TR4 nuclear receptor functions as a tumor suppressor for prostate tumorigenesis via modulation of DNA damage/repair system

Abstract

Testicular nuclear receptor 4 (TR4), a member of the nuclear receptor superfamily, plays important roles in metabolism, fertility and aging. The linkage of TR4 functions in cancer progression, however, remains unclear. Using three different mouse models, we found TR4 could prevent or delay prostate cancer (PCa)/prostatic intraepithelial neoplasia development. Knocking down TR4 in human RWPE1 and mouse mPrE normal prostate cells promoted tumorigenesis under carcinogen challenge, suggesting TR4 may play a suppressor role in PCa initiation. Mechanism dissection in both in vitro cell lines and in vivo mice studies found that knocking down TR4 led to increased DNA damage with altered DNA repair system that involved the modulation of ATM expression at the transcriptional level, and addition of ATM partially interrupted the TR4 small interfering RNA-induced tumorigenesis in cell transformation assays. Immunohistochemical staining in human PCa tissue microarrays revealed ATM expression is highly correlated with TR4 expression. Together, these results suggest TR4 may function as a tumor suppressor to prevent or delay prostate tumorigenesis via regulating ATM expression at the transcriptional level.

Fig. 1.

Loss of TR4 promotes prostate tumorigenesis. (A) TR4^{− /−} mice show PIN phenotype, whereas TR4^+/+ show normal prostate structure. The black arrow indicates PIN lesion. (B) PTEN^+/−-TR4^+/− mice develop PCa, whereas PTEN^+/−-TR4^+/+ show minor PIN lesions. The black arrow indicates prostate tumor. (C) TRAMP-TR4^+/− show tumor formation or severe PIN lesions, whereas TRAMP-TR4^+/+ show minor PIN lesions. Quantitations are shown in the right panels and the P values were calculated by Fisher’s exact test.

Fig. 2.

TR4 inhibits both mouse and human prostate epithelial cells growth and colony formation. (A) Quantitative PCR showed RWPE1 cells TR4 messenger RNA (mRNA) level was knocked down by stably transfecting TR4 small interfering RNA (upper left panel). RWPE1_siTR4 and RWPE1_scr cells were treated with carcinogen MNU at 100 μg/ml for 2h each cycle. After three cycles of treatment, the cells were passaged another five times. RWPE1_siTR4 cells had increased growth (upper right panel) and colony formation (lower left panel). P value is 0.0007 in colony formation assay by Student’s t-test (lower right panel). (B) Quantitative PCR showed mPrE cells TR4 mRNA level was knocked down by stably transfected TR4 small interfering RNA (upper left panel). mPrE_siTR4 and mPrE_scr cells were treated with carcinogen MNU at 100 μg/ml for 2h each cycle. After 30 cycles of treatment, the cells were passaged another five times. mPrE_siTR4 cells had increased growth (upper right panel) and colony formation (lower left panel). P value is 0.0016 in colony formation assay by Student’s t-test (lower right panel).

Fig. 3.

Reduction of TR4 increases DNA damage but decreases DNA repair gene ATM expression both in vitro and in vivo. (A) Knocking down TR4 in RWPE1 cells (RWPE1_siTR4) increased DNA damage marker γH2AX foci formation compared with scramble siRNA control cell line (RWPE1_scr) upon carcinogen MNU 100 μg/ml treatment for three cycles. From left to right panels are 4′,6-diamidino-2-phenylindole (DAPI) staining for nuclei, γH2AX staining and merge with DAPI and γH2AX staining. (B and C) DNA damage marker γH2AX expression in prostate tissues increased in PTEN^+/−-TR4^+/− mice (B) as well as in TRAMP-TR4^+/− mice (C) compared with TR4^+/+ control mice using IHC staining. (D) RWPE1_siTR4 cells fail to induce DNA repair gene ATM expression compared with RWPE1_scr upon carcinogen MNU 100 μg/ml treatment for three cycles. From left to right panels are DAPI staining for nuclei, ATM staining and merge with DAPI and ATM staining. (E and F) DNA repair gene ATM expression in prostate tissues decreased in PTEN^+/−-TR4^+/− mice (E) as well as in TRAMP-TR4^+/− mice (F) compared with TR4^+/+ control mice using IHC staining. The density of γH2AX and ATM staining was calculated by ImageJ software by averaging six randomly selected fields. P values are < 0.0001 by Student’s t-test in both mouse models.

Fig. 4.

TR4 prevents prostate tumorigenesis by regulating DNA repair gene ATM transcription. (A) Scheme of putative TR4 binding element on ATM promoter. The sequence was determined by Multi-genome Analysis of Positions and Patterns of Elements of Regulation search engine (MAPPER). The sequence matching model M00966 is marked in bold with underline. (B and C) ATM promoter was cloned into luciferase reporter vector pGL3 and was transfected along with internal control pRL-tk into RWPE1 cells. The results show that knocking down TR4 reduced ATM-Luc activity in RWPE1 cell, whereas overexpression increased ATM-Luc activity. Y-axis FL/RL indicates firefly luciferase activity normalized by internal control renilla luciferase activity. The results were normalized by internal control and were repeated three times. P values are 0.0068 and <0.0001 in TR4 knockdown and TR4 overexpression groups, respectively, via Student’s t-test, respectively. (D) Chromatin immunoprecipitation assay shows TR4 can physically bind to ATM promoter region shown in Figure 6A. 1×10⁶ cells per 200 μl of SDS Lysis Buffer were sonicated and DNA was pulled down by either TR4 antibody or normal mouse IgG. DNA from each group was washed, eluted and amplified by specific primers flanking the sequences shown in A. Input sample (1% of total sample) was gathered before immunoprecipitation was performed.

Fig. 5.

ATM interrupts loss of TR4-induced prostate tumorigenesis. (A and B) Quantitative PCR shows ATM mRNA overexpression in both RWPE1_siTR4 and mPrE_siTR4 cell lines. RWPE1siTR4_ATM and mPrEsiTR4_ATM represent ATM overexpression in these two cell lines. (C–F) Stably overexpressing functional ATM partially interrupted TR4 siRNA-induced cell growth (C and D) and colony formation (E and F, upper panels) after carcinogen MNU treatment in both RWPE1 and mPrE cell lines. Quantitations are shown in the lower panels of E and F and the P values were calculated by Student’s t-test.

Fig. 6.

ATM expression level is highly correlated with TR4 in human TMA. The representative IHC staining pictures are shown in the top panels. IHC in human prostate TMA shows correlations of ATM versus TR4 expression. When PCa samples were divided into TR4 positive versus TR4 negative, ATM expression is significantly reduced in the TR4-negative group. The P values were calculated by Fisher’s exact test.