BERKELEY, CA (UroToday.com) - Adrenal androgens have been widely reported to contribute to the pool of active androgen receptor (AR) ligands in prostate tumors.
To date, the downstream metabolism of weak adrenal androgens dehydroepiandrosterone, dehydroepiandrosterone sulphate, and androstenedione (A4) has been shown to yield active androgens via the activity of the 5α-reductase (SRD5A) and 17β-hydroxysteroid dehydrogenase (17βHSD) enzymes. Unique to the adrenal is also 11β-hydroxyandrostenedione (11OHA4), which was first isolated from human adrenal tissue in 1955.[1] This steroid had been overlooked for more than 50 years even though early research had shown that it could be further metabolized to 11β-hydroxy-5α-androstanedione (11OH-5α-dione), by the 5α-reduction of the C4/C5 double bond, and to 11β-hydroxyandrosterone (11OHAST).[2] It was only in 1989 that SRD5A was cloned and characterized and shown to convert both testosterone (T) and A4 to dihydrotestosterone (DHT) and 5α-androstanedione, respectively,[3] of which the latter is the preferred precursor of DHT in the alternate pathway.[4] The physiological role of 11OHA4 was being questioned in the eighties as the steroid had been shown to be a weak androgen. It was thought that 11OHA4 production may be a mechanism by which adrenal androgens are inactivated. It was also suggested that 11OHA4 could be used as a marker for adrenal A4 production, allowing for the discrimination between adrenal and gonadal androgen production in clinical conditions characterized by adrenal androgen excess. Interest in 11OHA4 waned, and in time the steroid was forgotten.
Attention is being paid to 11OHA4 once more – it was quantified for the first time in 2012 and shown to be one of the major metabolites produced in H295R adrenal cells, with basal levels increasing 4.5-fold to 390 nM upon stimulation.[5] 11-ketoandrostendione (11KA4) was also detected, albeit at low levels. In a subsequent study, analyses of adrenal vein samples pre- and post-ACTH stimulation, showed basal 11OHA4 being produced at levels 2-fold higher than A4, with ACTH increasing 11OHA4 levels to 811 nM. Low levels of 11KA4, 11β-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT) were also detected under both basal and stimulated conditions.[6] It is interesting to note that the levels of 11OHA4 were ± 100-fold higher than T and its derivatives under both basal conditions and ACTH stimulation.
We explored the downstream metabolism of 11OHA4 and 11KA4 by 17βHSD and found that reactions involving 11OHA4 are not readily catalyzed by 17βHSD type 3 or type 5, while reactions involving 11KA4 are. Both 11OHA4 and 11KA4 are reduced by SRD5A to form 11OH-5α-dione and 11keto-5α-androstanedione (11K-5α-dione) while 11OHT and 11KT are also metabolized by SRD5A to produce 11β-hydroxydihydrotestosterone (11OHDHT) and 11-ketodihydrotestosterone (11KDHT), respectively.[7, 8] We identified the 5α-reduced C11 metabolites by accurate mass determination as none of these steroids are presently available commercially. Fortunately, limits of detection and quantification have been significantly improved using technology such as liquid and gas chromatography coupled with tandem mass spectrometry (LC- MS, GC-MS) with sample analyses also being significantly more efficient using ultra-performance liquid chromatography-MS/MS (UPLC-MS/MS), both in terms of throughput and the number of metabolites which can be analysed in a single chromatographic step.
