Lessons Learned About Prostatic Transformation from the Age-Related Methylation of 5α-Reductase Type 2 Gene

Abstract

This commentary highlights the article by Ge et al, which proposes the use of methylation and expression of SRD5A2 as a gene signature to tailor therapies for prostatic diseases.

A nearly universal plight among aging males throughout the world is the hyperplastic growth of the transition zone within the prostate, known as benign prostatic hyperplasia (BPH).¹ With approximately 50% of males throughout the world developing BPH, it is the most common neoplasia in humans. Although the etiology of BPH is not fully resolved, it is known that its maintenance requires a continuous supply of androgen. This is documented by the observation that testicular suppression via luteinizing hormone releasing hormone superagonist reversibly reduces prostatic size and clinical BPH symptoms.² Likewise, the normal prostate is dependent on adequate chronic androgenic stimulation for both its fetal development and adult maintenance.³

Treatment with 5α-Reductase Inhibitors in BPH

In both BPH and normal prostate, androgen stimulation requires irreversible conversion within the prostate of circulating testosterone into the more potent androgen, dihydrotestosterone, via enzymatic activity of 5α-steroid reductase (SRD5A). There are at least three SRD5A genetic isoforms expressed within the prostate^4,5; however, only germline inheritance of loss-of-function mutations in the SRD5A2 gene is known to retard normal prostate development, thus preventing BPH.³ On the basis of these observations, orally active drugs (eg, finasteride and dutasteride), which reversibly inhibit SRD5A1 and irreversibly inhibit SRD5A2 enzyme,⁶ have been clinically developed for the treatment of BPH. Chronic daily treatment with these 5α-reductase inhibitors results in a decrease in prostate size by approximately 25% within 4 to 6 months and is further associated with improvement in clinical symptoms.^7,8 Interestingly, approximately 30% of patients do not respond to such chronic 5α-reductase inhibitor treatment.^7,8

This overall response rate is intriguing because Niu et al⁹ reported that prostatic expression of the SRD5A2 gene is variable and similarly absent in one third of aging men with BPH and that this down-regulation is associated with hypermethylation of CpG islands in the promoter of SRD5A2 gene detected using methylation-specific pull-down PCR. In the current issue of The American Journal of Pathology, this group proposes an inflammation-driven process involving DNA methyltransferase 1–dependent tumor necrosis factor-α/NF-κB/IL-6 signaling pathway as a mechanism for such epigenetic silencing of the SRD5A2 gene in BPH.¹⁰ In this study, transurethral resected BPH tissue from patients without 5α-reductase inhibitor treatment was analyzed. Therefore, the study limits assessing whether the hypermethylation of SRD5A2 gene may identify patients who will lack clinical response to inhibition of 5α-reductase. To address this important clinical issue, needle biopsy tissue could be analyzed from BPH patients who are administered 5α-reductase inhibitors to test prospectively if SRD5A2 gene hypermethylation may predict clinical response.

What Does Hypermethylation of the SRD5A2 Gene Inform About Prostatic Neoplasia?

The determination of whether hypermethylation of SRD5A2 gene may predict clinical response to treatment with 5α-reductase inhibitor will be significant. CpG island hypermethylation of gene regulatory elements is common in cancers (eg, CpG hypermethylation of the 5′ promoter region of pi-class glutathione-S-transferase gene is the most common and earliest genetic change in human prostate carcinogenesis¹¹). Until recently, it was not appreciated that in many tissues, stochastic methylation drift occurs in many genes during aging because of the imperfect maintenance of epigenetic marks of methylation driven by chronic inflammation.¹² This drift generates epigenetic mosaicism in aging stem cells. Although the methylation-specific pull-down PCR used in the discussed study is an exquisitely sensitive assay for detecting methylation drift, it does not allow determination of whether such methylation changes are passenger versus driver genetic changes in BPH tissue.¹⁰ If such hypermethylation-induced epigenetic silencing of the SRD5A2 gene provided no growth advantage, then these would be passenger changes and should be detected as polyclonal variation within an epigenetic mosaicism in BPH tissue. In contrast, if chronic inflammation-induced epigenetic silencing of SRD5A2 drives neoplastic expansion of the effected stem cells and their progeny, then it should be detected as clonally derived. To resolve this issue of whether the epigenetic silencing of SRD5A2 is a passenger versus driver in the development of prostatic neoplasia, the clonality of such SRD5A2 hypermethylation needs to be evaluated using either bisulfite sequencing or methyl-binding domain–single-nucleotide polymorphism technology.¹³

