Endocrine-resistant Breast Cancer Research Articles

Rapid Induction of the Unfolded Protein Response and Apoptosis by Estrogen Mimic TTC-352 for the Treatment of Endocrine-Resistant Breast Cancer.

Patients with long-term estrogen-deprived breast cancer, after resistance to tamoxifen or aromatase inhibitors develops, can experience tumor regression when treated with estrogens. Estrogen's antitumor effect is attributed to apoptosis via the estrogen receptor (ER). Estrogen treatment can have unpleasant gynecologic and nongynecologic adverse events; thus, the development of safer estrogenic agents remains a clinical priority. Here, we study synthetic selective estrogen mimics (SEM) BMI-135 and TTC-352, and the naturally occurring estrogen estetrol (E4), which are proposed as safer estrogenic agents compared with 17β-estradiol (E2), for the treatment of endocrine-resistant breast cancer. TTC-352 and E4 are being evaluated in breast cancer clinical trials. Cell viability assays, real-time PCR, immunoblotting, ERE DNA pulldowns, mass spectrometry, X-ray crystallography, docking and molecular dynamic simulations, live cell imaging, and Annexin V staining were conducted in 11 biologically different breast cancer models. Results were compared with the potent full agonist E2, less potent full agonist E4, the benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. We report ERα's regulation and coregulators' binding profiles with SEMs and E4 We describe TTC-352's pharmacology as a weak full agonist and antitumor molecular mechanisms. This study highlights TTC-352's benzothiophene scaffold that yields an H-bond with Glu353, which allows Asp351-to-helix 12 (H12) interaction, sealing ERα's ligand-binding domain, recruiting E2-enriched coactivators, and triggering rapid ERα-induced unfolded protein response (UPR) and apoptosis, as the basis of its anticancer properties. BPTPE's phenolic OH yields an H-Bond with Thr347, which disrupts Asp351-to-H12 interaction, delaying UPR and apoptosis and increasing clonal evolution risk.

An Overview of Phytochemical and Biological Activities: Ficus deltoidea Jack and Other Ficus spp.

ABSTRACTFicus deltoidea Jack (Moraceae) is a well-known medicinal plant used in customary medication among the Malay people to reduce and mend sicknesses such as ulcers, psoriasis, cytotoxicity, cardioprotective, inflammation, jaundice, vitiligo, hemorrhage, diabetes, convulsion, hepatitis, dysentery injuries, wounds, and stiffness. Ficus deltoidea contains a wide variety of bioactive compounds from different phytochemical groups such as alkaloids, phenols, flavonoids, saponins, sterols, terpenes, carbohydrates, and proteins. The genus Ficus has several hundreds of species, which shows excellent therapeutic effects and a wide variety of helpful properties for human welfare. Searching information was collected by using electronic databases including Web of Science, Science Direct, Springer, SciFinder, PubMed, Scopus, Medline, Embase, and Google Scholar. This review is, therefore, an effort to give a detailed survey of the literature on its pharmacognosy, phytochemistry, phytochemical, and pharmacological properties of Ficus and its important species. This summary could be beneficial for future research aiming to exploit the therapeutic potential of Ficus and its useful medicinal species.

Abemaciclib, a third CDK 4/6 inhibitor for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer

ABSTRACT Introduction The field of metastatic luminal breast cancer (hormone receptor positive, HER-2 negative) is dynamic and evolving, harboring some of the most significant therapeutic advances in medical oncology. Over the last decade, many pivotal trials showed excellent results with drastic improvements in survival as well as the quality of life of metastatic luminal breast cancer patients. Areas covered The successful inhibition of the cyclinD/cyclin-dependent kinases 4 and 6 (CDK4/6)–retinoblastoma protein (RB) pathway with potent CDK4/6 inhibitors improved the outcome of advanced luminal breast cancers. Abemaciclib is the third CDK 4/6 inhibitor arriving to the market after palbociclib and ribociclib. Here, we describe the biology of the CDK4/6 pathway and summarize clinical data of previously published pivotal trials emphasizing the efficacy and toxicity of abemaciclib. The aim was to define its place in the current guidelines and to make a brief comparison with other available drugs of same class in the absence of cross trials comparison. Expert opinion As there are no available biomarkers to predict response or resistance to abemaciclib, the promising overall survival data of MONARCH-2 could possibly impact the clinician’s choice to optimize treatment for endocrine-resistant metastatic breast cancer.

Comprehensive Transcriptomic Analysis Reveals Dysregulated Competing Endogenous RNA Network in Endocrine Resistant Breast Cancer Cells.

