Genotype of metabolic enzymes and the benefit of tamoxifen in postmenopausal breast cancer patients

BACKGROUND: Tamoxifen is widely used as endocrine therapy for oestrogen-receptor-positive breast cancer. However, many of these patients experience recurrence despite tamoxifen therapy by incompletely understood mechanisms. In the present report we propose that tamoxifen resistance may be due to differences in activity of metabolic enzymes as a result of genetic polymorphism. Cytochrome P450 2D6 (CYP2D6) and sulfotransferase 1A1 (SULT1A1) are polymorphic and are involved in the metabolism of tamoxifen. The CYP2D6*4 and SULT1A1*2 genotypes result in decreased enzyme activity. We therefore investigated the genotypes of CYP2D6 and SULT1A1 in 226 breast cancer patients participating in a trial of adjuvant tamoxifen treatment in order to validate the benefit from the therapy. METHODS: The patients were genotyped using PCR followed by cleavage with restriction enzymes. RESULTS: Carriers of the CYP2D6*4 allele demonstrated a decreased risk of recurrence when treated with tamoxifen (relative risk = 0.28, 95% confidence interval = 0.11–0.74, P = 0.0089). A similar pattern was seen among the SULT1A1*1 homozygotes (relative risk = 0.48, 95% confidence interval = 0.21–1.12, P = 0.074). The combination of CYP2D6*4 and/or SULT1A1*1/*1 genotypes comprised 60% of the patients and showed a 62% decreased risk of distant recurrence with tamoxifen (relative risk = 0.38, 95% confidence interval = 0.19–0.74, P = 0.0041). CONCLUSION: The present study suggests that genotype of metabolic enzymes might be useful as a guide for adjuvant endocrine treatment of postmenopausal breast cancer patients. However, results are in contradiction to prior hypotheses and the present sample size is relatively small. Findings therefore need to be confirmed in a larger cohort

The estrogen receptor-α A908G (K303R) mutation occurs at a low frequency in invasive breast tumors: results from a population-based study

INTRODUCTION: Evidence suggests that alterations in estrogen signaling pathways, including estrogen receptor-α (ER-α), occur during breast cancer development. A point mutation in ER-α (nucleotide A908G), producing an amino acid change from lysine to arginine at codon 303 (K303R) results in receptor hypersensitivity to estrogen. This mutation was initially reported in one-third of hyperplastic benign breast lesions, although several recent studies failed to detect it in benign or malignant breast tissues. METHODS: We screened 653 microdissected, newly diagnosed invasive breast tumors from patients in the Carolina Breast Cancer Study, a population-based case-control study of breast cancer in African American and white women in North Carolina, for the presence of the ER-α A908G mutation by using single-strand conformational polymorphism (SSCP) analysis and (33)P-cycle sequencing. RESULTS: We detected the ER-α A908G mutation in 37 of 653 (5.7%) breast tumors. The absence of this mutation in germline DNA confirmed it to be somatic. Three tumors exhibited only the mutant G base at nucleotide 908 on sequencing, indicating that the wild-type ER-α allele had been lost. The ER-α A908G mutation was found more frequently in higher-grade breast tumors (odds ratio (OR) 2.83; 95% confidence interval (CI) 1.09 to 7.34, grade II compared with grade I), and in mixed lobular/ductal tumors (OR 2.10; 95% CI 0.86 to 5.12) compared with ductal carcinomas, although the latter finding was not statistically significant. CONCLUSION: This population-based study, the largest so far to screen for the ER-α A908G mutation in breast cancer, confirms the presence of the mutant in invasive breast tumors. The mutation was associated with higher tumor grade and mixed lobular/ductal breast tumor histology

PAR1 is selectively over expressed in high grade breast cancer patients: a cohort study

