Functional Silencing of HSD17B2 in Prostate Cancer Promotes Disease Progression

Published OnlineFirst September 18, 2018; DOI: 10.1158/1078-0432.CCR-18-2392

Translational Cancer Mechanisms and Therapy Clinical Cancer Research Functional Silencing of HSD17B2 in Prostate Cancer Promotes Disease Progression Xiaomei Gao1,2, Charles Dai3, Shengsong Huang4, Jingjie Tang1,2, Guoyuan Chen1, Jianneng Li3, Ziqi Zhu3, Xuyou Zhu5, Shuirong Zhou1,2, Yuanyuan Gao1,2, Zemin Hou1,2, Zijun Fang1,2, Chengdang Xu4, Jianyang Wang1,2, Denglong Wu4, Nima Shariﬁ3,6,7, and Zhenfei Li1,2

Abstract

Purpose: Steroidogenic enzymes are essential for prostate (DHT) to each of their upstream precursors. HSD17B2 over- cancer development. Enzymes inactivating potent androgens expression suppressed androgen-induced cell proliferation were not investigated thoroughly, which leads to limited inter- and xenograft growth. Multiple mechanisms were involved ference strategies for prostate cancer therapy. Here we charac- in HSD17B2 functional silencing including DNA methylation terizedtheclinical relevance,significance, andregulation mech- and mRNA alternative splicing. DNA methylation decreased anism of enzyme HSD17B2 in prostate cancer development. the HSD17B2 mRNA level. Two new catalytic-deficient iso- Experimental Design: HSD17B2 expression was detected forms, generated by alternative splicing, bound to wild-type with patient specimens and prostate cancer cell lines. Function 17bHSD2 and promoted its degradation. Splicing factors of HSD17B2 in steroidogenesis, androgen receptor (AR) sig- SRSF1 and SRSF5 participated in the generation of new naling, and tumor growth was investigated with prostate isoforms. cancer cell lines and a xenograft model. DNA methylation Conclusions: Our findings provide evidence of the clinical and mRNA alternative splicing were investigated to unveil the relevance, significance, and regulation of HSD17B2 in prostate mechanisms of HSD17B2 regulation. cancer progression, which might provide new strategies for Results: HSD17B2 expression was reduced as prostate can- clinical management by targeting the functional silencing cer progressed. 17bHSD2 decreased potent androgen produc- mechanisms of HSD17B2. tion by converting testosterone (T) or dihydrotestosterone See related commentary by Mostaghel, p. 1139

Introduction mote prostate cancer progression (3). Thus, steroidogenic enzymes involved in androgen metabolism are essential targets Prostate cancer is the most common cancer in the United for prostate cancer treatment or biomarkers for disease diag- States and the fastest increasing cancer in China in men (1, 2). nosis (4, 5). Androgens activate androgen receptor (AR) signaling to pro- Testosterone (T) produced by testis is the major androgen- stimulating prostate cancer development until androgen dep- 1State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular rivation therapy (ADT; ref. 6). ADT resistance occurs and Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese disease progresses into castration-resistant prostate cancer Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, (CRPC) by utilizing adrenal precursors, such as DHEA P.R. China. 2CAS Key Laboratory of Systems Biology, CAS Center for Excellence or androstenedione (AD), to generate potent androgen in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, DHT (3, 7, 8). Steroidogenic enzymes that accelerate DHT Chinese Academy of Sciences, Shanghai, P.R. China. 3Department of Cancer synthesis have been investigated thoroughly in driving prostate Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio. 4Depart- ment of Urology, Tongji Hospital, Tongji University School of Medicine, Shanghai, cancer development and treatment resistance (9, 10). CYP17A P.R. China. 5Department of Pathology, Tongji Hospital, Tongji University School is required for conversion of cholesterol to DHEA, providing of Medicine, Shanghai, P.R. China. 6Department of Urology, Glickman Urological androgen precursors to prostate cancer (7). Abiraterone, target- and Kidney Institute, Cleveland Clinic, Cleveland, Ohio. 7Department of Hema- ing CYP17A, is used for treatment of CRPC and castration- tology and Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio. sensitive prostate cancer (11–14). 3bHSD1 is the rate-limiting Note: Supplementary data for this article are available at Clinical Cancer enzyme for DHEA to DHT metabolism. A SNP in 3bHSD1 Research Online (http://clincancerres.aacrjournals.org/). increases its protein stability and leads to worse outcomes after X. Gao, C. Dai, S. Huang, and J. Tang contributed equally to this article. ADT (15–19). Increased expression of AKR1C3 has been Corresponding Authors: Zhenfei Li, State Key Laboratory of Cell Biology, CAS reported as a mechanism of drug resistance. AKR1C3 catalyzes Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochem- AD or 5a-androstanedione (5a-dione) to T or DHT, respec- istry and Cell Biology, Chinese Academy of Sciences; University of Chinese tively, by modifying the 17-keto group to 17b-OH which is Academy of Sciences, Shanghai 200031, P.R. China. Phone: 86-21-54921339; essential for AR activation (20, 21). Increasing AKR1C3 leads E-mail: [email protected]; Nima Shariﬁ, [email protected]; and Denglong Wu, to more efﬁcient steroidogensis, which could subdue the [email protected] response to abiraterone or enzalutamide (22, 23). Thus, ste- doi: 10.1158/1078-0432.CCR-18-2392 roidogenic enzymes promoting androgen synthesis obtain 2018 American Association for Cancer Research. oncogenic function in disease development.

