Methylation of SRD5A2 promoter predicts a better outcome for castration-resistant prostate cancer patients undergoing androgen deprivation therapy.

PURPOSE: To determine whether SRD5A2 promoter methylation is associated with cancer progression during androgen deprivation therapy (ADT) in CRPC. PATIENTS AND METHODS: In a Local CRPC cohort, 42 prostatic specimens were collected from patients who were diagnosed as CRPC and underwent transurethral resection of the prostate (TURP) at Massachusetts General Hospital (MGH). In a metastatic CRPC (Met CRPC) cohort, 12 metastatic biopsies were collected from CRPC patients who would be treated with abiraterone plus dutasteride (Clinical Trial NCT01393730). As controls, 36 benign prostatic specimens were collected from patients undergoing prostate reduction surgery for symptoms of bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH). The methylation status of cytosine-phosphate-guanine (CpG) site(s) at SRD5A2 promoter regions was tested.
RESULTS: Compared with benign prostatic tissue, CRPC samples demonstrated higher SRD5A2 methylation in the whole promoter region (Local CRPC cohort: P < 0.001; Met CRPC cohort: P <0.05). In Local CRPC cohort, a higher ratio of methylation was correlated with better OS (R2 = 0.33, P = 0.013). Hypermethylation of specific regions (nucleotides -434 to -4 [CpG# -39 to CpG# -2]) was associated with a better OS (11.3±5.8 vs 6.4±4.4 years, P = 0.001) and PFS (8.4±5.4 vs 4.5±3.9 years, P = 0.003) with cutoff value of 37.9%. Multivariate analysis showed that SRD5A2 methylation was associated with OS independently (whole promoter region: P = 0.035; specific region: P = 0.02).
CONCLUSION: Our study demonstrate that SRD5A2 methylation in promoter regions, specifically at CpG# -39 to -2, is significantly associated with better survival for CRPC patients treated with ADT. Recognition of epigenetic modifications of SRD5A2 may affect the choices and sequence of available therapies for management of CRPC.

Introduction

Advanced castration resistant prostate cancer (CRPC) accounts for majority of 31,000 deaths each year in the United States, and prognostic tools that determine patients’ overall survival (OS) are lacking [1, 2]. Oral inhibitors targeting CYP-17 (by abiraterone) and the androgen receptor (AR) (by enzalutamide, apalutamide, darolutamide) have increased survival in CRPC in phase III studies [3-8]. However, resistance to AR-directed therapies remains a challenge, which indicates a complexity in the progression from invasive cancer to castration-resistant disease. Persistent AR signaling despite AR-axis inhibition is a critical mechanism of resistance in patients with metastatic CRPC (Met CRPC) [9]. Thus, a better understanding of the drivers in resistance is needed to develop therapeutic strategies that offer patients long-term clinical benefit.

Predictive biomarkers identifying CRPC patients who respond optimally to androgen deprivation therapy (ADT) is of great clinical importance. One mechanism of CRPC development is intratumoral biosynthesis of dihydrotestosterone (DHT) from adrenal precursors, which requires three enzymatic steps. 5-α reduction catalyzed by steroid 5-α reductase (SRD5A) is one of the three steps [10]. SRD5A has three isoforms, SRD5A1, SRD5A2 and SRD5A3. A recent study demonstrated that the expression of SRD5A1 and SRD5A2 are inversely modulated by AR activation, i.e., SRD5A1 transcription is activated but SRD5A2 is repressed, favoring a transcriptional isoform switch that correlates with SRD5A expression patterns in human tumors [11]. In metastatic prostate cancer (PCa) models, in vitro studies have shown that SRD5A1 is upregulated in the Met CRPC setting and is directly regulated by androgens [11-13]. In addition, patients carrying the more active GG genotype in SRD5A2 rs523349 exhibited a higher risk of the progression and death, suggesting that high 5α-reductase activity due to SRD5A2 rs523349 polymorphism may contribute to resistance to ADT in CRPC [14]. The mechanism underlying this transcriptional regulatory switch is still unknown.

In benign prostatic tissue, expression of SRD5A2 protein is variable and negatively correlated with methylation of the SRD5A2 promoter. Our recent studies show that 30% of adult prostates without malignancy do not express the SRD5A2 gene or protein, and that somatic suppression of SRD5A2 during adulthood is dependent on epigenetic changes associated with methylation of the promoter region of the SRD5A2 gene [15-17]. In this study, we tested SRD5A2 promoter methylation of 42 prostatic specimens and 12 metastatic biopsies and found significant hypermethylation of SRD5A2 promoter region for CRPC compared with benign prostatic specimens. Contrary to common belief that DNA methylation occurs during tumor initiation and progression [18], we demonstrated that hypermethylation of SRD5A2 in the whole promoter region (cytosine-phosphate-guanine [CpG]# -72 to +65) was correlated with better OS and progression free survival (PFS) of CRPC patients, and hypermethylation of promoter subset, CpG sites (CpG # -39 to -2), was best correlated with OS and PFS of CRPC patients. Our study suggests that SRD5A2 methylation in promoter regions, specifically at CpG# -39 to -2, a condition that favors an estrogenic as opposed to an androgenic milieu in the prostate, is significantly associated with better survival for CRPC patients treated with ADT. Recognition of epigenetic modifications of SRD5A2, which affects the prostatic hormonal environment, may affect the choices and sequence of available therapies for management of CRPC.

