functional silencing of hsd17b2 in prostate cancer promotes … · jingjie tang. 1, 2,#, guoyuan...

1 / 25

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,*,

Zhenfei Li1,2,*.

1 State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell

Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of

Sciences; University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai

200031, P. R. China

2 CAS Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell

Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of

Sciences, Shanghai, China

3 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic,

Cleveland, OH

4 Department of Urology, Tongji Hospital, Tongji University School of Medicine,

Shanghai 200065, China.

5 Department of Pathology, Tongji Hospital, Tongji University School of Medicine,

Shanghai 200065, China.

6 Department of Urology, Glickman Urological and Kidney Institute, Cleveland Clinic,

Cleveland, OH

7 Department of Hematology and Oncology, Taussig Cancer Institute, Cleveland Clinic,

Cleveland, OH

Running title: HSD17B2 function and regulation in prostate cancer

Association for Cancer Research. by guest on August 26, 2020. Copyright 2018 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

https://bloodcancerdiscov.aacrjournals.org

2 / 25

Abbreviations list: SNP: single nucleotide polymorphism; T: testosterone; DHT:

dihydrotestosterone; AR: androgen receptor; 5α-dione: androstanedione; DHEA:

dehydroepiandrosterone; AD: androstenedione; HE: hematoxylin and eosin; IHC:

immunohistochemistry; Dox: doxycycline.

# These authors contributed equally

* Co-correspondence authors

Zhenfei Li

320 Yue-yang Road, Shanghai, China 200031

Phone: +86-21-54921339

Email: [email protected]

Nima Sharifi

9500 Euclid Avenue, Cleveland, OH 44195Phone: 216 445-9750

FAX: 216 445-6269


Denglong Wu

389 Xin-cun Road, Shanghai, China 200065

Phone: +86-21-66111532


mailto:[email protected]



3 / 25


Conflict of interest:

The authors declare no potential conflict of interest.




4 / 25

Translational Relevance

Androgens sustain prostate cancer development. Steroidogenic enzymes promoting

androgen synthesis are well established therapeutic targets and predictive biomarkers,

while enzymes inactivating androgens have not been investigated thoroughly. Here we

show the clinical relevance and significance of HSD17B2 in prostate cancer, providing

potential biomarker for disease diagnosis. Mechanisms of HSD17B2 functional

silencing were further investigated to provide potential novel strategies for disease

intervention.



5 / 25

Abstract

Purpose: Steroidogenic enzymes are essential for prostate cancer development.

Enzymes inactivating potent androgens were not investigated thoroughly, which leads

to limited interfere strategies for prostate cancer therapy. Here we characterized the

clinical relevance, significance and regulation mechanism of enzyme HSD17B2 in

prostate cancer development.

Experimental Design: HSD17B2 expression was detected with patient specimens and

prostate cancer cell lines. Function of HSD17B2 in steroidogenesis, AR signaling and

tumor growth was investigated with prostate cancer cell lines and xenograft model.

DNA methylation and mRNA alternative splicing were investigated to unveil the

mechanisms of HSD17B2 regulation.

Results: HSD17B2 expression was reduced as prostate cancer progresses. 17βHSD2

decreased potent androgen production by converting testosterone (T) or

dihydrotestosterone (DHT) to their upstream precursors. HSD17B2 overexpression

suppressed androgen-induced cell proliferation and xenograft growth. Multiple

mechanisms were involved in HSD17B2 functional silencing including DNA methylation,

androgen stimulation and mRNA alternative splicing. DNA methylation and T

stimulation decreased HSD17B2 mRNA or protein level respectively. Two new

catalytic-deficient isoforms, generated by alternative splicing, bound to wild type

17βHSD2 and promoted its degradation. Splicing factors SRSF1 and SRSF5 participated

in the generation of new isoforms.

Conclusion: Our findings provide evidence of the clinical relevance, significance and

regulation of HSD17B2 in prostate cancer progression, which might provide new

strategies for clinical management by targeting the functional silencing mechanisms

of HSD17B2.



6 / 25

Introduction

Prostate cancer is the most common cancer in the US and the fastest increasing cancer

in China in men (1; 2). Androgens activate androgen receptor (AR) signaling to promote

prostate cancer progression (3). Thus steroidogenic enzymes involved in androgen

metabolism are essential targets for prostate cancer treatment or biomarkers for

disease diagnosis (4; 5).

Testosterone (T) produced by testis is the major androgen stimulating prostate cancer

development until androgen deprivation therapy (ADT)(6). ADT resistance occurs and

disease progresses into castration resistant prostate cancer (CRPC) by utilizing adrenal

precursors, such as dehydroepiandrosterone (DHEA) or androstenedione (AD), to

generate potent androgen dihydrotestosterone (DHT)(3; 7; 8). Steroidogenic enzymes

which accelerate DHT synthesis have been investigated thoroughly in driving prostate

cancer development and treatment resistance (9; 10). CYP17A is required for

conversion of cholesterol to DHEA, providing androgen precursors to prostate

cancer(7). Abiraterone, targeting CYP17A, is used for treatment of CRPC and

castration-sensitive prostate cancer (11-14). 3βHSD1 is the rate-limiting enzyme for

DHEA to DHT metabolism. A single nucleotide polymorphism (SNP) in 3βHSD1

increases its protein stability and leads to worse outcomes after ADT (15-19). Increased

expression of AKR1C3 has been reported as a mechanism of drug resistance. AKR1C3

catalyzes AD or 5α-androstanedione (5α-dione) to T or DHT, respectively, by modifying

the 17-keto group to 17β-OH which is essential for AR activation (20; 21). Increasing

AKR1C3 leads to more efficient steroidogensis, which could subdue the response to

abiraterone or enzalutamide (22; 23). Thus, steroidogenic enzymes promoting

androgen synthesis obtain oncogenic function in disease development.