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50-TCAAGCCCCAAAAAGGGGAC-30 and gD: 50-TGTCCATTTG- Translational Relevance 0 GAGCACCGAG-3 ) were inserted into CRISPR plasmid back- Androgens sustain prostate cancer development. Steroido- bone, lentiCRISPR v2 [a generous gift from Dr. Feng Zhang genic enzymes promoting androgen synthesis are well- (Addgene Plasmid #52961)] according to the protocol they established therapeutic targets and predictive biomarkers, provided (28). Then they were used to generate the HSD17B2 while enzymes inactivating androgens have not been investi- knockout MDA-PCa-2b stable cell line by using a lentiviral gated thoroughly. Here we show the clinical relevance and system. 293T cells were cotransfected with 10 mg each of con- significance of HSD17B2 in prostate cancer, providing poten- structed plasmids (containing gRNAs), pMD2.G, and psPAX2 tial biomarker for disease diagnosis. Mechanisms of HSD17B2 vector for 48 hours to package the virus. Then the virus was functional silencing were further investigated to provide concentrated by using PEG-it Virus Precipitation Solution (Sys- potential novel strategies for disease intervention. tem Biosciences) according to the provided protocol. Next, MDA- PCa-2b cells were infected with the concentrated virus for 24 hours with addition of polybrene (10 mg/mL), followed by selection with 2 mg/mL puromycin for nearly 2 weeks. However, steroidogenic enzymes that inactivate androgens have not been investigated thoroughly. The regulation of andro- High-performance liquid chromatography gen-inactivation enzymes is not well understood as is their role in Cells were seeded and incubated in 24-well plates with 0.2 b million cells/well for nearly 24 hours and then treated with tumor progression. The enzyme 17 HSD2 catalyzes the reverse 3 reaction of AKR1C3. It catalyzes 17b-OH to 17-keto and leads to indicated drugs and a mixture of radioactive ([ H]-labeled) and – fi nonradioactive steroids (final concentration, 50 nmol/L T and 10 androgen inactivation (24 27). The signi cance and regulation of 17bHSD2 in prostate cancer remains elusive. nmol/L DHT; nearly 1,000,000 cpm/well; PerkinElmer) at 37 C. Here we show HSD17B2 expression and function is reduced in Aliquots of medium were collected at the indicated time and b prostate cancer. Overexpression of HSD17B2 blocks potent treated with 300 units of -glucuronidase (Novoprotein Scien- fi m androgen synthesis and thus suppresses AR signaling and cell ti cInc.)at37C for 2 hours, extracted with 500 Lethyl growth. In vivo,17bHSD2 inhibits xenograft proliferation as a acetate:isooctane (1:1), dried under freeze dryer (Martin Christ tumor suppressor. The expression of HSD17B2 in prostate cancer Gefriertrocknungsanlagen). is regulated by DNA methylation, androgen stimulation and HPLC analysis was performed on a Waters Acquity ARC m mRNA alternative splicing. Our data demonstrate the tumor HPLC. Dried samples were reconstituted in 100 Lof50% suppressor role of 17bHSD2 and its regulation mechanisms in methanol and injected into the HPLC. Metabolites were sep- prostate cancer, shedding light on disease interruption through arated on CORTECS C18 reverse-phase column 4.6 50 mm, m HSD17B2. 2.7 mol/L(Waters)usingamethanol/watergradientat40C. The column effluent was analyzed using b-RAM model 3 in-line Materials and Methods radioactivity detector (LABLOGIC). All HPLC studies were run in triplicate and repeated at least three times in independent Cell lines experiments. Cell lines LNCaP, PC3, Du145, and HEK293T cells were purchased from the ATCC and maintained in RPMI1640 (LNCaP, Gene expression and immunoblotting PC3, and Du145) or DMEM (HEK293T) with 10% FBS. Dr. Cells were starved for at least 48 hours with phenol red-free Charles Sawyers (Memorial Sloan Kettering Cancer Center, New and serum free-medium and treated with the indicated andro- York, NY) kindly provided LAPC4 cells, which were grown in gens (100 nmol/L DHEA CAS#53-43-0, 10 nmol/L AD CAS#63- Iscove's Modified Dulbecco's Medium with 10% FBS. VCaP was 05-8, 10 nmol/L T CAS#58-22-0, 10 nmol/L 5a-dione CAS#846- kindly provided by Dr. Jun Qin (SIBS, Shanghai, China). All 46-8, and 1 nmol/L DHT CAS#521-18-6) purchased from experiments with LNCaP and VCaP were done in plates coated MedChemExpress or Steraloids. Cell to cDNA Kit (EZBioscience) with poly-DL-ornithine (Sigma-Aldrich). Cell lines was authen- was used for cDNA synthesis directly from cells. Quantitative PCR ticated by Hybribio and determined to be mycoplasma free (qPCR) experiment was conducted in Bio-Rad CFX96 (Bio-Rad) 0 0 with primers 5 -GGGAGCAAACAGGATTAGATACCCT-3 and using EZBioscience 2 SYBR Green qPCR master mix 0 0 5 - TGCACCATCTGTCACTCTGTTAACCTC-3 . (EZBioscience). The primers for qPCR were described in Supple- mentary Table S1. Plasmids construction and transfection Total protein was extracted from cells with RIPA buffer contain- Lentiviral vectors pCDH-puro or pLVX-tight-puro was ing protease inhibitors (Piece, Prod#88666). The following pri- used for HSD17B2 isoforms cloning. HSD17B2 shRNA and mary antibodies were used: anti-17bHSD2 (1:1,000; Santa Cruz), nontarget shRNA control (pLKO.1 TRC, Mission RNAi) were anti-FLAG (1:5,000; Sigma), anti-b-actin (1:1,000; Cell Signal constructed. The constructs were confirmed by DNA sequenc- Tech). ing. The primer sequences are presented in Supplementary Table S1. Pyrosequencing Virus particles were harvested 48 hours after transfection in DNA was extracted from cells with QIAamp DNA Mini Kit HEK-293T cells using PEI (Promega). Human prostate cancer cell (QIAGEN) and pyrosequencing was performed by Genergy Bio- lines were infected and selected with puromycin (Sigma Aldrich). technology. Briefly, genomic DNA was treated with bisulfite The function of all vectors was validated by Western blot analysis. conversion using Qiagen EpiTect Bisulfite Kit (QIAGEN). Primers Control gRNA (50-ATCTGCCATGGCGTCCTGGC-30) and selectively amplified either methylated or unmethylated DNA HSD17B2 gRNAs (gA: 50-ACTGTCCCACATAGTACTGT-30, gB: were used. PCR products were sequenced on PyroMark Q96 ID

