Tamoxifen Resistance In Patients Research Articles

Mammalian enabled protein enhances tamoxifen sensitivity of the hormone receptor-positive breast cancer patients by suppressing the AKT signaling pathway

BackgroundMammalian enabled (MENA) protein is a member of the enabled/vasodilator stimulated phosphoprotein (Ena/VASP) protein family, which regulates cytoplasmic actin network assembly. It plays a significant role in breast cancer invasion, migration, and resistance against targeted therapy and chemotherapy. However, its role in the efficacy of endocrine therapy for the hormone receptor-positive (HR+) breast cancer patients is not known. This study investigated the role of MENA in the resistance against tamoxifen therapy in patients with HR+ breast cancer and the underlying mechanisms.MethodsMENA expression levels in the clinical HR+ breast cancer samples (n = 119) were estimated using immunohistochemistry (IHC) to determine its association with the clinicopathological features, tamoxifen resistance, and survival outcomes. Western blotting (WB) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis was performed to estimate the MENA protein and mRNA levels in the tamoxifen-sensitive and -resistant HR+ breast cancer cell lines. Furthermore, CCK8, colony formation, and the transwell invasion and migration assays were used to analyze the effects of MENA knockdown on the biological behavior and tamoxifen sensitivity of the HR+ breast cancer cell lines. Xenograft tumor experiments were performed in the nude mice to determine the tumor growth rates and tamoxifen sensitivity of the control and MENA knockdown HR+ breast cancer cells in the presence and absence of tamoxifen treatment. Furthermore, we estimated the growth rates of organoids derived from the HR+ breast cancer patients (n = 10) with high and low MENA expression levels when treated with tamoxifen.ResultsHR+ breast cancer patients with low MENA expression demonstrated tamoxifen resistance and poorer prognosis compared to those with high MENA expression. Univariate and multivariate Cox regression analysis demonstrated that MENA expression was an independent predictor of tamoxifen resistance in patients with HR+ breast cancer. MENA knockdown HR+ breast cancer cells showed significantly reduced tamoxifen sensitivity in the in vitro experiments and the in vivo xenograft tumor mouse model compared with the corresponding controls. Furthermore, MENA knockdown increased the in vitro invasion and migration of the HR+ breast cancer cells. HR+ breast cancer organoids with low MENA expression demonstrated reduced tamoxifen sensitivity than those with higher MENA expression. Mechanistically, P-AKT levels were significantly upregulated in the MENA-knockdown HR + breast cancer cells treated with or without 4-OHT compared with the corresponding controls.ConclusionsThis study demonstrated that downregulation of MENA promoted tamoxifen resistance in the HR+ breast cancer tissues and cells by enhancing the AKT signaling pathway. Therefore, MENA is a promising prediction biomarker for determining tamoxifen sensitivity in patients with HR+ breast cancer.

Expression analysis elucidates the roles of Nicastrin, Notch4, and Hes1 in prognosis and endocrine-therapy resistance in ER-positive breast cancer patients.

Although some proposed mechanisms responsible for tamoxifen resistance have already been present, further study is needed to determine the mechanisms underlying tamoxifen resistance more clearly. The critical role of Notch signaling has been described in promoting resistance in therapeutics, but there is little information about its role in tamoxifen resistance progression. In the present study, the expression of Notch pathway genes, including Notch4, nicastrin and the Notch downstream target Hes1 was evaluated using quantitative RT-PCR in 36 tamoxifen-resistant (TAM-R) and 36 tamoxifen-sensitive (TAM-S) patients. Expression data were correlated with the clinical outcome and survival of patients. mRNA levels of Notch4 (fold change = 2.7), nicastrin (fold change = 6.71), and Hes1 (fold change= 7.07) were significantly higher in TAM-R breast carcinoma patients compared to sensitive cases. We confirmed all these genes were co-expressed. Hence, it seems that Notch signaling is involved in tamoxifen resistance in our TAM-R patients. Obtained results showed that Hes1, nicastrin, and Notch4 mRNA upregulation was correlated with the N stage. The extracapsular nodal extension was associated with nicastrin and Notch4 overexpression. Moreover, nicastrin overexpression was correlated with perineural invasion. Hes1 upregulation was also associated with nipple involvement. Finally, the Cox regression proportional hazard test revealed that overexpression of nicastrin was an independent worse survival factor. Presumably, upregulation of the Notch pathway may be involved in tamoxifen resistance in breast cancer patients.