Materials and methods

Patient specimens

With the approval of institutional review board at Massachusetts General Hospital (MGH) and Dana-Farber Cancer Institute, a total of 58 CRPC specimens and 36 benign prostatic specimens were used for methylation testing. Written consent was obtained from the study participants for the two cohorts.The Local CRPC cohort patients were diagnosed as CRPC between 2004 and 2013. Forty two formalin-fixed paraffin-embedded (FFPE) prostatic specimens were collected from patients who underwent transurethral resection of the prostate (TURP) at MGH to relieve patients’ urinary outflow obstruction from locally advanced PCa. All patients had received primary or secondary ADT. Pathology samples were reviewed by an expert genitourinary pathologist (CLW). As controls, 12 frozen benign prostatic specimens were collected from patients who underwent TURP for symptomatic benign prostatic hyperplasia (BPH) at MGH. The metastatic cohort included 12 metastatic biopsies that were collected from CRPC patients who were treated with abiraterone plus dutasteride (Clinical Trial NCT01393730). For comparison with this cohort, 24 benign prostatic specimens were collected from patients who underwent TURP for symptomatic BPH at MGH.

DNA methylation test: Targeted Next-Gen Bisulfite Sequencing (tNGBS) technique

Test procedure is described in Supplementary Information.

Immunohistochemistry

IHC was completed as previously described [16, 17]. More detail is described in the Supplementary Information.

TCGA prostate cancer data mining

The data of SRD5A2 promoter methylation and expression of SRD5A2 in normal prostatic specimens and primary PCa specimens from The Cancer Genome Atlas (TCGA) database: https://tcga-data.nci.nih.gov/tcga/ was extracted. The promoter methylation and expression of SRD5A2 between normal and tumor tissues were compared via the UALCAN website: http://ualcan.path.uab.edu [19]. The association between SRD5A2 promoter methylation and SRD5A2 expression was analyzed via the MEXPRESS website: https://mexpress.be [20].

Statistical analysis

Continuous variables were reported as the means ± SD. The mean differences of average methylation ratios of SRD5A2 promoter between CRPC and benign prostatic tissues were compared using the Student’s t-test. A heatmap was generated to compare the average methylation ratios between the two groups and identify the specific promoter regions with the most significant difference. R package “pheatmap” (version 1.0.12, https://cran.r-project.org/web/packages/pheatmap; R software 3.6.0) was used to draw the heatmap of methylation sites in the two cohorts, respectively. Those methylation sites were automatically clustered according to the “Euclidean” distance. With a cutoff value of the ratio of SRD5A2 promoter methylation, CRPC patients were further sub-grouped as SRD5A2-hypermethylation group and SRD5A2-hypomethylation group. Cutoff values were determined by X-tail software (version 3.6.1; Yale University, New Haven, USA). OS and PFS data of the CRPC patients were retrieved and calculated, then the ratio of SRD5A2 promoter methylation was correlated to the prognosis of disease with survival curve and correlation analysis. Hazard ratios (HRs) of SRD5A2 promoter methylation in predicting the OS and PFS of CRPC patients receiving ADT were calculated using the Cox proportional hazards regression model and tested using the log-rank test. Multivariable analyses were performed, including GS, PSA, and ratio of SRD5A2 methylation as covariates. A P value of < 0.05 was considered as statistical significance. All the statistical analyses were conducted by R (version 3.5.2; The R Foundation, Vienna, Austria) and GraphPad Prism (version 7.00; GraphPad Software, Inc., La Jolla, USA).

Results

Hypermethylation of the SRD5A2 promoter region for CRPC

Several independent studies have shown that expression of SRD5A1 is increased and SRD5A2 is decreased in the transition from hormone-naive PCa to CRPC [1, 13, 21, 22]. However, the mechanism and clinical significance of changes associated with SRD5A2 in CRPC is poorly understood. To explore whether epigenetic changes of SRD5A2 affects the therapeutic efficacy of ADT, we tested and compared SRD5A2 promoter methylation ratios between CRPC and benign prostatic tissues using the NGBS technique. We found that the methylation ratios of main SRD5A2 promoter regions were higher for CRPC prostatic tissues compared with benign prostatic tissues in the local CRPC cohort (Fig 1A). To identify methylation in the SRD5A2 specific promoter region, we performed unsupervised cluster analysis of the ratio of SRD5A2 promoter methylation on all CpG methylation sites. The CpG methylation sites were clustered into three modules, CpG# -72 to -42; CpG#-40 to -35 / CpG# -31 to -19; CpG# -34 to -32 / CpG# -18 to 65 (Fig 1C).

Fig 1

Hypermethylation of the SRD5A2 promoter region in CRPC.

