Endocrine Therapy In Estrogen Receptor Research Articles

Primary efficacy analyses of NeoRHEA, neoadjuvant biomarker research study of palbociclib combined with endocrine therapy in estrogen receptor positive/HER2 negative breast cancer.

522 Background: CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) are standard of care in the treatment of estrogen receptor (ER) positive, HER2-negative (ER+/HER2-) advanced breast cancer (BC). We tested palbociclib + ET as neoadjuvant therapy to identify baseline biomarkers of no response. Methods: NeoRHEA (NCT03065621) was a phase II, single arm trial evaluating 4 months of neoadjuvant palbociclib and ET in pre- or post-menopausal women with ER+/HER2- early BC. The primary objective was to identify biomarkers of no response by locally-assessed ultrasound (US) using RNA-seq on baseline (pretreatment) tumor biopsies. No response to treatment was defined as stable or progressive disease (SD or PD) at post-treatment US based on WHO criteria. Secondary endpoints included residual cancer burden (RCB) of 3 and absence of complete cell cycle arrest (CCCA) defined as Ki67 by immunohistochemistry (IHC) > 2.7% at surgery. We evaluated baseline clinicopathological characteristics, RNA-seq derived PAM50 subtypes and mRNA expression of ESR1, Ki67, CCNE1, RB1 and CDK6 single genes according to US response and CCCA. We also performed gene set enrichment analysis (GSEA) for 50 gene sets related to cancer hallmarks according to US response and CCCA. Results: 73 of 100 patients enrolled had baseline frozen tumors with enough cellularity and successful RNA-sequencing performed. Among the 73 patients, 70% were post-menopausal, 84% had cT2 and 70% cN0 tumors, 75% had invasive ductal carcinoma and 66% had histological grade 2 tumors. RNA-seq derived PAM50 subtypes were Luminal A, Luminal B, and non-Luminal in 56%, 36% and 8% of patients, respectively. Among the 73 patients, 31 (42%) had absence of US response (28 and 3 patients had stable and progressive disease by US, respectively) and 23 (31%) had RCB 3 tumors. Fifty-three of 73 patients had Ki67 by IHC available at surgery out of whom, 14 (26%) had absence of CCCA. Neither baseline clinicopathological characteristics nor PAM50 subtypes nor expression of ESR1, Ki67, CCNE1 and RB1 were associated with absence of US response or absence of CCCA. Interestingly, we observed higher baseline mRNA expression of CDK6 in patients with absence of US response (p = 0.039) or those with no CCCA (p = 0.004). We observed an enrichment in inflammatory / IFN-γ response and proliferation-related, G2M checkpoint gene sets in patients with US response (NES: 2.06, 2.30, 1.47; FDR: 7.04e-9, 5.48e-17, 0.0193 respectively) and an enrichment in early estrogen response gene set in patients with absence of US response (NES: -1.78, FDR: 0.0006). No signaling pathway was enriched in patients without CCCA. Conclusions: High pre-treatment mRNA expression of the CDK6 gene was associated with no US response or absence of CCCA in patients treated with neoadjuvant palbociclib and ET. Independent validation is needed. Clinical trial information: NCT03065621 .

Structure-guided identification of novel dual-targeting estrogen receptor α degraders with aromatase inhibitory activity for the treatment of endocrine-resistant breast cancer

Drug resistance is a major challenge in conventional endocrine therapy for estrogen receptor (ER) positive breast cancer (BC). BC is a multifactorial disease, in which simultaneous aromatase (ARO) inhibition and ERα degradation may effectively inhibit the signal transduction of both proteins, thus potentially overcoming drug resistance caused by overexpression or mutation of target proteins. In this study, guided by the X-ray structure of a hit compound 30a in complex with ER-Y537S, a structure-based optimization was performed to get a series of multiacting inhibitors targeting both ERα and ARO, and finally a novel class of potent selective estrogen receptor degraders (SERDs) based on a three-dimensional oxabicycloheptene sulfonamide (OBHSA) scaffold equipped with aromatase inhibitor (AI) activity were identified. Of these dual-targeting SERD-AI hybrids, compound 31q incorporating a 1H-1,2,4-triazole moiety showed excellent ERα degradation activity, ARO inhibitory activity and remarkable antiproliferative activity against BC resistant cells. Furthermore, 31q manifested efficient tumor suppression in MCF-7 tumor xenograft models. Taken together, our study reported for the first time the highly efficient dual-targeting SERD-AI hybrid compounds, which may lay the foundation of translational research for improved treatment of endocrine-resistant BC.