Having confirmed in LNCaP cells that 11OHA4 is metabolized to 11KA4, 11KT and 11OH-5α-dione, indicative of 11βHSD2 and SRD5A activity - with 11KA4 being converted to 11KT by 17βHSD - we investigated a second tier of downstream metabolism. Once produced in LNCaP cells, 11OH-5α-dione and 11OHDHT were both metabolized by 11βHSD2 to 11K5α-dione and 11KDHT respectively, with 11K5α-dione also being metabolized by 17βHSD to 11KDHT, further adding to the complexity of the steroid milieu. Subsequent AR transactivation studies showed that the 5α-reduction of 11OHT and 11KT, yielding 11OHDHT and 11KDHT, respectively, resulted in significant increases in androgenic activity. At physiologically relevant concentrations of 1 nM, 11KT and 11OHDHT act as partial AR agonists while 11KDHT acts as a full agonist, comparable to DHT.[7, 8]
Underlying these findings are the roles of the adrenal enzymes, cytochrome P450 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) as well as 11βHSD2. We have shown that 11βHSD type 1 and 2 catalyze the interconversion of 11KA4/11OHA4 and 11KT/11OHT. These enzymes have classically been associated with the activation/inactivation of glucocorticoids. 11βHSD2 was previously shown to be expressed in LNCaP cells, assaying the inactivation of cortisol.[9, 10] The fact that both enzymes catalyze reactions involving 11-hydroxy and 11-keto androgens provide new perspectives on the activation of steroid receptor ligands - with 11βHSD2 catalyzing the formation of 11KT and 11KDHT which are more active AR ligands than their 11-hydroxy precursor steroids. This is a reversal of the ligand inactivation role of 11βHSD2 whereby the enzyme converts the C11 hydroxy group of cortisol and corticosterone to a keto group, modulating the binding of active glucocorticoids to the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). Little is known about the role of 11βHSD2 in the interplay between the MR, GR and AR in prostate cancer and their responses to steroids influencing transcriptional activation. In addition, supplying 11βHSD2 with androgen substrates are CYP11B1 and CYP11B2, catalyzing the production of 11OHA4 and 11OHT. The physiological relevance of CYP11B2’s catalytic activity towards T in the adrenal is perhaps questionable as this enzyme is expressed in the zona glomerulosa where it catalyses the production of mineralocorticoids. It has, however, been reported that both CYP11B1 and CYP11B2 are expressed in prostate carcinomas.[11, 12] It is thus possible that androgens may be hydroxylated by CYP11B in prostate cancer tissue, and subsequently converted by 11βHSD2 to more active androgens.
11OHA4, exhibiting negligible androgenic activity itself, has not been considered a specific target in treatment regimes, even though its metabolism yields androgens which may be contributing significantly to CRPC. This steroid navigates through the same catalytic pathways in which DHT is produced, albeit with an additional hydroxy or keto group at the C11 position of the steroid molecule. What remains to be determined is whether the C11 substitutions hinder the inactivation by 3α-hydroxysteroiddehydrogenase (3αHSD), and the subsequent glucuronidation and elimination of these metabolites.
Our present understanding of steroid metabolism has been limited to that which we have come to accept as the known pathways, confined to those steroids which we have been able to analyze. It would seem that current treatments targeting specific steroidogenic enzymes, based on what we accept as known, have their drawbacks - together with the desired outcome of a clinical approach, the potential unwanted outcomes thereof need to be addressed. In abiraterone we have yet another inhibitor of a steroidogenic enzyme - inhibiting steroid 17α-hydroxylase/17,20-lyase (CYP17A1). The catalytic characteristics of CYP17A1 and clinical implications of genetic mutations have been studied for decades. In the adrenal, CYP17A1 catalyses the steroid shunt into the mineralocorticoid, glucocorticoid, and adrenal androgen pathways, and it comes as no surprise that its inhibition results in mineralocorticoid excess and cardiovascular risk. In addition, increased metabolites in the mineralocorticoid pathway will feed into other pathways. It is possible to extend pathways with new analytical technologies, placing existing as well as novel steroids within these pathways. The identification of novel steroids has been greatly facilitated by state-of-the-art analytical tools - automated accurate mass determination of an unknown steroid has replaced numerous chromatographic steps, extraction and crystallization, melting point determination followed by infrared spectrometric analysis, which once was the order of the day when identifying novel steroids. A better, more comprehensive understanding of steroid metabolism could result in improved clinical approaches in CRPC as well as more accurate assessments of predicted outcomes.
