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. 2019 Jan 1;25(1):426-439.
doi: 10.1158/1078-0432.CCR-18-1431. Epub 2018 Sep 4.

Contribution of Adrenal Glands to Intratumor Androgens and Growth of Castration-Resistant Prostate Cancer

Elahe A Mostaghel  1   2 , Ailin Zhang  2 , Susana Hernandez  2 , Brett T Marck  3 , Xiaotun Zhang  4 , Daniel Tamae  5 , Heather E Biehl  2 , Maria Tretiakova  6 , Jon Bartlett  2 , John Burns  2 , Ruth Dumpit  2 , Lisa Ang  2 , Alvin M Matsumoto  3 , Trevor M Penning  5 , Steven P Balk  7 , Colm Morrissey  4 , Eva Corey  4 , Lawrence D True  6 , Peter S Nelson  2
Affiliations

Contribution of Adrenal Glands to Intratumor Androgens and Growth of Castration-Resistant Prostate Cancer

Elahe A Mostaghel et al. Clin Cancer Res. .

Abstract

Purpose: Tumor androgens in castration-resistant prostate cancer (CRPC) reflect de novo intratumoral synthesis or adrenal androgens. We used C.B.-17 SCID mice in which we observed adrenal CYP17A activity to isolate the impact of adrenal steroids on CRPC tumors in vivo.

Experimental design: We evaluated tumor growth and androgens in LuCaP35CR and LuCaP96CR xenografts in response to adrenalectomy (ADX). We assessed protein expression of key steroidogenic enzymes in 185 CRPC metastases from 42 patients.

Results: Adrenal glands of intact and castrated mice expressed CYP17A. Serum DHEA, androstenedione (AED), and testosterone (T) in castrated mice became undetectable after ADX (all P < 0.05). ADX prolonged median survival (days) in both CRPC models (33 vs. 179; 25 vs. 301) and suppressed tumor steroids versus castration alone (T 0.64 pg/mg vs. 0.03 pg/mg; DHT 2.3 pg/mg vs. 0.23 pg/mg; and T 0.81 pg/mg vs. 0.03 pg/mg, DHT 1.3 pg/mg vs. 0.04 pg/mg; all P ≤ 0.001). A subset of tumors recurred with increased steroid levels, and/or induction of androgen receptor (AR), truncated AR variants, and glucocorticoid receptor (GR). Metastases from 19 of 35 patients with AR positive tumors concurrently expressed enzymes for adrenal androgen utilization and nine expressed enzymes for de novo steroidogenesis (HSD3B1, CYP17A, AKR1C3, and HSD17B3).

Conclusions: Mice are appropriate for evaluating adrenal impact of steroidogenesis inhibitors. A subset of ADX-resistant CRPC tumors demonstrate de novo androgen synthesis. Tumor growth and androgens were suppressed more strongly by surgical ADX than prior studies using abiraterone, suggesting reduction in adrenally-derived androgens beyond that achieved by abiraterone may have clinical benefit. Proof-of-concept studies with agents capable of achieving true "nonsurgical ADX" are warranted.

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Conflict of interest statement

The authors declare no financial conflicts of interest.