Tamoxifen Citrate- loaded synthetic high-density lipoproteins: Assessment of cellular toxicity in breast cancer cells

Tamoxifen Citrate (TC) is the standard endocrine therapy for estrogen receptor (ER) positive breast cancer. TC is a selective estrogen receptor modulator (SERM) whose estrogenic properties in uterus have been linked to increased side
 effects like blood clots, endometrial polyps and cancer. Therefore, significant amount of research has been carried out to develop tamoxifen loaded nano-formulations with a preferential accumulation in tumor tissue rather than healthy tissues.
 Synthetic high-density lipoproteins (sHDL) are novel nanocarriers with inherent active-targeting ability towards tumor cells through the ligand–receptor interaction between apolipoprotein A-I (Apo A-I) and scavenger receptor class B type I (SR-BI) overexpressed in various malignant cells. The current study was carried out to investigate whether encapsulation of TC in sHDL could improve the cytotoxic effect of TC against malignant cells. For this purpose, the cytotoxicity of TC-sHDL was evaluated in MCF-7 cell line in vitro. MTT assay demonstrated the increased cytotoxicity of TC-sHDL against cancer cells as compared with the cytotoxic effect of the free drug.

Bioinformatic Evaluation of Features on Cis-regulatory Elements at 6q25.1.

Eukaryotic non-coding regulatory features contribute significantly to cellular plasticity which on aberration leads to cellular malignancy. Enhancers are cis-regulatory elements that contribute to the development of resistance to endocrine therapy in estrogen receptor (ER)-positive breast cancer leading to poor clinical outcome. ER is vital for therapeutic targets in ER-positive breast cancer. Here, we review and report the different regulatory features present on ER with the objective to delineate potential mechanisms which may contribute to development of resistance. The UCSC Genome Browser, data mining, and bioinformatics tools were used to review enhancers, transcription factors (TFs), histone marks, long non-coding RNAs (lncRNAs), and variants residing in the non-coding region of the ER gene. We report 7 enhancers, 3 of which were rich in TF-binding sites and histone marks in a cell line-specific manner. Furthermore, some enhancers contain estrogen resistance variants and sites for lncRNA. Our review speculates putative models suggesting potential aberrations in gene regulation and expression if these regulatory landscapes and assemblies are altered. This review gives an interesting perspective in designing integrated in vitro studies including non-coding elements to study development of endocrine resistance in ER-positive breast cancer.

Neratinib plus trastuzumab is superior to pertuzumab plus trastuzumab in HER2-positive breast cancer xenograft models

Lapatinib (L) plus trastuzumab (T), with endocrine therapy for estrogen receptor (ER)+ tumors, but without chemotherapy, yielded meaningful response in HER2+ breast cancer (BC) neoadjuvant trials. The irreversible/pan-HER inhibitor neratinib (N) has proven more potent than L. However, the efficacy of N+T in comparison to pertuzumab (P) + T or L + T (without chemotherapy) remains less studied. To address this, mice bearing HER2+ BT474-AZ (ER+) cell and BCM-3963 patient-derived BC xenografts were randomized to vehicle, N, T, P, N+T, or P+T, with simultaneous estrogen deprivation for BT474-AZ. Time to tumor regression/progression and incidence/time to complete response (CR) were determined. Changes in key HER pathway and proliferative markers were assessed by immunohistochemistry and western blot of short-term-treated tumors. In the BT474-AZ model, while all N, P, T, N + T, and P + T treated tumors regressed, N + T-treated tumors regressed faster than P, T, and P + T. Further, N + T was superior to N and T alone in accelerating CR. In the BCM-3963 model, which was refractory to T, P, and P + T, while N and N + T yielded 100% CR, N + T accelerated the CR compared to N. Ki67, phosphorylated (p) AKT, pS6, and pERK levels were largely inhibited by N and N + T, but not by T, P, or P + T. Phosphorylated HER receptor levels were also markedly inhibited by N and N + T, but not by P + T or L + T. Our findings establish the efficacy of combining N with T and support clinical testing to investigate the efficacy of N + T with or without chemotherapy in the neoadjuvant setting for HER2+ BC.