On the other hand, steroidogenic enzymes which inactivate androgens have not been

investigated thoroughly. The regulation of androgen-inactivation enzymes is not well

understood as is their role in tumor progression. The enzyme 17βHSD2 catalyzes the

reverse reaction of AKR1C3. It catalyzes 17β-OH to 17-keto and leads to androgen

inactivation (24-27). The significance and regulation of 17βHSD2 in prostate cancer



7 / 25

remains elusive.

Here we show HSD17B2 expression and function is reduced in prostate cancer.

Overexpression of HSD17B2 blocks potent androgen synthesis and thus suppresses AR

signaling and cell growth. In vivo, 17βHSD2 inhibits xenograft proliferation as a tumor

suppressor. The expression of HSD17B2 in prostate cancer is regulated by DNA

methylation, androgen stimulation and mRNA alternative splicing. Our data

demonstrate the tumor suppressor role of 17βHSD2 and its regulation mechanisms in

prostate cancer, shedding light on disease interruption through HSD17B2.



8 / 25

Materials and methods

Cell lines

Cell lines LNCaP, PC3, Du145 and HEK293T cells were purchased from the American

Type Culture Collection (Manassas, VA) and maintained in RPMI-1640 (LNCaP, PC3 and

Du145) or DMEM (HEK293T) with 10% FBS. Dr. Charles Sawyers (Memorial Sloan

Kettering Cancer Center, New York, NY) kindly provided LAPC4 cells, which were grown

in Iscove’s Modified Dulbecco’s Medium with 10% FBS. VCaP was kindly provided by

Dr. Jun Qin (SIBS, Shanghai, China). All experiments with LNCaP and VCaP were done

in plates coated with poly-DL-ornithine (Sigma-Aldrich, St. Louis, MO). Cell lines was

authenticated by Hybribio (Guangzhou, China) and determined to be mycoplasma free

with primers 5’-GGGAGCAAACAGGATTAGATACCCT-3’ and 5’-

TGCACCATCTGTCACTCTGTTAACCTC-3’.

Plasmids construction and transfection

Lentiviral vectors pCDH-puro or pLVX-tight-puro was used for HSD17B2 isoforms

cloning. HSD17B2 shRNA and non-target shRNA control (pLKO.1 TRC, Mission RNAi)

were constructed. The constructs were confirmed by DNA sequencing. The primer

sequences are presented in supporting table 1.

Virus particles were harvested 48h after transfection in HEK-293T cells using PEI

(Promega). Human prostate cancer cell lines were infected and selected with

puromycin (Sigma Aldrich, St. Louis, Missouri, USA). The function of all vectors was

validated by western blot.

Control gRNA (5’-ATCTGCCATGGCGTCCTGGC-3’) and HSD17B2 gRNAs (gA: 5’-

ACTGTCCCACATAGTACTGT-3’, gB: 5’-TCAAGCCCCAAAAAGGGGAC-3’ and gD: 5’-

TGTCCATTTGGAGCACCGAG-3’) were inserted into CRISPR plasmid backbone,

lentiCRISPR v2, (a generous gift from Dr. Feng Zhang (Addgene Plasmid #52961))

according to the protocol they provided(28). Then they were used to generate the

HSD17B2 knock out MDA-PCa-2b stable cell line by using a lentiviral system. 293T cells



9 / 25

were cotransfected with 10 mg each of constructed plasmids (containing gRNAs),

pMD2.G, and psPAX2 vector for 48 hours to package the virus. Then the virus was

concentrated by using PEG-it Virus Precipitation Solution (System 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 µg/ml), followed by

selection with 2 µg/ml puromycin for ~2 weeks.

HPLC

Cells were seeded and incubated in 24-well plates with 0.2 million cells/well for ~24 h

and then treated with indicated drugs and a mixture of radioactive ([3H]-labeled) and

non-radioactive steroids (final concentration, 50 nM T and 10nM DHT; ~1,000,000

cpm/well; PerkinElmer, Waltham, MA) at 37°C. Aliquots of medium were collected at

the indicated time and treated with 300 units of β-glucuronidase (Novoprotein

Scientific Inc, China) at 37°C for 2h, extracted with 500 L ethyl acetate:isooctane (1:1),

dried under freeze dryer (Martin Christ Gefriertrocknungsanlagen, Germany).

High-performance liquid chromatography (HPLC) analysis was performed on a Waters

Acquity ARC HPLC. Dried samples were reconstituted in 100 µl of 50% methanol and

injected into the HPLC. Metabolites were separated on CORTECS C18 reverse-phase

column 4.6×50 mm, 2.7 μM (Waters, Ireland) using a methanol/water gradient at 40°C.

