Tamoxifen-treated Breast Cancer Patients Research Articles

CYP2D6 genotyping and the clinical impact on outcomes in breast cancer tamoxifen-treated patients.

Aims: To report the distribution of allele frequencies of CYP2D6 gene and to evaluate their influence on the clinical outcomes of a group of breast cancer patients receiving adjuvant tamoxifen treatment from Uruguay. Patients & methods: 199samples were genotyped through real-time polymerase chain reaction assays. Metabolization profiles were inferred from the genotypes. Correlations were evaluated using Pearson's χ2 test. Results: Phenotype frequencies were0.65 normal (NM), 0.30 intermediate (IM)and 0.05 poor metabolizers (PM). Similar clinical outcomes between NM and (PM+IM) patient groups (odds ratio=1.011, 95% CI=0.2703-3.7826;p=0.987) were found. Conclusion: CYP2D6 allele frequencies were analyzed for the first time in a cohort from Uruguay. Results did not support any impact of CYP2D6 gene polymorphisms on clinical outcomes.

EZH2 Expression and Survival for ER+/tamoxifen Treated Breast Cancer Patients with rs2302427 C>G: A Novel Prognostic and Risk Predictive Biomarker

Overexpression of the EZH2 gene silences several genes involved in DNA repair, cell-cell adhesion, and tumor suppressor genes, resulting in the development of several types of cancers. In the present study, a genetic polymorphism analysis was performed by selecting three SNPs (rs.2302427C>G, rs.3757441C>T, and rs.6950683T>C) of the EZH2 gene based on our previous in silico studies. A total of 250 breast cancer patients and 250 healthy individuals were recruited for the study. Patients with pre-operative breast cancer with different clinical-pathological variables and age-matched healthy women were recruited for the EZH2 gene expression analysis. The genetic polymorphism analysis revealed two SNPs (rs.2302427C>G and rs.6950683T>C) of the three studied SNPs of the EZH2 gene have a protective role in all three genetic models. The haplotype analysis predicted that two haplotypes ACGT and ACGC were significantly associated with a lower risk of breast cancer. Three significant findings of the SNP rs.2302427C>G (Asp193His) i.e., protective role against breast cancer, survival advantage in ER+/tamoxifen treated breast cancer patients, and decreased expression due to the presence of mutant GG genotype, suggests considering it as an important prognostic biomarker for a good survival outcome of breast cancer patients treated with ER+/tamoxifen. Compared with other studies, the other SNP rs.3757441T>C was observed to have a protective effect in breast cancer biology but plays an antagonistic role in colorectal cancer (CRC) biology. To our knowledge, this is the first detailed study on computationally validated EZH2 SNPs in breast cancer.

EXPRESSION PATTERN OF miR-125b-2, -155, -221, AND -320a IS ASSOCIATED WITH RESPONSE OF BREAST CANCER PATIENTS TO TAMOXIFEN.

Hormonal therapy is one of themain methods of comprehensive treatment of patients with locally advanced breast cancer(BC). Despite theintensive search for molecules associated with theaggressiveness of thetumor process, currently there are no reliable markers predicting response to neoadjuvant hormonal therapy(NHT). To investigate thecorrelation between miR-125b-2, -155, -221, -320a expression in tumor tissue and HER2/neu status and response to tamoxifen treatment in BC patients. Expression levels of miR-125b-2, -155, -221, and -320a were analyzed in biopsy samples of 50BC patients using a real-time polymerase chain reaction. We found that levels of miR-125b-2, -155, -221, and -320a were 1.72, 1.65, 1.85, and 2.89times higher in BC biopsy samples expressing estrogen/progesterone receptors and HER2/neu compared with HER2/neu-negative luminal tumors. Patients with a luminal BC showing higher levels of miR-125b-2and miR-320a expression before therapy demonstrated better response to NHT with tamoxifen. A strong correlation was calculated for miR-221expression and response to NHT(r = 0.61). Thehigh levels of miR-125b-2, -155, -221, and -320a in tumor tissue are associated with theHER2/neu-positive status of luminal BC subtypes. Tumor samples of patients showing thelow response to NHT with tamoxifen are characterized by lower expression of miR-125b-2and -320a. Hence, miR-125b-2and -320a could be considered as putative predictive biomarkers associated with tamoxifen sensitivity of hormone-dependent BC.