PELP-1 regulates adverse responses to endocrine therapy in Estrogen Receptor (ER) positive breast cancer

Introduction: Endocrine therapy has played an important role in the management of ER positive breast cancer over recent decades. Despite this, not all patients respond equally to endocrine intervention, which can lead to resistance, associated disease relapse and progression. Previous reports suggest that endocrine agents themselves may induce an invasive phenotype in ER positive breast cancers with low/aberrant expression of E-cadherin. Here we investigate this phenomenon further and provide data supporting a role for the ER co-receptor, PELP-1, in mediating an adverse response to endocrine agents.Materials and Methods: The effects of tamoxifen, fulvestrant and estrogen withdrawal (as a model for aromatase inhibitor therapy) on the invasive and migratory capacity of endocrine-sensitive MCF-7 and T47D cells, in the presence or absence of functional E-cadherin and/or PELP-1 (using siRNA knockdown), was assessed via Matrigel invasion and Boyden chamber migration assays. The effects of these endocrine therapies alongside E-cadherin/PELP-1 modulation on cell proliferation were further assessed by MTT assay. Western blotting using phospho-specific antibodies was performed to investigate signalling pathway changes associated with endocrine-induced changes in invasion and migration.Results: Both tamoxifen and fulvestrant induced a pro-invasive and pro-migratory phenotype in ER positive breast cancer cells displaying a high basal expression of PELP-1, which was augmented in the context of poor cell-cell contact. This process occurred in a Src-dependent manner with Src inhibition reversing endocrine induced invasion/migration. While this adverse response was observed using both tamoxifen and fulvestrant therapy, it was not observed under conditions of estrogen withdrawal.Conclusions: Our data confirms previous reports that anti-estrogens induce an adverse cell phenotype in ER+ breast cancer, particularly in the absence of homotypic cell contact. These results implicate E-cadherin and PELP-1 as potential biomarkers when deciding upon optimum adjuvant endocrine therapy, whereby tumours with high PELP-1/low E-cadherin expression may benefit from estrogen withdrawal therapy via aromatase inhibition, as opposed to ER modulation/antagonism.

Amphiregulin retains ERα expression in acquired aromatase inhibitor resistant breast cancer cells.

Acquired resistance to aromatase inhibitors (AIs) is a significant clinical issue in endocrine therapy for estrogen receptor (ER) positive breast cancer which accounts for the majority of breast cancer. Despite estrogen production being suppressed, ERα signaling remains active and plays a key role in most AI-resistant breast tumors. Here, we found that amphiregulin (AREG), an ERα transcriptional target and EGF receptor (EGFR) ligand, is crucial for maintaining ERα expression and signaling in acquired AI-resistant breast cancer cells. AREG was deregulated and critical for cell viability in ER+ AI-resistant breast cancer cells, and ectopic expression of AREG in hormone responsive breast cancer cells promoted endocrine resistance. RNA-sequencing and reverse phase protein array analyses revealed that AREG maintains ERα expression and signaling by activation of PI3K/Akt/mTOR signaling and upregulation of forkhead box M1 (FOXM1) and serum- and glucocorticoid-inducible kinase 3 (SGK3) expression. Our study uncovers a previously unappreciated role of AREG in maintaining ERα expression and signaling, and establishes the AREG-ERα crosstalk as a driver of acquired AI resistance in breast cancer.

Validation of CTS5 on a Retrospective Cohort of Real-Life Pre- and Postmenopausal Patients Diagnosed With Estrogen Receptor–Positive Breast Cancers: Is It Prognostic?

BackgroundMore than 50% of estrogen receptor (ER)-positive breast cancer (BC) distant recurrences (DR) develop after the completion of 5 years of adjuvant endocrine therapy (ET). Its extension is beneficial on disease-free survival and overall survival but increases therapy-related side effects. Selecting patients who could benefit the most from an extended regimen has become an increasing need. Clinical Treatment Score at 5 Years (CTS5) is a prognostic tool using clinicopathologic data to estimate DR risk after 5 years of ET for ER+ BC. We sought to validate the prognostic value of CTS5 in a retrospective cohort of real-life pre- and postmenopausal patients diagnosed with ER+ BC. Patients and MethodsCTS5 was calculated for 603 patients diagnosed with ER+ BC at Umberto I Hospital of Turin and DR-free after 5 years of ET. Primary endpoint was late DR (LDR) rate. ResultsMedian follow-up was 8 years (range, 6-26 years). The 426 postmenopausal women were categorized by CTS5 as follows: 152 low risk, 139 intermediate risk, and 135 high risk. LDR rates were 3.9%, 7.2%, and 15.6%, respectively. CTS5 results were prognostic for LDR: patients with CTS5-high showed a fourfold risk of developing an LDR compared to patients with CTS5-low (hazard ratio, 4.48; 95% confidence interval, 1.80-11.1). The same analysis was conducted for the 177 premenopausal women: 88 low risk, 40 intermediate risk, and 49 high risk. LDR rate were 5.6%, 7.5%, and 20.4%, respectively, proving CTS5 to be prognostic for premenopausal patients as well (CTS5-high vs. CTS5-low: hazard ratio, 3.40; 95% confidence interval, 1.06-11.0). ConclusionCTS5 was shown to be prognostic of the risk of LDR in our population of real-life pre- and postmenopausal patients. Our results support its use in clinical practice to better tailor the prescription of extended ET.

