, precipitation assay in VCaP cells demonstrated that similar to knockdown of ERG in the presence of PTEN depletion increased previous study (31), ERG interacted with AR (Fig. 4D). However, CCND1, CDK1, and SKP2 protein levels in VCaP cells (Supple- no interaction was detected between ERG and RB, and similar mentary Fig. S5A). Thus, these data provide futher support to the results were obtained in PC-3 cells stably expressing T1-E4 ERG hypothesis and the validation of ERG regulation of a few repre- (Fig. 4D), excluding the possibility that ERG may recruit RB to sentative gene targets defined by the integrated RNA-seq and repress cell-cycle genes in a manner similar to AR (46). However, ChIP-seq analyses. Our data that ERG bound to the promoter of because key cell-cycle regulators such as CCND1 and CDK1 were Ccnd1 and Cdk1 genes and repressed their expression suggests identified as transcriptionally repressed target genes of ERG, it is ERG is a potent upstream regulator of RB hypophosphorylation possible that ERG causes a reduction in RB phosphorylation and and activation. This notion is further supported by a recent report cell-cycle progression by directly downregulating cell-cycle genes. that in spite of the androgen-stimulating effect of RB hyperpho- This hypothesis is consistent with decreased expression of a subset sphorylation in TMPRSS2-ERG–negative LNCaP cells, RB remains of cell-cycle genes in ARhigh/KRThigh adenocarcinomas in TMT mice hypophosphorylated in TMPRSS2-ERG–positive VCaP cells even (Fig. 3) and in human LNCaP-RF and PC-3 cells stably expressing after androgen stimulation (46). ERG (Fig. 4B). Moreover, ERG-mediated upregulation of AR and downregulation of EMT genes were reversed by depletion of RB in Human prostate cancer cell lines recapitulate ERG-mediated ERG-expressing LNCaP-RF cells (Fig. 4E). Similar effects were repression of the cell cycle through the RB pathway observed in PC-3 cells (Fig. 4E). These results along with the ERG To delineate the relationship between ERG expression and ChIP-seq data (Fig. 3) suggest that ERG functions as an upstream PTEN/TP53 alteration in tumor cell proliferation, cellular iden- activator of RB by specifically binding to the promoter and repres- tity, and antiandrogen resistance, we surveyed human prostate sing expression of a subset of cell-cycle–driving genes. cancer cell lines including VCaP, C4-2, LNCaP, LNCaP-RF, and PC-3 (Fig. 4A). Among the cell lines surveyed, VCaP cells had the E2F1 activates expression of EMT-promoting factors in ERG- highest level of AR protein, hypophosphorylated RB, minimal negative, PTEN/TP53–altered tumor cells expression of cell-cycle–promoting proteins CCND1, CDK1, and It has been reported recently that E2F1 promotes prostate SKP2, and low levels of mesenchymal-related proteins TWIST1 cancer cell metastasis and enhanced mesenchymal-like

Figure 3. ERG expression downregulates key cell-cycle–driving genes and maintains both AR pathway and epithelial gene expression in mouse prostate tumors. A, Venn diagram indicating overlap between up- or downregulated genes in DMT ARlow/KRTlow (n ¼ 2) versus TMT ARhigh/KRThigh (n ¼ 3) tumors and ERG target genes identiﬁed by ChIP-sequencing (13). Fisher exact test (assuming human genome code for 27,000 genes, estimated from RefSeq) for ERG ChIP-seq versus downregulated genes: P < 0.001; for ERG ChIP-seq versus upregulated genes: P < 9.088e23. B, Gene Ontology analysis of 972 ERG target genes downregulated in TMT ARhigh/KRThigh tumors, ranked by P value. C, GSEA for all 2,595 up- or downregulated ERG target genes (mouse gene names converted to human homologs using NCBI Homology Map). D, Heatmap showing differentially expressed genes between two DMT ARlow/KRTlow and three TMT ARhigh/KRThigh tumors, highlighting a subset of genes involved in cell cycle, AR pathway, and epithelial-to-mesenchymal transition (EMT). E, RNA-seq and ChIP-seq track views from UCSC Genome Browser for two ERG predicted target genes (Ccnd1, Cdk1) with ERG-binding peaks and two predicted passenger genes (Sox11, Twist1) without ERG-binding peaks. Peaks underlined with black bars and boxed with a dashed red line indicate signiﬁcant ERG ChIP-seq peaks with P ¼ 1e5 or lower as determined by MACS. ERG ChIP-seq input tracks shown as a control for true ERG peaks. H3K4me3 peaks shown to indicate gene promoter regions. H3K4me1 peaks shown to indicate gene enhancer regions. F, qRT-PCR of n ¼ 2 DMT ARlow/KRTlow and n ¼ 3 TMT ARhigh/KRThigh tumors for Ccnd1, Cdk1, Twist1, and Sox11. Relative to Gapdh.