The column effluent was analyzed using β-RAM model 3 in-line radioactivity detector

(LABLOGIC, USA). All HPLC studies were run in triplicate and repeated at least 3 times

in independent experiments.

Gene expression and immunoblotting

Cells were starved for at least 48h with phenol red-free and serum free-medium and

treated with the indicated androgens (100nM DHEA CAS#53-43-0, 10nM AD CAS#63-

05-8, 10nM T CAS#58-22-0, 10nM 5a-dione CAS#846-46-8 and 1nM DHT CAS#521-18-

6). Cell to cDNA Kit (EZBioscience) was used for cDNA synthesis directly from cells.

Quantitative PCR (qPCR) experiment was conducted in Bio-Rad CFX96 (Bio-Rad) using



10 / 25

EZBioscience 2 SYBR Green qPCR master mix (EZBioscience). The primers for qPCR

were described in supplementary table 1.

Total protein was extracted from cells with RIPA buffer containing protease inhibitors

(Piece, Prod#88666).The following primary antibodies were used: anti-17βHSD2

(1:1000, Santa Cruz), anti-FLAG (1:5000, Sigma), anti-β-actin (1:1000, Cell Signal Tech).

Pyrosequencing

DNA was extracted from cells with QIAamp DNA Mini Kit (QIAGEN) and

pyrosequencing was performed by Genergy Biotechnology (Shanghai, China). Briefly,

genomic DNA was treated with bisulfite conversion using Qiagen EpiTect® Bisulfite Kit

(QIAGEN). Primers selectively amplified either methylated or unmethylated DNA were

used. PCR products were sequenced on PyroMark Q96 ID (QIAGEN). The primers for

PCR were described in supplementary table 1.

Cell proliferation assay

Cell proliferation assay was performed with cell counting kit-8 (Dojindo, Kumamoto,

Japan) in accordance with the manufacturer’s instructions. Briefly, 10,000 cells/well

dispensed in 100 µl aliquots were seeded in a 96-well plate and starved for 48h.

Androgen was added as indicated. The viable cells were measured after 4 and 7 days

according to the manufacturer’s protocol. The absorbance was read at 450 nm using a

microplate reader (BioTeK, USA) to estimate the viable cells in each well. The growth

curve was calculated by Graphpad Prism 5.0 software (San Diego, CA, USA).

Mouse xenograft studies

Male B-NDG® (B-NSG) mice (B:Biocytogen; N:NOD background; D:DNAPK (Prkdc) null;

G:IL2rgknockout) mice, 4 to 6 weeks of age were obtained from Beijing Biocytogen. All

mouse studies were conducted under a protocol approved by the Institutional Animal

Care and Use Committee (SIBCB-S373-1802-006). 1107 LNCaP-HSD17B2 stable cells

were subcutaneously implanted into the right flank of the intact mice with Matrigel



11 / 25

(Corning, #354234). Mice were randomly assigned into two groups when the

xenografts reached approximately 50 to 100 mm3 (length × width × width × 0.5):

sucrose control (5% sucrose, orally, n=10) and doxycycline (Sigma, dox, 2 mg/ml and

5% sucrose, orally, n=10). Sucrose and dox-containing water were replaced every two

days. Tumor growth was observed every 2-3 days for 21 days by measuring the two-

dimensional longest axis and shortest axis with a caliper. At the end of experiment, the

animals were sacrificed, xenografts were collected for further analysis. The difference

between treatment groups was assessed by Kaplan-Meier survival analysis using a log-

rank test in SigmaStat 3.5.

Xenograft tissue metabolism

40-50 mg xenograft tissues were seeded and incubated in 24-well plates and then

treated with a mixture of radioactive ([3H]-labeled) and non-radioactive steroids (final

concentration, 50 nM T; ~1,000,000 cpm/well; PerkinElmer, Waltham, MA) at 37°C.

Immunohistochemistry (IHC)

Patient specimens were collected at Tongji hospital with patient consent under a

hospital review board approved protocol. Consent was obtained from each patient or

related guardian. Experiments were carried out in accordance with Declaration of

Helsinki. Benign prostate tissues were collected from bladder cancer patients after

radical cystectomy. Prostate cancer tissues were collected from patients with localized

prostate cancer receiving only radical prostatectomy treatment. Xenograft samples or

patient samples were fixed in 4% formaldehyde solution and embedded in paraffin. 5

µm thick sections were cut from paraffin-embedded tissue blocks, deparaffinized and

rehydrated in ethanol, and then subjected to antigen retrieval by microwaving in

sodium citrate (pH 6) for 15 minutes. Endogenous peroxidase activity was blocked

using 3% hydrogen peroxide in PBS for 15 min. Sections were blocked with normal goat

serum for 30 min at room temperature, followed by incubation with primary

antibodies at 4°C overnight. The following primary antibodies were used: anti-

17βHSD2 (1:25, Santa Cruz), anti-Ki67 (1:400, Cell Signal Tech). After washing with PBS



12 / 25

three times on the second day, corresponding secondary antibodies were applied, and

samples were further incubated at room temperature for 30min. Slides were visualized

with DAB staining according the manufacturer’s instructions (GK500710, Genetech).

