Quantitation of androgen receptor gene expression in sporadic breast tumors by real-time RT–PCR: evidence that MYC is an AR-regulated gene

Ivan Bie`che 0 1 2 3 Beatrice Parfait 0 2 3 Sengu l Tozlu 0 1 2 Rosette Lidereau 0 1 2 Michel Vidaud 0 2 3 0 Abbreviations: AR , androgen receptor; ER, estrogen receptor; PR, progester- one receptor; MPA, medroxyprogesterone acetate; SBR, Scarff-Bloom- Richardson 1 Laboratoire d'Oncoge ne tique-INSERM E0017, Centre Rene Huguenin , St-Cloud , France 2 Mole culaire-UPRES JE 2195, Faculte des Sciences Pharmaceutiques et Biologiques, Universite Rene Descartes-Paris V , 4 Avenue de l'Observatoire, F-75006 Paris , France 3 Laboratoire de Ge ne tique Mole culaire-UPRES JE 2195, Faculte des Sciences Pharmaceutiques et Biologiques, Universite Rene Descartes-Paris V , Paris Little is known of the function and clinical significance of the androgen receptor (AR) in human breast cancer. Paradoxically, synthetic progestins, such as medroxyprogesterone acetate, are used for second line hormone therapy of breast cancer following tamoxifen failure. A sensitive and accurate assay for AR expression in breast tumors is thus required. Here we have developed and validated a real-time RT-PCR assay to quantify AR gene expression at the mRNA level in a series of 131 patients with unilateral invasive primary breast tumors. AR expression varied widely in tumor tissues (by at least 3 orders of magnitude), being underexpressed in 24/131 (18.3%) and overexpressed in 45/131 (34.4%) relative to normal breast tissues. We observed links (or trends) between AR status and age, menopausal status, Scarff-Bloom-Richardson histopathological grade, lymph node status and estrogen receptor and progesterone receptor status. High AR mRNA levels were negatively linked to MYC gene overexpression (P 8 10-6), confirming previous in vitro studies. Our results also suggest a role of the ARA70 gene (which encodes a major AR co-activator) in the AR pathway dysregulation observed in breast cancer. This simple, rapid and semi-automated method will be useful for screening cancer patients for altered AR expression and for predicting the response to androgen therapy in AR-related cancer patients. - The role of estrogen receptor (ER) and the progesterone receptor (PR) in human breast cancer is well established. Considerably less is known about the functional role and clinical significance of androgen receptor (AR) expression in this setting. Biochemical and immunohistochemical studies show that AR-positive tumors are more frequent (7090%) than ER-positive and PR-positive tumors (6080 and 50 70%, respectively) (14). Although ER, PR and AR are frequently co-expressed in breast tumors, ~10% of AR-positive tumors and, perhaps more importantly, 25% of AR-positive tumor metastases can be negative for ER and PR (1,5). Androgens have been shown to regulate the proliferation of AR-positive breast cancer cell lines in culture (6). Synthetic progestins, such as medroxyprogesterone acetate (MPA), are used as second line hormone therapy for breast cancer following tamoxifen failure (7). Birrell et al. (8) suggested that the antiproliferative effect of MPA in advanced cancer is mediated by AR. In vitro studies confirmed that MPA inhibits the proliferation of ER-negative and PR-negative cell lines via AR (9). Taken together, these findings suggest that AR determination may give additional predictive information on the response to endocrine treatments in breast cancer. AR expression has mainly been studied by means of a cytosol steroid-binding assay and immunohistochemistry. Although the former measures the status and functionality of the protein, it has several methodological shortcomings (1) and is time consuming. Furthermore, it requires the use of radioactive reagents and large amounts of tumor tissue, so that it is rarely used routinely in clinical laboratories. Immunohistochemical methods suffer from a lack of inter-laboratory standardization and cannot quantify the full range of alterations. However, this method also gives information concerning the status of the protein, but above all measures alterations on an individual cell basis. We quantified AR mRNA expression in a series of 131 patients with unilateral invasive primary breast tumors, using real-time quantitative RTPCR assay. This recent method of nucleic acid quantification in homogeneous solutions has the potential to become a standard in terms of its performance, accuracy, sensitivity, wide dynamic range, high throughput capacity and inter-laboratory agreement, and also yields statistical confidence values (10). We examined the relationship between AR expression status and classical clinical and pathological parameters, including patient outcome. AR mRNA levels were interpreted according to ER, ER and PR transcript levels measured using the same methodology and on the same homogeneous total RNA solutions. We also sought relationships between AR expression and that of genes known to be altered in breast cancer (RB1, CCND1, MYC and ERBB2), as well as several major genes involved in different steps of the AR pathway dysregulation observed in prostate cancer, i.e. the ARA70 gene (which codes for a major AR co-activator) (11), two well-known AR-responsive genes in prostate cancer (PAP, coding for prostatic acid phosphatase, and PSA, coding for prostatespecific antigen) (12) and DNMT1, a DNA methyltransferase gene that is altered in tumors (13), because loss of AR expression is associated with methylation of the AR promoter in prostate cancer cells (14). Materials and methods Disease-free survival No. of events (%)a Histological gradec I II aFirst relapses (local and/or regional recurrences and/or metastases). bLog rank test. cSBR classification. Information available for 122 patients. dInformation available for 124 patients. The samples were examined histologically for the presence of tumor cells. A tumor sample was considered suitable for this study if the proportion of tumor cells was 60%. Immediately following surgery the tumor samples were stored in liquid nitrogen until RNA extraction. The patients (mean age 58.2 years, range 3491) met the following criteria: primary unilateral non-metastatic breast carcinoma on which complete clinical, histological and biological data were available; no radiotherapy or chemotherapy before surgery. The main prognostic factors are presented in Table I. The median follow-up was 8.1 years (range 1.015.9). Forty-seven patients relapsed (the distribution of first relapse events was as follows: 13 local and/ or regional recurrences, 30 metastases and 4 both). Specimens of adjacent normal breast tissue from nine of the breast cancer patients and normal breast tissue from three women undergoing cosmetic breast surgery were used as sources of normal RNA. Real time RTPCR Theoretical basis. Quantitative values were obtained from the threshold cycle number at which the increase in the signal associated with exponential growth of PCR products begins to be detected using PE Biosystems analysis software, according to the manufactu (...truncated)