A new survival analysis model in adjuvant Tamoxifen-treated breast cancer patients using manifold-based semi-supervised learning

Breast cancer researchers are very interested in studying and analyzing the survival time of patients. However, choosing the appropriate model for survival time analysis is the main challenge in the survival analysis of these patients. Using a dataset of patients with breast cancer, a new survival analysis model is proposed in this study. First, using the Cox-PH model, the whole selected data was categorized into high-risk and low-risk groups. The classified data was given to the AFT model in the next step to estimate the survival time. In some instances, the value of the metabolic status of the Tamoxifen feature was missing. Next, using the information obtained from the first and second steps, in the proposed Manifold-based semi-supervised learning module, the value of missing data was estimated. Using all labeled data, the final survival analysis was performed, and the relative hazard obtained. The comparison of the proposed model via other previous models in survival analysis demonstrates that the proposed model is 26% more accurate than when only the Cox-PH model was used and 28% more accurate than when only the AFT model was used in the testing set. Furthermore, the findings demonstrate that when the Manifold-based SSL model was used in the learning module, the accuracy was 19% higher than the Co-training SSL method and 12% higher than the SVMSSL method. The advantages of the proposed survival analysis method are high capability for identifying the survival risk classes of the patient and high predictive accuracy for the relative hazard.

Identification of the estrogen receptor beta as a possible new tamoxifen-sensitive target in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype. Despite the proven efficacy of combined immunochemotherapy (R-CHOP) in the majority of patients, ~40% of DLBCL patients do not respond or will relapse and consequently have a very poor prognosis. The development of targeted therapies has not improved patient survival, underscoring the need for new treatment approaches. Using an unbiased genome-wide CD20 guilt-by-association approach in more than 1800 DLBCL patients, we previously identified the estrogen receptor beta (ERβ) as a new target in DLBCL. Here, we demonstrate that ERβ is expressed at significantly higher levels in DLBCL compared to normal B cells, and ERβ plays a role in the protection against apoptosis in DLBCL. Targeting of the ERβ with the selective estrogen receptor modulator tamoxifen reduces cell viability in all tested DLBCL cell lines. Tamoxifen-induced cell death was significantly decreased in an ERβ knock-out cell line. The activity of tamoxifen was confirmed in a xenograft human lymphoma model, as tumor growth decreased, and survival significantly improved. Finally, tamoxifen-treated breast cancer (BC) patients showed a significantly reduced risk of 38% for DLBCL compared to BC patients who did not receive tamoxifen. Our findings provide a rationale to investigate tamoxifen, a hormonal drug with a good safety profile, in DLBCL patients.

Efficacy of sonohysterography and hysteroscopy for evaluation of endometrial lesions in tamoxifen treated patients: a systematic review

Objective: This review aims to evaluate the incidence of endometrial lesions in tamoxifen-treated breast cancer patients identified by hysteroscopy (HS) and sonohysterography (SIS) and the diagnostic accuracy of the two methods to detect them. Methods: A systematic review of the literature concerning the role of HS and SIS for evaluation of the endometrium in tamoxifen-treated breast cancer patients was performed. We searched MEDLINE (PubMed), EMBASE, Cochrane Central Register of Controlled Trials, IBECS, BIOSIS, Web of Science, SCOPUS, congress abstracts, and Grey literature (Google Scholar; British Library). The search terms used were “hysteroscopy”, “hysterosonography”, “sonohysterography” combined with “tamoxifen”; 89 citations were identified and selected in the initial screening. Results: 28 studies were included in the systematic review. There were 61 citations excluded because they were review articles (n = 9) or case report (n = 5) and non-English articles (n = 8), and had too little information in the full text (n = 39). Similar accuracy between SIS and HS in detection of endometrial tamoxifen-related lesions was found. Conclusions: SIS may represent a minimally invasive, simple, safe, well-tolerated and cost-effective alternative to HS, associated with few contraindications and no potential complications.

Functional relationship of SNP (Ala490Thr) of an epigenetic gene EZH2 results in the progression and poor survival of ER+/tamoxifen treated breast cancer patients