ESR1 Mutations Are Not a Common Mechanism of Endocrine Resistance in Patients With Estrogen Receptor-Positive Breast Cancer Treated With Neoadjuvant Aromatase Inhibitor Therapy.

Introduction: Mutations in the ESR1 gene (ESR1m) are important mechanisms of resistance to endocrine therapy in estrogen receptor–positive (ER+) metastatic breast cancer and have been studied as a potential therapeutic target, as well as a predictive and prognostic biomarker. Nonetheless, the role of ESR1m as a possible mechanism of primary endocrine resistance, as well as whether it also occurs in tumors that are resistant to ET administered in early-stage disease as (neo)adjuvant, has not been adequately studied. In this study, we evaluated the prevalence of ESR1m in tumor samples from patients with ER+ breast cancer resistant to neoadjuvant aromatase inhibitor therapy.Methods: We followed a prospective cohort of patients with ER+ HER2– stages II and III breast cancer treated with neoadjuvant endocrine therapy (NET). Tumor samples from patients with a pattern of primary endocrine resistance [defined as a Preoperative Endocrine Prognostic Index (PEPI) score of ≥4] were identified and analyzed for the presence of ESR1m.Results: One hundred twenty-seven patients were included in the cohort, of which 100 (79%) had completed NET and underwent surgery. Among these patients, the PEPI score ranged from 0 to 3 in 70% (70/100), whereas 30% (30/100) had a PEPI score of 4 or more. Twenty-three of these patients were included in the analysis. ESR1 mutations were not identified in any of the 23 patients with early-stage ER+ breast cancer resistant to NET.Discussion: Growing evidence supports the notion that there are different mechanisms for primary and secondary endocrine resistance. Our study suggests that ESR1 mutations do not evolve rapidly and do not represent a common mechanism of primary endocrine resistance in the neoadjuvant setting. Therefore, ESR1m should be considered a mechanism of acquired endocrine resistance in the context of advanced disease. Further research should be conducted to identify factors associated with intrinsic resistance to ET.

The Central Contributions of Breast Cancer Stem Cells in Developing Resistance to Endocrine Therapy in Estrogen Receptor (ER)-Positive Breast Cancer.

Breast cancer stem cells (BCSC) play critical roles in the acquisition of resistance to endocrine therapy in estrogen receptor (ER)-positive (ER + ve) breast cancer (BC). The resistance results from complex alterations involving ER, growth factor receptors, NOTCH, Wnt/β-catenin, hedgehog, YAP/TAZ, and the tumor microenvironment. These mechanisms are likely converged on regulating BCSCs, which then drive the development of endocrine therapy resistance. In this regard, hormone therapies enrich BCSCs in ER + ve BCs under both pre-clinical and clinical settings along with upregulation of the core components of “stemness” transcriptional factors including SOX2, NANOG, and OCT4. SOX2 initiates a set of reactions involving SOX9, Wnt, FXY3D, and Src tyrosine kinase; these reactions stimulate BCSCs and contribute to endocrine resistance. The central contributions of BCSCs to endocrine resistance regulated by complex mechanisms offer a unified strategy to counter the resistance. ER + ve BCs constitute approximately 75% of BCs to which hormone therapy is the major therapeutic approach. Likewise, resistance to endocrine therapy remains the major challenge in the management of patients with ER + ve BC. In this review we will discuss evidence supporting a central role of BCSCs in developing endocrine resistance and outline the strategy of targeting BCSCs to reduce hormone therapy resistance.

A combinatorial biomarker predicts pathologic complete response to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2+ breast cancer.