BackgroundTamoxifen and fulvestrant, both approved for endocrine therapy, have remarkably increased the prognosis of hormone receptor-positive breast cancer patients. However, acquired resistance to endocrine therapy greatly reduces its clinical efficacy. Accumulating evidence suggests a pivotal role of non-coding RNAs (ncRNAs) in breast cancer endocrine resistance, but the specific functions of ncRNAs in tamoxifen and fulvestrant resistance remain largely unknown.MethodsMicroarray analysis was performed for endocrine therapy sensitive (MCF-7), tamoxifen-resistant (LCC2), and dual tamoxifen and fulvestrant-resistant (LCC9) breast cancer cells. Gene ontology and pathway analysis were conducted for functional prediction of the unannotated differentially expressed ncRNAs. Competing endogenous RNA regulatory networks were constructed.ResultsWe discovered a total of 3,129 long non-coding RNAs (lncRNAs), 13,556 circular RNAs (circRNAs), 132 microRNAs, and 3358 mRNAs that were significantly differentially expressed. We constructed co-expression networks for lncRNA-mRNA, circRNA-mRNA, and microRNA-mRNA. In addition, we established lncRNA-microRNA-mRNA and circRNA-microRNA-mRNA regulatory networks to depict ncRNA crosstalk and transcriptomic regulation of endocrine resistance.ConclusionsOur study delineates a comprehensive profiling of ncRNAs in tamoxifen and fulvestrant resistant breast cancer cells, which enriches our understanding of endocrine resistance and sheds new light on identifying novel endocrine resistance biomarkers and potential therapeutic targets to overcome endocrine resistance.

Activating transcription factor-2 (ATF2) is a key determinant of resistance to endocrine treatment in an in vitro model of breast cancer

BackgroundActivating transcription factor-2 (ATF2), a member of the leucine zipper family of DNA binding proteins, has been implicated as a tumour suppressor in breast cancer. However, its exact role in breast cancer endocrine resistance is still unclear. We have previously shown that silencing of ATF2 leads to a loss in the growth-inhibitory effects of tamoxifen in the oestrogen receptor (ER)-positive, tamoxifen-sensitive MCF7 cell line and highlighted that this multi-faceted transcription factor is key to the effects of tamoxifen in an endocrine sensitive model. In this work, we explored further the in vitro role of ATF2 in defining the resistance to endocrine treatment.Materials and methodsWe knocked down ATF2 in TAMR, LCC2 and LCC9 tamoxifen-resistant breast cancer cell lines as well as the parental tamoxifen sensitive MCF7 cell line and investigated the effects on growth, colony formation and cell migration. We also performed a microarray gene expression profiling (Illumina Human HT12_v4) to explore alterations in gene expression between MCF7 and TAMRs after ATF2 silencing and confirmed gene expression changes by quantitative RT-PCR.ResultsBy silencing ATF2, we observed a significant growth reduction of TAMR, LCC2 and LCC9 with no such effect observed with the parental MCF7 cells. ATF2 silencing was also associated with a significant inhibition of TAMR, LCC2 and LCC9 cell migration and colony formation. Interestingly, knockdown of ATF2 enhanced the levels of ER and ER-regulated genes, TFF1, GREB1, NCOA3 and PGR, in TAMR cells both at RNA and protein levels. Microarray gene expression identified a number of genes known to mediate tamoxifen resistance, to be differentially regulated by ATF2 in TAMR in relation to the parental MCF7 cells. Moreover, differential pathway analysis confirmed enhanced ER activity after ATF2 knockdown in TAMR cells.ConclusionThese data demonstrate that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity.

Insulin receptor substrate-1 (IRS-1) mediates progesterone receptor-driven stemness and endocrine resistance in oestrogen receptor+ breast cancer

BackgroundProgesterone receptors (PR) are potent modifiers of endocrine responses. In aberrant signalling cancer contexts, phosphorylation events dramatically alter steroid hormone receptor action.MethodsThe transcriptomes of primary tumours and metastases in mice harbouring ER+ breast cancer patient-derived xenografts (PDXs) were analysed following single-cell RNAseq. In vitro assays were employed to delineate mechanisms of endocrine resistance and stemness.ResultsA 16-gene phospho-Ser294 PR (p-PR) signature predicted poor outcome in ER+ breast cancer. Relative to primary PDX tumours, metastatic lesions expressed abundant p-PR and exhibited an activated PR gene programme with elevated expression of PGR and IRS-1. Breast cancer models of activated PR lost the expression of IGF1R and acquired insulin hypersensitivity with tamoxifen insensitivity. Activated p-PR+ breast cancer cells formed increased tumourspheres with enlarged ALDH+ and CD24−/CD44 populations. E2 induced PR/IRS-1 interaction and exchange of IGF1Rβ for IRS-1 in p-PR-containing transcriptional complexes. Inhibition of IRS-1 or IR and inducible IRS-1 knockdown reduced tumourspheres. Endocrine-resistant models of luminal B breast cancer induced p-PR in 3D cultures and required PR and IRS-1 for tumoursphere formation.ConclusionsPhospho-PR-B cooperates with IRS-1 to promote outgrowth of endocrine-resistant and stem-like breast cancer cells. Targeting phospho-PR/IRS-1 crosstalk may block the emergence of endocrine resistance.