Glutamine synthetase regulates the immune microenvironment and cancer development through the inflammatory pathway

Although adjuvant tamoxifen therapy is beneficial to estrogen receptor-positive (ER+) breast cancer patients, a significant number of patients still develop metastasis or undergo recurrence. Therefore, identifying novel diagnostic and prognostic biomarkers for these patients is urgently needed. Predictive markers and therapeutic strategies for tamoxifen-resistant ER+ breast cancer are not clear, and micro (mi)RNAs have recently become a focal research point in cancer studies owing to their regulation of gene expressions, metabolism, and many other physiological processes. Therefore, systematic investigation is required to understand the modulation of gene expression in tamoxifen-resistant patients. High-throughput technology uses a holistic approach to observe differences among expression profiles of thousands of genes, which provides a comprehensive level to extensively investigate functional genomics and biological processes. Through a bioinformatics analysis, we revealed that glutamine synthetase/glutamate-ammonia ligase (GLUL) might play essential roles in the recurrence of tamoxifen-resistant ER+ patients. GLUL increases intracellular glutamine usage via glutaminolysis, and further active metabolism-related downstream molecules in cancer cell. However, how GLUL regulates the tumor microenvironment for tamoxifen-resistant ER+ breast cancer remains unexplored. Analysis of MetaCore pathway database demonstrated that GLUL is involved in the cell cycle, immune response, interleukin (IL)-4-induced regulators of cell growth, differentiation, and metabolism-related pathways. Experimental data also confirmed that the knockdown of GLUL in breast cancer cell lines decreased cell proliferation and influenced expressions of specific downstream molecules. Through a Connectivity Map (CMap) analysis, we revealed that certain drugs/molecules, including omeprazole, methacholine chloride, ioversol, fulvestrant, difenidol, cycloserine, and MK-801, may serve as potential treatments for tamoxifen-resistant breast cancer patients. These drugs may be tested in combination with current therapies in tamoxifen-resistant breast cancer patients. Collectively, our study demonstrated the crucial roles of GLUL, which provide new targets for the treatment of tamoxifen-resistant breast cancer patients.

Metabolic/hypoxial axis predicts tamoxifen resistance in breast cancer

We sought in our cross-sectional study to investigate the role of metabolic/hypoxial axis in the development of tamoxifen (TMX) resistance in BC patients. Quantification of plasma LncRNA Taurine upregulated-1 (TUG-1), miRNA 186-5p (miR-186), serum Sirtuin-3 (SIRT3), Peroxisome Proliferator Activator Receptor alpha (PPAR-1 α) and Hypoxia Inducible Factor-1 (HIF-1α) was done in a cohort of patients divided into TMX-sensitive and TMX-resistant candidates. Multiple logistic regression and Receiver Operating Characteristic curve were developed for significant predictors. Plasma TUG-1 and miR-186 were significantly elevated in TMX resistant patients. Serum proteins SIRT3, PPAR-1 α and HIF-1α were deficient in TMX resistant patients compared to TMX sensitive patients, respectively. miR-186 was associated with respiratory symptoms, while, HIF-1α was associated with metastases in TMX resistant patients. Strong correlations were found between all parameters. A predictive model was constructed with TUG-1 and HIF-1α to estimate TMX resistance in BC patients with 88.3% sensitivity and 91.6% specificity. Hypoxia and metabolic dysregulations play important role in the development of TMX resistance in BC patients. Correlation between hypoxia, carcinogenesis and patient’s mortality have led to more aggressive phenotypes, increased risk of metastasis and resistance to TMX.

Low concentrations of 17β-estradiol exacerbate tamoxifen resistance in breast cancer treatment through membrane estrogen receptor-mediated signaling pathways.

The present study aims to discover the influences of tamoxifen and 17β-estradiol (E2) on tamoxifen-resistant (TamR) patients in vitro. Herein, we established a stabilized TamR MCF-7 cell line at 1μM via gradient concentrations of tamoxifen cultivation. The expression changes of four ER subtypes (ERα66, ERβ, ERα36 and GPR30) were found to bring about tamoxifen resistance. Moreover, the generation of tamoxifen resistance involved in apoptosis escape via a reactive oxygen species-regulated p53 signaling pathway. Interestingly, E2 at environmental concentrations (0.1-10nM) could activate the expression of both ERα36 and GPR30, and then stimulate the phosphorylation of ERK1/2 and Akt, resulting in cell growth promotion. Cell migration and invasion promotion, apoptosis inhibition, and cell cycle G1-S progression are involved in such proliferative effects. Conversely, the application of specific antagonists of ERα36 and GPR30 could restore tamoxifen's sensitivity as well as partially offset E2-mediated proliferation. In short, overexpression of ERα36 and GPR30 not only ablate tamoxifen responsiveness but also could promote tumor progression of TamR breast cancer under estrogen conditions. These results provided novel insights into underlying mechanisms of tamoxifen resistance and the negative effects of steroid estrogens at environmental concentrations on TamR MCF-7 cells, thus generating new thoughts for future management of ER-positive breast cancer.

