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Carcinogenesis vol.35 no.6 pp.1399–1406, 2014
Advance Access publication February 28, 2014
TR4 nuclear receptor functions as a tumor suppressor for prostate tumorigenesis via
modulation of DNA damage/repair system
Shin-Jen Lin1–4, Soo Ok Lee1–4, Yi-Fen Lee1–4,
Hiroshi Miyamoto1–4, Dong-Rong Yang1–4, Gonghui Li1–4
and Chawnshang Chang1–5,*
1George Whipple Lab for Cancer Research, 2Department of Pathology and
3Department of Urology, University of Rochester Medical Center, Rochester,
NY 14642, USA, 4Department of Radiation Oncology, and the Wilmot
Cancer Center, University of Rochester Medical Center, Rochester, NY
14642, USA and 5Sex Hormone Research Center, China Medical University/
Hospital, Taichung 404, Taiwan
*To whom correspondence should be addressed. George Whipple Lab for
Cancer Research, University of Rochester Medical Center, Box 626, 601
Elmwood Ave, Rochester, NY 14642, USA Tel: +1 585 2734500;
Fax: +1 585 7564133;
Testicular nuclear receptor 4 (TR4), a member of the nuclear
receptor superfamily, plays important roles in metabolism, fertil-
ity 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 tran-
scriptional level, and addition of ATM partially interrupted the
TR4 small interfering RNA-induced tumorigenesis in cell transfor-
mation assays. Immunohistochemical staining in human PCa tis-
sue 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.
Prostate cancer (PCa) is one of the most common cancers in the USA,
the highest in incidence and second highest in mortality in men (1). It
starts with prostate luminal epithelial cells malignant transformation
into small clumps of tumor cells confined in normal prostate glands,
named prostatic intraepithelial neoplasia (PIN). Over time these cells
with PIN then spread to the stromal compartment forming a prostate
tumor, and then gradually invade into blood vessels and metastasized
into bones or other organs. The etiology of PCa is complicated with
factors including genetic background (2), inherited factors (3), spe-
cific genes (4), diet (5) and vitamins (6), which might all be able to
contribute to PCa development. However, the molecular mechanisms
involved in PCa development remain unclear.
Testicular nuclear receptor 4 (TR4) belongs to the nuclear receptor
superfamily and was first cloned from human and rat in 1994 (7). TR4
is ubiquitously expressed in all tissues (49 mouse messenger RNAs in
39 tissues) examined (8). The TR4 knockout (TR4−/−) mouse was first
generated in 2004 by using traditional homologous recombination
strategy to delete the TR4 gene (9). The phenotypes of TR4−/− mouse
include growth retardation, defects in reproduction and maternal
behavior in female, disturbed development of cerebellum, impaired
oligodendrocytes and reduced myelination, delayed spermatogenesis
and reduced sperm count (9–12).
Here, we found the loss of TR4 in three different mouse models all
led to increased PIN and/or prostatic carcinoma formation. Molecular
mechanism dissections suggested TR4 might function as a tumor sup-
pressor via alteration of the signals involved in the DNA damage and
Materials and methods
All animal procedures were approved by the Animal Care and Use Committee
of the University of Rochester. TR4 heterozygous (TR4+/−) mice were made
by Lexicon Genetics (9). TR4 knockout (TR4−/−) mice were generated by mat-
ing male and female TR4+/− mice. PTEN heterozygous (PTEN+/−) mice were
purchased from National Cancer Institute. The TRAMP mice were purchased
from Jackson Laboratory. PTEN+/−-TR4+/− mice were generated by mating
male PTEN+/− mice with female TR4+/− mice. TRAMP TR4+/− mice were
generated by mating male TR4+/− mice with female TRAMP mice. The back-
ground of all mice used was C57BL/6J (B6).
Human or mouse tissues were collected and fixed with 10% formalin followed
by paraffin embedding. Samples were sliced to 5 μm thickness. We used the
primary antibodies of anti-TR4 (Perseus Proteomics), anti-ATM (Santa Cruz),
anti-γH2AX (Millipore) and anti-phospho-ATM serine 1981 (Millipore). The
primary antibody was recognized by the biotinylated secondary antibody and
visualized by Vectastain ABC peroxidase system and peroxidase substrate
DAB kit (Vector Laboratories). The positive staining signals in mouse tissues
were quantitated by Image J software.