Selective Estrogen Receptor Degraders (SERDs): A Promising Strategy for Estrogen Receptor Positive Endocrine-Resistant Breast Cancer.

Estrogen receptor (ER) plays important roles in gene transcription and the proliferation of ER positive breast cancers. Selective modulation of ER has been a therapeutic target for this specific type of breast cancer for more than 30 years. Selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs) have been demonstrated to be effective therapeutic approaches for ER positive breast cancers. Unfortunately, 30-50% of ER positive tumors become resistant to SERM/AI treatment after 3-5 years. Fulvestrant, the only approved selective estrogen receptor degrader (SERD), is currently an important therapeutic approach for the treatment of endocrine-resistant breast cancers. The poor pharmacokinetic properties of fulvestrant have inspired the development of a new generation of oral SERDs to overcome drug resistance. In this review, we describe recent advances in ERα structure, functions, and mechanisms of endocrine resistance and summarize the development of oral SERDs in both academic and industrial areas.

Time-Resolved Profiling Reveals ATF3 as a Novel Mediator of Endocrine Resistance in Breast Cancer.

Simple SummaryBreast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express estrogen receptor α (ERα) are the vast majority and are mostly treated with targeted endocrine therapy. However, resistance development poses an urgent clinical problem and the mechanisms of this phenomenon are not fully understood. In this study we identified ATF3 as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. Our work adds a new piece to the complex puzzle of the molecular mechanisms at the basis of endocrine therapy resistance and proposes ATF3 and the downstream pathways as putative targets for novel combinatorial treatment strategies.Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express Estrogen Receptor α account for around 70% of cases and are mostly treated with targeted endocrine therapy. However, depending on the degree of severity of the disease at diagnosis, 10 to 40% of these tumors eventually relapse due to resistance development. Even though recent novel approaches as the combination with CDK4/6 inhibitors increased the overall survival of relapsing patients, this remains relatively short and there is a urgent need to find alternative targetable pathways. In this study we profiled the early phases of the resistance development process to uncover drivers of this phenomenon. Time-resolved analysis revealed that ATF3, a member of the ATF/CREB family of transcription factors, acts as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. ATF3 was found to be essential in controlling crucial processes such as proliferation, cell cycle, and apoptosis during the early response to treatment through the regulation of MAPK/AKT signaling pathways. Its essential role was confirmed in vivo in a mouse model, and elevated expression of ATF3 was verified in patient datasets, adding clinical relevance to our findings. This study proposes ATF3 as a novel mediator of endocrine resistance development in breast cancer and elucidates its role in the regulation of downstream pathways activities.

The Relationship between Leptin, the Leptin Receptor and FGFR1 in Primary Human Breast Tumors.

Obesity is associated with increased breast cancer risk and poorer cancer outcomes; however, the precise etiology of these observations has not been fully identified. Our previous research suggests that adipose tissue-derived fibroblast growth factor-2 (FGF2) promotes the malignant transformation of epithelial cells through the activation of fibroblast growth factor receptor-1 (FGFR1). FGF2 is increased in the context of obesity, and increased sera levels have been associated with endocrine-resistant breast cancer. Leptin is a marker of obesity and promotes breast carcinogenesis through several mechanisms. In this study, we leverage public gene expression datasets to evaluate the associations between FGFR1, leptin, and the leptin receptor (LepR) in breast cancer. We show a positive association between FGFR1 and leptin protein copy number in primary breast tumors. These observations coincided with a positive association between Janus kinase 2 (Jak2) mRNA with both leptin receptor (LepR) mRNA and FGFR1 mRNA. Moreover, two separate Jak2 inhibitors attenuated both leptin+FGF2-stimulated and mouse adipose tissue-stimulated MCF-10A transformation. These results demonstrate how elevated sera FGF2 and leptin in obese patients may promote cancer progression in tumors that express elevated FGFR1 and LepR through Jak2 signaling. Therefore, Jak2 is a potential therapeutic target for FGFR1 amplified breast cancer, especially in the context of obesity.

Correction to "Pharmacology and Molecular Mechanisms of Clinically Relevant Estrogen Estetrol and Estrogen Mimic BMI-135 for the Treatment of Endocrine-Resistant Breast Cancer".