EZH2 is a classic histone methyltransferase that causes the trimethylation of H3K27 and consequently suppresses the cancer preventive genes. The role of elevated levels of EZH2 expression in breast cancer aggressiveness and poor prognosis is very well established. The present study focusses on the insight of the clinically important SNPs of EZH2 gene in determining the structure-function relationship towards breast cancer susceptibility. For this purpose the EZH2 SNPs (rs41277434A>C and rs201135441C>T (A490T)) were computationally explored and further the prediction outcomes were validated by performing population-based association and pharmacogenetic study in north Indian region of Punjab. The results of the present analysis provided the novel insight of rs201135441C>T (A490T) mutation, that A>T change i.e. nonpolar amino acid to polar amino acid stabilizes the enzyme-substrate (EZH2-Histone) complex which in turn promotes trimethylation over histone 3 (H3) at lysine residue 27 (H3K27me3) and this might be leading to the methylation of the promoter region of various cancer preventive genes, hence may increase the risk of breast cancer susceptibility. Further the association based study of SNP rs201135441C>T (A490T) support the in silico outcomes by revealing that the T mutant allele of rs201135441 has significantly increased the risk of breast cancer susceptibility and also reduce the overall survival and progression free survival of ER+/tamoxifen treated breast cancer patients and triple negative breast cancer (TNBC) patients, respectively. To the best of our knowledge, this is the first study on EZH2 polymorphism with breast cancer. In conclusion, these findings suggest that these variations in the EZH2 gene may have strong clinical significance as they can be targeted for prognosis, prevention and in drug development.

Impact of systemic adjuvant therapy and CYP2D6 activity on mammographic density in a cohort of tamoxifen-treated breast cancer patients

PurposeChange in mammographic density has been suggested to be a proxy of tamoxifen response. We investigated the effect of additional adjuvant systemic therapy and CYP2D6 activity on MD change in a cohort of tamoxifen-treated pre- and postmenopausal breast cancer patients.MethodsSwedish breast cancer patients (n = 699) operated 2006–2014, genotyped for CYP2D6, having at least three months postoperative tamoxifen treatment, a baseline, and at least one follow-up digital mammogram were included in the study. Other systemic adjuvant treatment included chemotherapy, goserelin, and aromatase inhibitors. Change in MD, dense area, was assessed using the automated STRATUS method. Patients were stratified on baseline characteristics, treatments, and CYP2D6 activity (poor, intermediate, extensive, and ultrarapid). Relative density change was calculated at year 1, 2, and 5 during follow-up in relation to treatments and CYP2D6 activity.ResultsMean relative DA decreased under the follow-up period, with a more pronounced MD reduction in premenopausal patients. No significant effect of chemotherapy, aromatase inhibitors, goserelin, or CYP2D6 activity on DA change was found. DA did not revert to baseline levels after tamoxifen discontinuation.ConclusionOur results indicate that other systemic adjuvant therapy does not further reduce MD in tamoxifen-treated breast cancer patients. We could not confirm the previously suggested association between CYP2D6 activity and MD reduction in a clinical setting with multimodality adjuvant treatment. No rebound effect on MD decline after tamoxifen discontinuation was evident.

Diagnostic Accuracy of Transvaginal Ultrasound Versus Hysteroscopy in Evaluation of Endometrial Pathology in Breast Cancer Women Receiving Tamoxifen: Correlation with Histopathological Examination

ABSTRACT Background: Multiple endometrial lesions have been reported in tamoxifen -treated breast cancer patients. As a consequence, a thorough uterine cavity examination is often necssary. Objective: To compare the efficacy of transvaginal ultrasound versus hysteroscopy in detecting the endometrial changes in tamoxifen-treated breast cancer patients, and to correlate the findings of both methods with histopathological examination of endometrial biopsies. Patients and methods: Twenty seven patients who had mastactomies for breast cancer and receiving tamoxifen (TAM) were enrolled in this prospective study. They subjected to transvaginal ultrasonography (TVUS) and hysteroscopy for endometrial assessment. Endometrial biopsy was taken from all participants for hitstopathological examination. The ultrasongraphic and hysteroscopic findings were compared and correlated with the histopathological results. Results: The diagnostic accuracy of TVUS versus hysteroscopy in detecting endometrial changes in breast cancer TAM treated women was as follows;TVUS had a sensitivity (82.4%), specificity (30.0%), PPV (66.7%), NPV (50.0%) and accuracy (63%) ;while, the hysteroscope had sensitivity (94.1%), specificity (60.0%), PPV (80.0%), NPV (85.7%) and accuracy (81.5%). Conclusion: Because of certain discrepancies between TVUS and histopathological results in TAM treated women, TVUS is not the best tool for evaluating the endometrium in those patients. The utility of the hysteroscopic image processing helps in the triage of women who require endometrial surveillance, so it will reduce the pathologist's workload and the patient's apprehension while waiting for histopathology result. Nevertheless hysteroscope cannot establish a conclusive diagnosis of premalignant or malignant lesions, but it provides a direct view for targeted biopsies to create a definitive diagnosis.

High expression of TRAF4 predicts poor prognosis in tamoxifen-treated breast cancer and promotes tamoxifen resistance.

