Targeting the stromal androgen receptor in primary prostate tumors at earlier stages

Abstract

To differentiate roles of androgen receptor (AR) in prostate stromal and epithelial cells, we have generated inducible-(ind)ARKO-TRAMP and prostate epithelial-specific ARKO TRAMP (pes-ARKO-TRAMP) mouse models, in which the AR was knocked down in both prostate epithelium and stroma or was knocked out in the prostate epithelium, respectively. We found that loss of AR in both mouse models resulted in poorly differentiated primary tumors with expanded intermediate cell populations. Interestingly, knockdown of both epithelial and stromal AR in ind-ARKO-TRAMP mice at earlier stages resulted in smaller primary prostate tumors with lower proliferation rates, and knockout of AR in pes-ARKO-TRAMP mice resulted in larger primary prostate tumors with higher proliferation rates. The differential proliferation rates, yet with similarly expanded intermediate cell populations, indicated that the prostate stromal AR might play a more dominant role than the epithelial AR to promote primary tumor proliferation at an early stage of tumor. Tissue recombination of human prostate stromal cell lines (WPMY1-v or WPMY1-ARsi) with human prostate cancer epithelial cell lines (PC3-v or PC3-AR9) further demonstrated that the AR might function as a suppressor in epithelial cells and a proliferator in stromal cells in the primary prostate tumors. The dual roles of the AR in prostate epithelium and stroma may require us to reevaluate the target and timing of androgen-deprivation therapy for prostate cancer patients and may suggest a need to develop new drugs to selectively target stromal AR in the primary prostate tumors at earlier stages.

Fig. 1.

Knockin of AR in prostate cancer PC3-AR9 cells suppressed tumor growth, and knockdown of AR in the prostate stromal WPMY1-ARsi cells suppressed the recombined epithelial PC3 tumor growth using in vivo orthotopic tumor implantation strategy. (A) Orthotopically implanted PC3-AR9 cells generated smaller tumors than tumors generated by PC3-v at 12 wk after implantation (arrows). Tumor cell proliferation signals were determined by Ki67 staining. (B) PC3-v or PC3-AR9 were recombined with WPMY1-v or WPMY1-ARsi cells for orthotopic implantation in anterior prostates of male nude mice. PC3-v cells recombined with WPMY1-v generated larger tumors than those from PC3-v recombined with WPMY1-ARsi cells (arrows). Twelve weeks after implantation, tumors were harvested to analyze the size, histology (by H&E staining) and proliferation (by Ki67 staining; quantitative results were attached). The differences are described in the text. Data are presented as mean ± SD; *P < 0.05.

Fig. 2.

AR knockout led to changes of prostate tumor growth in TRAMP mice. (A) We observed general visual changes of the reproductive organs among 16-wk-old pes-ARKO-TRAMP, WT TRAMP mice, pI-pC ind-ARKO-TRAMP, and pI-pC injected WT TRAMP. pes-ARKO-TRAMP mice had enlarged prostates (arrows) compared with WT TRAMP mice, without alteration of other reproductive organs. Ind-ARKO-TRAMP with pI-pC induction of AR knockdown at 12-wk-old had significantly smaller reproductive organs, including prostates, seminal vesicles, and testes. (B) Serum testosterone (T) levels were detected sequentially at 12 wk (before pI-pC injection), 16, 20, and 24 wk. The serum T levels remained unchanged in pes-ARKO-TRAMP and were significantly reduced in ind-ARKO-TRAMP mice.

Fig. 3.

Inducing AR knockdown in ind-ARKO-TRAMP mice led to changed cell populations in prostate tumor. (A) We observed more intermediate cell-like populations (arrows indicate yellow color, overlapped by green and red signals) in ind-ARKO-TRAMP mice after pI-pC injection at 12-wk-old, mice compared with pI-pC injected WT TRAMP mice by double immunofluorescence staining of CK5 (green) and CK8 (red) of ventral prostate tumors. (B) ind-ARKO-TRAMP tumors expressed higher CD44-positive cells (arrows) compared with pI-pC injected WT TRAMP tumors. Data are presented as mean ± SD; *P < 0.05; **P < 0.01.

Fig. 4.

AR-negative role in the growth of epithelium tumor was dominated by AR stroma function, which may positively stimulate epithelium proliferation. (A) The gross visual observation of the ventral prostates at 16 (lane 1) and 20 wk (lane 3). pes-ARKO-TRAMP mice generated larger tumors (arrowheads) than WT TRAMP mice, whereas ind-ARKO-TRAMP generated much smaller tumors than their WT TRAMP littermate mice. Histological analysis of different lobes of prostates at 16 and 20 wk (Lower). pes-ARKO-TRAMP and ind-ARKO-TRAMP tumor are more poorly differentiated than WT controls (loose). (B) We killed mice at different time points of 16, 20, and 24 wk and measured tumor size differences. The animal number of each group is indicated. (C) We demonstrated tumor growth differences by Ki67 IHC staining (Upper) and by BrdU incorporation (Lower) in 16-wk-old prostates. We i.p. injected mice with BrdU every 6 h 4 times and killed mice 24 h later. Tissue sections were stained by the BrdU-detecting kit (Zymed). (D) Double immunofluorescent staining of BrdU (green) and CK5 (red) on mouse prostate cancers. BrdU proliferation signals are reduced with CK5-positive cells and are increased in ind-ARKO-TRAMP mice (arrows indicate prostate tumor cells with positive nuclear BrdU green fluroscence staining). Although ind-ARKO-TRAMP also had a higher percentage of CK5 positive cells, the proliferation in their prostate was still low as compared with WT TRAMP mice.

Fig. 5.

Tumor metastasis is delayed in ind-ARKO-TRAMP mice. (A) Twenty-four-week-old ind-ARKO-TRAMP mice, with decreased (50–60%) AR expression in both prostate epithelium and stroma, developed smaller and less-aggressive metastatic tumors (arrowheads) compared with tumors from the WT TRAMP littermates. The size of metastatic tumors among different groups followed the sequence: pes-ARKO-TRAMP > WT TRAMP (with and without pI-pC)> ind-ARKO-TRAMP. The WT TRAMP mice with or without injection of only pl-pC developed similar sizes of metastatic tumors; only data of WT TRAMP mice without injection are shown. (B) Differences in tumor malignancies were demonstrated by comparing metastases from TRAMP mice with different AR-status when the original tumors reached to 1-cm diameter in different mouse groups. At the age of ≈22 wk, WT TRAMP mice developed 1-cm-diameter-size tumors, and those tumors were well differentiated with small pelvic lymph node metastases. As early as 18 wk, pes-ARKO-TRAMP tumors reached the similar size, and the mice had much larger tumor metastases to lymph nodes and multiple organs. It took 36 wk for the ind-ARKO-TRAMP to form the 1-cm-diameter tumors, which invaded into the seminal vesicle (lower arrowhead) and migrated to the liver (upper arrowhead). (C) Histological analysis of tumor sections. H&E staining showed WT TRAMP primary tumors were better differentiated than pes-ARKO-TRAMP and ind-ARKO-TRAMP tumors. (D) Survival rates were statistically different among WT TRAMP, castrated TRAMP, ind-ARKO-TRAMP, and pes-ARKO-TRAMP mice.