Correction to "Pharmacology and Molecular Mechanisms of Clinically Relevant Estrogen Estetrol and Estrogen Mimic BMI-135 for the Treatment of Endocrine-Resistant Breast Cancer".

The 3D genomic landscape of differential response to EGFR/HER2 inhibition in endocrine-resistant breast cancer cells

BackgroundRecent studies suggested that crosstalk between ERα and EGFR/HER2 pathways plays a critical role in mediating endocrine therapy resistance. Several inhibitors targeting EGFR/HER2 signaling, including FDA-approved lapatinib and gefitinib as well as a novel dual tyrosine kinase inhibitor (TKI) sapitinib, showed greater therapeutic efficacies. However, how 3D chromatin landscape responds to the inhibition of EGFR/HER2 pathway remains to be elucidated. MethodsIn this study, we conducted in situ Hi-C and RNA-seq in two ERα+ breast cancer cell systems, 1) parental MCF7 cells and its associated tamoxifen-resistant MCF7TR cells; and 2) parental T47D cells and its associated tamoxifen-resistant T47DTR cells, before and after the treatment of sapitinib. ResultsWe identified differential responses in topologically associated domains (TADs), looping genes and expressed genes. Interestingly, we found that many differential TADs and looping genes are reversible after sapitinib treatment, indicating that EGFR/HER2 signaling may play a role in reshaping and rewiring the high order genome organization. We further examined and recapitulated the reversible looping genes in 3D spheroids of breast cancer cells, demonstrating that 3D cell culture spheroid of breast cancer cells could be a potential preclinical breast cancer model for studying 3D chromatin regulation. ConclusionsOur study has provided significant insights into our understanding of 3D genomic landscape changes in response to EGFR/HER2 Inhibition in endocrine-resistant breast cancer cells. Our data provides a rich resource for further evaluating chromatin structural responses to EGFR/HER2 targeted therapies in endocrine-resistant breast cancer cells. Our analyses suggest that these alterations of chromatin structures and transcriptional programs may provide new avenues for intervention or designing of patient selection for targeted endocrine treatment.

Constitutively active androgen receptor supports the metastatic phenotype of endocrine-resistant hormone receptor-positive breast cancer

BackgroundHormone receptor positive (HR+) breast cancer (BCa) is the most frequently diagnosed subtype. Acquired and intrinsic resistance to conventional endocrine therapy (ET) commonly occurs and prompts incurable metastatic disease. Hence, ET-resistant (ET-R) HR+ BCa presents a therapeutic challenge. Previous studies show elevated androgen receptor (AR) that supports resistance to ET tamoxifen and correlates with HR+ BCa metastasis. Yet surprisingly, studies with AR-blocker enzalutamide (Enz) in ET-R HR+ BCa present conflicting results. We now report that a constitutively active, unique from canonical Enz-targeted, AR accumulates in endocrine resistant HR+ BCa cells.MethodsAR protein profiles in acquired and intrinsic ET-R HR + -BCa were defined with cell-free modification tests, in-house in-vivo SUMOylation assays, and PLA imaging. Genomic activity of native AR and modified-AR mimetic was tested with reporter assays and limited transcriptome analysis. Spheroid growth and migration studies were used to evaluate inhibitory actions of Enz and combinatorial therapy.ResultsSustained higher molecular weight SUMO-modified AR (SUMO-AR) persists in acquired and intrinsic ET-R BCa cell lines. Concurrently, SUMO isoforms and global SUMO-modified proteome also accumulates in the same cell lines. We identified AR as a novel substrate for the SUMO-E3 ligase HSPB1/Hsp27. Independent of ligand, SUMO-AR is resilient to ubiquitin-mediated proteasomal degradation, enriched in the nucleus, readily chromatin-bound, and transcriptionally active. Constitutive SUMO-AR initiates a gene-expression profile that favors epithelial-mesenchymal transition. Enz combined with a SUMO inhibitor attenuates migration and metastatic phenotype of ET-R HR+ BCa.ConclusionTargeting both unmodified and SUMO-modified AR prevents the metastatic progression of HR+ BCa with ET-R.1cz6dozgZCQes7_eQqKRNVVideo abstract

Endocrine Resistance in Breast Cancer: The Role of Estrogen Receptor Stability.

