Long-term Estrogen Deprivation Research Articles

Abstract PO4-23-08: Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer

Abstract Although clinical evidence indicates that treatment with estrogens elicits anti-cancer effects in ~30% of patients with advanced endocrine-resistant ER+ breast cancer, the underlying mechanism of action is unclear and this treatment remains under-utilized. ER+ breast cancer cells with acquired resistance to long-term estrogen deprivation (LTED) were used to model resistance to aromatase inhibitors (AIs) seen clinically. Genome-wide CRISPR/Cas9 knockout screening and transcriptomic profiling of LTED cells highlighted DNA Damage Response as a required pathway for therapeutic response to the estrogen 17b-estradiol. Tumor specimens from 2 patients with advanced ER+ breast cancer showed increased DNA damage response upon treatment with 17b-estradiol therapy compared to baseline. LTED cells treated with 17b-estradiol exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis, while parental cells did not. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops), and RNase H1-mediated prevention of R-loop formation abrogated 17b-estradiol-induced DNA damage. Pharmacological suppression of the DNA damage response via poly(ADP-ribose) polymerase (PARP) inhibition with olaparib enhanced 17b-estradiol-induced DNA damage. PARP inhibition synergized with 17b-estradiol to suppress growth and prevent tumor recurrence in both BRCA1/2-mutant and BRCA1/2-wild-type cell line and patient-derived xenograft models. We therefore conclude that 17b-estradiol-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells, and inhibition of the DNA damage response using drugs such as PARP inhibitors can exacerbate transcriptional stress and enhance therapeutic response to 17b-estradiol. This concept is being explored clinically in the PHOEBE trial testing the combination of 17b-estradiol and olaparib (NCT05900895). These findings also warrant further study of other DNA damage response inhibitors in advanced ER+ breast cancer. Moreover, these data indicate that PARP inhibitors may have applications beyond homologous recombination-deficient tumors. Citation Format: Nicole Traphagen, Gary Schwartz, Steven Tau, Alyssa Roberts, Amanda Jiang, Sarah Hosford, Jonathan Marotti, Abigail Goen, Bianca Romo, Anneka Johnson, Emily Duffy, Eugene Demidenko, Paul Heverly, Yaron Mosesson, Shannon Soucy, Fred Kolling, Todd Miller. Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-08.

Acetyl-CoA carboxylase 1 controls a lipid droplet-peroxisome axis and is a vulnerability of endocrine-resistant ER+ breast cancer.

Targeting aromatase deprives ER+ breast cancers of estrogens and is an effective therapeutic approach for these tumors. However, drug resistance is an unmet clinical need. Lipidomic analysis of long-term estrogen-deprived (LTED) ER+ breast cancer cells, a model of aromatase inhibitor resistance, revealed enhanced intracellular lipid storage. Functional metabolic analysis showed that lipid droplets together with peroxisomes, which we showed to be enriched and active in the LTED cells, controlled redox homeostasis and conferred metabolic adaptability to the resistant tumors. This reprogramming was controlled by acetyl-CoA-carboxylase-1 (ACC1), whose targeting selectively impaired LTED survival. However, the addition of branched- and very long-chain fatty acids reverted ACC1 inhibition, a process that was mediated by peroxisome function and redox homeostasis. The therapeutic relevance of these findings was validated in aromatase inhibitor-treated patient-derived samples. Last, targeting ACC1 reduced tumor growth of resistant patient-derived xenografts, thus identifying a targetable hub to combat the acquisition of estrogen independence in ER+ breast cancers.

Unraveling Vulnerabilities in Endocrine Therapy-Resistant HER2+/ER+ Breast Cancer.

Breast tumors overexpressing human epidermal growth factor receptor (HER2) confer intrinsic resistance to endocrine therapy (ET), and patients with HER2/estrogen receptor-positive (HER2+/ER+) breast cancer (BCa) are less responsive to ET than HER2-/ER+. However, real-world evidence reveals that a large subset of patients with HER2+/ER+ receive ET as monotherapy, positioning this treatment pattern as a clinical challenge. In the present study, we developed and characterized 2 in vitro models of ET-resistant (ETR) HER2+/ER+ BCa to identify possible therapeutic vulnerabilities. To mimic ETR to aromatase inhibitors (AIs), we developed 2 long-term estrogen deprivation (LTED) cell lines from BT-474 (BT474) and MDA-MB-361 (MM361). Growth assays, PAM50 subtyping, and genomic and transcriptomic analyses, followed by validation and functional studies, were used to identify targetable differences between ET-responsive parental and ETR-LTED HER2+/ER+ cells. Compared to their parental cells, MM361 LTEDs grew faster, lost ER, and increased HER2 expression, whereas BT474 LTEDs grew slower and maintained ER and HER2 expression. Both LTED variants had reduced responsiveness to fulvestrant. Whole-genome sequencing of aggressive MM361 LTEDs identified mutations in genes encoding transcription factors and chromatin modifiers. Single-cell RNA sequencing demonstrated a shift towards non-luminal phenotypes, and revealed metabolic remodeling of MM361 LTEDs, with upregulated lipid metabolism and ferroptosis-associated antioxidant genes, including GPX4. Combining a GPX4 inhibitor with anti-HER2 agents induced significant cell death in both MM361 and BT474 LTEDs. The BT474 and MM361 AI-resistant models capture distinct phenotypes of HER2+/ER+ BCa and identify altered lipid metabolism and ferroptosis remodeling as vulnerabilities of this type of ETR BCa.