(A) The ratio of SRD5A2 promoter methylation on individual CpG sites in the promoter region in CRPC and benign prostatic tissues in the Local CRPC cohort. (B) The ratio of SRD5A2 DNA promoter methylation on individual CpG sites in CRPC and benign prostatic tissues in the Met CRPC cohort. (C) Unsupervised cluster analysis of the ratio of SRD5A2 promoter methylation on all CpG methylation sites in the Local CRPC cohort. The CpG methylation sites were clustered into 3 modules, CpG# -72 to -42; CpG# -40 to -35 and -31 to -19; CpG# -34 to -32 and -18 to 65. (D) Unsupervised cluster analysis of the ratio of SRD5A2 promoter methylation on all the CpG methylation sites in the Met CRPC cohort. The methylation sites were clustered into 2 modules, CpG# -72 to -35, -31; CpG# -34 to -32 and -30 to 65. (E) The average methylation level of all tested CpG methylation sites in CRPC and benign prostatic tissues in the Local CRPC cohort. (F) The average methylation level of all tested CpG methylation sites in CRPC and benign prostatic tissues in the Met CRPC cohort. (G) The average methylation level of module 1: CpG# -72 to -40 in CRPC and benign prostatic tissues in the Local CRPC cohort. (H) The average methylation level of module 1: CpG# -72 to -40 in CRPC and benign prostatic tissues in the Met CRPC cohort. (I) The average methylation level of module 2: CpG# -39 to 65 in CRPC and benign prostatic tissues in the Local CRPC cohort. (J) The average methylation level of module 2: CpG# -39 to 65 in CRPC and benign prostatic tissues in the Met CRPC cohort.

Validating our finding in Met CRPC tissues demonstrated a similar finding as the Local CRPC cohort, i.e., higher methylation ratio of SRD5A2 for CRPC patients compared with that in the benign control (Fig 1B). In addition, unsupervised cluster analysis generated two modules, CpG# -72 to -35; CpG# -34 to -32 / CpG# -30 to 65 (Fig 1D). After analysis of the Local and Met CRPC cohorts, we identified CpG# -39 to 65 as the important region that is differentially methylated between CRPC vs. benign prostatic samples.

We further verified our finding with Student’s t-test based quantitative analyses. The average methylation ratios of the whole SRD5A2 promoter region (CpG#-72 to 65) and specific promoter region CpG#-39 to 65 of CRPC tissues were significantly higher than that of benign prostatic tissues in the Local CRPC cohort (Fig 1E and 1I) and in the Met CRPC cohort (Fig 1F and 1J). However, there was no significant difference in the average methylation ratios of region CpG#-72 to -40 between CRPC and benign control groups (Fig 1G and 1H).

Higher ratio of SRD5A2 promoter methylation was correlated with better prognosis of CRPC patients receiving ADT

We next addressed whether the SRD5A2 promoter methylation is correlated to the clinical efficiency of ADT for CRPC patients. CRPC patients were sub-grouped as the SRD5A2 hypermethylation group and the SRD5A2 hypomethylation group using the best cutoff value of methylation ratios. In the Local CRPC cohort, data of the whole promoter region of SRD5A2 (CpG#-72 to 65) was generated as a Heatmap (Fig 2A), which revealed the different levels of methylation between the two groups.

Fig 2

Hypermethylation of SRD5A2 in the whole promoter region (CpG # -72 to 65) was correlated with OS and PFS of CRPC patients in the Local CRPC cohort.

Patients were divided into a hypermethylation group and a hypomethylation group with a cutoff value of 46.5%, based on the average methylation level of all methylation sites. (A) Unsupervised cluster analysis of the ratio of SRD5A2 promoter methylation on all CpG methylation sites. (B) The difference of OS between the hypermethylation group and hypomethylation group. (C) The correlation between methylation level and OS. (D & E) Patients were divided into a hypermethylation group and a hypomethylation group with a cutoff value of 42%, based on the average methylation level of all methylation sites. (D) The difference of PFS between the two groups. (E) The correlation between methylation level and PFS.

Next, we retrieved OS and PFS data of the CRPC patients and correlated the ratio of SRD5A2 promoter methylation to the prognosis of disease with a survival curve and correlation analysis. We found that a higher methylation ratio was significantly correlated with better OS of CRPC patients receiving ADT (S1 and S2 Tables, 12.0 ± 6.3 vs 8.0 ± 4.8 years, P = 0.013; Fig 2B and 2C) and better PFS (S1 and S2 Tables, 8.4 ± 5.6 vs 5.3 ± 4.2 years, P = 0.045; Fig 2D and 2E). Similarly, in the Met CRPC cohort, the Heatmap demonstrated the difference of methylation in whole promoter region of SRD5A2 (CpG#-72 to 65) between the two groups (S2A Fig), and a higher methylation ratio was significantly associated with better OS (18.2 ± 5.2 vs 7.9 ± 4.3 years, P = 0.01, S2B and S2C Fig) and better PFS (10.0 ± 4.8 vs 5.0 ± 1.2 months, P = 0.002, S2D and S2E Fig). Together, our data suggest that SRD5A2 whole promoter region methylation both in prostatic tissue and metastatic biopsies predicts a better outcome of ADT treatment for CRPC patients.