Subsequently, they were counterstained with hematoxylin (G1080, Solarbio) and

mounted in dimethyl benzene.

The staining intensity was divided into four grades: no staining (0), weak (1), moderate

(2) and strong (3). The final IHC scoring was performed from the multiplication

between intensity and proportion scores of positive cells.

Chemical derivatization

Samples were derivatized by using Amplifex™ Keto Reagent (29; 30). Freeze-dried

samples were reconstituted in 50 μL of Amplifex™ Keto working solution before diluted

with 150 μL of 70% methanol. 200 μL of the final sample was injected onto the LC

system.

Mass spectrometry

Samples were analyzed on a high-performance liquid chromatography station (Agilent,

Germany) with a G4204A pumps, a G1367E auto-sampler, a G1316A column oven and

a triple quadrupole 6490 (Agilent, Singapore) equipped with an ESI source operating

in negative and positive ionization modes. The mobile phase for HPLC-MS/MS analyses

was a mixture of water (A) and methanol (B) both containing 0.1% formic acid at 0.3

ml min-1 with a gradient elution: 0 min 45% B, 9 min 54% B, 9.51 min 90% B, 12.5 min

90% B, 12.51 min 45% B and kept at 45% B until the end of the run (15 min). Separation

of drug metabolites was achieved using an Eclipse plus C18 RRHD analytical column

3.0 mm×50 mm, 1.8 µM (Agilent, USA) at 40°C. The injection volume was 10 μl,

performed with auto-sampler. Androgen was ionized using electrospray ionization in

positive ion mode (ESI). The temperature of the drying gas in the ionization source was

200 °C. The gas flow was 14l min-1, the nebulizer pressure was 20 psi and the capillary

voltage was 3000 V (negative and positive). The analytes were quantified using



13 / 25

multiple reaction monitoring (MRM) with the mass transitions and parameters for

each compound as listed in supplementary table 2. Methanol and water were LC–MS

grade and all were from Fisher Scientific.

Statistical analysis

All data were shown as mean ± standard deviation. Statistical analyses between groups

were performed using Student’s t test and One-Way ANOVA. The correlation was

determined by Pearson analysis. Survival was calculated by the Kaplan–Meier method

and differences were analyzed by the log-rank test. p-value < 0.05 was considered

statistically significant. All analyses were performed using Graphpad Prism 5.0

software.



14 / 25

Results

Expression of HSD17B2 declines with prostate cancer development. To investigate its

function in prostate cancer, we first detected the expression of HSD17B2 with patient

specimens. 10 radical prostatectomy tissues from patients with localized prostate

cancer were stained for 17βHSD2. 10 benign prostates from bladder cancer patients

with radical cystectomy were used as benign control. Routine pathological

examination methods such as hematoxylin and eosin (HE) staining were used to

distinguish the tumor tissue. The immunohistochemistry (IHC) staining results

indicated that prostate cancer had diminished expression of 17βHSD2 (Fig. 1 a and b,

supplementary table 3). In the prostate cancer specimens, benign adjacent to tumor

tissue had relatively higher expression of HSD17B2 compared with the related

cancerous tissue (Fig. 1 c and d, supplementary table 3). Data mining with public data

sets also indicated a decreasing level of HSD17B2 expression in prostate cancer

compared with normal counterparts (Fig. 1 e & f)(31; 32). Consistently, HSD17B2 gene

deletion was frequently found in both primary and metastatic prostate cancer, which

may partially explain the low expression of HSD17B2 in prostate cancer (Fig. 1 g & h)(33;

34). Together all these data indicate the decreasing expression of HSD17B2 and its

potential tumor suppressor role in prostate cancer.

17βHSD2 suppresses the conversion from testis and adrenal originated precursor to

DHT. We further detected HSD17B2 expression in different cancer cell lines. PC3 and

MDA-Pca-2b had the highest mRNA expression, while LNCaP, LAPC4 and VCaP had

limited HSD17B2 (Fig. 2a). Thus, in PC3 and MDA-Pca-2b cells T or DHT was rapidly

converted to AD or 5α-dione respectively but not in LNCaP or LAPC4 cells (Fig. 2 b & c,

Supplementary Fig. 1). Stable cell lines with doxycycline (dox)-inducible HSD17B2

expression were generated in LNCaP and VCaP with lentivirus. The expression of

HSD17B2 in LNCaP or VCaP inactivated T or DHT robustly (Fig. 2 d & e). We suspected

that HSD17B2 elimination, which frequently occurs with disease progression, would

promote adrenal precursors such as AD to be converted to potent androgens such as

DHT. Two different CRISPR constructs were used to knock out HSD17B2 in MDA-Pca-



15 / 25

2b (Supplementary Fig.2). More DHT was generated in the HSD17B2 knockout cell line,

indicating a potential mechanism of androgen accumulation in CRPC (Fig. 2f). Together,

the data demonstrate that down regulation of HSD17B2 facilitates DHT production in

prostate cancer cell lines.

17βHSD2 inhibits AR signaling and tumor growth in vivo. Due to its ability to

inactivate androgens, the expression of HSD17B2 was likely to affect AR signaling and

tumor growth. Induction of HSD17B2 expression in LNCaP and VCaP cells led to

impaired T or DHT stimulated AR target gene expression (Fig.3 a & b). Its expression

also suppressed testis or adrenal originated androgen induced cell proliferation (Fig.