<p>Abstract</p> <p>Background</p> <p>The protease-activated receptor (PAR1) expression is correlated with the degree of invasiveness in cell lines. Nevertheless it has never been directed involved in breast cancer patients progression. The aim of this study was to determine whether PAR1 expression could be used as predictor of metastases and mortality.</p> <p>Methods</p> <p>In a cohort of patients with infiltrating ductal carcinoma studied longitudinally since 1996 and until 2007, PAR1 over-expression was assessed by immunoblotting, immunohistochemistry, and flow citometry. Chi-square and log rank tests were used to determine whether there was a statistical association between PAR1 overexpression and metastases, mortality, and survival. Multivariate analysis was performed including HER1, stage, ER and nodes status to evaluate PAR1 as an independent prognostic factor.</p> <p>Results</p> <p>Follow up was 95 months (range: 2–130 months). We assayed PAR1 in a cohort of patients composed of 136 patients; we found PAR1 expression assayed by immunoblotting was selectively associated with high grade patients (50 cases of the study cohort; P = 0.001). Twenty-nine of 50 (58%) patients overexpressed PAR1, and 23 of these (46%) developed metastases. HER1, stage, ER and PAR1 overexpression were robustly correlated (Cox regression, P = 0.002, P = 0.024 and P = 0.002 respectively). Twenty-one of the 50 patients (42%) expressed both receptors (PAR1 and HER1 P = 0.0004). We also found a statistically significant correlation between PAR1 overexpression and increased mortality (P = 0.0001) and development of metastases (P = 0.0009).</p> <p>Conclusion</p> <p>Our data suggest PAR1 overexpression may be involved in the development of metastases in breast cancer patient and is associated with undifferentiated cellular progression of the tumor. Further studies are needed to understand PAR1 mechanism of action and in a near future assay its potential use as risk factor for metastasis development in high grade breast cancer patients.</p

17β-Oestradiol treatment modulates nitric oxide synthase activity in MDA231 tumour with implications on growth and radiation response

The putative oestrogen receptor negative human breast cancer cell line MDA231, when grown as tumours in mice continually receiving 17β-oestradiol, showed substantially increased growth rate when compared to control animals. Further, we observed that 17β-oestradiol treatment could both increase the growth rate of established MDA231 tumours as well as decreasing the time taken for initiating tumour growth. We have also demonstrated that this increase in growth rate is accompanied by a four-fold increase in nitric oxide synthase activity, which was predominantly the inducible form. Inducible-nitric oxide synthase expression in these tumours was confirmed by immunohistochemical analysis and appeared localized primarily in areas between viable and necrotic regions of the tumour (an area that is presumably hypoxic). Prophylactic treatment with the nitric oxide synthase inhibitor nitro-L-arginine methyl ester resulted in significant reduction in this apparent 17β-oestradiol-mediated growth promoting effect. Tumours derived from mice receiving 17β-oestradiol-treatment were characterized by a significantly lower fraction of perfused blood vessels and an indication of an increased hypoxic fraction. Consistent with these observations, 17β-oestradiol-treated tumours were less radio-responsive compared to control tumours when treated with a single radiation dose of 15 Gy. Our data suggests that long-term treatment with oestrogen could significantly alter the tumour oxygenation status during breast tumour progression, thus affecting response to radiotherapy

Estrogen receptor transcription and transactivation: Basic aspects of estrogen action

Estrogen signaling has turned out to be much more complex and exciting than previously thought; the paradigm shift in our understanding of estrogen action came in 1996, when the presence of a new estrogen receptor (ER), ERβ, was reported. An intricate interplay between the classical ERα and the novel ERβ is of paramount importance for the final biological effect of estrogen in different target cells