Tamoxifen is the main adjuvant endocrine therapeutic agent for patients with estrogen receptor positive breast cancer. However, the resistance to tamoxifen has become a serious clinical challenge and the underlying mechanisms are still poorly understood. TRAF4 is a member of tumor necrosis factor receptor-associated factor family and its role in tamoxifen resistance has not been found. In this study, we aimed to explore the roles of TRAF4 in tamoxifen-treated breast cancer and tamoxifen resistance. Through high-throughput sequencing and differential gene expression analyses, TRAF4 was identified as the research object in this study. The prognosis significance of TRAF4 was studied based on 155 tamoxifen-treated breast cancer patients obtained from Gene Expression Omnibus (GEO) database. We then investigated the TRAF4 expression level in tamoxifen-resistant and the tamoxifen-sensitive breast cancer cell lines with western blot and real-time quantitative PCR. The loss- and gain-of-function assay of TRAF4 in a tamoxifen-resistant cell line was evaluated using colony formation experiments and cell count kit-8 assay. We identified that TRAF4 was overexpressed in tamoxifen-resistant breast cancer cell line and TRAF4 overexpression was associated with worse overall survival (hazard ratio = 2.538, P = 0.017) and cancer-specific survival (hazard ratio = 2.713, P = 0.036) in tamoxifen-treated patients. Knockdown of TRAF4 reversed tamoxifen resistance, while overexpression of TRAF4 increased tamoxifen resistance, which confirmed the role of TRAF4 in tamoxifen resistance. Taken together, our study demonstrated that TRAF4 could be a novel prognostic biomarker for tamoxifen-treated breast cancer patients and a potential therapeutic target for tamoxifen resistance.

Digital Image Analysis of Ki-67 Stained Tissue Microarrays and Recurrence in Tamoxifen-Treated Breast Cancer Patients.

PurposeThe proliferation marker Ki-67 has been used as a prognostic marker to separate low- and high-risk breast cancer subtypes and guide treatment decisions for adjuvant chemotherapy. The association of Ki-67 with response to tamoxifen therapy is unclear. High-throughput automated scoring of Ki-67 might enable standardization of quantification and definition of clinical cut-off values. We hypothesized that digital image analysis (DIA) of Ki-67 can be used to evaluate proliferation in breast cancer tumors, and that Ki-67 may be associated with tamoxifen resistance in early-stage breast cancer.Patients and MethodsHere, we apply DIA technology from Visiopharm using a custom designed algorithm for quantifying the expression of Ki-67, in a case–control study nested in the Danish Breast Cancer Group clinical database, consisting of stages I, II, or III breast cancer patients of 35–69 years of age, diagnosed during 1985–2001, in the Jutland peninsula, Denmark. We assessed DIA-Ki-67 score on tissue microarrays (TMAs) from breast cancer patients in a case–control study including 541 ER-positive and 300 ER-negative recurrent cases and their non-recurrent controls, matched on ER-status, cancer stage, menopausal status, year of diagnosis, and county of residence. We used logistic regression to estimate odds ratios and associated 95% confidence intervals to determine the association of Ki-67 expression with recurrence risk, adjusting for matching factors, chemotherapy, type of surgery, receipt of radiation therapy, age category, and comorbidity.ResultsKi-67 was not associated with increased risk of recurrence in tamoxifen-treated patients (ORadj =0.72, 95% CI 0.54, 0.96) or ER-negative patients (ORadj =0.85, 95% CI 0.54, 1.34).ConclusionOur findings suggest that Ki-67 digital image analysis in TMAs is not associated with increased risk of recurrence among tamoxifen-treated ER-positive breast cancer or ER-negative breast cancer patients. Overall, our findings do not support an increased risk of recurrence associated with Ki-67 expression.

SRC Promotes Tamoxifen Resistance in Breast Cancer via Up-Regulating SIRT1.