RWPE1 cell line was obtained from the American Type Culture collection
(ATCC, Rockwell, MD) and maintained in complete keratinocyte serum-free
medium, supplemented with 1% penicillin/streptomycin, 50 mg/ml bovine
pituitary extract and 5 ng/ml epidermal growth factor (Life Technologies,
Barcelona, Spain). The spontaneously immortalized mouse prostatic epi-
thelial cell line, mPrE, was a generous gift from Dr M.Jiang (13) and main-
tained in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine
serum and 1% Antibiotic-Antimycotic solution (Invitrogen). Stable cell lines
expressing scramble small interfering RNA (scr) or small interfering RNA
(CGGGAGAAACCAAGCAATT) against TR4 (siTR4) were established by
transfecting pCDNA6/TR and pSuperior.retro.puro plasmids into RWPE1
or mPrE cells and selected for stable cell lines by treatment with blasticidin
(12 μg/ml) and puromycin (1.2 μg/ml) for 2 weeks. Tetracycline (1 μg/ml)
was added in order to induce siRNA expression. Primary cultures of mouse
embryonic fibroblasts (MEFs) were prepared from embryos at embryonic day
14.5 with TR4+/+ or TR4−/− genotypes and maintained in RPMI 1640 medium
(GIBCO) supplemented with 10% fetal bovine serum.
RNA extraction and quantitative PCR
RNA was extracted from cell lines using TRIzol® reagent (Invitrogen) and
converted to complementary DNA by Superscript III transcriptase (Invitrogen).
Quantitative PCR was performed using Bio-Rad CFX96 system with SYBR
green (Bio-Rad) to determine the level of messenger RNA expression of a gene
of interest. Expression levels were normalized to the expression of β-actin RNA.
Cell proliferation assay
Cells were subcultured to 10% confluence then treated with 100 μM N-methyl-
N-nitrosourea (MNU) or vehicle control dimethyl sulfoxide for 2 h. Then the
cells were grown to reach 100% confluence. Repeat the procedure of MNU
treatment for total three cycles. After three cycles, then we passaged the cells
five more times. The treated RWPE1 and mPrE cells were seeded in 24-well
plates (3000 cells/well) and cultured for 24, 48, 72 or 96 h. After each time
point, cell numbers were calculated by staining with 3-(4,5-dimethylthiazole-
2-yl)-2,5-diphenyl tetrazolium bromide agent.
Cell transformation/colony-forming assay
Cells were treated with 100 μM MNU or vehicle control dimethyl sulfoxide
for 3 cycles (RWPE1) or 30 cycles (mPrE) followed by five passages. Cells
Abbreviations: IHC, immunohistochemistry; MEF, mouse embryonic fibro-
blast; MNU, N-methyl-N-nitrosourea; PCa, prostate cancer; PIN, prostatic
intraepithelial neoplasia; TMA, tissue microarray; TR4, testicular nuclear
by guest on December 30, 2015
S.-J.Lin et al.
of prostate tumorigenesis. Indeed, results from our PTEN+/− mouse
model did support such a conclusion showing more PI3K/AKT signals
in PTEN+/− mice could reduce FOXO3a expression, which might then
lead to suppress TR4 expression. The consequence of such suppres-
sion then allowed PTEN+/−-TR4−/− mice to develop PIN more easily
The microarray data in Supplementary Table S1, available at
Carcinogenesis Online listed many genes involved in DNA repair
system were modulated upon loss of TR4. Other than ATM, that we
chose to further dissect to determine the mechanism by which TR4
suppressed prostate tumorigenesis, we believe other TR4 modulated
genes might also play important roles to influence prostate tumori-
genesis, especially since the prostate carcinogenesis is a long-term
process in order to accumulate enough mutations via DNA instability
(average age at time of diagnosis is 70, ref. 29). For example, muta-
tions in BRCA1 and BRCA2, important risk factors for breast/ovarian
cancers that play important roles in DNA damage response (30), have
also been linked to PCa development (4). In addition, the vitamin D
receptor, another nuclear receptor (31), could also regulate ATM sign-
aling to prevent PCa tumorigenesis (32), as well as the PTEN tumor
suppressor (33) also could maintain the chromosomal integrity (34).
In summary, we identified TR4 as a new prostate tumor suppressor
gene and mechanism dissection found TR4 might go through modu-
lation of the DNA repair system to suppress prostate tumorigenesis.
IHC data from human PCa samples also supported the linkage of TR4
expression with ATM expression and its roles in DNA damage. These
findings may provide us a new potential therapeutic target to better
battle PCa in the future.
Supplementary Table S1 and Figures S1 and S2 can be found at http://
National Institutes of Health Grants (CA156700, CA127548,
DK73414); Taiwan Department of Health Clinical Trial and Research
Center of Excellence Grant (DOH99-TD-B-111-004; China Medical
University, Taichung, Taiwan).
We thank K.Wolf for help in editing the manuscript.
Conflict of Interest Statement: The authors have nothing to disclose.
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Received August 13, 2013; revised February 18, 2014;
accepted February 19, 2014
by guest on December 30, 2015