3c). A dox-induced LNCaP-HSD17B2 stable cell line was used for xenograft experiments

in intact NDG mice. When tumors reached ~100mm3, one group of mice was given dox

plus sucrose in water and the control group had only sucrose without dox. The dox

treated group had relative smaller tumors and took a longer time to reach ~500 mm3

(5 fold), indicating the tumor suppressor role of HSD17B2 in vivo (Fig. 3 d & e).

Xenograft tumors were collected at the end of the mouse experiment. Significant

higher mRNA and protein level of HSD17B2 were found in the dox treated group (Fig.

3 f & g). The IHC results also demonstrated that high level of 17βHSD2 correlated with

low level of Ki-67, a cell proliferation marker, in the dox treated group (Fig. 3h and

Supplementary Fig.3). Intratumoral T levels were also detected and less T was

accumulated in the dox treated xenograft (Fig. 3i). Furthermore, fresh xenograft was

treated with [3H] T and rapid conversion from T to AD was detected after dox

treatment (Fig.3j). These data demonstrate the tumor suppressor role of HSD17B2 in

prostate cancer in vivo.

Multiple mechanisms of HSD17B2 regulation in prostate cancer. We further

investigated the regulation mechanisms of HSD17B2 in prostate cancer. Gene deletion

is one mechanism of silencing HSD17B2 expression (Fig. 1 g & h). We also examined

the DNA methylation status of the HSD17B2 promoter. Limited CpG islands are located

across the HSD17B2 promoter and two of them are in the binding site of SP1, a

transcription factor facilitating HSD17B2 transcription (Fig. 4a)(35). It has been



16 / 25

reported that SP1 has a higher affinity to the unmethylated DNA fragment, thus DNA

methylation would diminish its binding to HSD17B2 promoter (36). The

pyrosequencing results indicated that PC3 had the least DNA methylation in SP1

binding site, which correlated with its highest HSD17B2 expression (Fig. 4b). When

treated with 5-azacytidine, an inhibitor of DNA methyltransferase, HSD17B2

expression in DU145 increased significantly but not in PC3, confirming that DNA

methylation blocks HSD17B2 transcription in DU145 (Fig. 4c). To evaluate the effect of

androgens on 17βHSD2, we treated PC3 with different androgens and found T, as the

substrate of 17βHSD2, decreased but not increased its abundance (Fig. 4d). While AD,

the product of 17βHSD2, increased its protein level slightly (Fig. 4d). The effect of DHEA

was elusive and varied between different experiments, possibly due to its metabolism

to downstream androgens. Also the effect of androgen stimulation was limited at the

protein level only but not mRNA level (Supplementary Fig.4). In summary, DNA

methylation and androgen stimulation would affect HSD17B2 expression in prostate

cancer.

When we detected 17βHSD2 protein by immunoblotting, extra bands in C4-2 and

other cell lines were found, indicating a potential regulatory mechanism related with

alternative splicing (Fig.4e). We used a mixed cDNA library from various prostate

cancer cell lines to clone HSD17B2. Three different isoforms were identified in prostate

cancer cells. Compared with the wild type (L isoform), the middle-length (M) isoform

of HSD17B2 was lack of exon 2 (213 base pair) and the short-length (S) isoform was

lack of exon 2 and 3 (186 base pair) (Fig. 4f). The missing of exons did not interrupt the

protein translation. A primer pair located in exon 1 and 4 respectively was used to

confirm the existence of these isoforms in different prostate cancer cell lines. Three

different PCR products confirmed the existence of the isoforms, consistent with the

cloning constructs and immunoblotting results (Fig. 4g).

We further investigated the function of the new isoforms. Overexpression of the M or

S isoforms showed no effect on T metabolism, unlike the wild type one (Fig. 5a). It was

possibly because loss of exon 2 or 3 abolished the catalytic domain. It has been



17 / 25

reported that 17βHSD2 forms a dimer to execute its function, so the interaction

between different isoforms was examined (37). The results indicated that every

isoform could interact with itself or the other two (Fig. 5b). Besides, the M or S

isoforms were not as stable as the wild type one. When treated with cycloheximide

(CHX), an inhibitor of protein biosynthesis, the protein level of M and S isoform,

especially S isoform, disappeared quickly (Fig. 5c). The protease inhibitor, MG132

facilitated M and S isoform accumulation but not the wild type (Fig. 5d). Thus we

hypothesized that M and S isoform could promote wild type 17βHSD2 degradation by

binding L isoform as a heterodimer. When co-expressed with M or S isoform in 293T

cell lines, the L isoform had a shorter retention time (Fig. 5e). A dox induced M or S

expression stable cell line was generated in PC3. The induction of M or S isoform

expression would decrease endogenous wild type 17βHSD2 protein level (Fig. 5f).

Furthermore, the induction of M or S isoform expression would rescue the suppression

of AR signaling caused by wild type 17βHSD2 overexpression in LNCaP (Fig. 5g). The

splicing factors participated into the generation of these isoforms were investigated.