BackgroundEndocrine therapy plays a key role in estrogen receptor-positive breast cancer patients; but, tamoxifen resistance could be a real difficulty for these patients. Several attempts have been made to explore the mechanism and new therapies for these patients. We intend to clarify the expression change of SRC and SIRT1 in tamoxifen-resistant breast cancer cells and explore their functions on tamoxifen resistance.MethodsSRC and SIRT1 expressions were analyzed by RNA sequencing, qPCR and Western blotting. Loss and gain of function of SRC and SIRT1 were utilized to indicate their oncogenic roles in tamoxifen resistance in vitro and in vivo. Kaplan–Meier analysis and receiver operating characteristic curve were used to evaluate the survival and the predicted effects of SRC and SIRT1 on patients’ prognosis.ResultsHigh expressions of SRC and/or SIRT1 were found in tamoxifen-resistant cells and related to poor overall survival (p<0.05 for SRC, p<0.001 for SIRT1, p<0.001 for SRC and SIRT1) and cancer-specific survival (p<0.05 for SRC, p<0.01 for SIRT1, p<0.01 for SRC and SIRT1) of tamoxifen-treated breast cancer patients. Down-regulation of SRC (p<0.01) or SIRT1 (p<0.05) separately reversed the resistance to tamoxifen and the minimal concentration of SRC inhibitor KX-01 (p<0.05) or SIRT1 inhibitor EX527 (p<0.001) could also suppress cell proliferation. The expression level of SIRT1 was positively correlated with that of SRC. Overexpression of SRC significantly promotes the cell resistance to tamoxifen inhibited by SIRT1 (p<0.01). In vivo experiments confirmed the effects of SRC on tumor growth by over- or down-regulating SRC expression (p<0.001 and p<0.001, respectively).ConclusionSRC and SIRT1 are both up-regulated in tamoxifen-resistant breast cancer cells and related to a poor prognosis in tamoxifen-treated breast cancer. Moreover, SRC could promote tamoxifen resistance by up-regulating SIRT1. SRC and SIRT1 might be novel therapeutic targets in tamoxifen-resistant breast cancer and the interaction between SRC and SIRT1 needs to be further explored.

Hormonal status affects plasma exposure of tamoxifen and its main metabolites in tamoxifen-treated breast cancer patients

BackgroundTamoxifen is considered a prodrug of its active metabolite endoxifen, which is dependent on the CYP2D6 and CYP3A enzymes. Tamoxifen pharmacokinetic variability influences endoxifen exposure and, consequently, its clinical outcome. This study investigated the impact of hormonal status on the pharmacokinetics of tamoxifen and its metabolites in TAM-treated breast cancer patients.MethodsTAM-treated breast cancer patients (n = 40) previously believed to have CYP3A activity within the normal range based on oral midazolam and phenotyped as CYP2D6 normal metabolizers using oral metoprolol were divided into two groups according to premenopausal (n = 20; aged 35–50 years) or postmenopausal (n = 20; aged 60–79 years) status. All patients were treated with 20 mg/day tamoxifen for at least three months. Serial plasma samples were collected within the 24 h dose interval for analysis of unchanged tamoxifen, endoxifen, 4-hydroxytamoxifen and N-desmethyltamoxifen quantified by LC-MS/MS. CYP activities were assessed using midazolam apparent clearance (CYP3A) and the metoprolol/alfa-hydroxymetoprolol plasma metabolic ratio (CYP2D6). CYP3A4, CYP3A5 and CYP2D6 SNPs and copy number variation were investigated using TaqMan assays.ResultsPostmenopausal status increased steady-state plasma concentrations (Css) of tamoxifen (116.95 vs 201.23 ng/mL), endoxifen (8.01 vs 18.87 ng/mL), N-desmethyltamoxifen (485.16 vs 843.88 ng/mL) and 4-hydroxytamoxifen (2.67 vs 4.11 ng/mL). The final regression models included hormonal status as the only predictor for Css of tamoxifen [β-coef ± SE, p-value (75.03 ± 17.71, p = 0.0001)] and 4-hydroxytamoxifen (1.7822 ± 0.4385, p = 0.0002), while endoxifen Css included hormonal status (8.578 ± 3.402, p = 0.02) and race (11.945 ± 2.836, p = 0.007). For N-desmethyltamoxifen Css, the final model was correlated with hormonal status (286.259 ± 76.766, p = 0.0007) and weight (− 8.585 ± 3.060, p = 0.008).ConclusionThe premenopausal status was associated with decreased endoxifen plasma concentrations by 135% compared to postmenopausal status. Thus, the endoxifen plasma concentrations should be monitored mainly in the premenopausal period to maintain plasma levels above the efficacy threshold value.Trial registrationRBR-7tqc7k.

Younger tamoxifen-treated breast cancer patients also had higher risk of endometrial cancer and the risk could be reduced by sequenced aromatase inhibitor use: A population-based study in Taiwan.