Role of SALL4 and Nodal in the prognosis and tamoxifen resistance of estrogen receptor-positive breast cancer.

Despite the discovery of a number of different mechanisms underlying tamoxifen resistance, its molecular pathway is not completely clear. The upregulation of SALL4 and Nodal has been reported in breast cancer. Nevertheless, their role in tamoxifen resistance has not been investigated. In the present study, we compared Nodal and SALL4 expression in 72 tamoxifen sensitive (TAMS) and tamoxifen-resistant (TAMR) patients. Afterward, the correlation of expression data with clinicopathological features and survival of patients was studied. Results showed that both SALL4 and Nodal were significantly upregulated in TAMR compared to TAMS patients. Besides, there was a positive association between Nodal and SALL4 expression. Furthermore, we evaluated their correlation with the expression of Oct4, Nanog and Sox2 stemness markers. The results demonstrated that in most tissue samples there was a positive correlation between Nodal and SALL4 expression with these stemness markers. Besides, the overexpression of SALL4 and Nodal significantly correlated with the N stage. Moreover, the overexpression of SALL4 was associated with extracapsular invasion and lymphatic invasion. High level expressions of SALL4 and Nodal had a significant association with worse disease-free survival (DFS) rates. In addition, increased level of Nodal expression provides a superior predictor factor for DFS. The multivariate Cox regression analysis also revealed that for DFS, perineural invasion (PNI) was independently an unfavorable prognostic value. These findings suggest that the high expression of SALL4 and Nodal could contribute to tamoxifen resistance and worse survival rates in tamoxifen-treated ER+ breast cancer patients.

Identification of Ten Mitosis Genes Associated with Tamoxifen Resistance in Breast Cancer

BackgroundEndocrine therapy is the backbone therapy in estrogen receptor α (ER)-positive breast cancer, and tamoxifen resistance is a great challenge for endocrine therapy. Tamoxifen-resistant and sensitive samples from the international public repository, the Gene Expression Omnibus (GEO) database, were used to identify therapeutic biomarkers associated with tamoxifen resistance.Materials and MethodsIn this study, integrated analysis was used to identify tamoxifen resistance-associated genes. Differentially expressed genes (DEGs) were identified. Gene ontology and pathway analysis were then analyzed. Weighted correlation network analysis (WGCNA) was performed to find modules correlated with tamoxifen resistance. Protein–protein interaction (PPI) network was used to find hub genes. Genes of prognostic significance were further validated in another GEO dataset and cohort from Shanghai Ruijin Hospital using RT-PCR.ResultsA total of 441 genes were down-regulated and 123 genes were up-regulated in tamoxifen-resistant samples. Those up-regulated genes were mostly enriched in the cell cycle pathway. Then, WGCNA was performed, and the brown module was correlated with tamoxifen resistance. An overlap of 81 genes was identified between differentially expressed genes (DEGs) and genes in the brown module. These genes were also enriched in the cell cycle. Twelve hub genes were identified using PPI network, which were involved in the mitosis phase of the cell cycle. Finally, 10 of these 12 genes were validated to be up-regulated in tamoxifen-resistant patients and were associated with poor prognosis in ER-positive patients.ConclusionOur study suggested mitosis-related genes are mainly involved in tamoxifen resistance, and high expression of these genes could predict poor prognosis of patients receiving tamoxifen. These genes may be potential targets to improve efficacy of endocrine therapy in breast cancer, and inhibitors targeted these genes could be used in endocrine-resistant patients.

PAX2 promoter methylation and AIB1 overexpression promote tamoxifen resistance in breast carcinoma patients.

Disease recurrence is an important obstacle in estrogen receptor positive (ER+) tamoxifen treated breast carcinoma patients. Tamoxifen resistance-related molecular mechanisms are not fully understood. Alteration in DNA methylation which contributes to transcriptional regulation of cancer-related genes plays a crucial role in tamoxifen response. In the present study, the contribution of promoter methylation and mRNA expression of PAX2 and AIB1 in the development of breast carcinoma and tamoxifen refractory was assessed. Methylation specific-high resolution melting (MS-HRM) analysis and Real-time quantitative PCR (RT-qPCR) experiment were performed to analyze the promoter methylation and mRNA expression levels of PAX2 and AIB1 genes in 102 breast tumors and adjacent normal breast specimens. We indicated that PAX2 expression is decreased in breast tissues due to hypermethylation in its promoter region. Compared to the adjacent normal tissues, the tumors exhibited significantly lower relative mRNA levels of PAX2 and increased expression of AIB1. Aberrant promoter methylation of PAX2 and overexpression of AIB1 was observed in tamoxifen resistance patients compared to the sensitive ones. Cox regression analysis exhibited that the increased promoter methylation status of PAX2 and overexpression of AIB1 remained as unfavorable identifiers which influence patients' survival independently. Our results revealed that the aberration in PAX2 promoter methylation and AIB1 overexpression are associated with the tamoxifen response in breast carcinoma patients. Further research is needed to demonstrate the potential of using PAX2 and AIB1 expression and their methylation-mediated regulation as predictive or prognostic biomarkers or as a new target therapy for better disease management.