By overexpressing different splicing factors in PC3, M and S isoforms could only be

detected when cells were transfected with SRSF1 or SRSF5 (Fig. 5h). Also higher

expression of SRSF1 or SRSF5 correlated with low expression of HSD17B2 from

different public data sets (Fig. 5 i&j)(38; 39). Taken together, these data demonstrate

that mRNA alternative splicing produces two new catalytic-deficient isoforms

degrades wild type 17βHSD2, resulting in functional silencing of HSD17B2 in both

mRNA level and protein level.

Discussion

Steroidogenesis plays a critical role in the development and progression of prostate

cancer (40; 41). In benign prostate tissue and throughout various stages of prostate

cancer, steroidogenic enzymes convert T of gonadal origin or androgens of adrenal

origin to DHT, which potently activates AR signaling to sustain tissue development or

cancer progression (3). Steroidogenic enzymes which promote DHT synthesis, such as

CYP17A, 3βHSD1 and AKR1C3, are involved in therapy resistance. However enzymes



18 / 25

which inactivate DHT and T have not received much attention.

17βHSD2 catalyzes the conversion from T or DHT to AD or 5a-dione, respectively by

modifying the 17β-OH moiety to 17-keto (24). Its expression is gradually reduced as

disease progresses, indicating its tumor suppressor function. Our data demonstrate

that 17βHSD2 inhibits gonadal and adrenal androgen conversion to DHT, indicating its

important role in both localized prostate cancer and metastatic CRPC. Overexpression

of HSD17B2 in prostate cancer cell lines diminishes AR signaling and suppresses

androgen-induced cell proliferation and xenograft growth. Thus, it might also serve as

a prostate cancer prognostic biomarker.

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 promoter blocks the binding of SP1 and restrains HSD17B2 expression,

showing the importance of epigenetic regulation in prostate cancer. Surprisingly, T as

the substrate of 17βHSD2 reduces its abundance, indicating a positive-feedback loop

to stimulate prostate cancer development. As more potent androgen accumulated in

prostate cancer, it will not only sustain the tumor proliferation but also suppress the

androgen inactivation machinery by reducing 17βHSD2 protein level.

Another regulation mechanism is mRNA alternative splicing. The new isoforms

generated by mRNA alternative splicing have no enzymatic activity due to the

destruction of the catalytic domain. The new isoforms also bind to wild type 17βHSD2

and promote its degradation, thus enhancing androgen-induced AR signaling in

prostate cancer. The significance of the alternative splicing is not only cutting down

the mRNA level of the wild type HSD17B2 but only decreasing its protein abundance.

SRSF1 and SRSF5 are involved in HSD17B2 mRNA alternative splicing and might serve

as novel therapeutic targets or biomarkers.

In summary, we have found HSD17B2 expression decreases with prostate cancer

progression. 17βHSD2 inactivates the potent androgens, T and DHT, to restrain tumor

growth. Functional silencing of HSD17B2 in prostate cancer is achieved through



19 / 25

multiple mechanisms including gene deletion, DNA methylation, circulating androgen

stimulation and mRNA alternative splicing (Fig. 6). Two new isoforms generated by

splicing factors SRSF1 and SRSF5 bind to wild type 17βHSD2 to promote its degradation.

Our work unveils the essential role of HSD17B2 in prostate cancer progression and the

novel mechanisms of its regulation, which might provide new strategies for clinical

management.

Acknowledgements

We thank the staff members of Mass Spectrometry at National Facility for Protein in

Shanghai (NFPS), Zhangjiang Lab, China for providing technical support and assistance

in data collection and analysis. This work has been supported in part by funding from

National Key R&D program of China (2018YFA0508200 to Z.L.), Strategic Priority

Research Program of Chinese Academy of Sciences, Grant No. XDB19000000 (to Z.L.),

the National Natural Science Foundation of China (81722033 and 31771575 to Z.L.,

81672526 to D.W., 81872075 to J.T. ), Prostate Cancer Foundation Young Investigator

Award (#15YOUN11 to Z.L.), Youth Innovation Promotion Association (Chinese

Academy of Sciences, to J.T.), a Prostate Cancer Foundation Challenge Award (to N.S.),

and, grants from the National Cancer Institute (R01CA168899, R01CA172382, and

R01CA190289; to N.S.). The authors claim no conflict of interest.

References

1. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, et al. Cancer statistics in China, 2015. CA: a cancer

journal for clinicians 66:115-32

2. Siegel RL, Miller KD, Jemal A. 2018. Cancer statistics, 2018. CA: a cancer journal for clinicians

68:7-30

3. Sharifi N, Auchus RJ. 2012. Steroid biosynthesis and prostate cancer. Steroids 77:719-26

4. Armstrong CM, Gao AC. 2015. Drug resistance in castration resistant prostate cancer:

resistance mechanisms and emerging treatment strategies. American journal of clinical and

experimental urology 3:64-76

5. Penning TM, Byrns MC. 2009. Steroid hormone transforming aldo-keto reductases and cancer.

Ann N Y Acad Sci 1155:33-42

6. Huggins C, Hodges CV. 1972. Studies on prostatic cancer. I. The effect of castration, of estrogen

and androgen injection on serum phosphatases in metastatic carcinoma of the prostate. CA: a