HER2-positive (+) breast cancers, defined by HER2 overexpression and/or amplification, are often addicted to HER2 to maintain their malignant phenotype. Yet, some HER2+ tumors do not benefit from anti-HER2 therapy. We hypothesize that HER2 amplification levels and PI3K pathway activation are key determinants of response to HER2-targeted treatments without chemotherapy. Baseline HER2+ tumors from patients treated with neoadjuvant lapatinib plus trastuzumab [with endocrine therapy for estrogen receptor (ER)+ tumors] in TBCRC006 (NCT00548184) were evaluated in a central laboratory for HER2 amplification by fluorescence in situ hybridization (FISH) (n = 56). HER2 copy number (CN) and FISH ratios, and PI3K pathway status, defined by PIK3CA mutations or PTEN levels by immunohistochemistry were available for 41 tumors. Results were correlated with pathologic complete response (pCR; no residual invasive tumor in breast). Thirteen of the 56 patients (23%) achieved pCR. None of the 11 patients with HER2 ratio <4 and/or CN <10 achieved pCR, whereas 13/45 patients (29%) with HER2 ratio ≥4 and/or CN ≥10 attained pCR (P = 0.0513). Of the 18 patients with tumors expressing high PTEN or wild-type (WT) PIK3CA (intact PI3K pathway), 7 (39%) achieved pCR, compared with 1/23 (4%) with PI3K pathway alterations (P = 0.0133). Seven of the 16 patients (44%) with HER2 ratio ≥4 and intact PI3K pathway achieved pCR, whereas only 1/25 (4%) patients not meeting these criteria achieved pCR (P = 0.0031). Our findings suggest that there is a clinical subtype in breast cancer with high HER2 amplification and intact PI3K pathway that is especially sensitive to HER2-targeted therapies without chemotherapy. A combination of HER2 FISH ratio and PI3K pathway status warrants validation to identify patients who may be treated with HER2-targeted therapy without chemotherapy.

183TiP - Neoadjuvant biomarker research study of palbociclib combined with endocrine therapy in estrogen receptor positive/HER2 negative breast cancer: The phase II NeoRHEA trial

183TiP - Neoadjuvant biomarker research study of palbociclib combined with endocrine therapy in estrogen receptor positive/HER2 negative breast cancer: The phase II NeoRHEA trial

ER+ Breast Cancers Resistant to Prolonged Neoadjuvant Letrozole Exhibit an E2F4 Transcriptional Program Sensitive to CDK4/6 Inhibitors.

Purpose: This study aimed to identify biomarkers of resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancers treated with prolonged neoadjuvant letrozole.Experimental Design: We performed targeted DNA and RNA sequencing in 68 ER+ breast cancers from patients treated with preoperative letrozole (median, 7 months).Results: Twenty-four tumors (35%) exhibited a PEPI score ≥4 and/or recurred after a median of 58 months and were considered endocrine resistant. Integration of the 47 most upregulated genes (log FC > 1, FDR < 0.03) in letrozole-resistant tumors with transcription-binding data showed significant overlap with 20 E2F4-regulated genes (P = 2.56E-15). In patients treated with the CDK4/6 inhibitor palbociclib before surgery, treatment significantly decreased expression of 24 of the 47 most upregulated genes in letrozole-resistant tumors, including 18 of the 20 E2F4 target genes. In long-term estrogen-deprived ER+ breast cancer cells, palbociclib also downregulated all 20 E2F4 target genes and P-RB levels, whereas the ER downregulator fulvestrant or paclitaxel only partially suppressed expression of this set of genes and had no effect on P-RB. Finally, an E2F4 activation signature was strongly associated with resistance to aromatase inhibitors in the ACOSOG Z1031B neoadjuvant trial and with an increased risk of relapse in adjuvant-treated ER+ tumors in METABRIC.Conclusions: In tumors resistant to prolonged neoadjuvant letrozole, we identified a gene expression signature of E2F4 target activation. CDK4/6 inhibition suppressed E2F4 target gene expression in estrogen-deprived ER+ breast cancer cells and in patients' ER+ tumors, suggesting a potential benefit of adjuvant CDK4/6 inhibitors in patients with ER+ breast cancer who fail to respond to preoperative estrogen deprivation. Clin Cancer Res; 24(11); 2517-29. ©2018 AACR.