Objective:Previous Western studies reported that older (≥50 years) breast cancer survivors with tamoxifen treatment had higher risk of endometrial cancer. This study aims to disclose whether younger (<50 years) tamoxifen-treated breast cancer patients also had higher risk of endometrial cancer and to examine whether sequenced aromatase inhibitor (AI) use could reduce the risk.Materials and Methods:A population-based cohort of 39,216 newly diagnosed breast cancer patients was identified from Taiwan National Health Insurance Database from 1999 to 2012. The risk of endometrial cancer in nonusers (n = 14,588), tamoxifen-only (n = 19,302), and sequenced AI (n = 5326) users was compared with Cox regression analysis and was adjusted with age, diabetes, hypertension, and chemotherapy.Results:During the 14-year study period, 133 patients were diagnosed with subsequent endometrial cancers. Compared with nonusers, tamoxifen-only users had higher risk of endometrial cancer (14-year incidence 1.7% vs. 0.3%; adjusted hazard ratio [HR] 3.90; 95% confidence interval [CI], 2.37–6.42). This was observed in both older (≥50 years) and younger (40–50 years) age groups. Adjusted HR (95% CI) for the latter was 3.74 (1.65–8.48). This risk persisted after cessation of tamoxifen use. The risk of endometrial cancer was lower in sequenced AI when compared with tamoxifen-only users (adjusted HR 0.43; 95% CI, 0.25–0.72).Conclusions:Not only patients ≥50 years but also younger (40–49 years) patients with tamoxifen treatment had higher risk of subsequent endometrial cancer in this nation-wide cohort. We suggest regular gynecologic monitoring not only during active use but also during follow-up phase. Sequenced AI use may reduce the risk of endometrial cancer in tamoxifen-treated breast cancer patients.

Abstract 2121: Overcoming tamoxifen resistance and inhibiting metastatic recurrence in estrogen receptor-positive breast cancer

Abstract Estrogen receptor (ER)-positive breast cancer accounts for almost 75% of all breast cancers. Tamoxifen has been used for over 40 years to treat early, locally advanced and metastatic ER-positive breast cancer; however, patients develop resistance over time. Furthermore, metastatic recurrence, the major cause of death, is common in ER-positive breast cancer patients whose cancer progressed on tamoxifen and other hormone therapies. In this line, we and others reported epithelial-mesenchymal (EMT)-like changes upon acquisition of tamoxifen resistance hinting towards the co-occurrence of resistance and metastatic abilities of the cells. However, little is known about common molecular mediators of drug resistance and metastatic recurrence. Therefore, we addresses the question of how we concurrently overcome tamoxifen resistance and prevent lethal metastatic recurrence whereby eliminating the mortality associated with metastatic breast cancer. Combining whole-transcriptome sequencing and downstream pathway analysis, we identified cyclic AMP (cAMP) signaling to be the most significantly altered pathway in acquired tamoxifen resistant breast cancer cells. We found that PDE4D (Phosphodiesterase 4D), which hydrolyzes cAMP, was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. We demonstrated that PDE4D inhibition overcomes tamoxifen resistance in cell lines and tumor xenografts via cAMP induced endoplasmic reticulum stress and cell death. Importantly, our TamR cells have partial EMT and enrichment of TGF-beta signaling, and PDE4D inhibition in combination with tamoxifen inhibited cell migration in vitro and blocked TGF-beta induced EMT. RNA-Seq experiment also identified one of the highly oncogenic long non-coding RNAs (lncRNA) to be upregulated in TamR cells, and its targeting inhibits PDE4D and leads to tamoxifen sensitization. We showed that higher expressions of both PDE4D and the candidate lncRNA predict worse survival in tamoxifen-treated breast cancer patients. Currently, we are testing the tamoxifen sensitizer and metastasis blocker functions of PDE4D and our candidate lncRNA using patient-derived xenograft (PDX) models of metastatic ER-positive breast cancer. Overall, our results suggest that targeting PDE4D, or its upstream regulatory lncRNA, can simultaneously overcome tamoxifen resistance and prevent metastatic recurrence in ER-positive breast cancer. This is then expected to dramatically reduce mortality rates among ER-positive breast cancer patients in future. Citation Format: Pelin Gulizar Ersan, Ozge Saatci, Oguzhan Tarman, Rasmi Mishra, Nevin Belder, Unal Metin Tokat, Yasser Riazalhosseini, Ozgur Sahin. Overcoming tamoxifen resistance and inhibiting metastatic recurrence in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2121.