Figures

Figure 1.
Figure 1.. Serum steroid levels and adrenal CYP17A expression in male C.B-17 SCID mice.
A-G) Levels of the indicated steroids were measured by mass spectrometry in eugonadal male mice (intact, white bars, n=8) and age-matched mice at 12 weeks after castration alone (CX, gray bars, n=7) or castration plus adrenalectomy (ADX, blue bars, n=10). G) Methylation status of CYP17A in adrenal glands resected from intact (age 3 months) and castrated male mice (age 1–3 months), compared to a methylation control ranging from 100% methylated to un-methylated (0%). H) Protein levels of CYP17A by western blot in adrenal glands resected from intact and castrated male mice at the indicated age (in months). I) Levels of the indicated steroids in adrenal glands resected from (intact, white bars) and age-matched mice at 12 weeks after castration alone (CX, gray bars). Data in A-F and I are shown as mean and standard deviation and represent a minimum of 6 animals per group. P values calculated using the Mann-Whitney rank test between the indicated groups (P values <0.05 and <0.10 were considered as significant and trending towards significance, respectively).
Figure 2.
Figure 2.. Impact of adrenalectomy on growth of CRPC tumor models in vivo.
A) Individual tumor volume curves for the LuCaP96CR patient derived xenograft (PDX) model in mice treated with castration alone (CX, black curves) or castration plus adrenalectomy (CX + ADX, blue curves). B) Kaplan Meier analysis of progression free survival in CX vs. CX + ADX treated LuCaP96CR tumors (defined as tumor size <750mm3) with comparison of curves using the Mantel-Haenszel logrank test. C) Individual tumor growth curves for the LuCaP35CR patient derived xenograft (PDX) model in mice treated with CX (black curves) or CX + ADX, among which a subset of tumors regrew slowly (Slow, blue curves), while a subset (Rapid, red curves) regrew with kinetics similar to the CX only group (black curves). D) Kaplan Meier analysis of progression free survival in CX vs. CX + ADX treated LuCaP35CR tumors. E) Kaplan Meier analysis of progression free survival in LuCaP35CR tumors treated with CX + ADX, comparing median survival in the tumor subsets with Slow (blue) vs Rapid (red) re-growth.
Figure 3.
Figure 3.. Impact of adrenal inhibition on tumor steroid levels in CRPC tumor models in vivo.
Levels of the indicated steroids were measured by mass spectrometry in tumors resected from the A) LuCaP96CR and B) LuCaP35CR PDX studies shown in Figure 2, in mice treated with castration alone (CX, gray bars) or castration plus adrenalectomy (ADX, blue bars), at short time points (shaded blue bars) or at end of study (EOS, open blue bars). For LuCaP35CR the subset of EOS tumors that regrew slowly (S, blue dots) vs rapidly (R, red dots) are indicated. Data are shown as mean and standard deviation. P values calculated using the Mann-Whitney rank test between the indicated groups (P values <0.05 and <0.10 were considered as significant and trending towards significance, respectively). The impact of ABI (red bars, prior studies) vs ADX (blue bars, current study) on the percent change in C) Testosterone and D) DHT compared to castration alone is shown for LuCaP35CR (closed bars) and LuCaP96CR (open bars). P values for the difference in % change between ABI and ADX treated tumors are given under the horizontal bars at the bottom of each graph. P values for the difference in tumor steroids in each treatment group vs Cx alone are given in diagonal font at the top of each graph.
Figure 4.
Figure 4.. Impact of adrenalectomy on immunohistochemical (IHC) staining for AR, ARV7, PSA and GR in CRPC tumor models in vivo.
IHC staining for the indicated protein in tumors resected from the A) LuCaP96CR and B) LuCaP35CR PDX studies shown in Figure 3 in mice treated with castration alone (CX, gray bars) or castration plus adrenalectomy (ADX, blue bars), at short time points (shaded blue bars) or at end of study (EOS, open blue bars). For LuCaP35CR the subset of EOS tumors that regrew slowly (S, blue dots) vs rapidly (R, red dots) are indicated. Staining for AR, ARV7 and PSA quantified as the average nuclear (AR, ARV7) or cytoplasmic (PSA) staining intensity within tumor epithelium multiplied by the percentage of positive nuclei (AR, ARV7) or cells with positive cytoplasm (PSA) in tumor epithelium (denoted as AvgNuclearOD*%PosNuclei or AvgCytoOD*%PosCyto). Semi-quantitative scoring for nuclear GR expression calculated by multiplying the intensity level (0 for no stain, 1 for faint stain, and 2 for intense stain) by the percentage of cells (0–100%) at each intensity level and totaling the results, ranging from 0 (no staining in any cell) to 200 (intense staining in 100% of the cells). Data are shown as mean and standard deviation. P values calculated using the Mann-Whitney rank test between the indicated groups (P values <0.05 and <0.10 were considered as significant and trending towards significance, respectively). C) Representative examples of the indicated stains for LuCaP96CR (top) and LuCaP35CR (bottom) PDX models, showing each stain in an EOS tumor from a CX only or CX plus ADX treatment arm.
Figure 5.
Figure 5.. Expression of steroidogenic enzymes in CRPC metastases.
The heatmap summarizes staining observed on a TMA containing multiple bone and soft tissue CRPC metastases from 43 men collected via rapid autopsy. Each column represents serial staining of the same metastasis for the indicated proteins, ranging from negative expression (dark blue) to high expression (dark red). Vertical lines demarcate the set of metastases from each patient. Tumors are grouped based on negative (A) or positive (B, C) AR expression (in the majority of tumors in an individual patient), and then by steroidogenic potential. A) AR negative tumors with negative PSA staining and low expression of steroidogenic enzymes. Tumors from the two patients indicated had neuroendocrine histology. B) AR positive tumors with negative (top) or positive (bottom) PSA expression and low steroidogenic potential (based on low HSD3B1 expression). C) AR positive tumors with high steroidogenic potential. Tumors are grouped based on potential for adrenal androgen conversion (coordinate expression of HSD3B1and HSD17B3/AKR1C3 with low CYP17A1, top), or potential for de novo steroidogenesis (coordinate expression of CYP17A1, HSD3B1 and HSD17B3/AKR1C3, bottom). Among the group with high steroidogenic potential, patients in whom the soft tissue metastases appear distinctly different than the bone metastases are indicated in red. Representative IHC stains of the metastases labeled a-e in panel C are shown in Supplementary Figure 6.
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