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(QIAGEN). The primers for PCR were described in Supplementary anti-17bHSD2 (1:25; Santa Cruz), anti-Ki67 (1:400; Cell Signal Table S1. Tech). After washing with PBS three times on the second day, corresponding secondary antibodies were applied, and samples Cell proliferation assay were further incubated at room temperature for 30 minutes. Cell proliferation assay was performed with cell counting kit-8 Slides were visualized with DAB staining according the man- (Dojindo) in accordance with the manufacturer's instructions. ufacturer's instructions (GK500710; Genetech). Subsequently, Briefly, 10,000 cells/well dispensed in 100 mL aliquots were they were counterstained with hematoxylin (G1080; Solarbio) seeded in a 96-well plate and starved for 48 hours. Androgen and mounted in dimethyl benzene. was added as indicated. The viable cells were measured after 4 and The staining intensity was divided into four grades: no staining 7 days according to the manufacturer's protocol. The absorbance (0), weak (1), moderate (2), and strong (3). The final IHC scoring was read at 450 nm using a microplate reader (BioTeK) to estimate was performed from the multiplication between intensity and the viable cells in each well. The growth curve was calculated by proportion scores of positive cells. Graphpad Prism 5.0 software (San Diego). Chemical derivatization Mouse xenograft studies Samples were derivatized by using Amplifex Keto Reagent Male B-NDG (B-NSG) mice [B:Biocytogen; N:NOD back- (29, 30). Freeze-dried samples were reconstituted in 50 mLof ground; D:DNAPK (Prkdc) null; G:IL2rgknockout] mice, 4 to 6 Amplifex Keto working solution before diluted with 150 mLof weeks of age were obtained from Beijing Biocytogen. All mouse 70% methanol. Two hundred microliters of the final sample was studies were conducted under a protocol approved by the Insti- injected onto the LC system. tutional Animal Care and Use Committee (SIBCB-S373-1802- 7 006). A total of 1 10 LNCaP-HSD17B2 stable cells were Mass spectrometry fl subcutaneously implanted into the right ank of the intact mice Samples were analyzed on a HPLC station (Agilent) with a with Matrigel (Corning, #354234). Mice were randomly assigned G4204A pumps, a G1367E auto-sampler, a G1316A column into two groups when the xenografts reached approximately 50 to oven, and a triple quadrupole 6490 (Agilent) equipped with an 3 100 mm (length width width 0.5): sucrose control (5% ESI source operating in negative and positive ionization modes. ¼ sucrose, orally, n 10) and doxycycline (Dox; Sigma, 2 mg/mL The mobile phase for HPLC-MS/MS analyses was a mixture of ¼ and 5% sucrose, orally, n 10). Sucrose and dox-containing water water (A) and methanol (B) both containing 0.1% formic acid at were replaced every 2 days. Tumor growth was observed every 2 to 0.3 mL/min with a gradient elution: 0 minutes 45% B, 9 minutes 3 days for 21 days by measuring the two-dimensional longest axis 54% B, 9.51 minutes 90% B, 12.5 minutes 90% B, 12.51 minutes and shortest axis with a caliper. At the end of experiment, the 45% B, and kept at 45% B until the end of the run (15 minutes). fi animals were sacri ced, xenografts were collected for further Separation of drug metabolites was achieved using an Eclipse plus analysis. The difference between treatment groups was assessed C18 RRHD analytical column 3.0 mm 50 mm, 1.8 mmol/L – by Kaplan Meier survival analysis using a log-rank test in Sigma- (Agilent) at 40C. The injection volume was 10 mL, performed Stat 3.5. with auto-sampler. Androgen was ionized using electrospray ionization in positive ion mode (ESI). The temperature of Xenograft tissue metabolism the drying gas in the ionization source was 200C. The gas flow A total of 40 to 50 mg xenograft tissues were seeded and was 14 L/min, the nebulizer pressure was 20 psi and the capillary incubated in 24-well plates and then treated with a mixture of voltage was 3,000 V (negative and positive). The analytes were 3 fi radioactive ([ H]-labeled) and nonradioactive steroids ( nal quantified using multiple reaction monitoring (MRM) with the concentration, 50 nmol/L T; 1,000,000 cpm/well; PerkinElmer) mass transitions and parameters for each compound as listed in at 37 C. Supplementary Table S2. Methanol and water were LC/MS grade and all were from Fisher Scientific. IHC Patient specimens were collected at Tongji hospital with Statistical analysis patient consent under a hospital review board approved pro- All data were shown as mean SD. Statistical analyses between tocol. Consent was obtained from each patient or related groups were performed using Student t test and one-way ANOVA. guardian. Experiments were carried out in accordance with The correlation was determined by Pearson analysis. Survival was Declaration of Helsinki. Benign prostate tissues were collected calculated by the Kaplan–Meier method and differences were from patients with bladder cancer after radical cystectomy. analyzed by the log-rank test. P-value < 0.05 was considered Prostate cancer tissues were collected from patients with local- statistically significant. All analyses were performed using ized prostate cancer receiving only radical prostatectomy treat- Graphpad Prism 5.0 software. ment. Xenograft samples or patient samples were fixed in 4% formaldehyde solution and embedded in paraffin. Five-mm- thick sections were cut from paraffin-embedded tissue blocks, Results deparaffinized and rehydrated in ethanol, and then subjected to Expression of HSD17B2 declines with prostate cancer antigen retrieval by microwaving in sodium citrate (pH 6) for development 15 minutes. Endogenous peroxidase activity was blocked using To investigate its function in prostate cancer, we first detected 3% hydrogen peroxide in PBS for 15 minutes. Sections were the expression of HSD17B2 with patient specimens. Ten radical blocked with normal goat serum for 30 minutes at room prostatectomy tissues from patients with localized prostate cancer temperature, followed by incubation with primary antibodies were stained for 17bHSD2. Ten benign prostates from bladder at 4C overnight. The following primary antibodies were used: cancer patients with radical cystectomy were used as benign