Implications of Tamoxifen Resistance in Palbociclib Efficacy for Patients with Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer: Subgroup Analyses of KCSG-BR15-10 (YoungPEARL).

PurposeYoungPEARL (KCSG-BR15-10) trial demonstrated a significant progression-free survival (PFS) benefit for premenopausal patients with hormone receptor–positive/human epidermal growth factor receptor 2–negative (HR+/HER2−) metastatic breast cancer (MBC) for palbociclib plus exemestane with ovarian function suppression compared to capecitabine. However, the number of tamoxifen-sensitive premenopausal patients was small because most recurrences occurred early during adjuvant endocrine therapy (ET), with tamoxifen being the only drug used; hence, the data for these patients were limited. Here we present a subgroup analysis according to tamoxifen sensitivity from the YoungPEARL study.Materials and MethodsPatients were randomized 1:1 to receive palbociclib+ET (oral exemestane 25 mg/day for 28 days, palbociclib 125 mg/day for 21 days, plus leuprolide 3.75 mg subcutaneously every 4 weeks) or chemotherapy (oral capecitabine 1,250 mg/m2 twice daily for 14 days every 3 weeks). Tamoxifen resistance was defined as: relapse while on adjuvant tamoxifen, relapse within 12 months of completing adjuvant tamoxifen, or progression while on first-line tamoxifen within 6 months for MBC.ResultsIn total, 184 patients were randomized and 178 were included in the modified intention-to-treat population. PFS improvement in the palbociclib+ET group was observed in tamoxifen-sensitive patients (hazard ratio, 0.38; 95% confidence interval, 0.12 to 1.19). Furthermore, palbociclib+ET prolonged median PFS compared with capecitabine in tamoxifen-sensitive (20.5 months vs. 12.6 months) and tamoxifen-resistant (20.1 months vs. 14.5 months) patients. Palbociclib+ET demonstrated a higher rate of objective response, disease control, and clinical benefit in tamoxifen-sensitive patients.ConclusionThis post hoc exploratory analysis suggests that palbociclib+ET is a promising therapeutic option for premenopausal HR+/HER2− MBC patients irrespective of tamoxifen sensitivity.

Genome-wide analysis of therapeutic response uncovers molecular pathways governing tamoxifen resistance in ER+ breast cancer.

BackgroundPrioritization of breast cancer patients based on the risk of resistance to tamoxifen plays a significant role in personalized therapeutic planning and improving disease course and outcomes.MethodsIn this work, we demonstrate that a genome-wide pathway-centric computational framework elucidates molecular pathways as markers of tamoxifen resistance in ER+ breast cancer patients. In particular, we associated activity levels of molecular pathways with a wide spectrum of response to tamoxifen, which defined markers of tamoxifen resistance in patients with ER+ breast cancer.FindingsWe identified five biological pathways as markers of tamoxifen failure and demonstrated their ability to predict the risk of tamoxifen resistance in two independent patient cohorts (Test cohort1: log-rank p-value = 0.02, adjusted HR = 3.11; Test cohort2: log-rank p-value = 0.01, adjusted HR = 4.24). We have shown that these pathways are not markers of aggressiveness and outperform known markers of tamoxifen response. Furthermore, for adoption into clinic, we derived a list of pathway read-out genes and their associated scoring system, which assigns a risk of tamoxifen resistance for new incoming patients.InterpretationWe propose that the identified pathways and their read-out genes can be utilized to prioritize patients who would benefit from tamoxifen treatment and patients at risk of tamoxifen resistance that should be offered alternative regimens.FundingThis work was supported by the Rutgers SHP Dean's research grant, Rutgers start-up funds, Libyan Ministry of Higher Education and Scientific Research, and Katrina Kehlet Graduate Award from The NJ Chapter of the Healthcare Information Management Systems Society.