Higher ratio of SRD5A2 methylation in specific promoter regions was correlated with better prognosis of CRPC patients receiving ADT

Next, we sought to investigate if the methylation in more specific SRD5A2 promoter regions better predict the outcome of ADT treatment. We first focused our study on a specific promoter region CpG# -39 to 65 (Fig 1I and 1J). As expected, in the local CRPC cohort, a higher methylation ratio was significantly associated with better OS (P = 0.038, Fig 3A and 3B) and better PFS (P = 0.019, Fig 3C and 3D). Similarly, in the Met CRPC cohort, higher methylation was also significantly associated with better OS (P = 0.056, Fig 3E and 3F) and better PFS (P = 0.02, Fig 3G and 3H).

Fig 3

SRD5A2 hypermethylation of specific CpG sites (CpG # -39 to 65) in the promoter region was correlated with OS and PFS of CRPC patients.

Survival analysis was done based on clustering analysis of methylation site modules (CpG#-39 to 65 average methylation level). (A to D) In the Local CRPC cohort, patients were divided into a hypermethylation group and a hypomethylation group with a cutoff value of 23.3%, based on the average methylation level of specific CpG sites (CpG# -39 to 65). (A) The difference of OS between the two groups. (B) The correlation between SRD5A2 promoter methylation level and OS. (C) The difference of PFS between the two groups. (D) The correlation between methylation level and PFS. (E to H) In the Met CRPC cohort, patients were divided into a hypermethylation group and a hypomethylation group with a cutoff value of 28.0%, based on the average methylation level of specific CpG sites (CpG# -39 to 65). (E) The difference of OS between the two groups. (F) The correlation between methylation level and OS. (G) The difference of PFS between the two groups. (H) The correlation between methylation level and PFS.

Furthermore, we expected to find the best promoter regions, which might be more predictive of the outcome of ADT treatment. Among the series of promoter regions, the region located at CpG# -39 to -2 had the most statistically significant value in the Local CRPC cohort (S1 Table). Higher methylation of this specific region (nucleotides -434 to -4 [CpG# -39 to CpG# -2]) was associated with better OS (11.3 ± 5.8 vs 6.4 ± 4.4 years, P = 0.001) and PFS (8.4 ± 5.4 vs 4.5 ± 3.9 years, P = 0.003) with a cutoff value of 37.9% (S1 Table and Fig 4). Similarly, in the Met CRPC cohort, higher methylation of this specific region was also significantly associated with OS (8.4 ± 4.3 vs 14.6 ± 8.2 years, P = 0.04) and PFS (5.0 ± 1.2 vs 10.0 ± 4.8 years, P = 0.002) with a cutoff value of 47% (S2 Table and S3 Fig). Our data suggest that hypermethylation in specific region, nucleotides -434 to -4 (CpG# -39 to CpG# -2) might be a good marker to predict the ADT sensitivity for CRPC patients.

Fig 4

SRD5A2 hypermethylation of specific CpG sites (CpG # -39 to -2) in the promoter region was correlated with OS and PFS of CRPC patients in the Local CRPC cohort.

Patients were divided into a hypermethylation group and a hypomethylation group with a cutoff value of 37.9%, based on the average methylation level of specific CpG sites (CpG# -39 to -2). (A) Unsupervised cluster analysis of the ratio of SRD5A2 promoter methylation on CpG methylation sites (CpG # -39 to -2). (B) The difference of OS between the hypermethylation group and hypomethylation group. (C) The correlation between methylation level and OS. (D) The difference of PFS between the two groups. (E) The correlation between SRD5A2 methylation and PFS.

In addition, multivariable analysis demonstrated that SRD5A2 methylation both in the whole promoter region and specific region (CpG# -39 to -2) was associated with OS independent from GS and PSA (S3 Table, whole promoter region: P = 0.03; CpG# -39 to -2: P = 0.02).

SRD5A2 promoter methylation was negatively associated with SRD5A2 expression

Finally, we sought to investigate if SRD5A2 promoter methylation is associated with SRD5A2 expression. IHC data demonstrated that the protein expression of SRD5A2 was negatively correlated with its methylation ratio both in the whole promoter region (CpG# -72 to CpG# 65) (R = 0.235, P = 0.0011) and specific region (CpG# -39 to CpG# -2) (R = 0.287, P = 0.0003) (Fig 5A–5C).

Fig 5

Protein expression of SRD5A2 was negatively correlated with SRD5A2 promoter methylation.

(A to C) IHC with anti-SRD5A2 antibody. (A) Representative pictures of IHC. (B) The immunoreactive score was correlated with SRD5A2 promoter methylation on CpG# -72 to 65. (C) The immunoreactive score was correlated with SRD5A2 promoter methylation on CpG# -39 to -2. (D to F) TCGA data analysis. (D) SRD5A2 promoter methylation in primary PCa. (E) SRD5A2 expression in primary PCa. (F) The association between SRD5A2 promoter methylation and SRD5A2 expression. Blue rows represented the methylation levels of SRD5A2 promoter region, yellow rows represented SRD5A2 expression, and each column represented one sample.