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phenotypes (increased migration and invasion) by binding to the AR, our analyses were only focused on those samples with promoter and upregulating expression of the RHAMM gene expression of ERG gene fusion as demonstrated by RNA-seq or (HMMR; ref. 47). By analyzing E2F1 ChIP-seq data obtained in NanoString data (23, 39). In addition, we performed IHC analysis PC-3 cells (32), we found robust binding of E2F1 proteins in the on a human tissue microarray with 157 cores constructed from 51 loci of ERG-suppressed mesenchymal lineage-driving genes patients with metastatic CRPC undergoing standard-of-care clin- including SNAI1, TGFB2, TWIST1, TWIST2, and HMMR (Sup- ical biopsies at Mayo Clinic (Rochester, MN). This analysis plementary Fig. S5D). This observation was further confirmed by confirmed a strong association between AR and ERG expression ChIP-qPCR (Supplementary Fig. S5E). Most importantly, the (P ¼ 2.98e7, correlation ¼ 0.41; 95% CI ¼ 0.263–0.536; Fig. 5B effect of ERG and PTEN double knockdown on expression of and C; Supplementary Table S5). EMT-promoting genes and epithelial and mesenchymal cell markers in VCaP cells was abrogated by concomitant knockdown of ERG expression in PTEN/TP53 tumors regulates prostate tumor E2F1 by two independent shRNAs (Supplementary Fig. S5C). response to antiandrogen and anti-RB/E2F1 pathway drugs þ These data suggest that E2F1 mediates repression of downstream The above findings prompted us to hypothesize that ERG / þ mesenchymal lineage genes in the context of ERG /PTEN / ARhigh/KRThigh (TMT) adenocarcinoma cells would respond to p53null/mutant cells. In further support of the transcriptome results enzalutamide treatment, but ERG /ARlow/KRTlow (DMT) tumor in DMT and TMT mouse tumors, ectopic expression of T1-E4 ERG cells would not. Instead, ERG /ARlow/KRTlow (DMT) tumor cells in PC-3 cells reduced expression of CCND1, CDK1, TWIST1, and may rely heavily on RB hyperphosphorylation to maintain cell SOX11, as well as other key cell cycle, EMT, and neuroendocrine- proliferation, dedifferentiation, and antiandrogen-resistant phe- related genes (Fig. 4F; Supplementary Fig. S6A–S6C). ERG ChIP- notypes, and therefore this type of tumor may be highly respon- seq in VCaP cells and ChIP-qPCR in PC-3 cells stably expressing sive to RB-targeted therapy such as CDK4/6 inhibitors. Palbociclib T1-E4 ERG confirmed cell-cycle genes such as CCND1 and CDK1 (PD-0332991) is a CDK4/6 inhibitor that has been shown to be as direct ERG targets in human prostate cancer cells, but E2F1 effective in preclinical models of prostate and other cancer types, ChIP-seq and ChIP-qPCR in PC-3 cells demonstrated TWIST1 and and was recently approved by the FDA for treatment of breast other cell lineage–regulatory factors as downstream gene targets of cancer (46–50). Treatment of control LNCaP-RF cells (ERG / E2F1 (Fig. 4G and H; Supplementary Fig. S5D and S5E; Supple- ARlow) with enzalutamide alone had no overt effect on expression mentary Fig. S6D and S6E). It should be noted that there was an of cell-cycle genes, RB phosphorylation, and cell proliferation observed ERG ChIP-seq peak at the TGFB2 locus, although this (Fig. 6A and B; Supplementary Fig. S7A and S7B), confirming the binding could not be validated by ChIP-qPCR (Fig. 4G and H). antiandrogen-resistant nature of ERG /ARlow cells. However, Thus, our data cannot completely rule out the possibility that ERG palbociclib treatment, either alone or in combination with may also potentially regulate expression of this locus. Together, enzalutamide, significantly decreased expression of cell-cycle these data suggest that ERG directly binds to and regulates genes and inhibited proliferation in ERG /ARlow cells (Fig. 6A expression of a subset of cell-cycle genes in human PTEN/ and B; Supplementary Fig. S7A and S7B). It is interesting to TP53–mutated prostate cancer cells, which in turn leads to RB note that combination of enzalutamide and palbociclib signif- hypophosphorylation and inhibition of E2F1-mediated tran- icantly inhibited proliferation of the ERG /ARlow LNCaP-RF scription of mesenchymal-promoting genes. cells compared with palbociclib treatment alone (Fig. 6B), suggesting that palbociclib treatment may resensitize these cells ERG and AR expression is positively associated in human to enzalutamide treatment. As expected, LNCaP-RF cells stably þ prostate tumors expressing ERG (ERG /ARhigh) responded favorably to enzalu- In agreement with our observations in mouse models and tamide alone, and such effect was not enhanced in combina- human prostate cancer cell lines, genome analysis of CRPC tion with palbociclib (Fig. 6A and B; Supplementary Fig. S7A adenocarcinomas (CRPC-Ad) and neuroendocrine tumors and S7B). In further support of the finding that RB1 knockdown (CRPC-NE; ref. 23) revealed a significant association of ERG in ERG-positive cells abrogates the subsequent downregulation fusion gene expression with CRPC-Ad (ARhigh), but not CRPC- of cell lineage genes (Fig. 4E), RB1 knockdown in LNCaP-RF- NE tumors (ARlow; P ¼ 0.0485; OR ¼ 0.14; 95% CI ¼ 0.003– ERG T1-E4 cells also abolished enzalutamide sensitivity (Sup- 1.06; Fig. 5A). Because CRPC-NE tumors have low or absent AR plementary Fig. S7C and S7D). Sensitivity to enzalutamide in þ expression and TMPRSS2-ERG gene fusion expression is driven by ERG /ARhigh cells (LNCaP-RF-ERG T1-E4 and VCaP), but not