416 (PB-049) - PELP-1 regulates adverse endocrine responses to endocrine therapy in Estrogen Receptor (ER) positive breast cancer

416 (PB-049) - PELP-1 regulates adverse endocrine responses to endocrine therapy in Estrogen Receptor (ER) positive breast cancer

Abstract OT3-06-03: ABC-SE, Advanced Breast Cancer – Statins and Endocrine treatment

Abstract Full Title: A randomized, open-label, multi-center, phase II study to compare the efficacy and tolerability of atorvastatin 40 mg in addition to endocrine treatment in patients with estrogen receptor (ER) positive advanced breast cancer with focus on mechanisms of resistance. Background: The majority of metastatic breast cancer patients progress during endocrine therapy and eventually become resistant to treatment. Understanding how metastatic cancer cells adapt to different therapies is key for the development of improved treatment regimens. The effectiveness of endocrine therapy in ER+ tumors may be influenced by cholesterol through the cholesterol metabolite oxysterol 27-hydroxycholesterol, which acts as an ER ligand, harboring the ability to regulate ER-dependent tumor growth. Statin-mediated inhibition of the cholesterol pathway has been demonstrated to induce anti-neoplastic effects in both breast cancer cells and human breast cancer. Hence the goal of this study is to both understand the mechanisms of resistance to endocrine treatment and test the hypothesis that addition of statins will enhance the efficacy of endocrine treatment. Trial Design: A multi-center randomized, open-labelled, phase II trial in the first and second line metastatic treatment setting, comparing standard endocrine treatment (letrozole) with letrozole +/- atorvastatin (1:1). Upon progression in the first line setting, and as part of the translational studies of mechanisms of resistance to endocrine therapy, the patients receive second line endocrine treatment using fulvestrant. Eligibility criteria: 1) Patients diagnosed with ER positive/HER2 negative metastatic breast cancer, including locally advanced stage IV disease, requiring systemic endocrine treatment. 2) No Previous treatment for metastatic breast cancer, unless being considered for direct entry to the second part of the study with fulvestrant. Specific aims: To test the clinical efficacy of adding statins to endocrine treatment in advanced breast cancer. Primary endpoint: Clinical benefit rate, defined as the proportion of all randomly assigned patients who have the best overall response; complete response, partial response, or stable disease for at least 24 weeks following first-line letrozole treatment alone or in combination with atorvastatin. Translational endpoint: To elucidate mechanisms of resistance to endocrine treatment alone or in combination with statins in ER+ metastatic breast cancer. Statistical Methods:The primary endpoint of clinical benefit rate will be compared in the two groups using a logistic regression model where the odds ratios and associated 95% CIs and p-values will be reported. The secondary endpoint, progression-free-survival, will be analyzed in crude analysis using the Kaplan-Meier and Log-Rank test as well as the Cox regression hazards analysis with the latter allowing for confounder-controlled multivariate analysis. Present accrual and target accrual: The trial started recruiting as of October 10, 2016. The target accrual is 126 patients, whereof 17 are presently included in the trial. Contact information for people with a specific interest in the trial: Signe.Borgquist@med.lu.se Citation Format: Borgquist S, Ekholm M, Feldt M, Schyman T, Zackrisson S, Bosch A. ABC-SE, Advanced Breast Cancer – Statins and Endocrine treatment [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 OT3-06-03.

Abstract P3-03-11: Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1

Abstract Tamoxifen is the most widely used endocrine therapy in estrogen receptor (ER)- positive breast cancer patients. Tamoxifen resistance has been a major clinical problem and is accountable for relapse in about one third of these patients. Most of these recurrent patients will inevitably receive chemotherapy. However, the association between tamoxifen-resistance and chemosensitivity in breast cancer has never been explored. We found that both mRNA and protein expression of BARD1 and BRCA1 were significantly upregulated in tamoxifen-resistant MCF7 and T47D breast cancer cell lines, when compared with their parental lines. Furthermore, the tamoxifen-resistant cells were markedly more resistant to DNA-damaging chemotherapy including cisplatin and doxorubicin, but not to paclitaxel. Silencing BARD1 or BRCA1 by siRNAs or inhibition of BRCA1 phosphorylation by Dinaciclib restored the sensitivity to cisplatin and doxorubicin in tamoxifen-resistant cells. In addition, we identified that activated PI3K/AKT/ERK pathway in tamoxifen-resistant cells was responsible for the upregulation of BARD1 and BRCA1. BKM120, a PI3K inhibitor, decreased the expression of BARD1 and BRCA1 in tamoxifen-resistant cells and re-sensitized them to cisplatin and doxorubicin both in vitro and in xenografted mice. More importantly, higher BARD1 expression was significantly associated with poorer prognosis in breast cancer patients. BARD1 and BRCA1 were also significantly upregulated in recurrent breast cancer samples after tamoxifen compared with their primary counterparts. These results indicate an important role of BARD1 and BRCA1 in the chemoresistance of ER-positive breast cancer. Targeting PI3K/Akt pathway or BRCA1 phosphorylation may help to overcome the resistance to DNA-damaging chemotherapy in these tumors. Citation Format: Liu Q, Zhu Y, Liu Y. Tamoxifen-resistant breast cancer cells are resistant to DNA-damaging chemotherapy because of upregulated BARD1 and BRCA1 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-11.