Therapy of hormone receptor positive breast cancer (BCa) generally targets estrogen receptor (ER) function and signaling by reducing estrogen production or by blocking its interaction with the ER. Despite good long-term responses, resistance to treatment remains a significant issue, with approximately 40% of BCa patients developing resistance to ET. Mutations in the gene encoding ERα, ESR1, have been identified in BCa patients and are implicated as drivers of resistance and disease recurrence. Understanding the molecular consequences of these mutations on ER protein levels and its activity, which is tightly regulated, is vital. ER activity is in part controlled via its short protein half-life and therefore changes to its stability, either through mutations or alterations in pathways involved in protein stability, may play a role in therapy resistance. Understanding these connections and how ESR1 alterations could affect protein stability may identify novel biomarkers of resistance. This review explores the current reported data regarding posttranslational modifications (PTMs) of the ER and the potential impact of known resistance associated ESR1 mutations on ER regulation by affecting these PTMs in the context of ET resistance.

Point-activated ESR1Y541S has a dramatic effect on the development of sexually dimorphic organs.

Mutations in the estrogen receptor α (ERα) occur in endocrine-resistant metastatic breast cancer. However, a major gap persists with the lack of genetically tractable immune competent mouse models to study disease. Hence, we developed a Cre-inducible murine model expressing a point-activated ESR1Y541S (ESR1Y537S in humans) driven by its endogenous promoter. Germline expression of mutant ESR1Y541S reveals dramatic developmental defects in the reproductive organs, mammary glands, and bones of the mice. These observations provide critical insights into the tissue-specific roles of ERα during development and highlights the potential use of our model in further developmental and cancer studies.

Evidence for Enhanced Exosome Production in Aromatase Inhibitor-Resistant Breast Cancer Cells.

Aromatase inhibitors (AIs) represent the standard anti-hormonal therapy for post-menopausal estrogen receptor-positive breast cancer, but their efficacy is limited by the emergence of AI resistance (AIR). Exosomes act as vehicles to engender cancer progression and drug resistance. The goal of this work was to study exosome contribution in AIR mechanisms, using estrogen-dependent MCF-7 breast cancer cells as models and MCF-7 LTED (Long-Term Estrogen Deprived) subline, modeling AIR. We found that exosome secretion was significantly increased in MCF-7 LTED cells compared to MCF-7 cells. MCF-7 LTED cells also exhibited a higher amount of exosomal RNA and proteins than MCF-7 cells. Proteomic analysis revealed significant alterations in the cellular proteome. Indeed, we showed an enrichment of proteins frequently identified in exosomes in MCF-7 LTED cells. The most up-regulated proteins in MCF-7 LTED cells were represented by Rab GTPases, important vesicle transport-regulators in cancer, that are significantly mapped in “small GTPase-mediated signal transduction”, “protein transport” and “vesicle-mediated transport” Gene Ontology categories. Expression of selected Rab GTPases was validated by immunoblotting. Collectively, we evidence, for the first time, that AIR breast cancer cells display an increased capability to release exosomes, which may be associated with an enhanced Rab GTPase expression. These data provide the rationale for further studies directed at clarifying exosome’s role on endocrine therapy, with the aim to offer relevant markers and druggable therapeutic targets for the management of hormone-resistant breast cancers.

Abstract 5675: Metastatic organ-specific changes in ERα and XPO1 signaling and metabolic adaptations dictate the therapy responses in endocrine resistant breast cancers

Abstract Introduction: Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing a potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis with a 5-year relative overall survival rate of just 24%. The tumor microenvironment (TME) plays a key role in the behavior of cancer cells and their response to endocrine therapies. However, there is a critical need for relevant 3D models of different metastatic sites to use for studying the molecular mechanisms driving resistance in these tumor phenotypes. Methods: We established 3D cell culture systems in novel hydrogels derived from decellularized bone, lung and liver tissues - the major sites of metastasis of ER+ tumors, and compared with standard Matrigel culture. Endocrine responsive (MCF7 and T47D) and resistant (MCF7 and T47D cells with ESR1 Y537S or D538G mutations) breast cancer cell lines were treated with fulvestrant (FUL), tamoxifen (TAM) and palbociclib (PAL). Tumorigenicity was assessed using in vitro cell viability and colony formation assays and in vivo breast cancer cell line xenografts in athymic mice. ERα cistromes and transcriptomes were assessed using ChIP-Seq and RNA-Seq analysis. Metabolic changes were assessed using whole metabolite profiling by GC-MS, a Seahorse XF analyzer, and flux analysis with labeled glucose and glutamine. Results: We observed metastatic site-specific responses to each drug. For example, TAM blocked colony formation in Matrigel and lung hydrogel but increased colony formation in bone and liver hydrogels. FUL acted as an agonist and increased colony number and size in all three-tissue matrices, but not in Matrigel. PAL increased colony size and number in Matrigel and liver hydrogel but not in bone or lung hydrogel. Genome-wide ERα recruitment to chromatin was reduced in liver hydrogels, consistent with decreased clinical response to endocrine agents in patients with liver metastasis. We found that treatment with individual drugs activated novel metabolic pathways to increase survival in different metastatic sites. Using RNA-Seq analysis, we identified increased expression of nuclear export pathway components (XPO1, KPNA2, etc.) in various metastatic sites. Combining an FDA-approved XPO1 small molecule inhibitor, Selinexor (SEL) with the current therapies used for advanced breast cancers (FUL, TAM or PAL) decreased metabolic adaptations in response to individual drug treatments and prevented colony formation in hydrogels and tumor growth in xenograft models. Conclusions: Further developing and characterizing 3D metastatic site models will enable us to elucidate the underlying mechanisms of metastasis and resistance, and target these mechanisms with novel drug combinations in already metastasized patients. Targeting metastatic-site specific adaptations to regenerate new vulnerabilities in endocrine-resistant breast tumors is novel. Given the need for better strategies for improving therapy response of metastatic ER+ tumors, our findings show uncovering the role ERα-XPO1 crosstalk plays in metastatic breast cancer could lead to new combined therapies that reduce mortality. Citation Format: Qianying Zuo, Eylem Kulkoyluoglu Cotul, Ashlie Santaliz-Casiano, Ozan Berk Imir, Elif Tunc, Chengjian Mao, David J. Shapiro, Ben Ho Park, Yosef Landesman, Zeynep Madak-Erdogan. Metastatic organ-specific changes in ERα and XPO1 signaling and metabolic adaptations dictate the therapy responses in endocrine resistant breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5675.