2-Methoxyestradiol as an Antiproliferative Agent for Long-Term Estrogen-Deprived Breast Cancer Cells.

To identify effective treatment modalities for breast cancer with acquired resistance, we first compared the responsiveness of estrogen receptor-positive breast cancer MCF-7 cells and long-term estrogen-deprived (LTED) cells (a cell model of endocrine therapy-resistant breast cancer) derived from MCF-7 cells to G-1 and 2-methoxyestradiol (2-MeO-E2), which are microtubule-destabilizing agents and agonists of the G protein-coupled estrogen receptor 1 (GPER1). The expression of GPER1 in LTED cells was low (~0.44-fold), and LTED cells displayed approximately 1.5-fold faster proliferation than MCF-7 cells. Although G-1 induced comparable antiproliferative effects on both MCF-7 and LTED cells (IC50 values of >10 µM), 2-MeO-E2 exerted antiproliferative effects selective for LTED cells with an IC50 value of 0.93 μM (vs. 6.79 μM for MCF-7 cells) and induced G2/M cell cycle arrest. Moreover, we detected higher amounts of β-tubulin proteins in LTED cells than in MCF-7 cells. Among the β-tubulin (TUBB) isotype genes, the highest expression of TUBB2B (~3.2-fold) was detected in LTED cells compared to that in MCF-7 cells. Additionally, siTUBB2B restores 2-MeO-E2-mediated inhibition of LTED cell proliferation. Other microtubule-targeting agents, i.e., paclitaxel, nocodazole, and colchicine, were not selective for LTED cells. Therefore, 2-MeO-E2 can be an antiproliferative agent to suppress LTED cell proliferation.

Revisiting Estrogen for the Treatment of Endocrine-Resistant Breast Cancer: Novel Therapeutic Approaches.

Estrogen receptor (ER)-positive breast cancer is the most common subtype, representing 70-75% of all breast cancers. Several ER-targeted drugs commonly used include the selective estrogen receptor modulator (SERM), tamoxifen (TAM), aromatase inhibitors (AIs) and selective estrogen receptor degraders (SERDs). Through different mechanisms of action, all three drug classes reduce estrogen receptor signaling. Inevitably, resistance occurs, resulting in disease progression. The counterintuitive action of estrogen to inhibit ER-positive breast cancer was first observed over 80 years ago. High-dose estrogen and diethylstilbestrol (DES) were used to treat metastatic breast cancer accompanied by harsh side effects until the approval of TAM in the 1970s. After the development of TAM, randomized trials comparing TAM to estrogen found similar or slightly inferior efficacy but much better tolerability. After decades of research, it was learned that estrogen induces tumor regression only after a period of long-term estrogen deprivation, and the mechanisms of tumor regression were described. Despite the long history of breast cancer treatment with estrogen, this therapeutic modality is now revitalized due to the development of novel estrogenic compounds with improved side effect profiles, newly discovered predictive biomarkers, the development of non-estrogen small molecules and new combination therapeutic approaches.

