A Polymorphism in the TC21 Promoter Associates with an Unfavorable Tamoxifen Treatment Outcome in Breast Cancer MatjazˇRokavec, 1,3,5 Werner Schroth, 1,3 Sandra M.C. Amaral, 1,3,6 Peter Fritz, 1,3 Lydia Antoniadou, 1,3 DamjanGlavacˇ, 5 WolfgangSimon, 2 Matthias Schwab, 1,3,4 Michel Eichelbaum, 1,3 and Hiltrud Brauch 1,3 1 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and 2 Department of Gynecology, Robert Bosch Hospital, Stuttgart, Germany; 3 University of Tu ¨bingen and 4 Department of Clinical Pharmacology, University Hospital Tu ¨bingen, Tu ¨bingen, Germany; 5 Department of Molecular Genetics, Institute of Pathology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia; and 6 Faculdade de Cieˆncias Me ´dicas, Universidade Nova de Lisboa, Lisbon, Portugal Abstract Tamoxifen therapy is a standard in the treatment of estrogen receptor (ER)-positive breast cancer; however, its efficacy varies widely among patients. In addition to interpatient differences in the tamoxifen-metabolizing capacity, there is growing evidence that crosstalk between ER and growth factor signaling contributes to tamoxifen resistance. We focused on TC21, a member of the Ras superfamily, to investigate the influence of the TC21 À582C>T promoter polymorphism on TC21 expression and treatment outcome. Immunohistochem- ical analyses of breast tumors revealed a higher TC21 expression in ER-negative compared with ER-positive tumors. Expression in ER-positive tumors was higher in carriers of the T allele in an allele dose–dependent manner. Quantitative real-time PCR analyses showed that TC21 mRNA expression is decreased after transfection of ERA in ER-negative breast cancer cells MDA-MB-231, UACC893, and BT-20. In MCF7 ER- positive cells, TC21 expression decreased with 17B-estradiol treatment and increased after treatment with tamoxifen metabolites, 4-OH-tamoxifen, or endoxifen. In patients treated with adjuvant mono tamoxifen, high cytoplasmic TC21 tumor expression or the carriership of the À582T allele conferred increased recurrence rates [n = 45: hazard ratio (HR), 3.06; 95% confidence interval (95% CI), 1.16–8.05; n = 206: HR, 1.79; 95% CI, 1.08–3.00, respectively]. A combined analysis with the data of the known tamoxifen predictor CYP2D6 showed an improvement of outcome prediction compared with CYP2D6 or TC21 genotype status alone (per mutated gene HR, 2.35; 95% CI, 1.34–4.14). Our functional and patient-based results suggest that the TC21 À582C>T polymorphism improves prediction of tamoxifen treatment outcome in breast cancer. [Cancer Res 2008;68(23):9799–808] Introduction Recent developments in breast cancer treatment point to tamoxifen as a catalyst for the change to targeted therapy (1). Although 50% to 70% of all estrogen receptor (ER)-positive tumors are responsive to tamoxifen treatment, failure and tumor resistance represent major clinical problems, thus limiting the usefulness of tamoxifen therapy (2–5). Three possible mechanisms of drug resistance are currently under debate: Either the patient can influence the effectiveness of tamoxifen via alterations of drug metabolism called metabolic resistance, or the ER-positive tumor is or can become refractory to treatment called intrinsic or acquired resistance (5). Although tamoxifen itself is a prodrug, 4-hydroxy tamoxifen and endoxifen have been recognized as the clinically potent metabolites due to their 100 times higher affinity to ER (6, 7) and capability of breast cancer cell growth inhibition (8). To this end, it has become increasingly clear that the hosts cytochrome P450 2D6 (CYP2D6) enzyme activity, which is subject to variation (9) is critical for their formation (6, 10, 11). Detrimental pharmacogenetic effects have been reported in that individuals with a genetically determined impaired metabolizer phenotype had significantly less favorable recurrence-free time (RFT) and disease- free survival (12–14). Although this can be explained by interpatient differences in ER genomic activity, there is growing evidence that also ER nongenomic or membrane-initiated steroid signaling activities and crosstalk with growth factor signal transduction pathways may contribute to tamoxifen resistance. Activation of ER outside the nucleus leads to the activation of surface tyrosine kinase receptors (e.g., IGF-IR, epidermal growth factor receptor, and HER2) as well as interaction with cellular kinases and adaptor molecules [e.g., c-Src, Shc, p85a regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K)], which in turn lead to the activation of mitogen-activated protein kinase (MAPK) and AKT pathways known to orchestrate cell proliferation and survival (15–17). These signaling pathways in turn can activate ER itself or its coactivators and corepressors, thereby increasing the potential of genomic/nuclear ER activity (5, 18). The role of the nongenomic steroid signaling calls for strategies for the identifi- cation of relevant factors of this intricate crosstalk that may add to the understanding of disease outcome under tamoxifen. Among the key components of growth factor signaling are the Ras proteins for which an involvement in tamoxifen resistance has been suggested (19, 20). TC21 also known as R-Ras2 is a member of the Ras superfamily of GTP-binding proteins, which are major regulators of signaling pathways involved in cell division, migration, adhesion, differentiation, and apoptosis (21–24). Anal- ogous to the classic H-, N-, and K-Ras proteins, TC21 is the only other Ras member mutated in human cancers (25, 26). Its role in tumorigenesis and cell growth regulation has become evident from the following invitro observations: GTPase deficient TC21 mutants showed transforming activities in NIH3T3 mouse fibroblasts and Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Hiltrud Brauch, Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Auerbachstrasse 112, 70376 Stuttgart, Germany. Phone: 49-711- 8101-3705; Fax: 49-711-85-92-95; E-mail: hiltrud.brauch@ikp-stuttgart.de. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-0247 www.aacrjournals.org 9799 Cancer Res 2008; 68: (23). December 1, 2008 Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/68/23/9799/2592381/9799.pdf by guest on 24 June 2022