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control. Routine pathologic examination methods such as hema- sets also indicated a decreasing level of HSD17B2 expression in toxylin and eosin (HE) staining were used to distinguish the prostate cancer compared with normal counterparts (Fig. 1E and tumor tissue. The IHC staining results indicated that prostate F; refs. 31, 32). Consistently, HSD17B2 gene deletion was fre- cancer had diminished expression of 17bHSD2 (Fig. 1A and B; quently found in both primary and metastatic prostate cancer, Supplementary Table S3). In the prostate cancer specimens, which may partially explain the low expression of HSD17B2 in benign adjacent to tumor tissue had relatively higher expression prostate cancer (Fig. 1G and H; refs. 33, 34). Together all these of HSD17B2 compared with the related cancerous tissue (Fig. 1C data indicate the decreasing expression of HSD17B2 and its and D; Supplementary Table S3). Data mining with public data potential tumor suppressor role in prostate cancer.

A B #1707038 #1813067 #1712000 #1800416 HSD17B2 expression 1.5 P < 0.01

Benign 1.0 HSD2 β #1814145 #1716478 #1721684 #1732741 0.5 Anti-17 Relative expression 0.0

Benign Prostate cancer Prostate cancer

C Prostate cancer patients DE P < 0.01 #1734048 #1717198 P = 0.012 200 1.0

150

0.5 HSD2

β 100 HSD17B2 expression HSD17B2 expression Anti-17 0.0 50 Relative Adjacent Tumor Benign Adjacent Tumor Adjacent Tumor Prostate cancer (GSE70770, Whitington et al, Nature genetics 2016) FHG P < 0.01 12 Deletion 300 10 Mutation 8 200 Deep deletion Shallow deletionDiploid Gain 6 29 106 194 4 TCGA (primary)

100 4 8.7% 31.8% 58.3% 1.2%

2 10 99 39 2 SU2C (metastatic) Deletion frequency (%) HSD17B2 expression 0 6.7% 66% 26% 1.3% 0 Benign Prostate Prostate MICH cancer database TCGA SU2C (GSE71016, Zhang et al, TCGA 2015 Cancer research, 2016) FHCRC,2016MSKCC 2014 Broad/CornellBroad/Cornell 2013 2012

Figure 1. HSD17B2 expression declines with prostate cancer progression. A, IHC staining of 17bHSD2 with patient specimens. Specimens from radical cystectomy patients were selected as benign control, and specimens from radical prostatectomy patients were taken as tumor tissue. B, Relative expression level of HSD17B2 in benign and prostate cancer tissue. IHC scoring was performed from the multiplication between intensity and proportion scores of positive cells. P value was calculated with t test. C and D, HSD17B2 expression in benign adjacent to tumor and cancerous tissue. P value was calculated with paired t test. E and F, HSD17B2 expression in public data sets (GSE70770 and GSE71016). G, Frequency of HSD17B2 gene deletion in different databases. TCGA, The Cancer Genome Atlas. H, HSD17B2 gene deletion in primary and metastatic prostate cancer. Deep deletion, two copies of HSD17B2 deleted; shallow deletion, one copy of HSD17B2 deleted. SU2C, Stand Up To Cancer; TCGA, The Cancer Genome Atlas.