GPER mediates decreased chemosensitivity via regulation of ABCG2 expression and localization in tamoxifen-resistant breast cancer cells

Rescue chemotherapy is usually the preferred treatment for patients with advanced estrogen receptor-positive (ER+) breast cancer with endocrinotherapy resistance. However, these patients often simultaneously show a poor response to cytotoxic drugs, and thus the detailed mechanism of this resistance needs to be further investigated. Our previous research indicated that the G-protein-coupled estrogen receptor (GPER) is a novel mediator of the development of multidrug resistance, including resistance to both endocrinotherapy and chemotherapy, and ATP binding cassette subfamily G member 2 (ABCG2) has been identified as an engine that confers cancer cells with chemoresistance by expelling xenobiotics and chemotherapeutics. Here, we are the first to show that the expression levels of GPER and ABCG2 are markedly increased in tamoxifen-resistant ER + metastases compared to the corresponding primary tumors. A plasma membrane expression pattern of GPER and ABCG2 was observed in patients with metastases. Furthermore, both ER modulator tamoxifen, GPER-specific agonist G1 and pure ER antagonist ICI 182,780 significantly enhanced ABCG2 expression in tamoxifen-resistant breast cancer cells (MCF-7R) but not in tamoxifen-sensitive cells (MCF-7). The activated downstream GPER/EGFR/ERK and GPER/EGFR/AKT signaling pathways were responsible for regulating the expression and cell membrane localization of ABCG2, respectively, in MCF-7R cells. Interestingly, the above phenomenon could be alleviated by inhibitors of both the indicated signaling pathways and by knockdown of GPER in MCF-7R cells. More importantly, the tamoxifen-induced GPER/ABCG2 signaling axis was shown to play a pivotal role in the development of chemotherapy (doxorubicin) resistance both in vitro and in vivo. The clinical data further revealed that tamoxifen-resistant patients with high GPER/ABCG2 signaling activation had poor progression-free survival (PFS) when given rescue anthracycline chemotherapy. Therefore, our data provide novel insights into GPER-mediated chemoresistance and provide a rationale for the GPER/ABCG2 signaling axis being a promising target for reversing chemoresistance in patients with advanced ER + tamoxifen-resistant breast cancer.

Histone methyltransferases regulate the transcriptional expression of ER\u03b1 and the proliferation of tamoxifen-resistant breast cancer cells

PurposeAlthough tamoxifen remains the frontline treatment for ERα-positive breast cancers, resistance to this drug limits its clinical efficacy. Most tamoxifen-resistant patients retain ERα expression which may support growth and progression of breast cancers. Therefore, we investigated epigenetic regulation of ERα that may provide a rationale for targeting ERα in these patients.MethodsExpression levels of the mixed-lineage leukemia (MLL) family of proteins in tamoxifen-resistant breast cancer cells and publicly available breast cancer patient data sets were analyzed. Histone methylation levels in ERα promoter regions were assessed using chromatin immunoprecipitation. Expression levels of ERα and its target gene were analyzed using western blotting and real-time qPCR. Cell-cycle was analyzed by flow cytometry.ResultsThe expression of MLL3 and SET-domain-containing 1A (SET1A) were increased in tamoxifen-resistant breast cancers. An MLL3 chromatin immunoprecipitation-sequencing data analysis and chromatin immunoprecipitation experiments for MLL3 and SET1A suggested that these proteins bound to enhancer or intron regions of the ESR1 gene and regulated histone H3K4 methylation status. Depletion of MLL3 or SET1A downregulated the expression level of ERα and inhibited the growth of tamoxifen-resistant breast cancer cells. Additional treatment with fulvestrant resulted in a synergistic reduction of ERα levels and the growth of the cells.ConclusionsThe enhanced expression of MLL3 and SET1A in tamoxifen-resistant breast cancer cells supported the ERα-dependent growth of these cells by increasing ERα expression. Our results suggest that targeting these histone methyltransferases might provide an attractive strategy to overcome endocrine resistance.

Increased Expression of Gankyrin and Stemness Factor Oct-4 are Associated with Unfavorable Clinical Outcomes and Poor Benefit of Tamoxifen in Breast Carcinoma Patients.

Tamoxifen is the most important treatment component in estrogen receptor positive (ER+) breast carcinoma patients. Tamoxifen resistance incidence presents an important obstacle in clinical treatment. Mechanisms underlying tamoxifen refractory are not completely understood. Although elevated expression of Gankyrin (P28GANK) and stem cell markers Nanog, Oct-4 and Sox-2 have been reported in breast carcinoma, their role in tamoxifen resistance progression has not been explored. In the present study, P28GANK and stem cell markers Nanog, Oct-4 and Sox-2 expression were evaluated using quantitative RT-PCR and immunohistochemical technology in 72 breast carcinoma patients who received tamoxifen as adjuvant anti-hormone treatment. Expression data were correlated with the clinical outcome and survival of patients. Data analysis showed that P28GANK, Oct-4 and Sox-2 transcripts were significantly overexpressed in tamoxifen resistance patients. Immunohistochemical staining indicated that protein expression of P28GANK and Oct-4 were also significantly higher in tamoxifen resistance patients. We have shown a positive correlation between mRNA and protein expression of P28GANK, Oct-4 and Sox-2. Multivariate logistic regression analysis indicated that P28GANK (P = 0.002) and Oct-4 (P = 0.013) overexpression could be negative independent factors of disease outcome. Additionally, in the whole study group, multivariate Cox regression analysis revealed that high expression of P28GANK and Oct-4 remained significant and unfavorable predictive factors for patients' survival. These findings suggest that Gankyrin and Oct-4 overexpression could promote tamoxifen refractory in breast cancer patients. More studies are warranted to clarify the predictive role of these potential biomarkers for patients who don't benefit from tamoxifen treatment and their possible application as prognostic markers in ER+ tamoxifen-treated breast carcinoma patients.