Abstract P3-11-06: Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance

Abstract Current data using gene expression and DNA modification profiles yield limited information for evaluating the potential resistance to therapies. Recent studies show that the chromatin landscape is dynamic and defines cell identity and disease status. Thus, changes in open chromatin sites could provide important information on disease progression and identify potential therapeutic targets. The majority of breast cancers express estrogen receptor (ER) and initially respond to endocrine therapies blocking ER activity. However, endocrine therapy resistance (ETR) occurs de novo or follows an initial response. Recent studies have identified new drivers of ETR, such as mTOR and CDK4/6. Therapy with these inhibitors in combination with endocrine therapy have improved patient survival, but the molecular mechanism of ETR is not fully understood and additional therapeutic options for ETR are needed. To investigate the molecular mechanisms of ETR, we established in vitro Long-Term Estrogen Deprivation (LTED) model using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression upon LTED with ESR1 upregulation in some cell lines and downregulation in others. LTEDs with ESR1 upregulation were sensitive to fulvestrant, while the others were resistant. We analyzed time-course of chromatin landscape transitions during acquisition of ETR in ESR1 up- and downregulated LTEDs cells by Assay for Transposase Accessible Chromatin (ATAC-seq) combined with RNA-seq. Because transcription factors play a primary key role in gene expression and in cell-fate changes including response to therapies, we performed bioinformatic analysis to uncover differential open chromatin sites, select enhancer signatures and identify digital footprints of transcription factors. Unlike ChIP-seq, which identifies binding sites for a known transcription factor, ATAC-seq permits analysis of all sites potentially accessible to the transcriptional machinery and predict occupancy by ~700 different transcription factors at once. With these algorithms, we found that Grainyhead-like 2 (GRHL2) is associated with development of both ESR1 overexpressed and downregulated types of LTEDs. Furthermore, RNA-seq analysis showed that GRHL2 target genes are upregulated in LTEDs. Furthermore, in the METABRIC database, high GRHL2 expression is significantly associated with poor outcome in ER+ breast cancer patients treated with endocrine therapy. Taken together, these data suggest that GRHL2 may be a novel important transcription factor in endocrine therapy resistant breast cancer regardless of fulvestrant sensitivity. Our studies provide evidence that chromatin landscape analysis, coupled with the transcription factor network algorithm, is capable of identifying novel biomarkers or therapeutic targets, This approach will expand the range of translational research as it is applicable to many cancers and diseases. Citation Format: Saori Fujiwara, Songjoon Baek, Kaustubh Wagh, Diana Stavreva, Lyuba Varticovski, Gordon Hager. Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-06.

Repeated exposure to 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) accelerates ligand-independent activation of estrogen receptors in long-term estradiol-deprived MCF-7 cells

It was previously identified that there may be an active metabolite of bisphenol A (BPA), 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). An in vitro system was developed to detect MBP toxicity to the Michigan Cancer Foundation-7 (MCF-7) cells that had been repeatedly exposed to a low dose of the metabolite. MBP profoundly activated estrogen receptor (ER)-dependent transcription as a ligand, with an EC50 of 2.8 nM. Women are continuously exposed to numerous estrogenic environmental chemicals; but their susceptibility to these chemicals may be significantly altered after menopause. Long-term estrogen-deprived (LTED) cells, which display ligand-independent ER activation, are a postmenopausal breast cancer model derived from MCF-7 cells. In this study, we investigated the estrogenic effects of MBP on LTED cells in a repeated exposure in vitro model. The results suggest that i) nanomolar levels of MBP reciprocally disrupt the balanced expression of ERα and ERβ proteins, leading to the dominant expression of ERβ, ii) MBP stimulates ERs-mediated transcription without acting as an ERβ ligand, and iii) MBP utilizes mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling to evoke its estrogenic action. Moreover, the repeated exposure strategy was effective for detecting low-dose estrogenic-like effects caused by MBP in LTED cells.

Long noncoding RNA DIO3OS induces glycolytic-dominant metabolic reprogramming to promote aromatase inhibitor resistance in breast cancer

Aromatase inhibition is an efficient endocrine therapy to block ectopic estrogen production for postmenopausal estrogen receptor (ER)-positive breast cancer patients, but many develop resistance. Here, we show that aromatase inhibitor (AI)-resistant breast tumors display features of enhanced aerobic glycolysis with upregulation of long noncoding RNA (lncRNA) DIO3OS, which correlates with poor prognosis of breast cancer patients on AI therapies. Long-term estrogen deprivation induces DIO3OS expression in ER-positive breast tumor cells, which further enhances aerobic glycolysis and promotes estrogen-independent cell proliferation in vitro and in vivo. Mechanistically, DIO3OS interacts with polypyrimidine tract binding protein 1 (PTBP1) and stabilizes the mRNA of lactate dehydrogenase A (LDHA) by protecting the integrity of its 3’UTR, and subsequently upregulates LDHA expression and activates glycolytic metabolism in AI-resistant breast cancer cells. Our findings highlight the role of lncRNA in regulating the key enzyme of glycolytic metabolism in response to endocrine therapies and the potential of targeting DIO3OS to reverse AI resistance in ER-positive breast cancer.