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17bHSD2 suppresses the conversion from testis and adrenal 17bHSD2 inhibits AR signaling and tumor growth in vivo originated precursors to DHT Because of its ability to inactivate androgens, the expression We further detected HSD17B2 expression in different cancer of HSD17B2 was likely to affect AR signaling and tumor cell lines. PC3 and MDA-Pca-2b had the highest mRNA expres- growth. Induction of HSD17B2 expression in LNCaP and sion, whereas LNCaP, LAPC4, and VCaP had limited HSD17B2 VCaPcellsledtoimpairedT-orDHT-stimulatedARtarget (Fig. 2A). Thus, in PC3 and MDA-Pca-2b cells T or DHT was gene expression (Fig. 3A and B). Its expression also suppressed rapidly converted to AD or 5a-dione respectively but not in testis or adrenal originated androgen-induced cell prolifera- LNCaP or LAPC4 cells (Fig. 2B and C; Supplementary Fig. S1). tion (Fig. 3C). A dox-induced LNCaP-HSD17B2 stable cell line Stable cell lines with dox-inducible HSD17B2 expression were was used for xenograft experiments in intact NDG mice. When generated in LNCaP and VCaP with lentivirus. The expression tumors reached nearly 100 mm3, one group of mice was given of HSD17B2 inLNCaPorVCaPinactivatedTorDHTrobustly dox plus sucrose in water and the control group had only (Fig. 2D and E). We suspected that HSD17B2 elimination, sucrose without dox. The dox-treated group had relative which frequently occurs with disease progression, would pro- smaller tumors and took a longer time to reach 500 mm3 mote adrenal precursors such as AD to be converted to potent (ﬁve-fold), indicating the tumor suppressor role of HSD17B2 androgens such as DHT. Two different CRISPR constructs were in vivo (Fig. 3D and E). Xenograft tumors were collected at the used to knock out HSD17B2 in MDA-Pca-2b (Supplementary end of the mouse experiment. Signiﬁcant higher mRNA and Fig. S2). More DHT was generated in the HSD17B2 knockout protein level of HSD17B2 were found in the dox-treated group cell line, indicating a potential mechanism of androgen accu- (Fig. 3F and G). The IHC results also demonstrated that high mulation in CRPC (Fig. 2F). Together, the data demonstrate level of 17bHSD2 correlated with low level of Ki-67, a cell that down regulation of HSD17B2 facilitates DHT production proliferation marker, in the dox-treated group (Fig. 3H; Sup- in prostate cancer cell lines. plementary Fig. S3). Intratumoral T levels were also detected

A BC 250 PC3 LNCaP AD T 5α-dione DHT T AD DHT 5α-dione 200 100 80 100 100 Others

150 80 80 80 60 100 60 60 60 40 50 40 40 40 HSD17B2/RPLPO Steroids% Steroids% 20 0 20 20 20

C4-2 PC3 0 LAPC4VCAP DU14522RV1 0 0 0 RWPE1LNCaP 0.5 hour 1 hour 0.5 hour 1 hour 0 hour 7 hours 24 hours 48 hours 0 hour 7 hours 24 hours 48 hours

MDA-Pca-2b D F LNCaP-HSD17B2 stable celll line MDA-Pca-2b cell line 17βHSD2 17βHSD2 TAD DHT 5α-dione α 100 100 100 80 AD 5 -dione DHT 17βHSD2 80 80 80 60 100 80 5 60 60 60 80 60 4 40 3 T% 40 40 40 40 AD% 60

DHT% 2 WT

20 AD% 40 20 DHT% 20 20 20 5a-dione% 1

20 5 α -dione% 0 0 0 0 0 0 − − − − − − − − 0 Dox + + Dox + + Dox + + Dox + + 0 hour 48 hours 72 hours 0 hour 48 hours 72 hours 0 hour 48 hours 72 hours 0.5 hour 1 hour 0.5 hour 1 hour 0.5 hour 1 hour 0.5 hour 1 hour 100 80 6 5 E 80 60 4 VCaP-HSD17B2 stable celll line 60 40 3

β 17βHSD2 AD% 40 20 2 17 HSD2 DHT% TAD DHT 5α-dione 1 20 5 α -dione% 0 100 100 100 80 0 KO HSD17B2-1 0 0 hour 48 hours 72 hours 0 hour 48 hours 72 hours 0 hour 48 hours 72 hours 80 80 80 60 100 80 8 60 60 60 80 60 6 40 40 T% 40 40 60 4 40 AD% DHT% AD% 40 20 2 20 DHT%

20 20 20 5a-dione%

20 5 α -dione% 0 0 0 0 0 0 − KO HSD17B2-2 0 Dox − + − + Dox− + + Dox − + − + Dox − + − + 0 hour 48 hours 72 hours 0 hour 48 hours 72 hours 0 hour 48 hours 72 hours 0.5 hour 1 hour 0.5 hour 1 hour 0.5 hour 1 hour 0.5 hour 1 hour

Figure 2. 17bHSD2 suppresses the conversion from testis or adrenal androgens to DHT. A, Expression of HSD17B2 in different prostate cancer cell lines. B and C, T and DHT metabolism in PC3 (high expression of HSD17B2) and LNCaP (low expression of HSD17B2). D and E, Overexpression of HSD17B2 in LNCaP (D) or VCaP (E) led to inactivation of T and DHT. Dox (1 mg/mL) was used to induce the expression of HSD17B2 in the stable cell lines. F, Knockout of HSD17B2 facilitated DHT accumulation in MDA-Pca-2b. Adrenal precursor AD was used to treat MDA-Pca-2b cell lines. KO, knockout; WT, wild type.

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A C E LN-HSD17B2 stable cell Line VCaP-HSD17B2 stable cell line Xenograft growth > 5-fold 10 10 −Dox +Dox −Dox 150 −Dox +T +Dox +T +Dox Sucrose 8 8 −Dox +DHEA +Dox +DHEA 6 P = 0.0187 Dox 100 6 6 5 ** 4 4 4 FKBP5

TMPRSS2 3 ** 50 Progression 2 2 2 OD ratio 1 0 0 0 Ctr T DHT Ctr T DHT 0 0 5 10 15 20 days Day 1 Day 4 Day 7 B D F VCaP-HSD17B2 stable cell line LN-HSD17B2 xenograft LN-HSD17B2 xenograft 3.0 10 25 100 −Dox − Dox 80 2.5 8 +Dox 20 * +Dox 60 2.0 15 40 6 * 1.5 HSD17B2 20 4 10 * FKBP5 0 1.0 fold Relative TMPRSS2 * 5 2.5 .5 2 0 2.0 0.0 0 8 2 8 y 0 y 4 1.5 Ctr T DHT Ctr T DHT ay y 1 y 1 Da Da D Da Day 15Da 1.0 FKBP5 G Sucrose Dox Sucrose Dox 0.5 0.0 9 2 2 #39 #50 #57 #59 #65 #58 #32 #53 #60 #19 #6 #62 #75 #92 #23 #81 #64 #55 #93 #17 #5 3 ox ox # #6 #75 #81 #9 -D -D Anti-Flag 53-Dox #17-Dox#19 # #58-Dox#60 Anti-Actin I