References:
- Touchstone, H.C., Glazer, L., Cooper, D.Y., Roberts, J.M. The isolation of delta 4-androstene-11 beta-ol-3,17-dione from human adrenal incubates. J. Clin. Endocrinol. Metab. 15. (1955): 382–384.
- Masuda, M. Urinary ketosteroid excretion patterns in congenital adrenal hyperplasia. J. Clin. Endocrinol. Metab. 17. (1957): 1181–1190.
- Andersson, S., Bishop, R.W., Russell, D.W. Expression Cloning and Regulation of steroid 5α-Reductase, an Enzyme Essential for Male Sexual Differentiation. JBC. 264. no. 27 (1989): 16249-16255.
- Chang, K.H., Li, R., Papari-Zareei, M., Watumull, L., Zhao, Y.D., Auchus, R.J., Sharifi, N. Dihydrotestosterone synthesis bypasses testosterone to drive castrationresistant prostate cancer. Proc. Natl. Acad. Sci. USA 108. (2011): 13728–13733.
- Schloms, L., Storbeck, K.H., Swart, P., Gelderblom, W.C., Swart, A.C. The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: Quantification of steroid intermediates and end products in H295R cells. J. Steroid Biochem. Mol. Biol. 128. (2012) 128–138.
- Rege, J., Nakamura, Y., Satoh, F., Morimoto, R., Kennedy, M.R., Layman, L.C., Honma, S., Sasano, H., Rainey, W.E. Liquid chromatography-tandem mass spectrometry analysis of human adrenal vein 19-carbon steroids before and after ACTH stimulation. J. Clin. Endocrinol. Metab. 98. (2013): 1182–1188.
- Swart, A.C., Schloms, L., Storbeck, K.H., Bloem, L.M., Toit, T.D., Quanson, J.L., Rainey, W.E., Swart, P. 11β-Hydroxyandrostenedione, the product of androstenedione metabolism in the adrenal, is metabolized in LNCaP cells by 5alpha-reductase yielding 11beta-hydroxy-5alpha-androstanedione. J. Steroid Biochem. Mol. Biol. 138. (2013): 132–142.
- Storbeck, K.H., Bloem, L.M., Africander, D., Schloms, L., Swart, P., Swart, A.C. 11β -Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: A putative role in castration resistant prostate cancer? Mol. Cell Endocrinol. 377. (2013): 135–146.
- Page, N., Warriar, N., Govindan, M.V. 11β-Hydroxysteroid dehydrogenase and tissue specificity of androgen action in human prostate cancer cell LNCaP. J. Steroid Biochem. Mol. Biol. 49. (1994): 173-181.
- Dovio, A., Sartori, M.L., de Francia, S., Mussino, S., Perotti, P., Saba, L., Abbadessa, G., Racca, S., Angeli, A. Differential expression of determinants of glucocorticoid sensitivity in androgen-dependent and androgen-independent human prostate cancer cell lines. J. Steroid Biochem. Mol. Biol. 116. (2009): 29-36.
- Biancolella, M., Valentini, A., Minella, D., Vecchione, L., D’Amico, F., Chillemi, G., Gravina, P., Bueno, S., Prosperini, G., Desideri, A., Federici, G., Bernardini, S., Novelli, G. Effects of dutasteride on the expression of genes related to androgen metabolism and related pathway in human prostate cancer cell lines. Invest. New Drugs. 25. (2007): 491–497.
- Mitsiades, N., Sung, C.C., Schultz, N., Danila, D.C., He, B., Eedunuri, V.K., Fleisher, M., Sander, C., Sawyers, C.L., Scher, H.I. Distinct patterns of dysregulated expression of enzymes involved in androgen synthesis and metabolism in metastatic prostate cancer tumors. Cancer Res 72. (2012): 6142–6152
Written by:
Amanda C. Swart, PhD as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.
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