Identification of estrogen receptor down-regulators for endocrine resistant breast cancer

Resistance to endocrine therapies remains an impediment for the treatment of estrogen receptor (ER) positive breast cancer. ER down regulator Fulvestrant has showed great activity to overcome the endocrine resistance. However, Fulvestrant has poor bioavailability due to the hydrophobicity. Identification of novel ER down regulator is still important. Compounds 172 and 183 are two steroidal compounds with androgen scaffold but significantly down regulated ER in multiple breast cancer cell lines. RT-PCR results indicated that both compounds did not affect ER gene expression. Proteasome inhibitor MG132 could attenuate ER down regulation effect of the compounds, suggesting that the ER down regulation was via ubiquitin-proteasomal pathway. Furthermore, compounds 172 and 183 could downregulate ER in endocrine resistant breast cancer cell model long term estrogen deprivation (LTED) MCF-7 cells. Hydrophobicity of compounds 172 and 183 were determined and showed improved solubility compared to Fulvestrant. All these results suggested that compounds 172 and 183 could be potential lead compounds for drug development for the treatment of endocrine resistance breast cancer.

Dietary genistein and 17β-estradiol implants differentially influence locomotor and cognitive functions following transient focal ischemia in middle-aged ovariectomized rats at different lengths of estrogen deprivation

Genistein possesses estrogenic activity and has been considered a potential replacement for estrogen replacement therapy after menopause. In the current study, we investigated the neuroprotective effects of dietary genistein at varied lengths of estrogen deprivation in middle-aged ovariectomized Sprague-Dawley rats under ischemic conditions. Two weeks of treatment with dietary genistein at 42 mg/kg but not 17β-estradiol implants improved cognitive flexibility (Morris water maze test) after short-term estrogen deprivation (2 weeks) but not long-term estrogen deprivation (12 weeks). 17β-estradiol implants but not dietary genistein improved locomotor asymmetry (cylinder test) after long-term but not short-term estrogen deprivation. Dietary genistein but not 17β-estradiol implant improved early phase motor learning (rotarod test) after long-term estrogen deprivation. Neither 17β-estradiol implant nor dietary genistein reduced infarct size after either short-term or long-term estrogen deprivation. Genistein, however, reduced ionized calcium-binding adaptor molecule-1 (Iba1) expression, a marker of brain inflammation, at the ipsilateral side of stroke injury after short-term but not long-term estrogen deprivation. This study suggests that the neuroprotective effects of dietary genistein on motor and cognitive functions are distinctly influenced by the length of estrogen deprivation following focal ischemia. SignificanceThere is an increasing postmenopausal population opting for homeopathic medicines for the management of menopausal symptoms due to the perceived distrust in estrogen use as hormone replacement. Basic and clinical studies support the notion that early, but not delayed, hormone replacement after menopause is beneficial. Furthermore, evidence suggests that delaying hormone replacement augments the detrimental, rather than the beneficial effects of estrogens. Because of the active consideration of soy isoflavones including genistein as alternatives to estrogen replacement, it is necessary to understand the ramifications of soy isoflavones use when their administration is begun at various times after menopause.

ECM1 is associated with endocrine resistance in ER+ breast cancers

ABSTRACT Extracellular matrix protein 1 (ECM1) is associated with a poor prognosis of breast cancers. However, the role of ECM1 with endocrine resistance in estrogen receptor-positive (ER+) breast cancers has not been elucidated yet. We show that ECM1 promotes endocrine resistance in ER+ breast cancers. ECM1 is overexpressed in luminal breast cancer patients compared to the basal type of breast cancer. Significantly, higher expression of ECM1 is associated with poor response to endocrine therapies in luminal B breast cancer patients. We found that ECM1 is upregulated in CAMA1 and MDA-MB-361 cells grown in long-term estrogen-deprived (LTED) conditions. Moreover, the ablation of ECM1 significantly inhibited the proliferation of CAMA1 LTED and MDA-MB-361 LTED cells. Finally, an interrogation of a dataset containing transcriptome and proteome of breast cancer cell lines revealed that the level of ECM1 mRNA is positively correlated with that of phosphorylated Src. Based on these findings, we strongly suggest that ECM1 significantly contributes to the acquisition of endocrine resistance in ER+ breast cancers by the activation of Src.

Estrogen Receptor Complex to Trigger or Delay Estrogen-Induced Apoptosis in Long-Term Estrogen Deprived Breast Cancer.

