Antiestrogen Resistance Research Articles

MRNA-Lipid Nanoparticle-Mediated Restoration of PTPN14 Exhibits Antitumor Effects by Overcoming Anoikis Resistance in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) poses a challenging prognosis due to early metastasis driven by anoikis resistance. Identifying crucial regulators to overcome this resistance is vital for improving patient outcomes. In this study, a genome-wide CRISPR/Cas9 knockout screen in TNBC cells has identified tyrosine-protein phosphatase nonreceptor type 14 (PTPN14) as a key regulator of anoikis resistance. PTPN14 expression has shown a progressive decrease from normal breast tissue to metastatic tumors. Overexpressing PTPN14 has induced anoikis and inhibited cell proliferation in TNBC cells, while normal human breast cells are unaffected. Mechanistically, PTPN14 is identified as a key factor in dephosphorylating breast cancer antiestrogen resistance 3, a novel substrate, leading to the subsequent inhibition of PI3K/AKT and ERK signaling pathways. Local delivery of in vitro transcribed PTPN14 mRNA encapsulated by lipid nanoparticles in a TNBC mouse model has effectively inhibited tumor growth and metastasis, prolonging survival. The study underscores PTPN14 as a potential therapeutic target for metastatic TNBC, with the therapeutic strategy based on mRNA expression of PTPN14 demonstrating clinical application prospects in alleviating the burden of both primary tumors and metastatic disease.

Abstract PS17-09: An FGFR1/2 degrader overcomes antiestrogen resistance in ER+/FGFR1-amplified breast cancer

Abstract We previously reported that nuclear FGFR1 promotes antiestrogen resistance in ER+/FGFR1-amplified breast cancer. Nuclear FGFR1 activity was not affected by FGFR tyrosine kinase inhibitors (TKIs). Furthermore, pan-FGFR TKIs are not well tolerated. Therefore, new therapeutic strategies that inhibit nuclear FGFR1 while sparing FGFR3 and FGFR4 would be required to overcome antiestrogen resistance and limit toxicity. For this study, we used the proteolysis-targeting chimera (PROTAC) DGY-09-192, consisting of the FGFR TKI BCJ398 linked with a VHL recruiting ligand, to degrade FGFR1 and FGFR2. We aimed to determine whether selective degradation of FGFR1/2 overcomes endocrine resistance. Treatment with 50-100 nM DGY-09-192 strongly induced FGFR1 degradation and suppressed phosphorylation of FGFR1 and its downstream targets FRS2, AKT, and ERK1/2 in ER+/FGFR1-amplified CAMA1 and MDA-MB-134 breast cancer cells. FGFR1 degradation was evident within 4 h and lasted 48-96 h after drug washout. The proteasome inhibitor MG132 (10 µM) rescued both FGFR degradation and phosphorylation of downstream signaling molecules induced by DGY-09-192, confirming that FGFR1 degradation is dependent on ubiquitination and proteasomal degradation. At equivalent concentrations, DGY-09-192 suppressed phosphorylation of FGFR1 and its downstream targets more potently than BGJ398. Subcellular fractionation showed treatment with DGY-09-192 (100 nM) degraded FGFR1 in membrane, nuclear, and chromatin-bound fractions in both CAMA1 and MDA-MB-134 cells. Treatment of >CAMA1 cells with DGY-09-192 (>100 nM) downregulated nuclear FGFR1 target genes and canonical ERα target genes, such as CCND1, VEGFA, and CDK12 as measured by qRT-PCR whereas treatment with BGJ398 (100 nM) did not. Treatment with DGY-09-192 (500 nM) reversed resistance to the ER degrader fulvestrant in CAMA1 cells as measured by colony formation assays and using the Incucyte Live-Cell Analysis System. Each drug alone showed a modest effect on cell proliferation whereas the combination completed blocked cell growth (p < 0.0001). Finally, treatment of NOD scid gamma (NSG) mice with established ER+/FGFR1-amplified HCI-011 patient-derived xenografts with DGY-09-192 (40 mg/kg daily x2-6) induced FGFR1 degradation and blocked phosphorylation of downstream targets in the tumors. Studies using the combination of DGY-09-192 and fulvestrant in mice bearing established HCI-011 xenografts are in progress. We next investigated the effect of DGY-09-192 on cancer cells harboring FGFR1 and FGFR2 activating mutations. DGY-09-192 treatment induced FGFR2 degradation and blocked downstream signaling in MFE296 endometrial cancer and EFM-19 breast cancer cells that harbor the FGFR2N549K and FGFR2K659E hotspot mutations, respectively. In addition, DGY-09-192 strongly suppressed MFE296 cell proliferation. We are currently investigating whether DGY-09-192 blocks signaling and cell proliferation induced by the N546K and K656E hotspot FGFR1 mutations in ER+ breast cancer cells. Notably, a recent clinical study showed that tumors harboring these FGFR1 activating mutations are refractory to the pan-FGFR TKI pemigatinib. Conclusions: The FGFR1/2 PROTAC DGY-09-192 induced degradation of nuclear and membrane-bound FGFR1 and blocked FGFR1-induced signaling and nuclear activity in ER+/FGFR1-amplified breast cancer cells. The combination of DGY-09-192 and fulvestrant synergistically suppressed proliferation of these cells. Therefore, FGFR1 degradation represents a promising therapeutic strategy to block FGFR1 activity more completely and selectively in ER+ breast cancers harboring activating FGFR1 alterations. Citation Format: Yasuaki Uemoto, Chang-Ching Lin, Alberto Servetto, Bingnan Wang, Dan Ye, Fabiana Napolitano, Hima Milan Patel, Luigi Formisano, M Rosario Chica-Parrado, Emmanuel Bikorimana, Saurabh Mendiratta, Chuo Chen, Ariella Hanker, Carlos Arteaga. An FGFR1/2 degrader overcomes antiestrogen resistance in ER+/FGFR1-amplified 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 PS17-09.