High CDK6 Protects Cells from Fulvestrant-Mediated Apoptosis and is a Predictor of Resistance to Fulvestrant in Estrogen Receptor-Positive Metastatic Breast Cancer.

Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a major clinical problem. Recently, the CDK4/6 inhibitor palbociclib combined with letrozole or fulvestrant was approved for treatment of ER+ advanced breast cancer. However, the role of CDK4/6 in endocrine resistance and their potential as predictive biomarkers of endocrine treatment response remains undefined. We investigated the specific role of increased CDK6 expression in fulvestrant-resistant cells by gene knockdown and treatment with palbociclib, and evaluated the effect in cell proliferation, apoptosis, and kinase activity. Furthermore, we evaluated CDK6 expression in metastatic samples from breast cancer patients treated or not with fulvestrant. We found increased expression of CDK6 in two fulvestrant-resistant cell models versus sensitive cells. Reduction of CDK6 expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib resensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer patients treated with fulvestrant (N = 45 and 46) correlated significantly with shorter progression-free survival (PFS) on fulvestrant treatment (P = 0.0006 and 0.018), whereas no association was observed in patients receiving other first- or second-/third-line endocrine treatments (N = 68, P = 0.135 and 0.511, respectively). Our results indicate that upregulation of CDK6 may be an important mechanism in overcoming fulvestrant-mediated growth inhibition in breast cancer cells. Patients with advanced ER+ breast cancer exhibiting high CDK6 expression in the metastatic lesions show shorter PFS upon fulvestrant treatment and thus may benefit from the addition of CDK4/6 inhibitors in their therapeutic regimens. Clin Cancer Res; 22(22); 5514-26. ©2016 AACR.

Antiestrogen Resistant Cell Lines Expressing Estrogen Receptor α Mutations Upregulate the Unfolded Protein Response and are Killed by BHPI.

Outgrowth of metastases expressing ERα mutations Y537S and D538G is common after endocrine therapy for estrogen receptor α (ERα) positive breast cancer. The effect of replacing wild type ERα in breast cancer cells with these mutations was unclear. We used the CRISPR-Cas9 genome editing system and homology directed repair to isolate and characterize 14 T47D cell lines in which ERαY537S or ERαD538G replace one or both wild-type ERα genes. In 2-dimensional, and in quantitative anchorage-independent 3-dimensional cell culture, ERαY537S and ERαD538G cells exhibited estrogen-independent growth. A progestin further increased their already substantial proliferation in micromolar 4-hydroxytamoxifen and fulvestrant/ICI 182,780 (ICI). Our recently described ERα biomodulator, BHPI, which hyperactivates the unfolded protein response (UPR), completely blocked proliferation. In ERαY537S and ERαD538G cells, estrogen-ERα target genes were constitutively active and partially antiestrogen resistant. The UPR marker sp-XBP1 was constitutively activated in ERαY537S cells and further induced by progesterone in both cell lines. UPR-regulated genes associated with tamoxifen resistance, including the oncogenic chaperone BiP/GRP78, were upregulated. ICI displayed a greater than 2 fold reduction in its ability to induce ERαY537S and ERαD538G degradation. Progestins, UPR activation and perhaps reduced ICI-stimulated ERα degradation likely contribute to antiestrogen resistance seen in ERαY537S and ERαD538G cells.

Huaier extract synergizes with tamoxifen to induce autophagy and apoptosis in ER-positive breast cancer cells

Tamoxifen (TAM) is the most widely used endocrine therapy for estrogen receptor (ER)-positive breast cancer patients, but side effects and the gradual development of insensitivity limit its application. We investigated whether Huaier extract, a traditional Chinese medicine, in combination with TAM would improve treatment efficacy in ER-positive breast cancers. MTT, colony formation, and invasion and migration assays revealed that the combined treatment had stronger anticancer effects than either treatment alone. Huaier extract enhanced TAM-induced autophagy, apoptosis, and G0/G1 cell cycle arrest, as measured by acidic vesicular organelle (AVO) staining, TUNEL, flow cytometry, and western blot. Additionally, combined treatment inhibited tumorigenesis and metastasis by suppressing the AKT/mTOR signaling pathway. Huaier extract also enhanced the inhibitory effects of TAM on tumor growth in vivo in a xenograft mouse model. These results show that Huaier extract synergizes with TAM to induce autophagy and apoptosis in ER-positive breast cancer cells by suppressing the AKT/mTOR pathway.

