Androgen receptor expression and cellular proliferation during transition from androgen-dependent to recurrent growth after castration in the CWR22 prostate cancer xenograft

Abstract

Androgen receptor expression was analyzed in the CWR22 human prostate cancer xenograft model to better understand its role in prostate cancer recurrence after castration. In androgen-dependent tumors, 98.5% of tumor cell nuclei expressed androgen receptor with a mean optical density of 0.26 +/- 0.01. On day 2 after castration androgen deprivation decreased immunostained cells to 2% that stained weakly (mean optical density, 0.16 +/- 0.08). Cellular proliferation measured using Ki-67 revealed <1% immunostained cells on day 6. Androgen receptor immunostained cells increased to 63% on day 6 and 84% on day 32 although immunostaining remained weak. Cellular proliferation was undetectable beyond day 6 after castration until multiple foci of 5 to 20 proliferating cells became apparent on day 120. These foci expressed increased levels of prostate-specific antigen, an androgen receptor-regulated gene product. In tumors recurrent 150 days after castration androgen receptor-immunostaining intensity was similar to CWR22 tumors from intact mice although the percentage of cells immunostained was more variable. The appearance of proliferating tumor cells that expressed androgen receptor and prostate-specific antigen 120 days after castration suggests that these cells represent the origin of recurrent tumors.

Figures

Figure 1.

AR and Ki-67 immunohistochemistry of CWR22 tumors. AR expression was similar in androgen-dependent and recurrent tumors. AR-positive cells decreased to a minimum on day 2 after castration. Nuclear localization of AR returned on day 6 after castration but its intensity was lower than in androgen-dependent tumors. Higher percentages of AR-positive cells with lower levels of AR-staining intensity were recognized on days 32, 64, 90, and 120 after castration. Left: AR immunohistochemistry (scale bar, 10 μm). A: CWR22 before castration. B: Day 2 after castration. C: Day 6 after castration. D: Day 120 after castration. E: Recurrent CWR22 ∼150 days after castration. MIB-1 detection of the Ki-67 nuclear proliferation antigen showed similar rates of cellular proliferation in T-stimulated and recurrent tumors. Proliferation decreased on day 2 after castration and reached a level that was barely detectable on days 6 and 12 after castration. Proliferation was detectable as small nests of Ki-67-positive cells on day 120 after castration. Right: Ki-67 immunohistochemistry (scale bar, 20 μm). F: CWR22 before castration. G: Day 2 after castration. H: Day 6 after castration. I: Day 120 after castration. J: Recurrent CWR22 ∼150 days after castration.

Figure 2.

Western immunoblot analysis of AR protein in CWR22 tumors. An AR protein of 110 to 114 kd was present in lysates of androgen-dependent CWR22 tumors from intact mice. AR protein decreased after castration until tumor recurred ∼150 days after castration. AR protein levels found in recurrent tumors in the absence of androgens were similar to those found in the original androgen-dependent tumors. The position of the molecular mass marker (kd) is indicated. This experiment was performed with two to six different tumors at each time point with similar results.

Figure 3.

PSA and Ki-67 double immunohistochemistry of CWR22 tumors harvested from a mouse on day 120 after castration. Tissue PSA expression was visualized with diaminobenzidine tetrahydrochloride (cytoplasmic brown staining, white arrows) and Ki-67 expression was visualized with AEC (dark red nuclear staining, black arrows). Counterstaining was done with hematoxylin. Proliferating tumor cells emerged from the same foci where PSA was expressed (scale bar, 20 μm). Ki-67 immunostaining from the double immunohistochemistry showed a pattern of numerous blobs in the nucleoplasm rather than a typical uniform nuclear staining. Protease treatment during PSA immunohistochemistry may have degraded Ki-67 protein.

Figure 4.

Comparison of heterogeneity of AR expression and Ki-67 expression in CWR22 specimens from intact mice and on recurrence ∼150 days after castration. A: Mean percent AR positivity. B: MOD of AR. C: Mean percent Ki-67 positivity.