20 / 25

cancer journal for clinicians 22:232-40

7. Montgomery RB, Mostaghel EA, Vessella R, Hess DL, Kalhorn TF, et al. 2008. Maintenance of

intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant

tumor growth. Cancer research 68:4447-54

8. Mostaghel EA, Page ST, Lin DW, Fazli L, Coleman IM, et al. 2007. Intraprostatic androgens and

androgen-regulated gene expression persist after testosterone suppression: therapeutic

implications for castration-resistant prostate cancer. Cancer research 67:5033-41

9. Penning TM. 2015. Mechanisms of drug resistance that target the androgen axis in castration

resistant prostate cancer (CRPC). The Journal of steroid biochemistry and molecular biology

153:105-13

10. Cai C, Chen S, Ng P, Bubley GJ, Nelson PS, et al. 2011. Intratumoral de novo steroid synthesis

activates androgen receptor in castration-resistant prostate cancer and is upregulated by

treatment with CYP17A1 inhibitors. Cancer research 71:6503-13

11. de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, et al. 2011. Abiraterone and increased

survival in metastatic prostate cancer. The New England journal of medicine 364:1995-2005

12. Ryan CJ, Smith MR, de Bono JS, Molina A, Logothetis CJ, et al. 2013. Abiraterone in metastatic

prostate cancer without previous chemotherapy. The New England journal of medicine

368:138-48

13. James ND, de Bono JS, Spears MR, Clarke NW, Mason MD, et al. 2017. Abiraterone for Prostate

Cancer Not Previously Treated with Hormone Therapy. The New England journal of medicine

377:338-51

14. Fizazi K, Tran N, Fein L, Matsubara N, Rodriguez-Antolin A, et al. 2017. Abiraterone plus

Prednisone in Metastatic, Castration-Sensitive Prostate Cancer. The New England journal of

medicine 377:352-60

15. Chang KH, Li R, Kuri B, Lotan Y, Roehrborn CG, et al. 2013. A gain-of-function mutation in DHT

synthesis in castration-resistant prostate cancer. Cell 154:1074-84

16. Hearn JW, AbuAli G, Reichard CA, Reddy CA, Magi-Galluzzi C, et al. 2016. HSD3B1 and resistance

to androgen-deprivation therapy in prostate cancer: a retrospective, multicohort study. The

Lancet. Oncology 17:1435-44

17. Hettel D, Sharifi N. 2018. HSD3B1 status as a biomarker of androgen deprivation resistance and

implications for prostate cancer. Nature reviews. Urology 15:191-6

18. Hearn JWD, Xie W, Nakabayashi M, Almassi N, Reichard CA, et al. 2018. Association of HSD3B1

Genotype With Response to Androgen-Deprivation Therapy for Biochemical Recurrence After

Radiotherapy for Localized Prostate Cancer. JAMA oncology 4:558-62

19. Almassi N, Reichard C, Li J, Russell C, Perry J, et al. 2018. HSD3B1 and Response to a

Nonsteroidal CYP17A1 Inhibitor in Castration-Resistant Prostate Cancer. JAMA oncology 4:554-

7

20. Adeniji AO, Chen M, Penning TM. 2013. AKR1C3 as a target in castrate resistant prostate cancer.

The Journal of steroid biochemistry and molecular biology 137:136-49

21. Marchais-Oberwinkler S, Henn C, Moller G, Klein T, Negri M, et al. 2011. 17beta-Hydroxysteroid

dehydrogenases (17beta-HSDs) as therapeutic targets: protein structures, functions, and

recent progress in inhibitor development. The Journal of steroid biochemistry and molecular

biology 125:66-82

22. Liu C, Armstrong CM, Lou W, Lombard A, Evans CP, Gao AC. 2017. Inhibition of AKR1C3



21 / 25

Activation Overcomes Resistance to Abiraterone in Advanced Prostate Cancer. Molecular

cancer therapeutics 16:35-44

23. Liu C, Lou W, Zhu Y, Yang JC, Nadiminty N, et al. 2015. Intracrine Androgens and AKR1C3

Activation Confer Resistance to Enzalutamide in Prostate Cancer. Cancer research 75:1413-22

24. Lu ML, Huang YW, Lin SX. 2002. Purification, reconstitution, and steady-state kinetics of the

trans-membrane 17 beta-hydroxysteroid dehydrogenase 2. The Journal of biological chemistry

277:22123-30

25. Wu L, Einstein M, Geissler WM, Chan HK, Elliston KO, Andersson S. 1993. Expression cloning

and characterization of human 17 beta-hydroxysteroid dehydrogenase type 2, a microsomal

enzyme possessing 20 alpha-hydroxysteroid dehydrogenase activity. The Journal of biological

chemistry 268:12964-9

26. Casey ML, MacDonald PC, Andersson S. 1994. 17 beta-Hydroxysteroid dehydrogenase type 2:

chromosomal assignment and progestin regulation of gene expression in human endometrium.

The Journal of clinical investigation 94:2135-41

27. Castagnetta LA, Carruba G, Traina A, Granata OM, Markus M, et al. 1997. Expression of different

17beta-hydroxysteroid dehydrogenase types and their activities in human prostate cancer cells.

Endocrinology 138:4876-82

28. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, et al. 2014. Genome-scale CRISPR-Cas9

knockout screening in human cells. Science 343:84-7

29. Jin W, Jarvis M, Star-Weinstock M, Altemus M. 2013. A sensitive and selective LC-differential

mobility-mass spectrometric analysis of allopregnanolone and pregnanolone in human plasma.

