Targeting GPR30 with G-1: a new therapeutic target for castration-resistant prostate cancer

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   Figure 1

   G-1 inhibited growth and induced necrosis in the castration-resistant tumors. G-1 inhibited growth of the CR tumor (bottom panel) but not the androgen-sensitive tumors (top panel). When LNCaP xenografts grew to 150 mm³, mice were divided into two groups: intact and castrated. Intact animals received subcutaneous injections of a vehicle (2.5% DMSO and 5% ethanol) or G-1 (4 mg/kg) daily for 16 days. For the castrated group, mice were castrated and, when the tumor re-emerged, they were treated with a vehicle or G-1 daily for 16 days. Error bars represent mean±s.e.m., n=6–8/group, *P<0.05.
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   Figure 2

   G-1 induced massive necrosis and neutrophil infiltration in the CR tumors. (A) G-1 triggered massive necrosis in CR tumors. Tumor sections were stained with H&E, and the necrotic area was quantified as described in the Supplementary Methods. (B) G-1 induced significant necrosis associated with massive inflammation, which in turn was associated with neutrophil infiltration, both surrounding the necrotic area and within the viable area, in CR tumors only. The yellow arrow represents massive inflammation. Magnification: 20× (H&E, upper panel), 200× (H&E, lower panel), 100× (neutrophil IHC, upper panel), and 200× (neutrophil IHC, lower panel). Scale bars represent 50 μm in all micrographs. (C) G-1 reduced the microvessel area ratio in the intratumoral stromal region but not in the tumor capsule. Microvessel area ratio is calculated as the ratio of the microvessel area to the intratumoral stromal area or the capsule area. (D) Ki67 and cleaved caspase-3 staining of tumor cells was used to determine proliferation and apoptosis respectively. (E) G-1 did not induce toxicity in castrated mice as determined by the absence of changes in body weight (left panel) and in serum assays of organ damage marker enzymes (right panel). Error bars represent mean±s.e.m., n=6–8/group, *P<0.05; NS, not significant; H&E, hematoxylin and eosin; IHC, immunohistochemistry.
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   Figure 3

   G-1 induced unique changes in gene expression in castrated animals. (A) Heat map of hierarchically clustered differential gene expression in intact or castrated animals treated with a vehicle or G-1 blue, downregulated; yellow, upregulated; n=4 per group. A scheme for gene selection for Ingenuity Pathway Analysis is shown. (B) Quantitative real-time PCR analyses of the G-1-induced human and mouse genes in intact and castrated animals. Data were normalized to the levels of housekeeping genes: human-specific GAPDH (for human genes) or ActB (for mouse genes). Error bars represent mean±s.e.m., n=6. ^#P<0.05 compared with intact-vehicle treatment and *P<0.05 compared with castrated-vehicle treatment. NS, not significant.
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   Figure 4

   Androgen suppressed GPR30 expression via AR. (A) Androgen (white bars, 0.1 and 1 nM) suppressed GPR30 expression, and suppression was reversed by bicalutamide (black bars) in AR-positive LNCaP cells but not in AR-negative PC-3 cells. Cells were treated with androgen in the presence or absence of bicalutamide for 4 days. Prostate-specific antigen (PSA) was a positive control for the androgen-stimulated AR response gene. (B) siAR abolished the androgen-suppressed GPR30 expression in LNCaP cells. Error bars represent mean±s.d. of three independent experiments, **P<0.01. (C) Castration upregulated GPR30 expression in vivo. RNA was extracted, and GPR30 expression of the LNCaP xenograft in intact mice (AS tumor, n=9) was compared with that after castration of mice (CR tumor, n=9). Relative mRNA expression was compared with that of intact mouse no. 1. AR, androgen receptor; D, dihydroxytestosterone; PSA, prostate-specific antigen; R, R1881; siNT, siRNA-non-targeting; siAR, siRNA-AR.
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   Figure 5

   GPR30 staining in primary PC and CRPC metastases. A high level of GPR30 was detected in a larger proportion of metastatic CRPC specimens when compared with primary PC specimens.
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   Figure 6

   A schematic diagram showing G-1-induced innate antitumor response in castration-resistant LNCaP prostate cancer in vivo. For LNCaP xenografts in vehicle- or G-1-treated intact animals or vehicle-treated castrated animals, focal ischemic necrosis was detected in the tumor. However, in G-1-treated castrated animals, massive necrosis and neutrophil infiltration were detected in the necrotic area as well as within the viable area of the tumor. (Box) In human xenografts, the levels of expression of human-specific chemokine and inflammatory response genes were increased; in the mouse stroma, the levels of expression of a panel of murine-specific neutrophil-related cytokine genes were elevated. In both intact and castrated animals, macrophages resided in the tumor capsule and B cells localized to the intratumoral stroma of the LNCaP xenograft.

• © 2014 Society for Endocrinology