Figure 4. ERG binds to the promoter and regulates expression of cell-cycle genes in human PTEN/TP53–altered prostate cancer cells. A, Western blot analysis of expression of key AR pathway, cell-cycle, and EMT-related proteins in ﬁve prostate cancer cell lines. B, Western blot analysis of expression of key AR pathway, cell-cycle, and EMT-related proteins in LNCaP-RF and PC-3 cell lines after lentiviral-mediated expression of full-length (ERG-FL) or ERG (T1-E4). C, Cell proliferation as measured by SRB assay for LNCaP-RF and PC-3 cell lines with lentiviral-mediated expression of ERG-FL or ERG (T1-E4). D, Top, coimmunoprecipitation of endogenous ERG and RB in VCaP cells. ERG and AR coimmunoprecipitation shown as positive control. Bottom, coimmunoprecipitation of ERG and RB in PC-3 cells stably expressing ERG (T1-E4). E, Western blot analysis of expression of key AR pathway and EMT-related genes in LNCaP-RF and PC-3 cell lines after lentiviral-mediated expression of ERG (T1-E4) with or without RB1 knockdown. F, RT-qPCR of CCND1, CDK1, SKP2, FOXM1, TWIST1, TWIST2, SOX11, and TGFB2 in PC-3 cells with or without lentiviral-mediated expression of ERG (T1-E4). Relative to GAPDH. G, ERG ChIP-seq tracks in VCaP cells (GSE14092; ref. 31) and H3K4me3 (histone mark of promoters) ChIP-seq tracks in LNCaP cells (GSE43791) (33) from UCSC genome browser for CCND1, CDK1, SKP2, FOXM1, TWIST1, TWIST2, SOX11,andTGFB2. ERG ChIP-seq input tracks shown as a control for true ERG peaks. Peaks underlined with black bars and boxed with a dashed red line indicate signiﬁcant ERG ChIP-seq peaks with P ¼ 1e5 or lower as determined by MACS. Asterisk indicates ERG peak that could not be validated by ChIP-qPCR. H, ERG ChIP-qPCR of CCND1, CDK1, SKP2, FOXM1, TWIST1, TWIST2, SOX11, and TGFB2 in PC-3 cells with lentiviral-mediated expression of ERG (T1-E4).

