Abstract
We have used the autochthonous transgenic adenocarcinoma of mouse prostate (TRAMP) model to investigate the relationship between somatic mutation in the androgen receptor (AR) and the emergence of androgen-independent prostate cancer. Here we report the identification, isolation, and characterization of distinct classes of AR variants from spontaneous prostate tumors in the TRAMP model. Using cDNA cloning, single stranded conformation polymorphism and sequencing strategies, 15 unique somatic mutations in the AR were identified in prostate tumors obtained from eight TRAMP mice between 24 and 29 weeks of age. At least one mutation was isolated from each mouse. All mutations were single base substitutions, 10 were missense and 5 were silent. Nine mutations in the AR were identified in tumors of four mice that were castrated at 12 weeks of age. Interestingly, the majority of mutations (seven out of nine, 78%) identified in the androgen-independent tumors colocalized in the AR transactivation domain. The remaining mutations colocalized in the AR ligand binding domain. In general, the AR variants demonstrated promoter-, cell-, and cofactor-specific activities in response to various hormones. All AR variants isolated in this study maintained strong sensitivity for androgens, and four AR variants isolated from castrated mice demonstrated increased activities in the absence of ligand. The K638M and F677S variants demonstrated increased activities in response to androgen, and K638M also demonstrated increased response to estradiol. In the presence of AR coactivator ARA70 the E231G variant demonstrated increased activity in response to both androgen and estradiol. However, in the presence of AR coactivator ARA160 the E231G variant was selectively responsive to androgen. Collectively these analyses not only indicate that somatic mutations in the AR gene occur spontaneously in TRAMP tumors but also how changes in the hormonal environment may drive the selection of spontaneous somatic mutations that provide a growth advantage.
Highlights
Androgens are essential for the development and maintenance of the prostate and hormonal therapy
androgen receptor (AR) gene mutations have been identified in human prostate cancer, it has been difficult to perform a comprehensive analysis of their incidence and nature because of the paucity of clinical samples representing the earliest form of the disease and the genetic and pathologic heterogeneity of the disease
Spontaneous Mutation in AR in Primary Prostate Tumors—To investigate the possibility that somatic mutation in the AR occurred during the natural history of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, full-length AR cDNA was first amplified from tumor tissue by reverse transcription-polymerase chain reaction (PCR) using three overlapping primer sets and the error-free Vent DNA polymerase
Summary
Transgenic Animals—TRAMP mice, heterozygous for the PB-T antigen transgene, were maintained in a pure C57BL/6 background. 30-cycle PCR was performed on the plasmid DNA in reactions containing a 200 M concentration of each of the four deoxyribonucleotide triphosphates, 1.5 Ci of [␣-32P]dCTP (3,000 Ci/mmol, ICN Biochemicals, Irvine, CA), 10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton-100, 200 M appropriate sense and antisense primer sets, and 0.5 unit of Vent DNA polymerase. Expression Vectors—The plasmid pcDNA3/HA-mAR was constructed to express full-length wild type mouse AR with an amino-terminal hemagglutinin antigen (HA; Santa Cruz Biotechnology, Santa Cruz, CA) tag sequence and a consensus Kozak translation initiation sequence (see Fig. 2B). This was accomplished by inserting the mouse full-length AR cDNA and HA tag into CMV expression vector pcDNA3.1 (Invitrogen Corporation, Carlsbad, CA). Hormones—The synthetic androgen R1881 was purchased from NEN Life Science Products. 17-Estradiol, progesterone, and dexamethasone were purchased from Sigma
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