Abstract 3508: FOXA1 genetic alterations in whites versus blacks or African Americans in breast and prostate cancer

Abstract Forkhead box A1 (FOXA1) is a pioneer factor for the nuclear hormone receptors: estrogen receptor and androgen receptor. FOXA1 plays a major role inducing endocrine resistance in breast cancer (BC) and prostate cancer (PC), the two most prevalent cancers in the United States. In this study, we investigated FOXA1 gene alterations across different race and ethnicity using the TCGA PanCan Atlas dataset for BC and PC patients. The BC and PC dataset included 1084 and 494 patient samples, respectively, profiled for copy number alterations (CNA), gene expression, and mutations. In the BC dataset, the samples were from 877 non-Hispanic (81%), 38 Hispanic (3.5%) and 169 patients with no ethnicity data (15.5%). Among them, the majority of patients were White (n=751, 69.3%) followed by Black/African American (AA) (n=182, 16.8%), Asian (n=60, 5.5%), and American Indian or Alaska Native (n=1, 0.09%). Ninety BC patients (8.3%) had no race information. The PC dataset included 152 non-Hispanic (30.8%), 0 Hispanic, and 342 patients with no ethnicity data (69.2%). The samples obtained for the PC study were from White (n=147, 29.8%), Black/AA (n=7, 1.4%), and Asian (n=2, 0.4%) patients. Majority of the PC patients had no racial information (n=338, 68.4%) recorded. In the BC dataset, the incidence of FOXA1 alterations was 16/1070 (1.5%) CNA 24/1082 (2.2%) high mRNA expression, and 31/1066 (2.9%) mutations. Only amplifications were found within the BC patients. In the PC dataset, there were 15/489 (3.1%) CNA, 16/493 (3.2%) high mRNA expression, and 28/494 (5.7%) mutations reported in FOXA1. Deep deletion was found in one of the PC patients while the rest had amplifications. Due to insufficient numbers of Hispanic patients in the datasets, we compared the incidence of various FOXA1 alterations in White vs. Black/AA population using Fisher's exact test. Only FOXA1 mutation rate was significantly higher (p =0.03) in Blacks/AA (2/7, 28.6%) compared to Whites (5/147, 3.4%) in PC, but not in the BC dataset. Comparing the separate results of the FOXA1 CNA and gene overexpression White vs. Black/AA patients were statistically significant in both BC and PC datasets. A majority of FOXA1 mutations were missense mutations with a few frame shifts in BC and PC. The missense mutation reported in both BC and PC datasets were D226G, D226N, and H247Y. Additional studies are necessary to understand the functional significance of these mutations on the development of cancer. Complete and larger datasets that include the race and ethnicity information from diverse group of patients as well as tumor molecular subtyping are also needed for the assessment of the mechanism of health disparity in BC and PC in minority population in the United States. Citation Format: Jennifer Torres, Hariprasad Thangavel, Xiaoyong Fu, Rachel Schiff, Meghana V. Trivedi. FOXA1 genetic alterations in whites versus blacks or African Americans in breast and prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3508.