Then, we validated our finding using the publicly available TCGA dataset. The data of SRD5A2 promoter methylation from 502 primary PCa tissues and 50 normal control prostatic tissues were extracted from TCGA. We found that the promoter regions of SRD5A2 of PCa specimen were significantly hypermethylated compared with normal prostatic tissues (P < 0.0001, Fig 5D). To compare the expression of SRD5A2, 497 primary PCa tissues and 52 normal prostatic tissues were analyzed, and we found that the expression of SRD5A2 in primary PCa tissues was significantly lower than the normal prostatic tissues (P < 0.0001, Fig 5E). Furthermore, we found that the methylation levels of most sites of the SRD5A2 promoter were negatively correlated with expression of SRD5A2. Three of SRD5A2 promoter sites had the highest correlation with SRD5A2 expression, which were cg26638505 (CpG# -25, R = -0.489), cg06492144 (CpG# -35, R = -0.393) and cg08894761 (CpG# -25, R = -0.383), sites that overlap with CpG# -39 to CpG# -2 (Fig 5F). The data here further supports our finding that hypermethylation in specific region CpG# -39 to -2 might be a good marker to predict the ADT sensitivity for CRPC patients.

Discussion

SRD5A2 is the predominant isoform of 5-α reductase expressed in the prostate, whereas SRD5A1 represents less than 10% of total 5α-reductase levels in normal prostate cells [23]. In PCa cells, the expression of SRD5A2 decreases, but the expression of SRD5A1 increases [21-24]. These two enzymes have been associated with PCa initiation and PCa progression. For instance, studies on polymorphisms in SRD5A1 and SRD5A2 have suggested its association with PCa risk and PCa recurrence [25-27]. However, SRD5A2 rs9282858, the common nonsynonymous single nucleotide polymorphism was found not significantly associate with PCa risk [25]. Therefore, further studies are warranted to identify how the epigenetic change of these enzymes effects on PCa. To our knowledge, ours is the first study to analyze the association of epigenetic change of SRD5A2 with response to ADT for CRPC patients. In contradistinction to widely held assumptions that DNA methylation is correlated with cancer progression, our findings show that hypermethylation in the SRD5A2 promoter region, specifically CpG# -39 to -2, predicted better OS and PFS for patients with CRPC. The study suggests that quantitative SRD5A2 methylation analysis in a pre-treatment biopsy could allow identification of patients most likely to benefit and facilitate tailoring of ADT therapy. For example, PCa patients with SRD5A2 methylation may benefit less from intensified AR-directed therapy, or from early addition of docetaxel in castration-sensitive metastatic disease [28, 29]. Alternatively, patients with SRD5A2 methylation may respond differently to androgen signaling inhibitors or targeted therapies toward the estrogen receptor pathway may prove beneficial in this subset of patients.

Tumor cells can be activated by epigenetic alterations, and further use epigenetic processes to escape from chemotherapy and host immune surveillance. Therefore, targeting the epigenome, including DNA methylation and histone modifications has been a growing emphasis of recent drug discovery [18]. In our study, data from two cohorts demonstrated that there is hypermethylation of SRD5A2 promoter in CRPC compared with benign prostatic tissues (Fig 1). This result was further confirmed by analyzing TCGA data (Fig 5). Most interestingly, we found that SRD5A2 methylation in promoter regions, specifically at CpG# -39 to CpG# -2, is significantly associated with better survival for CRPC patients treated with ADT. Being consistent with the study for high-risk gliomas which showed that MGMT promoter methylation predicts better survival outcomes [30], our finding not only provides evidence that alteration of the epigenome is an important step in cancer progression, but also opens new windows of opportunities in personalized medicine for CRPC patients.

There are several methods to test for DNA methylation or a CpG island in the promoter region of a single gene. The most widely used methods include pyrosequencing, methylation-specific polymerase chain reaction (PCR), and direct Sanger sequencing. In this study we used the tNGBS technique to test the methylation of the SRD5A2 promoter region, which allowed us to analyze massive quantities of CpG sites for methylation [31, 32]. While we analyzed methylation of single CpG site in the SRD5A2 promoter region, our future study will identify whether single CpG site(s) methylation within a CpG island shore of the SRD5A2 gene contribute(s) to alteration in SRD5A2 expression and subsequent acquisition of treatment resistance.