Antiestrogen therapy of breast cancer has been a “gold standard” of treatment of estrogen receptor (ER)-positive breast cancer for decades. Resistance to antiestrogen therapy may develop, however, a vulnerability in long-term estrogen deprived (LTED) breast cancer cells was discovered. LTED breast cancer cells may undergo estrogen-induced apoptosis within a week of treatment with estrogen in vitro. This phenomenon has been also validated in vivo and in the clinic. The molecular ER-mediated mechanism of action of estrogen-induced apoptosis was deciphered, however, the relationship between the structure of estrogenic ligands and the activity of the ER in LTED breast cancer cells remained a mystery until recently. In this review we provide an overview of the structure-activity relationship of various estrogens with different chemical structures and the modulation of estrogen-induced apoptosis in LTED breast cancer cells resistant to antihormone therapy. We provide analysis of evidence gathered over more than a decade of structure-activity relationship studies by our group on the role of the change in the conformation of the estrogen receptor and the biological activities of different classes of estrogens and the receptor as well in LTED breast cancer.

Acetyl-CoA synthetases ACSS1 and ACSS2 are 4-hydroxytamoxifen responsive factors that promote survival in tamoxifen treated and estrogen deprived cells

Acetyl-CoA synthetases ACSS1 and ACSS2 promote conversion of acetate to acetyl-CoA for use in lipid synthesis, protein acetylation, and energy production. These enzymes are elevated in some cancers and important for cell survival under hypoxia and nutrient stress. 4-hydroxytamoxifen (4-OHT) can induce metabolic changes that increase cancer cell survival. An effect of 4-OHT on expression of ACSS1 or ACSS2 has not been reported. We found ACSS1 and ACSS2 are increased by 4-OHT in estrogen receptor-α positive (ER+) breast cancer cells and 4-OHT resistant derivative cells. ERα knockdown blocked ACSS1 induction by 4-OHT but not ACSS2. 4-OHT also induced ACSS2 but not ACSS1 expression in triple negative breast cancer cells. Long-term estrogen deprivation (LTED) is a model for acquired resistance to aromatase inhibitors. We found LTED cells and tumors express elevated levels of ACSS1 and/or ACSS2 and are especially sensitive to viability loss caused by depletion of ACSS1 and ACSS2 or treatment with an ACSS2-specific inhibitor. ACSS2 inhibitor also increased toxicity in cells treated with 4-OHT. We conclude ACSS1 and ACSS2 are 4-OHT regulated factors important for breast cancer cell survival in 4-OHT-treated and long-term estrogen deprived cells.

Abstract P4-02-05: Exploiting novel models of endocrine-resistant breast cancer to identify new therapeutic targets