H 2.0 HE 17βHSD2 Ki67 ** 1.8

1.6 Negative control 1.4

1.2

1.0 T in xenograft (ng/mg) T in xenograft 0.8 #53 +Dox −Dox (+Dox) J LN-HSD17B2 xenograft 100

80 #55 60 (+Dox) T% 40

0 #75 100 (+Sucrose) 80 60

AD% 40

#92 20 (+Sucrose) 0 Dox - -+-+-+-+-+---+- -++ ++

Figure 3. 17bHSD2 inhibits tumor proliferation. A and B, Overexpression of HSD17B2 in LNCaP (A) and VCaP (B) inhibited androgen-induced AR target gene expression. C, 17bHSD2 inhibited T- and DHEA-induced cell proliferation. Dox (1 mg/mL) was used to induce HSD17B2 expression in VCaP. , P < 0.01. D and E, HSD17B2 inhibited xenograft growth. Dox (2 mg/mL) was used to induce HSD17B2 expression. Growth curve (D) and Kaplan–Meier survival analysis (E, time for tumors reaching ﬁve-fold size) were used to show the results of tumor growth. , P < 0.05. F and G, HSD17B2 expression in xenograft. Both mRNA (F) and protein (G) level were detected in sucrose or dox-treated xenograft. Fresh xenograft tumors were collected for immediate mRNA or protein detection. H, IHC staining of xenograft. Ki67 was used as a proliferation marker. I, Concentration of T in xenograft. , P < 0.01. J, T metabolism in xenograft. Fresh xenograft was collected and treated with dox (xenografts from dox-treated group) or not (xenografts from sucrose-treated group). [3H]-T was used to treat xenograft tissues for metabolism assay.

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and less T was accumulated in the dox-treated xenograft (Fig. indicated that PC3 had the least DNA methylation in SP1 binding 3I). Furthermore, fresh xenograft was treated with [3H] T and site, which correlated with its highest HSD17B2 expression rapid conversion from T to AD was detected after dox treat- (Fig. 4B). When treated with 5-azacytidine, an inhibitor of ment (Fig. 3J). These data demonstrate the tumor suppressor DNA methyltransferase, HSD17B2 expression in DU145 role of HSD17B2 in prostate cancer in vivo. increased significantly but not in PC3, confirming that DNA methylation blocks HSD17B2 transcription in DU145 Multiple mechanisms of HSD17B2 regulation in prostate (Fig. 4C). In summary, DNA methylation would affect HSD17B2 cancer expression in prostate cancer. We further investigated the regulation mechanisms of When we detected 17bHSD2 protein by immunoblotting, HSD17B2 in prostate cancer. Gene deletion is one mechanism extra bands in C4-2 and other cell lines were found, indicat- of silencing HSD17B2 expression (Fig. 1G and H). We also ing a potential regulatory mechanism related with alternative examined the DNA methylation status of the HSD17B2 promoter. splicing (Fig. 4D). We used a mixed cDNA library from Limited CpG islands are located across the HSD17B2 promoter various prostate cancer cell lines to clone HSD17B2.Three and two of them are in the binding site of SP1, a transcription different isoforms were identified in prostate cancer cells. factor facilitating HSD17B2 transcription (Fig. 4A; ref. 35). It Compared with the wild type (L isoform), the middle-length has been reported that SP1 has a higher affinity to the unmethy- (M) isoform of HSD17B2 was lack of exon 2 (213 base pair) lated DNA fragment, thus DNA methylation would diminish its and the short-length (S) isoform was lack of exon 2 and 3 binding to HSD17B2 promoter (36). The pyrosequencing results (186 base pair; Fig. 4E). The missing of exons did not

A D SP1 binding site Site 1 Site 2 -161 -66 -60 TSS

HSD17B2 promoter Exon LNCaP C4-2 LAPC4 VCAP 22RV1 MDA-Pca-2bDU145 PC3 SP1 binding site L B M Site 1 Site 2 100 CpG island site 1 S 17 β HSD2 80

60 Actin LNCaP 40 E 20 Methylation% Wild type:1164 bp 0 Catalytic domain VCaP ~42 kD 12345 100 CpG island site 2 Middle:951 bp 80 PC3 60 ~36 kD 1345 40 Short:768 bp 20 Methylation% ~28 kD 1 45 0 DU145

PC3 VCaP LNCaP DU145

C F 12 Ctr LAPC4 LNCaP PC3 ALVA31 MDA-Pca-2bMixture μ 10 2.5 mol/L Aza 5 μmol/L Aza 8 600 bp (WT/ L) 6 400 bp (M) 4 HSD17B2 2 200 bp (S) 0 DU145 PC3

Figure 4. Multiple regulation mechanisms of HSD17B2 functional silencing. A, Schema of SP1 binding site and HSD17B2 promoter. B, DNA methylation of HSD17B2 promoter in prostate cancer cell lines. C, HSD17B2 expression after 5-azacytidine (Aza) treatment. HSD17B2 expression was normalized to RPLPO. Basal expression of HSD17B2 in each cell lines was taken as 1. D, Endogenous 17bHSD2 expression in different cell lines. E, Schema of different HSD17B2 isoforms. F, Amplicon of different isoforms in prostate cancer cell lines. Primers located in HSD17B2 exon 1 and exon 4 were used for PCR.