Abstract P2-06-14: The role of SLC7A5 (LAT1) in endocrine therapy-resistant breast cancer

Abstract Endocrine therapies are commonly used to treat estrogen receptor-positive (ER+) breast cancers, which comprise 70% of all new breast cancer cases. Unfortunately, emergence of resistance to these therapies presents a major clinical challenge. Cancer cells can adapt to the dysregulation of cellular metabolism induced by endocrine therapy in order to evade cell death.Central to this adaptation is the scavenging of free-formed amino acids from the tumor microenvironment. For example, we found 109 solute carrier (SLC) mRNAs to be differentially expressed between endocrine-sensitive and resistant cells. We began our mechanistic studies of these genes with SLC family 7 member 5 (SLC7A5 or LAT1). SLC7A5 is a key component of a transmembrane transporter, which can complex with CD98 and increase the uptake of large, neutral amino acids (such as leucine or tyrosine). We used a panel of endocrine therapy-resistant (LCC9) and sensitive (MCF7; LCC1) breast cancer cells. SLC7A5 expression was upregulated by estrogen in MCF7 and LCC1 cells; this induction was blocked by fulvestrant treatment. Basal expression of the SLC7A5 protein in the absence of estrogen was 2.75-fold higher in LCC9 cells compared with MCF7 cells; SLC7A5 mRNA expression was 71-fold higher. Fulvestrant treatment did not significantly alter SLC7A5 mRNA or protein expression in LCC9 cells. Inhibiting SLC7A5 function using either a pharmacological inhibitor (JPH203), or depleting expression using siRNA, led to significant suppression of LCC9 cell growth. Cell cycle analysis revealed that SLC7A5 depletion caused cells to accumulate in the G1-phase, with a concurrent reduction of cells in S-phase. In four publicly available datasets of ER+, tamoxifen treated breast cancer patients, high expression of SLC7A5 was significantly associated with poor relapse-free survival. This study uncovers a novel adaptive mechanism in endocrine therapy-resistant breast cancer cells that is facilitated by increased expression of SLC7A5, which enables them to supplement their increased metabolic needs and promoting cell growth. Blocking the functions of SLC7A5, perhaps in conjunction with inhibition of autophagy, may therefore offer a new avenue of potential therapeutic intervention against endocrine therapy-resistant breast cancers. Citation Format: Sevigny CM, Sengupta S, Luo Z, Jin L, Pearce D, Clarke R. The role of SLC7A5 (LAT1) in endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-14.

Event-free survival following early endometrial events in breast cancer patients treated with anti-hormonal therapy: A nationwide claims data study.

Tamoxifen, an anti-estrogen agent that can suppress breast cancer, has been reported to increase endometrium-related adverse events. There are no guidelines for screening tamoxifen-treated patients for endometrial disease. We analyzed nationwide claims data related to endometrial diseases to investigate patterns of endometrial disease in breast cancer patients who underwent hormonal treatment.We sourced claims data from the Health Insurance Review and Assessment Service in South Korea. Patients who made their first claim for an anti-hormonal agent between January 1, 2010 and December 31, 2012 were enrolled retrospectively. We analyzed patient characteristics and all claims related to endometrial disease, stratified by prescribed hormonal agents.Among a total of 32,496 enrolled patients, 19,603 used tamoxifen only and 10,101 were treated with an aromatase inhibitor (AI) alone. Endometrial events occurred in 15.4% (3028/19603) of the tamoxifen-only patients and 2.0% (201/10101) of the AI-only group. In patients diagnosed with breast cancer at the age of 50 or older, the hazard ratio (HR) of endometrial malignancy in the tamoxifen-only group compared to the AI-only group was 4.13 (95% CI 1.404–12.159, P = .010). The HR of curettage in the tamoxifen-only group was 31.0 (95% CI 19.668–48.831, P <.001).The occurrence of endometrial events among tamoxifen-treated breast cancer patients was higher than in patients treated with only AI, similar to previous studies. However, the HR of curettage was uniquely high, despite its invasiveness. Guidelines for screening endometrial disease and improvements of healthcare policy are required to appropriately manage high-risk patients.

Expression and clinicopathological significance of DNA methyltransferase 1, 3A and 3B in tamoxifen-treated breast cancer patients