Abstract Introduction: While endocrine therapy is an effective, well-tolerated treatment for estrogen receptor positive breast cancer (ER+BC), a large proportion of initial responders will develop hormone therapy resistance, and relapse. A major challenge in determining the mechanisms underlying endocrine therapy resistance is our limited ability to recapitulate inter- and intra-tumour heterogeneity in vitro. In this study we developed ER+BC cells resistant to estrogen deprivation, and to palbociclib treatment, and subjected these to 2D and 3D high-throughput drug and siRNA screens, in order to elucidate the pathways underpinning the development of endocrine resistance. Methods: ER+BC cell lines modelling relapse on aromatase inhibitor therapy were derived by long-term culture in the absence of exogenous estrogen, and were termed long-term estrogen deprived (LTED). Two of these lines were then used to generate additional resistance to palbociclib, thus modelling endocrine-resistant, palbociclib-resistant disease. These lines were then subjected to screens performed in 2D and 3D. Effects of the siRNAs and drug compounds were assessed by measuring cell viability, with a robust Z score of -2 taken as a threshold of significance. Table 1 outlines the key characteristics of the cell lines, the details of the drug screens performed, and the top 3 drugs with their corresponding targets. The siRNA screens were performed in 2D and 3D with a 709 kinome library. Results: The siRNA screens highlighted PI3K-AKT-mTOR signalling as being dysregulated in multiple 3D and 2D models, while the drug screens showed that compounds targeting phosphoinositide 3-kinase alpha (PI3Kα) were significant common hits. This is consistent with the recent success of the BYLieve trial in targeting PI3Kα. A second key area of dependency was in cell cycle regulation, with CDK7 and CDK9 found to be significant hits in multiple models. The 709 kinase screens in 2D and 3D demonstrated broadly similar results (43 vs 37 median number of significant hits 2D vs 3D; Z-score <-2), while drug screening in 3D (Table 1) provided increased discrimination in identifying key vulnerabilities. CDK7 and CDK9 were selected for further study. siRNA-mediated knockdown of CDK9 had a significant effect on cell viability in endocrine-sensitive, endocrine-resistant, and palbociclib-resistant settings, whereas CDK7 knockdown did not. However, the CDK7 inhibitors THZ1 and ICEC0942, and the CDK9 inhibitors NVP-2 and AZD4573, were equally effective in endocrine-sensitive, endocrine-resistant, and palbociclib-resistant settings. Importantly, presence of a PIK3CA mutation was required for entry into the BYLieve trial, while CDK7 and CDK9 inhibitors were effective in the PIK3CA wild-type and mutant setting. Furthermore, while the presence of the ESR1 mutation is associated with poorer PFS with certain therapies, such as exemestane, the CDK7 and CDK9 inhibitors were equally effective in the ESR1 mutant and wild-type setting. Finally, combination treatment of palbociclib with CDK7 and CDK9 inhibitors showed synergism in palbociclib-resistant models. Conclusions: Targeted inhibition of CDK7 and/or CDK9 is effective in the LTED palbociclib-resistant setting, thus opening future avenues for the treatment of advanced, endocrine-resistant, palbociclib-resistant breast cancer. Table 1.Cell line characteristics and drug screen findingsCell line and key characteristics2D drug screen of 396 compounds, at 3 concentrations*3D drug screen of 70 compounds at 1 concentrationNumber of significant drug screen hits (Z score <-2). The top 3 hits and their targets are shownMCF7 LTEDWT6812PIK3CA mutantDinaciclib (CDKs 2/5/1/9)CUDC-907 (PI3Kα/HDAC)ESR1 wild typeTHZ1 (CDK7)PIK-75 HCl (PI3K)PIK-75 HCl (PI3Kα)Flavopiridol (CDKs 1/2/4/6)MCF7 LTEDY537C849PIK3CA mutantCUDC-907 (PI3Kα/HDAC)CUDC-907 (PI3Kα/HDAC)Activating ESR1 mutation (Y537C)THZ1 (CDK7)BGT226 (PI3K/mTOR)PIK-75 HCl (PI3Kα)NVP-2 (CDK9)MCF7 LTEDY537C-PalboR(n/a)8As MCF7 LTEDY537CCUDC-907 (PI3Kα/HDAC)Palbociclib resistantPIK-75 HCl (PI3Kα)NVP-2 (CDK9)SUM44 LTEDY537S6717PIK3CA mutantDinaciclib (CDKs 2/5/1/9)NVP-2 (CDK9)Activating ESR1 mutation (Y537S)CUDC-907 (PI3Kα/HDAC)WYE-125132 (mTOR)E-cadherin nullBGT226 (PI3K/mTOR)CUDC-907 (PI3Kα/HDAC)HCC1428 LTED5114PIK3CA wild typeCUDC-907 (PI3Kα/HDAC)CUDC-907 (PI3Kα/HDAC)ESR1 wild typeBGT226 (PI3K/mTOR)BGT226 (PI3K/mTOR)Torin-2 (mTOR)Sapanisertib (mTOR)HCC1428 LTEDPalboR(n/a)11As HCC1428 LTEDCUDC-907 (PI3Kα/HDAC)Palbociclib resistantCUDC-101 (EGFR)BGT226 (PI3K/mTOR)T47D LTED5712PIK3CA mutantCUDC-907 (PI3Kα/HDAC)CUDC-907 (PI3Kα/HDAC)Loss of ER expression in adapting to LTEDDinaciclib (CDKs 2/1/5/9)PIK-75 HCl (PI3Kα)THZ1 (CDK7)CUDC-101 (EGFR)ZR75.1 LTED29(n/a)PTEN nullDinaciclib (CDKs 2/5/1/9)Loss of ER expression in adapting to LTEDTHZ1 (CDK7)Omipalisib (PI3K/mTOR)LTED: long-term estrogen deprived*Number of significant hits at 100 nM dose are shown Citation Format: Arany Soosainathan, Joanna Nikitorowicz-Buniak, Sunil Pancholi, Marjan Iravani, John Alexander, Syed Haider, Stephen R Johnston, Mitchell Dowsett, Lesley A Martin, Clare M Isacke. Exploiting novel models of endocrine-resistant breast cancer to identify new therapeutic targets [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-05.

Cholesterol Depletion Modulates Drug Resistance Pathways to Sensitize Resistant Breast Cancer Cells to Tamoxifen

Cancer drug resistance poses a significant risk of relapse and mortality. Adjuvant tamoxifen use has significantly reduced breast cancer mortality; however, many patients relapse due to acquired resistance. We aim to assess the potential of a cholesterol depletor (acetyl plumbagin) combined with tamoxifen to reduce cholesterol accumulation and cancer drug resistance. Cell viability, apoptosis and cholesterol staining was assessed following combination treatment. Gene and protein expression in cancer drug resistance and lipoprotein signalling pathways were assessed using RT2 Profiler™ PCR arrays and STRING networks. Combined treatment led to an increase in apoptosis and reduced intracellular cholesterol in MCF-7 and long-term estrogen deprived (LTED) cells compared to single compound treatments. Furthermore, the combination treatment perturbed several cholesterol-related and cancer-drug resistance pathways. The present study demonstrates the efficacy of tamoxifen combined with acetyl plumbagin in potentially disrupting the PI3K/Akt/PKB and Akt/mTORC1 signalling pathways in MCF-7 cells, reducing breast cancer cell proliferation and resistance.