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A B E 6 LN-Stable CHX L/Vector L/M L/S 5 Flag-L Flag-M Flag-S 100 μmol/L 0810664 081064 08104 (hours) 4 Myc-L ++ + + Myc-M + + + + 3 + + + + AD% Myc-S 2 Flag 1 Anti- 0 Myc 100 Actin 80 IP Flag 60 Anti-

T% Flag F 40 CHX S M Vector 20 100 μmol/L 02483 02483 0 83 24 (hours) 0 Dox - +- + - + - + Anti-

MS M S Flag C 3 hours 24 hours Myc 100 μmol/L

CHX 0 3 8 24 (hours) Input Actin Anti-Flag Anti- Flag L G LNCaP Anti-actin 10 Vector D 17βHSD2 8 17βHSD2 + Dox Anti-Flag LMS M − − − MG132 + + + 6 Anti-actin Anti-Flag 4

Anti-Flag 2 S Anti-actin PMEPA1/RPLPO Anti-actin 0 Ctr T DHT Ctr T DHT MS H IJ Prostate cancer (GSE68135) Prostate cancer (GSE21034) 50 4,000 r = -0.5077 r = -0.4401 1,200 r = -0.2274 r = -0.2404 2O H L M S Ctr SRSF1 SRSF5 P = 0.0030 40 P = 0.0042 3,000 P = 0.0149 P = 0.0051 1,000 30 800 L 2,000 M 20 SRSF1 SRSF5 S SRSF1 SRSF5 500 10 1,000 400

0 0 0 4 9 14 4 9 14 0 0 0 100 200 300 400 100 200 300 400 HSD17B2 HSD17B2 HSD17B2 HSD17B2

Figure 5. Function of new isoforms of 17bHSD2. A, M and S isoforms do not inactivate T. Dox was used to induce M or S expression in LNCaP stable cell line. B, Isoforms of 17bHSD2 interact with one another. C, Half-life of different 17bHSD2 isoforms. Plasmids were transfected into 293T cells, and cycloheximide (CHX) was added 18 hours after transfection. D, MG132 increased M and S isoform stability. Plasmids were transfected into the 293T cell line, and MG132 was added 24 hours after transfection. E and F, M and S isoforms promoted wild-type 17bHSD2 degradation. Transient overexpression of M or S in 293T (E) or stable-expression in PC3 (F) promoted wild-type 17bHSD2 degradation. G, M or S isoforms suppressed wild-type 17bHSD2 function in target gene regulation. LNCaP stable cell lines expressing dox-induced M or S isoform were transfected with wild-type HSD17B2 and treated with or without dox as indicated. T, 10 nmol/L; DHT, 1 nmol/L. H, SRSF1 and SRSF5 overexpression produced more M and S mRNA. Primers located in HSD17B2 exon 1 and exon 4 were used for PCR. I and J, Correlation between HSD17B2 and SRSF1/5 in different public data sets (GSE 21034, GSE 68135).

interrupt the protein translation. A primer pair located in been reported that 17bHSD2 forms a dimer to execute its exon 1 and 4 respectively was used to conﬁrm the existence of function, so the interaction between different isoforms was these isoforms in different prostate cancer cell lines. Three examined (37). The results indicated that every isoform could different PCR products conﬁrmed the existence of the iso- interact with itself or the other two (Fig. 5B). Besides, the M or S forms, consistent with the cloning constructs and immuno- isoforms were not as stable as the wild-type one. When treated blotting results (Fig. 4F). with cycloheximide (CHX), an inhibitor of protein biosynthe- We further investigated the function of the new isoforms. sis, the protein level of M and S isoform, especially S isoform, Overexpression of the M or S isoforms showed no effect on T disappeared quickly (Fig. 5C). The protease inhibitor, MG132 metabolism, unlike the wild-type one (Fig. 5A). It was possibly facilitated M and S isoform accumulation but not the wild type because loss of exon 2 or 3 abolished the catalytic domain. It has (Fig. 5D). Thus, we hypothesized that M and S isoform could

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HSD17B2 Function and Regulation in Prostate Cancer

AD 5α-dione

AAAAA 17βHSD2-L AAAAA 17βHSD2-L T AR

17βHSD2-L 17βHSD2-L 17βHSD2-M DHT 17βHSD2-S AR

AAAAA AAAAA Figure 6. Schema of HSD17B2 function and degradation regulation. 17bHSD2 inactivates T and DHT to prevent disease progression. DNA methylation, androgen stimulation, and alternative splicing were involved in AAAAA HSD17B2 functional silencing. CpG methylation HSD17B2-L isoform