Abstract 5676: Preclinical evaluation of estrogen receptor coregulator binding inhibitor ERX-245 in breast cancer

Abstract Background: Breast cancer (BC) is the most common cancer in American women. Majority of BC (70%) is estrogen receptor alpha (ERα) positive and these tumors initially respond to ER-targeted therapy, however, acquired therapy-resistance limit the utility of ERα-targeted therapy using aromatase inhibitors and antiestrogens. Importantly, both therapy-sensitive and therapy-resistant tumors retain ESR1 signaling, via interaction with critical oncogenic coregulator proteins. We recently developed a small organic molecule, ESR1 coregulator binding inhibitor ERX-11. The objective of this study is to develop better analogues of ERX-11 using medicinal chemistry approaches. Methods: Virtual screening of a quarter million compounds and medicinal chemistry approaches were used to design new analogues of ERX-11 and identified ERX-245 as potent analogue. Effect of ERX-245 was evaluated in vitro using multiple BC models that express wild type (WT) ERα (MCF-7, ZR-75) and BC models with acquired resistance (MCF-7-Tam, MCF-7-LTLT). Mechanistic studies were performed using RNA-Seq, Western blotting, qRT-PCR and reporter gene assays. The in vivo efficacy of ERX-245 was examined using xenograft, and xenograft-derived explant (XDEx) models. Results: We initially performed an intensive virtual screening of a quarter million compounds and selected several candidates that showed strong binding energy to ERα. We then designed and developed several analogues of ERX-11 using modeled structural interactions. Using this approach, we identified ERX-245 as a potential lead compound for interaction with ERα. Like the parental ERX-11, ERX-245 significantly reduced the cell viability of both WT and therapy-resistant BC cells with an IC50 of 300-500 nM with minimal activity in ER-negative models such as TNBC cells. In long-term colony formation assays, ERX-245 significantly reduced the colony formation ability of both ER-WT and therapy-resistant BC cells. In ERE reporter assays, ERX-245 significantly reduced the estrogen-mediated reporter activity. ERX-245 significantly reduced the invasion of endocrine-resistant BC cells. RNA sequencing revealed unique pathways blocked by ERX-245. PK studies indicated that ERX-245 is more polar and has better solubility and pharmacokinetic properties compared to ERX-11. Treatment of ERX-245 decreased the proliferation and increased apoptosis (TUNEL staining) in xenograft derived explant (XDEx) models. ERX-245 also showed potent activity against WT-ERα and therapy-resistant xenograft models. Conclusions: Collectively, these results suggest that the ERX-11 analogue ERX-245 has potential to therapeutically target endocrine therapy resistant BC. Citation Format: Suryavathi Viswanadhapalli, Shihong Ma, Tae Kyung Lee, Xihui Liu, Kara Kassees, Uday P. Pratap, Junhao Liu, Weiwei Tang, Rajeshwar R. Tekmal, Jung-Mo Ahn, Ganesh V. Raj, Ratna K. Vadlamudi. Preclinical evaluation of estrogen receptor coregulator binding inhibitor ERX-245 in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5676.

Abstract 1975: Development and validation of detection of ESR1 mutations in circulating tumor DNA using PNA-LNA mediated PCR clamping

Abstract Background: The occurrence of ESR1 mutations is rare in estrogen receptor (ER) positive primary breast cancer patients; however these mutations are frequently reported in ER positive recurrent breast cancer patients pretreated with endocrine therapy. ESR1 mutation is an essential mechanism of resistance to endocrine therapy and predictive biomarker as well as a potential therapeutic target. The measurement of ESR1 mutations in circulating tumor DNA (ctDNA) has been studied as a non-invasive method to quickly assess endocrine therapy resistant metastatic breast cancer patients. As a simple, inexpensive, low-invasive assay, we developed a novel detection system of ESR1 mutations using peptide nucleic acid-locked nucleic acid mediated PCR clamping (PNA-LNA PCR clamping) assay. Methods: We developed a novel detection system to screen mutations at hot spot codons (E380, L536, Y537 and D538) in ESR1. Major mutations, E380Q, Y537S and D538G, were detected using PNA-LNA PCR clamping assay, and the other mutations were detected by directly sequencing amplified product of the clamp PCR. To evaluate the developed system, we used available plasma samples from ER positive breast cancer patients diagnosed at national cancer center Japan. The patient samples were screened for 9 mutations at hotspots (E380Q, L536H, L536Q, L536P, L536R, Y537S, Y537N, Y537C, and D538G) in ESR1 by an NGS panel for plasma (ctDNA), Oncomine Pan-Cancer Cell-Free Assay, and then the results were compared with the data acquired by the developed system. Results: Cell free DNA was obtained from 68 patients with ER positive breast cancer. ESR1 mutations at hotspots were found in 8.8% (6/68) of the samples by ctDNA NGS. The detected mutations were as follows: 2 samples at E380Q, 1 L536H, 3 at Y537S, 2 at Y537N, 1 at Y537C, and 5 at D538G. The average number of mutations per sample was 2.33. The concordance rate between the developed system and ctDNA NGS was calculated using results from these 6 positive samples, together with 4 negative samples, at the 9 mutations totaling 90 sites. Among the 14 hotspot mutations detected by the NGS, 12 were also detected by the developed system, giving the sensitivity of 85.7%. At 76 negative sites judged by the NGS, 74 were also judged as negative by the developed system, giving specificity of 97.4%. Combining these numbers together, the calculated concordance rate was 95.6% (86/90). Conclusion: This system using PNA-LNA mediated PCR clamping assay is a simple, inexpensive, low-invasive assay, and could be a potential alternative to plasma genotyping for the detection of plasma ESR1mutation in breast cancer patients. Citation Format: Yuki Kojima, Kan Yonemori, Tomomi Yoshino, Shigehiro Yagishita, Emi Noguchi, Hiroshi Terasaki, Sachiro Asano, Akinobu Hamada, Kenji Tamura. Development and validation of detection of ESR1 mutations in circulating tumor DNA using PNA-LNA mediated PCR clamping [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1975.