OCT1 Is a Poor Prognostic Factor for Breast Cancer Patients and Promotes Cell Proliferation via Inducing NCAPH.

Octamer transcription factor 1 (OCT1) is a transcriptional factor reported to be a poor prognostic factor in various cancers. However, the clinical value of OCT1 in breast cancer is not fully understood. In the present study, an immunohistochemical study of OCT1 protein was performed using estrogen receptor (ER)-positive breast cancer tissues from 108 patients. Positive OCT1 immunoreactivity (IR) was associated with the shorter disease-free survival (DFS) of patients (p = 0.019). Knockdown of OCT1 inhibited cell proliferation in MCF-7 breast cancer cells as well as its derivative long-term estrogen-deprived (LTED) cells. On the other hand, the overexpression of OCT1 promoted cell proliferation in MCF-7 cells. Using microarray analysis, we identified the non-structural maintenance of chromosomes condensin I complex subunit H (NCAPH) as a novel OCT1-taget gene in MCF-7 cells. Immunohistochemical analysis showed that NCAPH IR was significantly positively associated with OCT1 IR (p < 0.001) and that positive NCAPH IR was significantly related to the poor DFS rate of patients (p = 0.041). The knockdown of NCAPH inhibited cell proliferation in MCF-7 and LTED cells. These results demonstrate that OCT1 and its target gene NCAPH are poor prognostic factors and potential therapeutic targets for patients with ER-positive breast cancer.

HOTAIR Facilitates Endocrine Resistance in Breast Cancer Through ESR1/miR-130b-3p Axis: Comprehensive Analysis of mRNA-miRNA-lncRNA Network

BackgroundTo summarize the regulatory role of mRNA-miRNA-lncRNA network associated with endocrine therapy resistance (ETR) in breast cancer.MethodsWe analyzed the differentially expressed genes (DEGs), differentially expressed lncRNAs (DELs), and differentially expressed miRNAs (DEMs) in long-term estrogen-deprived (LTED) estrogen receptor (ER)-positive breast cancer cells (LTED MCF7) (modeling relapse on endocrine therapy) and MCF7 cells in the presence of estrogen (E2) (modeling a patient at primary diagnosis) by mining GSE120929 and GSE120930 datasets. The mRNA-miRNA-lncRNA network was constructed by multiple bioinformatic tools. The prognosis of genes from the network was validated in breast cancer patients with following systemic treatment (endocrine therapy) by GEPIA, Kaplan–Meier plotter and UALCAN database.ResultsTotally, 769 DEGs, 33 DEMs, and 10 DELs were selected. The mRNA-miRNA-lncRNA network was established including 60 mRNA nodes, 6 miRNA nodes and 3 lncRNA nodes. A significant module containing 3 nodes and 3 edges was calculated based on the mRNA-miRNA-lncRNA network. The hub genes in the network are ABCG2, ESR1 and GJA1. ESR1/miR-130b-3p/HOTAIR are significantly correlated with the prognosis of breast cancer patients with endocrine therapy.ConclusionThis study provides a novel ETR-related mRNA-miRNA-lncRNA network. Further, we suggest that ESR1/miR-130b-3p/HOTAIR may be promising targets for clinical treatment of endocrine therapy-resistant breast cancer.

Targeting WEE1 Inhibits Growth of Breast Cancer Cells That Are Resistant to Endocrine Therapy and CDK4/6 Inhibitors

Despite the success of antiestrogens in extending overall survival of patients with estrogen receptor positive (ER+) breast tumors, resistance to these therapies is prevalent. ER+ tumors that progress on antiestrogens are treated with antiestrogens and CDK4/6 inhibitors. However, 20% of these tumors never respond to CDK4/6 inhibitors due to intrinsic resistance. Here, we used endocrine sensitive ER+ MCF7 and T47D breast cancer cells to generate long-term estrogen deprived (LTED) endocrine resistant cells that are intrinsically resistant to CDK4/6 inhibitors. Since treatment with antiestrogens arrests cells in the G1 phase of the cell cycle, we hypothesized that a defective G1 checkpoint allows resistant cells to escape this arrest but increases their dependency on G2 checkpoint for DNA repair and growth, and hence, targeting the G2 checkpoint will induce cell death. Indeed, inhibition of WEE1, a crucial G2 checkpoint regulator, with AZD1775 (Adavosertib), significantly decreased cell proliferation and increased G2/M arrest, apoptosis and gamma-H2AX levels (a marker for DNA double stranded breaks) in resistant cells compared with sensitive cells. Thus, targeting WEE1 is a promising anti-cancer therapeutic strategy in standard therapy resistant ER+ breast cancer.