Impact of estrogen receptor α on the tamoxifen response and prognosis in luminal-A-like and luminal-B-like breast cancer.

The luminal-A-like and luminal-B-like breast cancer groups have distinct biological features that lead to differences in the treatment response and clinical outcome. The aim of this study was to examine the value of the distribution pattern of ERα expression, ESR1 SNPs as well as ESR1 mRNA expression in predicting tamoxifen response and survival in patients with luminal-A-like and luminal-B-like breast cancer. A total of 135 patients with both subtypes were stratified into two groups depending on the tamoxifen response: tamoxifen-resistant patients (TR) and tamoxifen-sensitive patients (TS). ESR1 mRNA expression was measured by real-time quantitative reverse transcription-PCR. Three polymorphisms of ESR1 (rs2077647, rs2228480 and rs1801132) were genotyped using a TaqMan assay. The distribution pattern of ERα expression was analyzed immunohistochemically using the visual assessment of staining. The primary endpoint was progression-free survival (PFS). There was a significant decrease in ESR1 mRNA expression level in the TR group when compared to the TS group among patients with luminal-B-like subtype (P = 0.038). ESR1 2014AA mutant genotype of rs2228480 was more prevalent in the TR patients with luminal-B-like subtype than the TS group (P = 0.045). In the luminal-A-like group, tamoxifen-resistant tumors were more frequently heterogeneous for ERα expression than tamoxifen-sensitive tumors (P = 0.016). Multivariate analysis showed a strong association of lymph node status and the distribution pattern of ERα expression with tamoxifen responsiveness in this cohort of patients. In addition, a luminal-A-like patients with the heterogeneous ERα expression had a significantly shorter PFS time than those with the homogeneous ERα (P = 0.013). These results indicate that the heterogeneous expression of ERα is an accurate predictor of tamoxifen response and survival in luminal-A-like breast cancer patients. ESR1 rs2228480 may act as a marker with a high prognostic potential in luminal-B-like tumors.

Abstract 4320: Enhanced expression of histone methyltransferases increase ERα expression in tamoxifen-resistant breast cancer cells

Abstract Estrogen receptor (ER)-positive breast cancers depend on ER signaling for growth and progression. Although tamoxifen remains the frontline treatment for such cancers, resistance to this drug limits its clinical efficacy. Nevertheless, the majority of tamoxifen-resistant patients retain ERα expression, providing a rationale for targeting ERα in these patients. Here, we found that the expression of mixed lineage leukemia (MLL) family proteins are increased in tamoxifen-resistant breast cancer cells and patients. Specifically, mixed lineage leukemia 3 (MLL3) and SET domain containing 1A (SET1A) depletion downregulated the expression of ERα protein and mRNA levels in a ER-positive breast cancer cell. Chromatin Immunoprecipitation analysis revealed that the knock-down of MLL3 and SET1A reduce the histone H3K4 methylation status in ESR1 promoter regions. We also observed the positive correlation between ERα level and MLL3/SET1A level in breast cancer clinical data sets. Importantly, MLL3 and SET1A depletion downregulated the ERα and its downstream growth-related protein levels, as well as the growth of both ER-positive and tamoxifen-resistant breast cancer cells. Likewise, MLL3 and SET1A knockdown decreased the proportion of cells entering the cell cycle. Additional fulvestrant treatment resulted in synergistic reduction of ERα levels and growth of the tamoxifen-resistant breast cancer cells. Thus, enhanced expression of MLL3 and SET1A in tamoxifen-resistant breast cancer cells supported ERα-dependent growth of breast cancer cells, suggesting that MLL3 SET1A might provide an attractive target for overcoming endocrine resistance. Citation Format: Seungsu Kim, Min-Ho Lee, Mi-Ock Lee. Enhanced expression of histone methyltransferases increase ERα expression in tamoxifen-resistant breast cancer cells [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 4320.