Here, we present evidence that SRD5A2 promoter methylation could be used as a prognostic marker for CRPC patients treated with ADT. An explanation could be that SRD5A2 promoter hypermethylation causes the absence of SRD5A2 expression, and an androgenic to estrogenic switch in prostate tissue. This hypothesis is supported by our recent studies and others [17, 33–35]. In BPH tissues, we showed that the level of estradiol is dramatically elevated in prostatic samples with methylation of the SRD5A2 promoter, a condition that favors an estrogenic, as opposed to an androgenic, milieu in the prostate. The phosphorylation of estrogen receptor-α (ERα) in prostatic stroma is upregulated when SRD5A2 expression is absent [17]. During PCa initiation, not only androgens but also estrogens are required for malignant transformation [34, 35], suggesting that there is a role for AR/ERα cooperation in androgen targeted therapy resistance. Therefore, combination of treatments targeting both AR and ERα pathways may improve therapies for management of CRPC [34, 36]. Together, we postulate that SRD5A2 methylation in promoter regions, a condition that favors an estrogenic, as opposed to an androgenic milieu in the prostate, is significantly associated with better survival for CRPC patients treated with ADT. Further studies are warranted to examine whether hormonal interventions play a role in CRPC disease progression and treatment efficiency.

SRD5A2 DNA methylation profiling.

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Hypermethylation of SRD5A2 in the whole promoter region (CpG # -72 to 65) was correlated with overall survival (OS) and progression free survival (PFS) of castration-resistant prostate cancer (CRPC) patients in the metastatic (Met) CRPC cohort.

Patients were divided into a hypermethylation group and a hypomethylation group with a cutoff value of 40.0%, based on the average methylation level of all methylation sites. (A) Unsupervised cluster analysis of the ratio of SRD5A2 promoter methylation on all CpG methylation sites. (B) The difference of OS between the hypermethylation group and the hypomethylation group. (C) The correlation between methylation level and OS. (D & E) Patients were divided into a hypermethylation group and a hypomethylation group with a cutoff value of 32.5%, based on the average methylation level of all methylation sites. (D) The difference of PFS between hypermethylation group and hypomethylation group. (E) The correlation between SRD5A2 promoter methylation level and PFS.

(TIF)

Click here for additional data file.

In the Met CRPC cohort, patients were divided into a hypermethylation group and a hypomethylation group with a cutoff value of 47%, based on the average methylation level of specific CpG sites (CpG# -39 to -2).

(A) Unsupervised cluster analysis of the ratio of SRD5A2 promoter methylation. (B) The difference of OS between the two groups. (C) The correlation between methylation level and OS. (D & E) Patients were divided into a hypermethylation group and a hypomethylation group with a cutoff value of 37.8%, based on the average methylation level of specific CpG sites (CpG# -39 to -2). (D) The difference of PFS between the two groups. (E) The correlation between methylation level and PFS.

(TIF)

Click here for additional data file.

Supporting material and methods [16,17,37]

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(DOCX)

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Cutoff values of SRD5A2 promoter methylation in Local CRPC cohort.

(DOCX)

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Cutoff values of SRD5A2 promoter methylation in Met CRPC cohort.

(DOCX)

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Multivariable regression analyses.

(DOCX)

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15 Jan 2020

PONE-D-19-33856

Methylation of SRD5A2 promoter predicts a better outcome for castration-resistant prostate cancer patients undergoing androgen deprivation therapy

PLOS ONE

Dear Dr. Olumi:

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit and my decision is "Minor Revision" to fully meet  the PLOS ONE’s publication criteria (as it currently stands). Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewer #1:

Understanding prostate cancer tumor progression mechanism and potential targets for treatment is very important. The 5a-Reductase types 1 and 2, encoded by SRD5A1 and SRD5A2, are key enzymes that catalyze the conversion of testosterone to dihydrotestosterone, androgen receptor (AR) agonist in prostate cells. 5a-Reductase type 2 is the predominant isoform expressed in the normal prostate. However, its expression decreases during prostate cancer (PCa) progression, whereas SRD5A1 increases, and the mechanism underlying this transcriptional regulatory switch is still unknown. In this research manuscript, Wang, et al  reports that methylation of SRD5A2 promoter occurs frequently in CRPC patients treated with ADT, and provided  important patient data (from local cohorts and TCGA). Furthermore, authors demonstrated the negative correlation between promoter methylation of SRD5A2 and SRD5A2 expression.

Comments:

1. The major strength of the study is the patient data from two cohorts to validate their hypothesis and comparison of findings with the TCGA data. Paper is well written and all the experiments with perfect controls in place.

2. Authors shall provide explanation about the basis on which CpG methylation sites were clustered into 3 modules, (CpG# -72 to -42; CpG# -40 to -35 and -31 to -19; CpG# -34 to -32 and -18 to 65 in CRPC patients) whereas methylation sites were clustered into 2 modules (CpG# -72 to -35, -31; CpG# -34 to -32 and -30 to 65 in metastatic CRPC cohorts).

3. Have the authors found any relation between the expression of SRD5A2 and survival in local CRPC patient cohorts, although they have shown through the TCGA data analyzed by MEXPRESS?

4. In Figure 3, 4 and 5 only 3 H has a value of “r”=0.78 (positive correlation), in other panels, “r” shows weak correlation, would the authors explain why.

5. In Figure 5 B and C, “r” and “P” values are not shown. Authors shall add the “r” and “P” in figure 5 B and C.

6. Have authors found any correlation between Androgen Receptor expression and SRD5A2 expression in local patient cohorts?

