Distinct patterns of dysregulated expression of enzymes involved in androgen synthesis and metabolism in metastatic prostate cancer tumors

Abstract

Androgen receptor (AR) signaling persists in castration-resistant prostate carcinomas (CRPC), because of several mechanisms that include increased AR expression and intratumoral androgen metabolism. We investigated the mechanisms underlying aberrant expression of transcripts involved in androgen metabolism in CRPC. We compared gene expression profiles and DNA copy number alteration (CNA) data from 29 normal prostate tissue samples, 127 primary prostate carcinomas (PCa), and 19 metastatic PCas. Steroidogenic enzyme transcripts were evaluated by quantitative reverse transcriptase PCR in PCa cell lines and circulating tumor cells (CTC) from CRPC patients. Metastatic PCas expressed higher transcript levels for AR and several steroidogenic enzymes, including SRD5A1, SRD5A3, and AKR1C3, whereas expression of SRD5A2, CYP3A4, CYP3A5, and CYP3A7 was decreased. This aberrant expression was rarely associated with CNAs. Instead, our data suggest distinct patterns of coordinated aberrant enzyme expression. Inhibition of AR activity by itself stimulated AKR1C3 expression. The aberrant expression of the steroidogenic enzyme transcripts was detected in CTCs from CRPC patients. In conclusion, our findings identify substantial interpatient heterogeneity and distinct patterns of dysregulated expression of enzymes involved in intratumoral androgen metabolism in PCa. These steroidogenic enzymes represent targets for complete suppression of systemic and intratumoral androgen levels, an objective that is supported by the clinical efficacy of the CYP17 inhibitor abiraterone. A comprehensive AR axis-targeting approach via simultaneous, frontline enzymatic blockade, and/or transcriptional repression of several steroidogenic enzymes, in combination with GnRH analogs and potent antiandrogens, would represent a powerful future strategy for PCa management.

Fig. 1. Pathways of testosterone/DHT biosynthesis and metabolism, associated enzymes and their expression in metastatic PCa specimens

A. Cholesterol, the precursor of all steroidogenesis, is converted to DHT via several enzymatic steps: In the Δ5 pathway (named after the presence of a double carbon bond in the C5 position of the A steroid ring; steroids highlighted in green) and the Δ4 pathway (steroids highlighted in light red), testosterone is synthesized and then reduced by 5α-reductases to DHT, that has a ~5–10-fold higher affinity for AR. Androgen precursors can also be reduced before testosterone synthesis, generating an alternate pathway (“backdoor pathway”, steroids highlighted in light blue) that bypasses testosterone and leads to DHT. This pathway has been proposed to be active in prostate tissue, in particular prostate cancer (17). Recently, it was demonstrated that the dominant route of DHT synthesis in CRPC bypasses testosterone (23), and instead requires 5α-reduction of androstenedione by SRD5A1 to 5α-androstanedione (highlighted in yellow), which is then converted to DHT. Testosterone and DHT are oxidized (via cytochrome P450 3A oxidases) followed by conjugation to glucuronides (via uridine diphospho-glucuronosyl transferases UGT2B7, UGT2B15 and UGT2B17), that are then excreted. Enzymes involved in promoting testosterone/DHT synthesis are highlighted in green, while enzymes promoting their metabolism/inactivation are highlighted in dark blue. The target sites of clinically relevant inhibitors are also shown (Figure modified from (19)). B. Heatmap of outliers (red: overexpressed transcript, blue: underexpressed transcript) for AR and transcripts involved in androgen metabolism in the metastatic PCa specimens (outlier expression compared to the distribution of expression in normal prostate samples, see Methods and (26)).

Fig. 2

Heatmap of outliers (red: overexpressed transcript, blue: underexpressed transcript) for AR and transcripts involved in androgen metabolism in the primary PCa specimens (outlier expression compared to the distribution of expression in normal prostate samples, see Methods and (26)).

Fig. 3

Boxplots of average mRNA expression (log2-based) for AR, AKR1C3, SRD5A1, SRD5A3, SRD5A2, CYP3A4, CYP3A5, and CYP3A7 in normal prostate tissue, primary PCas and metastatic PCas. We found increased expression of AR, AKR1C3, SRD5A1, and SRD5A3, and decreased expression of SRD5A2, CYP3A4, CYP3A5 and CYP3A7 in metastatic PrCa. **: P<0.01 vs both normal tissue and primary carcinomas; *: P<0.01 vs normal tissue. Complete results are presented in Suppl. Table 3.

Fig. 4

Integration of expression outlier data with CNA analysis for AR and genes involved in androgen metabolism reveals that only a small subset of metastastic carcinoma specimens with altered mRNA expression (over- or under-expressor outliers) have gene copy gains or losses, respectively, that can account for the dysregulated mRNA levels. The majority of cases with dysregulated expression of transcripts involved in androgen metabolism are not associated with respective CNAs. Bars represent the percentage of metastastic carcinomas with outlier expression for each transcript involved in androgen metabolism (bars pointing up indicate overexpressor outliers, while bars pointing down indicate underexpressor outliers for each transcript). The white part of each bar indicates specimens with outlier level of expression that also exhibited DNA copy gain (for overexpressors) or loss (for underexpressors), respectively.

Fig. 5

Integration of expression outlier data with CNA analysis for AR and genes involved in androgen metabolism reveals that only a small subset of primary carcinoma specimens with altered mRNA expression (over- or under-expressor outliers) have gene copy gains or losses, respectively, that can account for the dysregulated mRNA levels. The majority of cases with dysregulated expression of transcripts involved in androgen metabolism are not associated with respective CNAs. Bars represent the percentage of primary carcinomas with outlier expression for each transcript involved in androgen metabolism (bars pointing up indicate overexpressor outliers, while bars pointing down indicate underexpressor outliers for each transcript). The white part of each bar indicates specimens with outlier level of expression that also exhibited DNA copy gain (for overexpressors) or loss (for underexpressors), respectively.

Fig. 6. Expression of the AKR1C family members is inversely related to androgen signaling

(A) Inverse correlation between expression levels of AKR1C1, AKR1C2, AKR1C3 and AKR1C4 vs an AR-regulated gene signature (indicative of AR signaling output) in metastatic PCas. All P values<0.001. (B) Exposure of LNCaP cells to androgen deprivation (medium supplemented with 10% charcoal-stripped serum, CSS) for 48 hrs potently upregulates expression of AKR1C3. This upregulation is suppressed by addition of the synthetic androgen R1881 (1 nM). Moreover, treatment of LNCaP cells (growing in medium supplemented with 10% regular FBS) with the anti-androgen enzalutamide (MDV3100) upregulates AKR1C3 expression. AKR1C3 mRNA levels were quantified by qRT-PCR, normalized to actin mRNA levels, and expressed as a % over values of control wells (grown in medium supplemented with 10% regular FBS)±SD (*=P<0.05, **=P<0.005).

Fig. 7

Multiplex qRT-PCR analysis of CTCs from CRPC patients for AKR1C3, SRD5A1, CYP17A1, AR, and KLK3 (PSA) transcripts reveals positivity in several CTC samples, confirming that these transcripts are expressed in the cancer cells in these tumors and providing a non-invasive method for monitoring of their expression. Results are presented as Ct (cycle threshold) values (i.e. the number of cycles required for the fluorescent signal to cross a previously defined threshold) in a heatmap. Ct values are inversely proportional to the amount of target nucleic acid in the sample. Therefore, low Ct values (orange or even yellow color) indicate strong expression of the target mRNA, while high Ct values (e.g. dark blue color) indicate weak expression. Each sample was run in duplicate. VCaP cells served as a positive control.