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Figure 5. ERG expression correlates with AR expression in human patient datasets. A, Fisher exact test to determine association between ERG fusion and CRPC- adenocarcinoma (CRPC-Ad) or CRPC-neuroendocrine (CRPC-NE) tumor subtypes from the Beltran cohort (23). B, Representative IHC images for AR and ERG from human tissue microarray of metastatic CRPC obtained from Mayo Clinic. C, Pearson product–moment correlation between AR and ERG IHC staining in clinical biopsies of metastatic CRPC in B.

þ ERG /ARlow (LNCaP-RF-EV) cells, was abrogated by androgen ERG /ARlow/KRTlow DMT and ERG /ARhigh/KRThigh TMT tumors deprivation of culture media (Supplementary Fig. S7E and and confirmed the findings from LNCaP-RF xenografts (Fig. 6D S7F), confirming AR pathway dependence in ERG-positive cells. and E; Supplementary Fig. S8D and S8E). Collectively, these data We further examined the responsiveness of ERG-positive highlight that PTEN/TP53–altered tumors with hyperphosphory- prostate cancer to antiandrogen therapy using in vivo models. lated RB are resistant to enzalutamide, but are sensitive to CDK4/6 Similar to the findings in vitro,ERG /ARlow LNCaP-RF xenograft inhibition alone or in combination with enzalutamide. In con- tumors were resistant to enzalutamide treatment (Fig. 6C; trast, ERG expression maintains antiandrogen sensitivity in Supplementary Fig. S8A–S8C). In contrast, treatment of these tumors even with PTEN/TP53 alteration and this effect is related tumors with palbociclib significantly decreased tumor volume, to ERG-induced inhibition of cell-cycle gene expression and Ki67 staining, and RB phosphorylation (Fig. 6C; Supplemen- restored AR signaling. tary Fig. S8A–S8C). Similar to the LNCaP-RF cell line study, combination of enzalutamide and palbociclib significantly decreased ERG /ARlow LNCaP-RF xenograft tumor volume Discussion compared with palbociclib treatment alone (Fig. 6C; Supple- The findings in this study emphasize that the unique combi- mentary Fig. S8A), which further highlights the potential effi- nation of genetic mutations present within a single prostate tumor þ cacy of combination treatment in these tumors. In ERG /ARhigh can greatly affect response to androgen- and AR-targeted thera- LNCaP-RF xenograft tumors, palbociclib treatment alone pies. In particular, our study of the novel Pten/Trp53/ERG triple- exerted little to no effect but both enzalutamide treatment mutant mouse model of prostate cancer recapitulates a trio of alone and in combination with palbociclib significantly genetic events that cooccur in a significant subtype of prostate reduced the tumor volume, Ki67 staining, and expression of tumors. Previous studies demonstrated that loss of Pten and Trp53 pRB S795 (Fig. 6C; Supplementary Fig. S8A–S8C). induces lineage plasticity in mouse prostate cancer, where pros- We attempted to perform similar studies using DMT and TMT tate-specific Pten and Trp53 double KO mice develop prostate spontaneous tumor models. However, we found it was quite adenocarcinoma at a young age and further evolve into ARlow/ challeging to crossbreed five different alleles together to simul- KRTlow tumors (8–10, 41). These data and ours support the taneously generate large cohorts of DMT and TMT mice at the hypothesis that Pten/Trp53-altered tumors may transition from same ages. Because of this technical difficulty, we performed an ARhigh/KRThigh adenocarcinoma to an altered ARlow/KRTlow similar drug treatment studies using allografts derived from state (Fig. 6F, left).