7. Authors have used local patent cohorts, please provide a link where data can be accessible if possible as per the PLosOne data sharing policy.

Reviewer #2:

The present study is a provacative analysis on the methylation status of the SDR5A2 promoter, which encodes an enzyme that metabolizes testosterone to the more potent androgen DHT, in castrate-resistant prostate cancer (CRPC)( specimens. Although the numbers of specimens analyzed in the present data are relatively small, the findings do show that there is a significant trend for promoter hypermethylation in CRPC specimens compared to benign prostate tissue. Most significantly, the higher ratio of promoter hypermethylation was positively associated with a significant increase in time to progression and patient survival when the patient data was interrogated. Immunohistochemistry analysis of SDR5A2 protein levels inversely correlated with the gene methylation levels, although this data has marked overlap and requires further follow-up. The data were further studied in the TCGA database which supported the primary data in this manuscript. Although the number of metastatic samples was very low in this study (n=12), the data indicate that the SDR5A2 methylation status is likewise able to predict patient survival.  The data are well discussed and fit well into emerging findings regarding the altered hormonal milieu in CRPC that drives androgen resistance.

Comments:

1. The paper would be improved by cell-based interrogations that replicate the specimen-based observations and support the conclusions, i.e. modifications of SDR5A2 methylation status change expression of this gene and alter its protein levels as well as steroid metabolism.

2. At present, the studies are merely correlative and direct studies are warranted.

________________________

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We look forward to receiving your revised manuscript.

Kind regards,

Mohammad Saleem,

University of Minnesota

Academic Editor, PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

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2. We noticed minor instances of text overlap with the following previous publication(s), which need to be addressed:

(1) https://www.mdpi.com/2073-4425/9/9/429/htm

(2) https://www.sciencedirect.com/science/article/abs/pii/S0046817719300747?via%3Dihub

(3) https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.7b00070

(4) https://onlinelibrary.wiley.com/doi/full/10.1002/cncr.30071

The text that needs to be addressed involves the Discussion section.

In your revision please ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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24 Jan 2020

Reviewer #1:

Understanding prostate cancer tumor progression mechanism and potential targets for treatment is

very important. The 5a-Reductase types 1 and 2, encoded by SRD5A1 and SRD5A2, are key enzymes

that catalyze the conversion of testosterone to dihydrotestosterone, androgen receptor (AR) agonist in

prostate cells. 5a-Reductase type 2 is the predominant isoform expressed in the normal prostate.

However, its expression decreases during prostate cancer (PCa) progression, whereas SRD5A1

increases, and the mechanism underlying this transcriptional regulatory switch is still unknown. In this

research manuscript, Wang, et al reports that methylation of SRD5A2 promoter occurs frequently in

CRPC patients treated with ADT, and provided important patient data (from local cohorts and TCGA).

Furthermore, authors demonstrated the negative correlation between promoter methylation of SRD5A2

and SRD5A2 expression.

Comments:

1. The major strength of the study is the patient data from two cohorts to validate their hypothesis and

comparison of findings with the TCGA data. Paper is well written and all the experiments with perfect

controls in place.

Response: Thanks for reviewer’s positive comments.

2. Authors shall provide explanation about the basis on which CpG methylation sites were clustered

into 3 modules, (CpG# -72 to -42; CpG# -40 to -35 and -31 to -19; CpG# -34 to -32 and -18 to 65 in

CRPC patients) whereas methylation sites were clustered into 2 modules (CpG# -72 to -35, -31; CpG#

-34 to -32 and -30 to 65 in metastatic CRPC cohorts).

Response: Thanks for this suggestion. In the section of Statistical Analysis, we explained: R

package “pheatmap” which was used to draw the heatmap of methylation sites in the two

cohorts, respectively. Those methylation sites were automatically clustered according to the

“Euclidean” distance.

3. Have the authors found any relation between the expression of SRD5A2 and survival in local CRPC

patient cohorts, although they have shown through the TCGA data analyzed by MEXPRESS?

Response: Our two cohorts include patients with metastatic CRPC. As a result, we have

focused our study on the methylation of SRD5A2 promoter and survival in patients with

metastatic CRPC. To address the reviewer’s concern, as mentioned by the reviewer, we utilized

the TCGA data to assess SRD5A2’s promoter methylation. In the near future studies, we plan to

evaluate if the expression of SRD5A2 is associated with survival of localized CRPC patients.

4. In Figure 3, 4 and 5 only 3H has a value of “r”=0.78 (positive correlation), in other panels, “r” shows

weak correlation, would the authors explain why.

Response: Thanks for this careful review. In Figure 3, 4 and 5, the majority of analyses showed

the significant correlations (P<0.05), which are consistent with the Kaplan Meier survival curve.

The low R value might be caused by the small number of subjects.

5. In Figure 5 B and C, “r” and “P” values are not shown. Authors shall add the “r” and “P” in figure 5 B

and C.

Response: Thanks for this suggestion. The “r” and “P” values in figure 5 B and C have been

added.