AAAAA HSD17B2-S isoform

AAAAA Disease HSD17B2 HSD17B2-M isoform AR progression

SRSF5

SRSF1

HSD17B2 pre-mRNA

promote wild-type 17bHSD2 degradation by binding L isoform and throughout various stages of prostate cancer, steroidogenic as a heterodimer. When coexpressed with M or S isoform in enzymes convert T of gonadal origin or androgens of adrenal 293T cell lines, the L isoform had a shorter retention time (Fig. origin to DHT, which potently activates AR signaling to sustain 5E). A dox-induced M or S expression stable cell line was tissue development or cancer progression (3). Steroidogenic generatedinPC3.TheinductionofMorSisoformexpression enzymes which promote DHT synthesis, such as CYP17A, would decrease endogenous wild-type 17bHSD2 protein level 3bHSD1 and AKR1C3, are involved in therapy resistance. How- (Fig.5F).Furthermore,theinductionofMorSisoformexpres- ever enzymes which inactivate DHT and T have not received much sion would rescue the suppression of AR signaling caused by attention. wild-type 17bHSD2 overexpression in LNCaP (Fig. 5G). The 17bHSD2 catalyzes the conversion from T or DHT to AD or splicing factors participated into the generation of these iso- 5a-dione, respectively by modifying the 17b-OH moiety to forms were investigated. By overexpressing different splicing 17-keto (24). Its expression is gradually reduced as disease factors in PC3, M and S isoforms could only be detected when progresses, indicating its tumor suppressor function. Our data cells were transfected with SRSF1 or SRSF5 (Fig. 5H). Also demonstrate that 17bHSD2 inhibits gonadal and adrenal higher expression of SRSF1 or SRSF5 correlated with low androgens' conversion to DHT, indicating its important role expression of HSD17B2 from different public data sets (Fig. in both localized prostate cancer and metastatic CRPC. Over- 5I and J; refs. 38, 39). Taken together, these data demonstrate expression of HSD17B2 in prostate cancer cell lines diminishes that mRNA alternative splicing produces two new catalytic- AR signaling and suppresses androgen-induced cell prolifera- deﬁcient isoforms degrades wild-type 17bHSD2, resulting in tion and xenograft growth. Thus, it might also serve as a functional silencing of HSD17B2 in both mRNA level and prostate cancer prognostic biomarker. protein level. Investigation of the regulatory mechanisms of HSD17B2 in prostate cancer will shed light on novel strategies for prostate cancer treatment. DNA methylation in the HSD17B2 promot- Discussion er blocks the binding of SP1 and restrains HSD17B2 expres- Steroidogenesis plays a critical role in the development and sion, showing the importance of epigenetic regulation in progression of prostate cancer (40, 41). In benign prostate tissue prostate cancer.

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Another regulation mechanism is mRNA alternative splicing. Acquisition of data (provided animals, acquired and managed patients, The new isoforms generated by mRNA alternative splicing have provided facilities, etc.): X. Gao, C. Dai, S. Huang, G. Chen, J. Li, Z. Fang, fi no enzymatic activity due to the destruction of the catalytic C. Xu, J. Wang, D. Wu, N. Shari ,Z.Li b Analysis and interpretation of data (e.g., statistical analysis, biostatistics, domain.Thenewisoformsalsobindtowild-type17 HSD2 computational analysis): X. Gao, C. Dai, S. Huang, J. Tang, X. Zhu, Z. Hou, and promote its degradation, thus enhancing androgen- C. Xu, D. Wu, N. Sharifi,Z.Li induced AR signaling in prostate cancer. The significance of Writing, review, and/or revision of the manuscript: X. Gao, C. Dai, J. Tang, J. Li, the alternative splicing is not only cutting down the mRNA level D. Wu, N. Sharifi,Z.Li of the wild-type HSD17B2 but only decreasing its protein Administrative, technical, or material support (i.e., reporting or organizing abundance. SRSF1 and SRSF5 are involved in HSD17B2 mRNA data, constructing databases): X. Gao, S. Huang, J. Tang, S. Zhou, Y. Gao, Z. Hou, Z. Fang, C. Xu, J. Wang, D. Wu, N. Sharifi,Z.Li alternative splicing and might serve as novel therapeutic targets Study supervision: N. Sharifi,Z.Li or biomarkers. Insummary,wehavefoundHSD17B2 expression decreases with prostate cancer progression. 17bHSD2 inactivates the Acknowledgments potent androgens, T and DHT, to restrain tumor growth. The authors thank the staff members of Mass Spectrometry at National Functional silencing of HSD17B2 in prostate cancer is achieved Facility for Protein in Shanghai (NFPS), Zhangjiang Lab, China, for providing through multiple mechanisms including gene deletion, DNA technical support and assistance in data collection and analysis. This work has been supported in part by funding from the National Key R&D program of methylation, circulating androgen stimulation and mRNA China (2018YFA0508200; to Z. Li), funding from the Strategic Priority Research alternative splicing (Fig. 6). Two new isoforms generated by Program of Chinese Academy of Sciences (XDB19000000; to Z. Li), funding splicing factors SRSF1 and SRSF5 bind to wild-type 17bHSD2 from the National Natural Science Foundation of China (81722033 and to promote its degradation. Our work unveils the essential role 31771575, to Z. Li; 81672526, to D. Wu; and 81872075, to J. Tang), a Prostate of HSD17B2 in prostate cancer progression and the novel Cancer Foundation Young Investigator Award (#15YOUN11; to Z. Li), funding mechanisms of its regulation, which might provide new strat- from the Youth Innovation Promotion Association (Chinese Academy of Sciences; to J. Tang), a Prostate Cancer Foundation Challenge Award egies for clinical management. (to N. Sharifi), and grants from the National Cancer Institute (R01CA168899, R01CA172382, and R01CA190289; to N. Sharifi). Disclosure of Potential Conflicts of Interest N. Sharifi is a consultant/advisory board member for Pfizer, Tolmar, Asana, The costs of publication of this article were defrayed in part by the and Sanofi. No potential conflicts of interest were disclosed by the other authors. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Authors' Contributions this fact. Conception and design: C. Dai, D. Wu, N. Sharifi,Z.Li Development of methodology: C. Dai, S. Huang, J. Tang, Z. Zhu, Y. Gao, C. Xu, Received July 25, 2018; revised September 8, 2018; accepted September 14, N. Sharifi,Z.Li 2018; published first September 18, 2018.

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Functional Silencing of HSD17B2 in Prostate Cancer Promotes Disease Progression

Xiaomei Gao, Charles Dai, Shengsong Huang, et al.

Clin Cancer Res 2019;25:1291-1301. Published OnlineFirst September 18, 2018.

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