Relation of EGFR/PI3K/AKT signaling components with tamoxifen efficacy in patients with estrogen-dependent breast cancer

Background. It is generally accepted that crosstalk between the growth factor receptor and ER pathways implicated in tamoxifen resistance. The aim of the study was to examine the relationship between mRNA level, protein expression and gene polymorphism of the EGFR/PI3K/AKT signaling components with tamoxifen efficacy in patients with estrogen-dependent breast cancer. Materials and methods. The study included 95 breast cancer patients who had received adjuvant tamoxifen, of which 31 patients developed recurrence/metastasis after tamoxifen treatment (tamoxifen resistance group), 64 patients did not develop any diseases progression (tamoxifen sensitive group) during the 5 years of follow-up. Genotypes for ESR1 (rs2077647, rs2228480, rs1801132), EGFR (rs1468727, rs2227983), AKT1 (rs1130233) and PTEN (rs11202592) were analyzed using a TaqMan assay. Using reverse transcription-PCR, the relative expression of mRNA for ESR1, EGFR, AKT1 and PTEN was determined. ERα, EGFR, Akt (pS473) and PTEN expression level was evaluated using immunohistochemistry. Progression-free survival (PFS) was estimated by Kaplan – Meier analysis. Results. The minor allele of ESR1 rs2077647 was more prevalent in tamoxifen sensitive tumors compared to tamoxifen resistant tumors (p = 0.044). We found high AKT1 mRNA expression level in tamoxifen sensitive group compared with tamoxifen resistance patients (7.27 ± 5.29 and 0.02 ± 0.01, respectively, p = 0.014). ESR1 rs2228480 was significantly associated with tamoxifen resistance (p = 0.028). EGFR and Akt (pS473) protein expression level was significantly higher in the tamoxifen resistance group compared to tamoxifen sensitive breast cancer patients (p = 0.006 and 0.037, respectively). Patients carrying mutant genotypes of ESR1 rs2228480 had a poorer progression-free survival than those carrying wild and heterozygous variants (log rank p = 0.043). Positive EGFR tumor expression as well as positive Akt (pS473) expression were significantly associated with shorter PFS (log rank p = 0.014 and 0.048, respectively). Conclusion. Polymorphic sites of the ESR1 gene, AKT1 mRNA expression, EGFR expression level and Akt (pS473) protein expression can be potential molecular markers associated with tumor sensitivity/resistance to tamoxifen treatment.

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.

BQ323636.1, a Novel Splice Variant to NCOR2, as a Predictor for Tamoxifen-Resistant Breast Cancer.

Purpose: Adjuvant tamoxifen treatment revolutionized the management of estrogen receptor (ER)-positive breast cancers to prevent cancer recurrence; however, drug resistance compromises its clinical efficacy. The mechanisms underlying tamoxifen resistance are not fully understood, and no robust biomarker is available to reliably predict those who will be resistant. Here, we study BQ323636.1, a novel splice variant of the NCOR2 gene, and evaluate its efficacy in predicting tamoxifen resistance in patients with breast cancer.Experimental Design: A monoclonal anti-BQ323636.1 antibody that specifically recognizes the unique epitope of this splice variant was generated for in vitro mechanistic studies and for in vivo analysis by immunohistochemistry on tissue microarrays of two independent cohorts of 358 patients with more than 10 years clinical follow-up data, who had ER-positive primary breast cancer and received adjuvant tamoxifen treatment. An orthotopic mouse model was also used.Results: Overexpression of BQ323636.1 conferred resistance to tamoxifen in both in vitro and in an orthotopic mouse model. Mechanistically, coimmunoprecipitation showed BQ323636.1 could bind to NCOR2 and inhibit the formation of corepressor complex for the suppression of ER signaling. Nuclear BQ3232636.1 overexpression in patients samples was significantly associated with tamoxifen resistance (P = 1.79 × 10-6, sensitivity 52.9%, specificity 72.0%). In tamoxifen-treated patients, nuclear BQ323636.1 overexpression was significantly correlated with cancer metastasis and disease relapse. Nuclear BQ323636.1 was also significantly associated with poorer overall survival (P = 1.13 × 10-4) and disease-specific survival (P = 4.02 × 10-5).Conclusions: These findings demonstrate that BQ323636.1 can be a reliable biomarker to predict tamoxifen resistance in patients with ER-positive breast cancer. Clin Cancer Res; 24(15); 3681-91. ©2018 AACRSee related commentary by Jordan, p. 3480.

Abstract P4-08-06: Phosphorylation of insulin-like growth factor-1 receptor (IGF-1R) is associated with tamoxifen resistance by activating the PI3K/MAPK pathway