6. Have authors found any correlation between Androgen Receptor expression and SRD5A2

expression in local patient cohorts?

Response: Thanks for this question. At this moment, we did not evaluate the correlation

between AR and SRD5A2 expression.

7. Authors have used local patient cohorts, please provide a link where data can be accessible if

possible as per the PLosOne data sharing policy.

Response: Thanks for this suggestion. We have made the raw informatics data accessible

online. Link: https://doi.org/10.7910/DVN/MPAM2H

Reviewer #2:

The present study is a provacative analysis on the methylation status of the SDR5A2 promoter, which

encodes an enzyme that metabolizes testosterone to the more potent androgen DHT, in castrateresistant

prostate cancer (CRPC)( specimens. Although the numbers of specimens analyzed in the

present data are relatively small, the findings do show that there is a significant trend for promoter

hypermethylation in CRPC specimens compared to benign prostate tissue. Most significantly, the

higher ratio of promoter hypermethylation was positively associated with a significant increase in time to

progression and patient survival when the patient data was interrogated. Immunohistochemistry

analysis of SDR5A2 protein levels inversely correlated with the gene methylation levels, although this

data has marked overlap and requires further follow-up. The data were further studied in the TCGA

database which supported the primary data in this manuscript. Although the number of metastatic

samples was very low in this study (n=12), the data indicate that the SDR5A2 methylation status is

likewise able to predict patient survival. The data are well discussed and fit well into emerging findings

regarding the altered hormonal milieu in CRPC that drives androgen resistance.

Comments:

1. The paper would be improved by cell-based interrogations that replicate the specimen-based

observations and support the conclusions, i.e. modifications of SDR5A2 methylation status change

expression of this gene and alter its protein levels as well as steroid metabolism.

Response: Thanks for the reviewer’s great suggestion on the mechanism study.

Utilizing prostate cancer LnCap, DU145 and other cell lines, we are working to

understand whether and how the modification of SRD5A2 methylation status alters

steroid metabolism. This suggestion will be part of future publications which is some

time away.

2. At present, the studies are merely correlative and direct studies are warranted.

Response: Again, we thank the reviewer’s for his/her insight. Currently, we are using cultured

cells to identify whether there is a cause and effect relationship between SRD5A2 methylation

and better survival for CRPC patients using in-vitro and in-vivo model systems.

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file

naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliati

ons.pdf

Response: We have carefully reviewed and revised the manuscript to meet PLOS ONE's style

requirements.

2. We noticed minor instances of text overlap with the following previous publication(s), which need to

be addressed:

(1) https://www.mdpi.com/2073-4425/9/9/429/htm

Response: It has been quoted and rephrased. Please see page 21.

(2) https://www.sciencedirect.com/science/article/abs/pii/S0046817719300747?via%3Dihub

Response: It has been addressed. Please see page 22.

(3) https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.7b00070

Response: This literature is not related to our study. Not sure where the text overlap is.

(4) https://onlinelibrary.wiley.com/doi/full/10.1002/cncr.30071

Response: It has been quoted and rephrased. Please see page 19.

The text that needs to be addressed involves the Discussion section.

In your revision please ensure you cite all your sources (including your own works), and quote or

rephrase any duplicated text outside the methods section. Further consideration is dependent on these

concerns being addressed.

Response: Thanks for this careful review. We have cited all the sources, quoted and rephrased

all the duplicated text.

3. Please provide additional details regarding participant consent.

In the ethics statement in the Methods and online submission information, please ensure that you have

specified (i) whether consent was informed and (ii) what type you obtained (for instance, written or

verbal, and if verbal, how it was documented and witnessed).

Response: We have specified in the Section of Patient specimen (page 8): Written consent was

obtained from the study participants for the two cohorts.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted

after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial

Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu),

and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and

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https://www.youtube.com/watch?v=_xcclfuvtxQ

Response: We have added and validated the ORCID for the corresponding author.

5. Please upload a copy of Figure 6, to which you refer in your text on page 16. If the figure is no

longer to be included as part of the submission please remove all reference to it within the text.

Response: Thanks for this careful review. This is the typo, which should refer to figure 5 on

current page 20.

6. Please include captions for your Supporting Information files at the end of your manuscript, and

update any in-text citations to match accordingly. Please see our Supporting Information guidelines for

more information: http://journals.plos.org/plosone/s/supporting-information

Response: Captions for the Supporting Information files have been included at the end of

manuscript, and in-text citations have been updated accordingly.

14 Feb 2020

Methylation of SRD5A2 promoter predicts a better outcome for castration-resistant prostate cancer patients undergoing androgen deprivation therapy

PONE-D-19-33856R1

Dear Dr. Olumi,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With Kind Regards,

Mohammad Saleem, Academic Editor-PLOS ONE

University of Minnesota-Minneapolis

19 Feb 2020

PONE-D-19-33856R1

Methylation of SRD5A2 promoter predicts a better outcome for castration-resistant prostate cancer patients undergoing androgen deprivation therapy

Dear Dr. Olumi:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. MOHAMMAD Saleem

Academic Editor

PLOS ONE