Analytical and bioanalytical chemistry 405:9497-508

30. Bussy U, Huertas M, Chung-Davidson YW, Li K, Li W. 2016. Chemical derivatization of

neurosteroids for their trace determination in sea lamprey by UPLC-MS/MS. Talanta 149:326-

34

31. Zhang L, Wang J, Wang Y, Zhang Y, Castro P, et al. 2016. MNX1 Is Oncogenically Upregulated in

African-American Prostate Cancer. Cancer research 76:6290-8

32. Whitington T, Gao P, Song W, Ross-Adams H, Lamb AD, et al. 2016. Gene regulatory

mechanisms underpinning prostate cancer susceptibility. Nature genetics 48:387-97

33. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, et al. 2013. Integrative analysis of complex

cancer genomics and clinical profiles using the cBioPortal. Science signaling 6:pl1

34. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, et al. 2012. The cBio cancer genomics portal:

an open platform for exploring multidimensional cancer genomics data. Cancer discovery

2:401-4

35. Cheng YH, Imir A, Suzuki T, Fenkci V, Yilmaz B, et al. 2006. SP1 and SP3 mediate progesterone-

dependent induction of the 17beta hydroxysteroid dehydrogenase type 2 gene in human

endometrium. Biology of reproduction 75:605-14

36. Zhu WG, Srinivasan K, Dai Z, Duan W, Druhan LJ, et al. 2003. Methylation of adjacent CpG sites

affects Sp1/Sp3 binding and activity in the p21(Cip1) promoter. Molecular and cellular biology

23:4056-65

37. Lin SX, Yang F, Jin JZ, Breton R, Zhu DW, et al. 1992. Subunit identity of the dimeric 17 beta-

hydroxysteroid dehydrogenase from human placenta. The Journal of biological chemistry

267:16182-7

38. Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, et al. 2010. Integrative genomic profiling



22 / 25

of human prostate cancer. Cancer cell 18:11-22

39. Prueitt RL, Wallace TA, Glynn SA, Yi M, Tang W, et al. 2016. An Immune-Inflammation Gene

Expression Signature in Prostate Tumors of Smokers. Cancer research 76:1055-65

40. Mostaghel EA, Montgomery B, Nelson PS. 2009. Castration-resistant prostate cancer: targeting

androgen metabolic pathways in recurrent disease. Urologic oncology 27:251-7

41. Dai C, Heemers, H., Sharifi, N. 2017. Androgen signaling in prostate cancer. Cold Spring Harb

Perspect Med



23 / 25

Figure 1. HSD17B2 expression declines with prostate cancer progression. (a) IHC

staining of 17βHSD2 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 database. (h) HSD17B2 gene deletion in primary and metastatic prostate

cancer. Deep deletion, 2 copies of HSD17B2 deleted; shallow deletion, 1 copy of

HSD17B2 deleted.

Figure 2. 17βHSD2 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. Doxycycline (Dox, 1 µg/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.

Figure 3. 17βHSD2 inhibits tumor proliferation. (a) and (b) Overexpression of

HSD17B2 in LNCaP (a) and VCaP (b) inhibited androgen induced AR target genes

expression. (c) 17βHSD2 inhibited T and DHEA induced cell proliferation. Dox (1 µg/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 5-

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



24 / 25

in sucrose or dox treated xenograft. Fresh xenograft tumors were collected for

immediate mRNA or protein detection. (h) Immunohistochemistry 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.

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

treatment. HSD17B2 expression was normalized to RPLPO. Basal expression of

HSD17B2 in each cell lines was taken as 1. (d) 17βHSD2 protein abundance after

androgen stimulation. PC3 cells were treated with indicated androgens for 8h before

collection for western blot. (e) Endogenous 17βHSD2 expression in different cell lines.

(f) Schema of different HSD17B2 isoforms. (g) Amplicon of different isoforms in

prostate cancer cell lines. Primers located in HSD17B2 exon 1 and exon 4 were used

for PCR.

Figure 5. Function of new isoforms of 17βHSD2. (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

17βHSD2 interact with one another. (c) Half-life of different 17βHSD2 isoforms.

Plasmids were transfected into 293T cells and cycloheximide (CHX) was added 18h

after transfection. (d) MG132 increased M and S isoform stability. Plasmids were

transfected into the 293T cell line and MG132 was added 24h after transfection. (e)

and (f) M and S isoforms promoted wild type 17βHSD2 degradation. Transient

overexpression of M or S in 293T (e) or stable-expression in PC3 (f) promoted wild type

17βHSD2 degradation. (g) M or S isoforms suppressed wild type 17βHSD2 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 nM; DHT, 1 nM. (h) SRSF1 and SRSF5 overexpression produced more M and S



25 / 25

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).

Figure 6. Schema of HSD17B2 function and regulation. 17βHSD2 inactivates T and

DHT to prevent disease progression. DNA methylation, androgen stimulation and

alternative splicing were involved in HSD17B2 functional silencing.



functional silencing of hsd17b2 in prostate cancer promotes … · jingjie tang. 1, 2,#, guoyuan...

Documents