Abstract PS18-09: Novel chimeric small molecule AC682 potently degrades estrogen receptor with oral anti-tumor efficacy superior to fulvestrant

Abstract Modulation of estrogen activity and/or synthesis has been the mainstay therapeutic strategy for estrogen receptor (ER)-positive breast cancer. Fulvestrant, which provides superior clinical benefit over aromatase inhibitors and selective ER modulators, achieves its potent ER signaling inhibition through unique ER degradation activity beyond direct antagonism. Despite this superiority, the efficacy of fulvestrant appears limited by poor drug exposure through intramuscular injection. Up to 50% of ER baseline levels remained after 6 months of fulvestrant treatment. Therefore, an oral ER degrader with optimal pharmacokinetic profile may lead to better convenience and efficacy than fulvestrant. Here we report preclinical characterization of our novel oral ER degrader development candidate, AC682, a chimeric small molecule containing both ligands of ER and E3 ligase discovered with our ACCU-Degron technology. Through engaging E3 ligase and ER simultaneously, AC682 induced potent ER degradation with a sub-nanomolar half-maximal degradation concentration (DC50) in multiple ER-positive breast cancer cell lines tested, including a tamoxifen-resistant long-term estrogen deprived (LTED) cell line and cell lines expressing clinically-relevant ESR1 variants (Y537S and D538G). Proteasome-dependent ER degradation by AC682 peaked after a few hours of treatment, resulting in diminished expression of ER-regulated genes and subsequent cell growth inhibition. In the in vivo setting, oral dosing of AC682 achieved substantial drug exposure and oral bioavailability, with well tolerance in multiple animal species. In estradiol-dependent MCF7 xenograft tumors, oral daily dosing of AC682 led to dose-dependent tumor growth inhibition/regression and concomitant tumor ER protein reduction more than 90% at study termination. In particular, 3 mg/kg oral daily dosing achieved tumor stasis. When administrated in combination with CDK4/6 inhibitor Palbociclib, AC682 showed clear synergy in both the estradiol-dependent MCF7 model and a tamoxifen-resistant MCF7 model. In a patient-derived xenograft model, which bears ESR1 Y537S mutation and is hormone-independent, AC682 significantly inhibited tumor growth whereas fulvestrant only showed weak efficacy. In conclusion, oral chimeric ER degrader AC682 demonstrated robust ER degradation and anti-tumor efficacy in preclinical models, supporting its continued investigation in the clinic. Citation Format: Wei He, Hui Zhang, Lorena Perkins, Lissette Bouza, Ke Liu, Yimin Qian, Jie Fan. Novel chimeric small molecule AC682 potently degrades estrogen receptor with oral anti-tumor efficacy superior to fulvestrant [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-09.

Perils of prolonged ovarian suppression and hypoestrogenism in the treatment of breast cancer: Is the risk of treatment worse than the risk of recurrence?

Premenopausal breast cancer is usually estrogen receptor positive, and hence, prolonged ovarian suppression by medical or surgical means to prevent recurrence has become standard of management to improve disease-free survival. Ten-year adjuvant tamoxifen therapy is associated with 3.5% fewer recurrences compared to five years. The SOFT trial demonstrated small but statistically significant incremental improvements in long-term disease-free survival by the addition of gonadotropin-releasing hormone analog treatment (triptorelin) to an aromatase inhibitor (exemestane). Profound hypoestrogenism in the premenopausal age group may not be well tolerated due to a host of bothersome side effects (primarily vasomotor symptoms, musculoskeletal complaints, genitourinary syndrome of menopause, and mood disorders). Prolonged hypoestrogenism in younger women is associated with premature development of cardiovascular disease, bone loss, cognitive decline, and all-cause mortality. This paper explores multi-system consequences of prolonged hypoestrogenism in premenopausal women derived from studies of women with and without breast cancer. Pretreatment counseling in estrogen receptor positive breast cancer should emphasize the benefit of prolonged estrogen suppression on breast cancer recurrence and established risks of lifelong hypoestrogenism on quality of life and all-cause mortality. Future genomic research may help identify the best candidates for extended ovarian suppression to avoid treating many women when only a minority benefit.