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Figure 6. Differential responses of ERG-positive and ERG-negative human xenograft and mouse allograft tumors with PTEN/TP53 alterations to enzalutamide and palbociclib. A, Western blot analysis of expression of key AR pathway, cell-cycle, and EMT-related proteins in LNCaP-RF cells with or without lentiviral-mediated ERG (T1-E4) expression after treatment with vehicle, enzalutamide (ENZ, 10 mmol/L), palbociclib (PD, 1 mmol/L), or combination (ENZ þ PD). B, Cell proliferation as measured by SRB assay for LNCaP-RF cells with or without lentiviral-mediated ERG (T1-E4) expression after treatment with vehicle, ENZ (10 mmol/L), PD (1 mmol/L), or combination. C, LNCaP-RF xenograft tumor volume with or without lentiviral-mediated ERG (T1-E4) expression during 3-week treatment with vehicle, ENZ (30 mg/kg/day), PD (100 mg/kg/day), or combination. Six xenografts (n ¼ 6) per cell line, per drug treatment. D, ERG/ARlow/KRTlow DMT and ERGþ/ARhigh/ KRThigh TMT allograft tumor volume during 3 weeks of treatment with vehicle, ENZ (30 mg/kg/day), PD (100 mg/kg/day), or combination. Five allografts (n ¼ 5) per genotype, per drug treatment. E, Characterization of allograft tumors from (D) after 3 weeks of treatment. Top, H&E. Subsequent rows, IHC for ERG, AR, pRB S795, and Ki67. F, A hypothetical model. In prostate cancer cells without the TMPRSS2-ERG fusion, PTEN deletion/mutation and TP53 deletion/mutation favor cell-cycle gene expression, CDK activation, and RB inhibition (hyperphosphorylation), which in turn lead to E2F1 activation and luminal-epithelial-to-mesenchymal cell identity transition, antiandrogen resistance, and increased CDK4/6 inhibitor sensitivity. In contrast, in prostate cancer cells harboring the TMPRSS2-ERG fusion, overexpression of ERG results in decreased expression of a subset of cell-cycle–promoting genes and RB activation (hypophosphorylation), thereby leading to E2F1 inhibition and maintenance of luminal epithelial cell identity, increased antiandrogen sensitivity, but CDK4/6 inhibitor resistance.

Further analysis of the novel Pten/Trp53/ERG model revealed mediated cellular reprogramming (Fig. 6F, right). Studies in that ERG binds to chromatin loci of a subset of cell-cycle–driving human prostate cancer cell lines also supported these ﬁndings. genes and decreases their expression in Pten/Trp53–altered mouse Most importantly, similar results were obtained through analysis prostate tumors, thereby preventing loss of RB activity and E2F1- of patient datasets and clinical samples. Although previous

www.aacrjournals.org Clin Cancer Res; 2018 OF13

Blee et al.

