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. 2011;17(7-8):657-64.
doi: 10.2119/molmed.2010.00143. Epub 2011 Feb 22.

Steroidogenic enzymes and stem cell markers are upregulated during androgen deprivation in prostate cancer

Minja J Pfeiffer  1 , Frank P Smit, John P M Sedelaar, Jack A Schalken
Affiliations

Steroidogenic enzymes and stem cell markers are upregulated during androgen deprivation in prostate cancer

Minja J Pfeiffer et al. Mol Med. 2011.

Abstract

Considerable levels of testosterone and dihydrotestosterone (DHT) are found in prostate cancer (PCa) tissue after androgen deprivation therapy. Treatment of surviving cancer-initiating cells and the ability to metabolize steroids from precursors may be the keystones for the appearance of recurrent tumors. To study this hypothesis, we assessed the expression of several steroidogenic enzymes and stem cell markers in clinical PCa samples and cell cultures during androgen depletion. Gene expression profiles were determined by microarray or qRT-PCR. In addition, we measured cell viability and analyzed stem cell marker expression in DuCaP cells by immunocytochemistry. Seventy patient samples from different stages of PCa, and the PCa cell line DuCaP were included in this study. The androgen receptor (AR) and enzymes (AKR1C3, HSD17B2, HSD17B3, UGT2B15 and UGT2B17 ) that are involved in the metabolism of adrenal steroids were upregulated in castration resistant prostate cancer (CRPC). In vitro, some DuCaP cells survived androgen depletion, and eventually gave rise to a culture adapted to these conditions. During and after this transition, most of the steroidogenic enzymes were upregulated. These cells also are enriched with stem/progenitor cell markers cytokeratin 5 (CK5) and ATP-binding cassette sub-family G member 2 (ABCG2). Similarly, putative stem/progenitor cell markers CK5, c-Kit, nestin, CD44, c-met, ALDH1A1, α2-integrin, CD133, ABCG2, CXCR4 and POU5F1 were upregulated in clinical CRPC. The upregulation of steroidogenic enzymes and stem cell markers in recurrent tumors suggests that cancer initiating cells can expand by adaptation to their T/DHT deprived environment. Therapies targeting the metabolism of adrenal steroids by the tumor may prove effective in preventing tumor regrowth.

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Figures

Figure 1
Figure 1
Schematic overview of the steroidogenic pathway. AMACR, α-methylacyl-CoA racemase; FASN, fatty acid synthase; SREB, G protein-coupled receptor; StAR, steroidogenic acute regulatory protein; CYP, cytochrome P450; CYB5, cytochrome b5; HSD, hydroxysteroid dehydrogenase; AKR, aldo-keto reductase; SRD, 5α-reductase; UGT, UDP glucuronosyltransferase; green boxes, adrenal androgens; blue box, testicular androgen; yellow box, tissue androgen.
Figure 2
Figure 2
mRNA expression of AR and enzymes involved in steroid metabolism in clinical samples of LG, HG, met and CRPC. Analysis was done by using the t test. *P < 0.05; **P < 0.01. Horizontal line, median value; open circle, outliers; small asterisk, extremes. AKR, aldo-keto reductase; HSD, hydroxysteroid dehydrogenase; SRD, 5α-reductase; UGT, UDP glucuronosyltransferase.
Figure 3
Figure 3
Relative expression values of enzymes involved in steroid metabolism and AR during hormone depletion up to 12 d in DuCaP cells. The mRNA expression of DuCaP cells grown in RPMI-1640 supplemented with 10% FCS or 10% CSS for 24–96 h and 12 d was assessed by qRT-PCR SYBR Green method. Relative expression values were calculated by using expression values in basic culture conditions (FCS 24h) as a reference. Enzymes CYP11A1, CYP17A1, HSD3B2 and HSD17B3, for which the Cp-values were high (Cp > 36), were left out of the figure. Experiment was done twice. Two-way ANOVA; Error bars, SEM; *P < 0.05; **P < 0.01. StAR, steroidogenic acute regulatory protein; FASN, fatty acid synthase; HSD, hydroxysteroid dehydrogenase; AKR, aldo-keto reductase; RDH, retinol dehydrogenase (oxidative 3α-HSD).
Figure 4
Figure 4
Relative expression values of enzymes involved in steroid metabolism and AR during continuous hormone depletion DuCaP cells. The mRNA expression of DuCaP cells grown in RPMI-1640 supplemented with 10% FCS or 10% CSS for several passages (P1–P2) was assessed by qRT-PCR SYBR Green method. Relative expression values were calculated by using expression values in hormone-depleted culture conditions (CSS) as a reference. Enzymes CYP11A1, CYP17A1 and HSD3B2, for which the Cp-values were high (Cp > 36), were left out of the figure. Experiment was done twice. Two-way ANOVA; Error bars, SEM; *P < 0.05; **P < 0.01. StAR, steroidogenic acute regulatory protein; FASN, fatty acid synthase; HSD, hydroxysteroid dehydrogenase; AKR, aldo-keto reductase; RDH, retinol dehydrogenase (oxidative 3α-HSD).
Figure 5
Figure 5
Proliferation of DuCaP cells in different hormonal conditions. Cells were grown in RPMI-1640 supplemented with 10% FCS or 10% CSS, or first 12 d with CSS after which FCS was re-administered the next 12 d. Cell viability was measured with a standard MTT assay. The graph shows optical density values at 595 nm. Experiment was done three times with triplicates. Two-way ANOVA. Line, trend line; error bars, SEM; **P < 0.01.
Figure 6
Figure 6
Immunohistochemical stainings for putative stem/progenitor cell markers in DuCaP. Cells were grown in RPMI-1640 supplemented with 10% FCS or 10% CSS for 7 d and stained for putative stem/progenitor cell markers. Positive staining was found for CK5, ABCG2 and α2-integrin. No staining was detected for c-Kit, nestin or CD133. Known positive tissues or cells were used as controls. Scale: 0.2 mm.
Figure 7
Figure 7
mRNA expression of putative stem cell markers in clinical samples of met and CRPC. Analysis was done by using the t test. Horizontal line, median value; open circle, outliers; asterisk, extremes. CD133, prominin 1.
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