Abstract Background IGF-1R is overexpressed in a substantial number of breast cancer cases. When phosphorylated, IGF-1R activates the PI3K and MAPK pathways. In preclinical studies, IGF-1R overexpression has been described to be associated with tamoxifen resistance. Expression of activated IGF-1R has not been established as a marker for endocrine resistance in the clinic. We tested the value of p-IGF-1R to predict adjuvant tamoxifen benefit in IGF-1R-positive tumors from ER+ breast cancer patients and its possible association with PI3K/MAPK activation. We also carried out cell line experiments to illustrate a potential causal link between IGF-1R activation status, PI3K/MAPK pathway activation and tamoxifen resistance. Methods Primary tumor blocks from 563 ER+ (stage I-III) postmenopausal patients previously randomized between tamoxifen (1 to 3 years) vs. no adjuvant therapy (IKA trial 1982-1993; Beelen et al, Breast Cancer Res, 2014) were recollected. Immunohistochemistry scoring on tissue microarrays included PTEN, p-IGF-1R, p-AKT(Thr308), p-AKT(Ser473) and p-p70S6K(Thr389) by cytoplasmic intensity (0-3), of p-4EBP1(Ser65) and p-ERK1/2(Thr202/204) by percentage of tumor cells with positive nuclear staining and of p-S6RP(Ser235/236) by percentage of tumor cells with positive membranous staining. Informative data on p-IGF-1R staining was available for 364 IGF-1R-positive tumors. Multivariate Cox models including standard prognostic factors were used to assess hazard ratios (HR) for recurrence-free interval for tamoxifen treatment, p-IGF-1R status (negative vs. positive) and their interaction. MCF-7 and T47D cell lines were used to validate the clinical findings. IncuCyte cell proliferation experiments were performed with various IGF-1R-related growth stimulating and inhibiting conditions. Western blots were carried out on cells under various growth conditions to analyze whether activation of IGF-1R would be associated with PI3K/MAPK pathway activation. Results Patients having tumors without p-IGF-1R expression derived significant benefit from tamoxifen (HR 0.4290, 95% CI 0.2356 - 0.7813; p = 0.00566), while those having tumors with p-IGF-1R expression had no benefit (HR 0.8051, 95% CI 0.2643 – 2.453; p = 0.70). The p for interaction was not significant (p = 0.32106). Tumors positive for p-IGF-1R had more expression of (phospho)proteins downstream of the PI3K/MAPK pathways. These results were supported by Western blots from the cell lines examined under various growth conditions. In both cell lines, linsitinib (a dual IGF-1R and insulin receptor inhibitor) was able to block IGF-1R signaling, preventing activation of the PI3K and MAPK pathways and abrogating cell proliferation in the presence of tamoxifen. Conclusions Postmenopausal breast cancer patients with p-IGF-1R-positive tumors appear to derive no benefit from adjuvant tamoxifen. Tumors with p-IGF-1R expression were associated with PI3K/MAPK pathway activation. In breast cancer cell lines with activated IGF-1R signaling, addition of linsitinib to endocrine therapy can restore sensitivity. This combination might be an interesting treatment option for tamoxifen-resistant patients. Citation Format: Kruger DT, Alexi X, Opdam M, Schuurman KG, Sanders J, van der Noort V, Boven E, Zwart W, Linn SC. Phosphorylation of insulin-like growth factor-1 receptor (IGF-1R) is associated with tamoxifen resistance by activating the PI3K/MAPK pathway [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 P4-08-06.

Tamoxifen Treatment in Correlation with Increased ET-1 Levels Is Associated with the Development of Breast Cancer Metastases

Background: In breast cancer patients, a correlation between endothelin-1 (ET-1) and lymph node metastasis was found. While breast cancer with a positive ER status can be treated with Tamoxifen, several studies describe increasing Tamoxifen resistance in patients. We analyzed the relationship between Tamoxifen, ET-1 overexpression, and ER leading to Tamoxifen resistance. Methods: Breast cancer cell lines were treated with Tamoxifen, ET-1, estrogen and combinations. Using qRT-PCR, immune-precipitation, Western blot, EMSA and immunohistology target gene expression and ER complex partners were investigated. Human biopsies and mastectomy specimens were immunohistologically studied for Vimentin 3, and ERβ. Results: Breast cancer cells stimulated with a combination of Tamoxifen and ET-1 downregulate ERα, while upregulating intracellular ET-1, and ERβ. Immunoprecipation of nuclear extracts with ET-1, ERα or ERβ agarose conjugated antibodies reveals a complex formation change replacing ERα by ERβ once Tamoxifen forms a complex with ET-1. ERβand ET-1 migrate into the nucleus. ET-1 stimulation upregulates metastases promoting target genes (IL-6, Wnt11), including a novel one, Vimentin 3. Tissue analyses show Vim3 and ERβ expression in metastases of ERα positive breast cancer, and in ERα negative biopsies/mastectomy specimens. Conclusion: We are the first to describe a complex consisting of Tamoxifen, ERβ and ET-1, whose nuclear transmigration causes an overexpression of target genes. This mechanism may explain Tamoxifen resistance. Future pathologic analyses should include estrogen beta receptor status as well as the ET-1 expression. This concept presents a new treatment approach for individualized medicine in breast cancer patients with increased ET-1 levels.