studies have suggested a potential role for ERG in repressing ERG (13) would be particularly useful to better characterize this neuroendocrine differentiation and partially rescuing AR function mechanism, although slightly less physiologically relevant. (12, 13), this study represents the first to demonstrate ERG- These findings in prostate cancer also raise the larger question mediated protection of the epithelial adenocarcinoma cell lineage of whether the mechanism defined in the current study might be in a clinically relevant mouse model with Pten/Trp53 mutations applicable to other RB alteration–related cancer types such as lung (Fig. 6F). cancer (genomic loss of RB1 promotes the transition from ade- We further demonstrated in Pten/Trp53–mutated mouse pros- nocarcinoma to small-cell lung cancer; ref. 54) and breast cancer tate cancer and xenograft models that while ERG-positive tumors (functional loss of RB due to HER2 amplification leads to for- are sensitive to antiandrogen treatment, ERG-negative tumors mation of nonluminal breast cancer). Nevertheless, our findings have no overt response to antiandrogens and instead respond support the evaluation of ERG fusion as a viable biomarker to well to the CDK4/6 inhibitor palbociclib. These findings were guide antiandrogen and RB pathway–targeted therapies for PTEN/ recapitulated in human cell lines. Together, these data reveal a TP53–mutated, RB1-intact prostate cancer. Studies such as these previously undefined role of ERG in maintaining neoplastic will be essential to combat lineage plasticity-mediated therapy epithelial cell identity and antiandrogen sensitivity in PTEN/ resistance in prostate cancer as well as other cancers. TP53–mutated prostate cancer and highlight that different therapeutic strategies are needed for PTEN/TP53–altered tumors with Disclosure of Potential Conflicts of Interest or without ERG (Fig. 6F). No potential conflicts of interest were disclosed. Despite a previous finding that ERG overexpression alone is sufficient for focal prostatic intraepithelial neoplasia (PIN) for- Authors' Contributions mation in mice with 129/Sv background (35), we did not observe Conception and design: W. Xu, H. Huang any PIN lesions in Pb-ERG mice during the course of our previous Development of methodology: A.M. Blee, Y. He, Y. Chen Acquisition of data (provided animals, acquired and managed patients, study (51) and this report (Fig. 1C) perhaps due to the different provided facilities, etc.): A.M. Blee, Y. He, Y. Yang, J. Dugdale, M. Kohli, genetic (C57BL6/129) background of mice we used. ERG expres- R. Jimenez sion levels in Pb-ERG mouse prostate tissue were comparable with Analysis and interpretation of data (e.g., statistical analysis, biostatistics, that in the TMPRSS2-ERG fusion-positive human VCaP cell line computational analysis): A.M. Blee, Y. He, Z. Ye, Y. Yan, T. Ma, Y. Chen, L. Wang (see Supplementary Fig. S2D). Nevertheless, our findings are Writing, review, and/or revision of the manuscript: A.M. Blee, Y. He, M. Kohli, consistent with other reports that ERG alone is not sufficient to R. Jimenez, Y. Chen, L. Wang, H. Huang Administrative, technical, or material support (i.e., reporting or organizing promote prostate tumorigenesis in mice within the studied time – Pb ERG data, constructing databases): A.M. Blee, Y. Pan, E. Kuehn frame (51 53). The - transgenic mouse model also pro- Study supervision: A.M. Blee, W. Xu, H. Huang vides the unique ability to study AR-dependent transgenic expression of ERG, which mimics AR-driven TMPRSS2-ERG expression Acknowledgments in human prostate tumors (35). For these reasons, this model This work was supported in part by grants from NIH (CA134514, CA130908, system is particularly relevant and analogous to human prostate CA203849, and CA193239; to H. Huang) and DOD (W81XWH-14-1-0486; to cancer. However, it is important to note that the reduced AR H. Huang). expression observed in approximately 50% of the tumors in TMT mice could contribute to absence of AR-dependent, Pb-promoter- The costs of publication of this article were defrayed in part by the ERG payment of page charges. This article must therefore be hereby marked driven expression of . The exact underlying molecular mech- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate anism warrants further investigation, and future studies will this fact. explore the exact cause-and-effect of reduced AR expression and low low absence of ERG expression in the AR /KRT subset of TMT Received February 26, 2018; revised May 4, 2018; accepted May 23, 2018; mice. Additional studies with a more robust knock-in model of published first May 29, 2018.

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ERG Controls Luminal Lineage and Antiandrogen Sensitivity

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www.aacrjournals.org Clin Cancer Res; 2018 OF15

TMPRSS2-ERG Controls Luminal Epithelial Lineage and Antiandrogen Sensitivity in PTEN and TP53-Mutated Prostate Cancer

Alexandra M. Blee, Yundong He, Yinhui Yang, et al.

Clin Cancer Res Published OnlineFirst May 29, 2018.

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