Progression of tamoxifen resistance remained as a crucial obstacle to treatment of estrogen receptor positive breast carcinoma patients. Recent studies demonstrated the importance of DNA methylation pattern on tamoxifen refractory. This study aimed to investigate the protein expression pattern and clinicopathological significance of DNA methyltransferase 1, 3A and 3B, as leading factors in regulation of DNA methylation process, in breast carcinoma patients with adjuvant tamoxifen therapy. Seventy two Formalin-Fixed Paraffin-Embedded (FFPE) breast tumor tissues of tamoxifen sensitive (TAMS) and tamoxifen resistance (TAM-R) patients were recruited for immunohistochemical experiments. DNMT1, DNMT3a, and DNMT3b expressions were observed in 86, 72.2 and 100% of tamoxifen resistance patients, respectively. Data analysis indicated that DNMTs were overexpressed in TAM-R tumors (P < 0.05). In TAM-S subgroup, DNMT1, DNMT3A and DNMT3B expression was associated with high histologic grade (P = 0.049, P = 0.01 and P = 0.02, respectively). DNMT3B expression was also correlated with lymphatic invasion (P = 0.034). In TAM-R subgroup, DNMT1 expression associated with extracapsular nodal extension (P = 0.019). DNMT3A and DNMT3B expression showed a significant association with high histologic grade (P = 0.001) and DNMT3A expression was also associated with HER-2 status (P = 0.027). Cox proportional hazard model demonstrated that overexpression of DNMT3B remained as an independent and unfavorable prognostic factor for disease free survival (P < 0.001). Taken together, these results suggest that DNMTs could be an effective factor in development of tamoxifen resistance in breast tumors.

Effect of CYP2C19 and CYP2D6 genotype on tamoxifen treatment outcome indicates endogenous and exogenous interplay.

We investigated the interaction of CYP2C19 and CYP2D6 genotype on clinical outcome in tamoxifen-treated breast cancer patients. A cohort of 306 patients on tamoxifen treatment for a minimum of 1 year were employed to analyze the effect of genotype-predicted phenotype on relapse-free survival. We show that the group with worst outcome and highest risk of relapse is that of 2C19↑-2D6↓ (hazard ratio: 2.94), when adjusting for age, Nottingham prognostic index and adjuvant chemotherapy. Furthermore, the effect of 2C19↑-2D6↓genotype-predicted phenotype is greatly enhanced in premenopausal patients (hazard ratio: 21.08). We hypothesize that poor bioactivation of tamoxifen in patients with low CYP2D6 activity and high CYP2C19 metabolism represents a tamoxifen-treated patient group that has the worst clinical outcome.

Abstract 4896: The role of SLC7A5 (LAT1) in endocrine therapy-resistant breast cancer

Abstract Breast cancer is the leading cancer diagnosis for women in the United States. Endocrine therapies, such as tamoxifen and aromatase inhibitors, are used to treat the estrogen receptor-positive (ER+) breast cancers that comprise 70% of all new cases. Unfortunately, emergence of resistance to these therapies presents a major clinical challenge. Cancer cells can adapt to dysregulate the bioenergetics of cellular metabolism and evade cell death. Solute carrier family 7 member 5 (SLC7A5 or LAT1) is expressed across the cell membrane and is a transporter of large, neutral amino acids (such as leucine or tyrosine) supporting cell proliferation. Using LCC9 breast cancer cells that are resistant to tamoxifen and fulvestrant, we studied the role of LAT1 in endocrine therapy-resistant cells as compared to sensitive MCF7 breast cancer cells. SLC7A5 expression was upregulated by estrogen in MCF7 cells that was blocked in the presence of fulvestrant treatment. A significant 2.75-fold upregulation of SLC7A5 protein and 71-fold upregulation of SLC7A5 mRNA was found in LCC9 cells (as compared with MCF7 cells) without any estrogen regulation. Fulvestrant treatment did not significantly alter SLC7A5 mRNA or protein expression. These data suggest that higher levels of SLC7A5 in resistant cells may help in transporting key amino acids from their microenvironment to support cell growth. Inhibiting the functions of SLC7A5 using a pharmacologic inhibitor, JPH203, or depleting its expression by using siRNA led to significant growth suppression of LCC9 cells. Cell cycle analysis revealed that SLC7A5 depletion increased G1 phase of cell cycle and reduced S phase cells. In five publicly available data sets, of estrogen receptor-positive and tamoxifen-treated breast cancer patients, high expression of SLC7A5 was associated with worse prognosis. Endocrine therapy resistance is partly driven by autophagy in breast cancer cells. We depleted SLC7A5 expression in LCC9s to better understand the role of this transporter in autophagy. This study uncovers a novel adaptive mechanism in endocrine therapy-resistant breast cancer cells that is facilitated by increased SLC7A5 mediated transport of large neutral amino acids enabling them to supplement their increased metabolic needs and promoting cell growth. Therefore, blocking the functions of SLC7A5, perhaps in conjunction with inhibition of autophagy, may offer an avenue of potential therapeutic intervention in endocrine therapy-resistant breast cancers. Citation Format: Catherine M. Sevigny, Surojeet Sengupta, Zhexun Luo, Lu Jin, Dominic Pearce, Robert Clarke. The role of SLC7A5 (LAT1) in endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4896.