Abstract ES04-2: Understanding nuclear receptor in breast cancer

Abstract Breast cancer cells have receptors on the surface, in the cytoplasm and in the nucleus. Chemical messengers such as steroid hormones bind to receptors, and this causes changes in the transcriptional program of the cells. Breast cancer cells may express two important steroid receptors: estrogen receptor s alpha and beta (ERs a and b, respectively), and/or progesterone receptor (PR). ER-positive cancer cells depend on estrogen for their growth, so they can be treated with drugs to block estrogen effects (e.g. tamoxifen), or which block estrogen synthesis (Cyp19 [aromatase] inhibitors or Cyp19 inhibitors which block both androgen and estrogen synthesis. Cells which express more of these receptors generally respond better to hormonal therapies. The ERs are from a large family of nuclear receptor transcription factors (TFs). When bound by hormone (they bind to DNA directly or they can interact with other transcription factors bound to DNA to regulate gene expression. ER and PR acts by recruiting a complex of coactivator or corepressor proteins that modulate ER functions. These coactivators are important regulatory molecules, and evidence suggests that some are oncogenes, capable of causing breast cancer and showing gene amplification and/or over-expression in a ER-psositiv breast cancers, thus affecting tumor growth and treatment. Many mechanisms for resistance to endocrine therapies have been postulated and the loss of ER over time although not common can happen. The evolution of receptor mutations over time perhaps in tumor subpopulations has also been postulated, and recent evidence suggests that they occur more frequently than once thought in ER-positive metastatic gtumors. In addition, the PR is frequently lost or altered, which can result from its activation from upstream growth factor receptors. Coactivators that are overexpressed can also modulate ER function so that tumors fail to respond to blocking ER therapies. The basic biology, function, and altered mechanism of action of ER/PR which occur during tumor evolution will be discussed in this educational session. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr ES04-2.</jats:p

Abstract P4-04-08: Therapeutic strategy for ERα mutation driven-endocrine resistance in ER-positive breast cancers

Abstract Background: Although estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy, 25% of these patients are at risk of relapse and the development of acquired endocrine resistance. Recently mutations in the ER gene (ESR1) have been identified which induce resistance to endocrine therapy. The most frequent ESR1 mutation, Y537S, promotes ligand-independent ER activity. It is known that ER regulates the cell cycle in a ligand-dependent manner. In this study, we examined the effects of the Y537S ESR1 mutation on cell cycle signaling and therapeutic response to checkpoint inhibitor. Material and Methods: MCF-7 cells expressing the Y537S ESR1 mutation were generated by CRISPR-Cas9 knock-in techniques. Cells were incubated in phenol red minus medium containing 5% charcoal-dextran treated serum for 5 days to remove exogenous hormones. Cell cycle and apoptosis were examined by Flow cytometry and Annexin-V assays. Proliferation was analyzed by BrdU incorporation. Cell senescence was determined using beta-galactosidase assays. Cell cycle checkpoint kinases were examined by western blot analysis. Cell growth was analyzed using soft agar or MTT assays. Results: Levels of p53 and apoptosis pathway proteins were significantly elevated in Y537S ESR1 mutant cells using RNA expression and reverse-phase protein microarrays. The ATM/ATR and Chk1/Chk2 mediated checkpoint signaling, the upstream pathway of p53, was activated in ESR1 Y537S mutation, which was repressed with fulvestrant treatment. ESR1 Y537 mutant cells accumulated about 5 fold in S phase and 1.7 fold in G2/M phase compared to control cells in estrogen deprived (ED) condition. BrdU incorporation was also increased about 2.5 fold compared with parental cells in estrogen-free medium. In addition, ESR1 Y537 mutant cells expressed a DNA double-strand break marker, gamma-H2AX protein in ED condition. Apoptosis and senescence were observed in ESR1 Y537S mutant cells in regular medium, however, apoptosis was not shown in ED medium. Chk1 inhibitor, PF477736 sensitized MCF-7 expressing ESR1 Y537S mutation to endocrine treatments such as fulvestrant, tamoxifen and AZD9496. Conclusion: Combination therapy with cell cycle checkpoint kinase inhibitor may lead better prognosis in ER mutation driven-endocrine resistance in postmenopausal breast cancers. Citation Format: Kim J-A, Fuqua SAW. Therapeutic strategy for ERα mutation driven-endocrine resistance in ER-positive breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-08.</jats:p