Abstract 4365: Estrogen receptor β agonists reverses letrozole therapy resistance of breast cancer through FOXO3 and FOXM1 mediated actions

Abstract Introduction: Breast cancer (BC) is the most common cancer in women worldwide and leading cause of cancer death. More than 70% of cancers are estrogen receptor alpha (ERα) positive. Despite effective therapeutic strategies for treating hormone receptor-positive (HR+) breast cancer, resistance to endocrine therapy that is either de novo or acquired still occurs. Understanding the molecular pathways that contribute to BC resistance will enable to develop new treatments for improving the efficacy of endocrine therapy. Studies have shown that estrogen receptor beta (ERβ) and its agonists inhibits the different types of cancers including BC. Herein, we investigated mechanism(s) for therapeutic efficacies of ERβ agonists in breast cancer. Methods: To investigate the mechanisms of ERβ agonists (S-Equol and LY500307), in the treatment of breast cancer, we used therapy sensitive (MCF7-Aro) and letrozole resistant (MCF7-Aro-LTLT) cells and ERβ knock down cell lines, generated by CRISPR-Cas9 technology. The wild type and ERβ knock down cells with or without treatment of ERβ agonists were analyzed, for cell proliferation, protein (Western), Gene expression array analysis, RNA-seq analysis, data-independent acquisition (DIA) mass spectrometry analyses and RNA (RT-qPCR) analysis. Xenograft and patient derived BC explants (PDEX) were used to test the antiproliferative activity of ERβ agonists. Results: Our results demonstrated that treatment with ERβ agonists, S-Equol and LY500307 inhibited the growth of both endocrine therapy sensitive and resistant breast cancer cells. Gene expression array analysis of LY500307 treated breast cancer cells showed the modulation of signaling molecules involved in cell death and cell cycle pathways. RNA-seq analysis showed that treatment with S-equol modulated the ERβ target genes involved in tumor progression and resistance to hormone therapies. Mass spectrometry based DIA analysis and Reactome pathway analysis revealed that S-equol treatment modulated the key proteins involved in DNA replication and cell cycle. Both ERβ agonists S-Equol and LY500307 inhibited the cell proliferation of parental cells whereas they could not elicits the inhibitory effect on the knock out cells. By using ERα positive PDX tumors, we have demonstrated that both the ERβ agonists inhibited the proliferation of human BC tumors. Our mechanistic studies confirmed that increased FOXO3 activation by S-equol in LTLT cells can reverse letrozole resistance. Our results showed that inhibiting FOXM1 by ERβ agonists significantly reduce the proliferation of letrozole-resistant cells. These changes were not observed in ERβ knockdown cells confirming the anti-proliferative role of ERβ in breast cancer progression. Conclusions: Collectively, our results suggest that ERβ agonists may offer a new targeted therapy for endocrine therapy resistant breast tumors. (Supported by BCRP 151884/DOD grant W81XWH-16-1-0294 and P30 CA-54174 NCI-Mays cancer center grant). Citation Format: Kumaraguruparan Ramasamy, Suryavathi Viswanadhapalli, M.A. Sureshkumar, Susan Weintraub, Ratna K. Vadlamudi, Rajeshwar R. Tekmal. Estrogen receptor β agonists reverses letrozole therapy resistance of breast cancer through FOXO3 and FOXM1 mediated actions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4365.