Abstract S3-03: Nuclear FGFR1 interaction with estrogen receptor (ER) α is associated with resistance to endocrine therapy in ER+/FGFR1-amplified breast cancer

Abstract Background: Estrogen receptor (ER)-positive breast cancers (BC) initially respond to antiestrogens but eventually become hormone-independent and recur. FGFR1 is amplified in ∼10% of ER+ BC and is associated with early recurrence on antiestrogen therapy. Notably, one third of FGFR1-amplified tumors have simultaneous amplification of CCND1, FGF3, FGF4 and FGF19 on chromosome 11q12-14. Herein, we investigated the mechanisms by which FGFR1 amplification confers resistance to antiestrogen therapy in ER+ BC cells. Results: We performed whole exome sequencing in tumor biopsies from 130 patients with an operable ER+/HER2- BC who had received letrozole for 10-21 days prior to surgery. Tumors were categorized by the natural log (ln) of post-letrozole Ki67 as sensitive (ln ≤1 or ≤2.7% Ki67+ cells; n=68) or resistant (ln ≥2 or ≥7.4%; n=18). We found amplifications in FGFR1 and/or 11q12-14 in 6/11 (55%) resistant tumors compared with 5/34 (15%) in sensitive tumors (p=0.006); all cases were confirmed by FGFR1-fluorescence in situ hydridization (FISH). Resistant tumors with FGFR1 and/or 11q12-14-amplification showed a marked increase in nuclear FGFR1 with letrozole. ER+/FGFR1-amplified CAMA1 and MDA134 cell lines also exhibited co-localization of ER and FGFR1 in the nucleus. Cell proliferation was partially reduced by estrogen deprivation, and FGFR1 siRNA further reduced cell growth in hormone-depleted medium. We generated CAMA1 and MDA134 cells resistant to long-term estrogen deprivation (LTED). These cells exhibited overexpression of FGF3/4/19 and ERα with a concomitant increase in ligand-independent ER transcriptional activity and growth. An ER-FGFR1 interaction was observed in the nucleus and cytosol of CAMA1 parental cells with enhanced interaction in CAMA1 LTED cells. Genetic (with siRNA) and pharmacologic (with lucitinib) inhibition of FGFR1 reduced a) nuclear localization of FGFR1; b) ER transcriptional activity; and c) cell proliferation. Nuclear localization and ER-FGFR1 interaction were disrupted by a kinase-deficient FGFR1. Conversely, addition of FGF3 ligand stimulated ER-FGFR1 interaction and ER transcriptional activity, suggesting FGFR activation can regulate ER function. Inhibition of FGF receptor-specific substrate (FRS2), a principal mediator of FGFR1 signal transduction to the MAPK and PI3K pathways, with siRNA or pharmacologic inhibition of PI3K with buparlisib or MEK with GSK1120212 did not reduce ER transcriptional activity suggesting that, in ER+/FGFR1-amplified cancer cells, ER function is not modulated by FGFR signal transducers. Finally, using chromatin immunoprecipitation (ChIP) we showed that FGFR1 binds directly to estrogen response elements (ERE). This association was reduced with lucitanib. We are currently investigating genes modulated by ER/FGFR1 in ER+ BC and the in vivo anti-tumor efficacy of dual inhibition of FGFR1 and ER in ER+/FGFR1-amplified patient-derived breast cancer xenografts. Conclusions: These data support a critical role of ER and FGFR1 interaction in endocrine resistance in ER+/FGFR1-amplified breast cancer. Targeting of FGFR1 in combination with antiestrogens may abrogate resistance to endocrine therapy in these tumors and is worthy of clinical investigation. Citation Format: Formisano L, Young CD, Bhola NE, Bulen B, Estrada VM, Wagle N, Van Allen E, Red Brewer ML, Jansen VM, Guerrero AL, Giltnane JM, Strcker T, Arteaga CL. Nuclear FGFR1 interaction with estrogen receptor (ER) α is associated with resistance to endocrine therapy in ER+/FGFR1-amplified breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S3-03.