This resolution is significant for several reasons. First, the stromal and epithelial compartments within the prostate are organized via adult stem cell units.¹ Second, chronic inflammation occurs in >75% of BPH tissues.¹⁴ Combination of these observations suggests that chronic inflammation could provide a perfect storm for stochastic methylation drift-induced epigenetic reprograming of stems cells within the prostate of aging males, needed as a driver of neoplastic growth.

Therefore, it is important to point out that in the study by Ge et al,¹⁰ transurethral resected BPH tissues were analyzed, which contain both stromal and epithelial cells, and that in the adult prostate, SRD5A2 gene is normally expressed by both of these cell types.^4,5 Thus, in the future studies, it will be important to fractionate such BPH tissue to evaluate the clonality of the CpG hypermethylation–induced epigenetic silencing of the SRD5A2 gene in both cell types. If it is clonal in either of these cell types, this would strongly suggest that down-regulation of SRD5A2 activity must have a growth advantage for stem cells and their progeny within the aging prostate. In addition, future studies need to evaluate whether the other SRD5A isoforms (ie, SRD5A1 and SRD5A3) expressed in prostate tissue^4,5 are also down-regulated. This is particularly important, because recent clinical trials have suggested that chronic (>5 years) treatment of aging males with oral 5-α reductase inhibitors decreases the incidence of low-grade (ie, Gleason score, 6), but enhances the incidence of higher-grade (ie, Gleason score, 7 to 10), localized prostate cancer.¹⁵

What Drives Chronic Inflammation in the Prostate?

In addition to the findings of Olumi et al,¹⁰ other studies also support the hypothesis that chronic inflammation induces epigenetic reprogramming, resulting in a growth advantage in both BPH and prostate cancer during aging. With regard to the development of BPH, there is an increase in the ratio of prostate stromal/epithelial area, going from a 3:1 ratio in nonhyperplastic normal prostates of young men to a ratio of 5:1 in BPH tissue of older men.¹⁶ This change is associated with an increase in cellular turnover in both the stromal and epithelial compartments of BPH versus normal prostate tissue.¹⁷ Two thirds of this stromal compartment in BPH tissue is composed of smooth muscle (SM),¹⁸ and BPH is associated with subtle epigenetic reprogramming in the phenotype of these SM cells, as demonstrated by their down-regulation in SM myosin heavy chain¹⁹ and up-regulation of α2 macroglobulin mRNA expression.²⁰ The mechanism for such changes is not fully resolved, but it is known that SM cells switch phenotype from contractive to proliferative in response to extrinsic and/or intrinsic stimuli, a process termed SM phenotype modulation.²¹

There are data supporting the concept that such SM phenotype modulation occurs in BPH due to a chronic immune inflammatory process.²² This idea is based on the fact that nearly all BPH specimens contain inflammatory infiltrates, but no bacterial or foreign antigens have been identified.^14,22 The infiltrate consists predominantly of chronically activated CD4⁺ T lymphocytes, which are permanently recruited to prostate tissue via elevated expression of IL-15 and interferon ɤ, proinflammatory cytokines produced by prostate SM and infiltrating T cells, respectively.^22,23 Dysregulation of the immune response in BPH is further compounded by elevated expression of the proinflammatory IL-17 by T cells stimulating enhanced production of IL-6 and IL-8, which themselves stimulate stromal growth, further increasing IL-15 levels.^22,23 These combinational events thus initiate a chronic inflammatory process. Such a chronic inflammatory process amplifies disruption of the barrier function of the epithelial tight junctions, allowing more autoantigens (eg, prostate-specific antigen and human glandular kallikrien-2) to leak into the prostate stromal compartment, inducing a vicious cycle of chronic inflammation within the prostate.^24,25

In conclusion, such chronic inflammation provides both a driving force for induction of epigenetic changes and a selective microenvironment for the outgrowth of neoplastic cells in the prostate of men as they age. Thus, inhibiting such chronic inflammation within the prostate of aging males is a promising approach for chemoprevention for both BPH and prostate cancer.^24,25