Deciphering the Role of BCAR3 in Cancer Progression: Gene Regulation, Signal Transduction, and Therapeutic Implications.

This review comprehensively explores the gene BCAR3, detailing its regulation at the gene, mRNA, and protein structure levels, and delineating its multifunctional roles in cellular signaling within cancer contexts. The discussion covers BCAR3's involvement in integrin signaling and its impact on cancer cell migration, its capability to induce anti-estrogen resistance, and its significant functions in cell cycle regulation. Further highlighted is BCAR3's modulation of immune responses within the tumor microenvironment, a novel area of interest that holds potential for innovative cancer therapies. Looking forward, this review outlines essential future research directions focusing on transcription factor binding studies, isoform-specific expression profiling, therapeutic targeting of BCAR3, and its role in immune cell function. Each segment builds towards a holistic understanding of BCAR3's operational mechanisms, presenting a critical evaluation of its therapeutic potential in oncology. This synthesis aims to not only extend current knowledge but also catalyze further research that could pivotally influence the development of targeted cancer treatments.

BCAR4 facilitates trastuzumab resistance and EMT inbreast cancer via sponging miR-665 and interacting withYAP1.

Breast cancer antiestrogen resistance 4 (BCAR4) has been suggested that can modulate cell behavior, resulting in tumorigenesis and chemoresistance. However, the underlying mechanisms of BCAR4 in trastuzumab resistance (TR) is still elusive. Here, we explored the function and the underlying mechanism of BCAR4 involving in TR. We found that BCAR4 is significantly upregulated in trastuzumab-resistant BC cells. Knockdown of BCAR4 could sensitize the BC cells to trastuzumab and suppress epithelial-mesenchymal transition (EMT). Mechanically, BCAR4 promotes yes-associated protein 1 (YAP1) expression by competitively sponging miR-665, to activated TGF-β signaling. Reciprocally, YAP1 could occupy the BCAR4 promoter to enhance its transcription, suggesting that there exists a positive feedback regulation between YAP1 and BCAR4. Targeting the BCAR4/miR-665/YAP1 axis may provide a novel insight of therapeutic approaches for TR in BC.

Autophagy and senescence facilitate the development of antiestrogen resistance in ER positive breast cancer.

Estrogen receptor positive (ER+) breast cancer is the most common breast cancer diagnosed annually in the US with endocrine-based therapy as standard-of-care for this breast cancer subtype. Endocrine therapy includes treatment with antiestrogens, such as selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators (SERDs), and aromatase inhibitors (AIs). Despite the appreciable remission achievable with these treatments, a substantial cohort of women will experience primary tumor recurrence, subsequent metastasis, and eventual death due to their disease. In these cases, the breast cancer cells have become resistant to endocrine therapy, with endocrine resistance identified as the major obstacle to the medical oncologist and patient. To combat the development of endocrine resistance, the treatment options for ER+, HER2 negative breast cancer now include CDK4/6 inhibitors used as adjuvants to antiestrogen treatment. In addition to the dysregulated activity of CDK4/6, a plethora of genetic and biochemical mechanisms have been identified that contribute to endocrine resistance. These mechanisms, which have been identified by lab-based studies utilizing appropriate cell and animal models of breast cancer, and by clinical studies in which gene expression profiles identify candidate endocrine resistance genes, are the subject of this review. In addition, we will discuss molecular targeting strategies now utilized in conjunction with endocrine therapy to combat the development of resistance or target resistant breast cancer cells. Of approaches currently being explored to improve endocrine treatment efficacy and patient outcome, two adaptive cell survival mechanisms, autophagy, and "reversible" senescence, are considered molecular targets. Autophagy and/or senescence induction have been identified in response to most antiestrogen treatments currently being used for the treatment of ER+ breast cancer and are often induced in response to CDK4/6 inhibitors. Unfortunately, effective strategies to target these cell survival pathways have not yet been successfully developed. Thus, there is an urgent need for the continued interrogation of autophagy and "reversible" senescence in clinically relevant breast cancer models with the long-term goal of identifying new molecular targets for improved treatment of ER+ breast cancer.

Abstract B007: Vandetanib abrogates antiestrogen resistance in MAPK-driven ER+ breast cancer by halting key intracellular signaling pathways and multiple druggable kinases

Abstract Introduction: Vandetanib has been shown to abrogate ER+ breast cancer growth via inhibition of MAPK; we sought to elucidate the effects of Vandetanib on MAPK-driven endocrine-resistant breast cancer. Methods: Chronically treated MCF7-TAM (Tamoxifen), MCF7-EXE (Exemestane), and wild-type MCF7 ER+ breast cancer cell lines were treated with Vandetanib and/or Tamoxifen. Cell viability assays were conducted with CellTiter-Glo. Cells were treated with Vandetanib (10 uM) and vehicle for 24 hours, and mRNA sequencing was performed. To elicit TKI-specific proteomic changes, cells were treated with control or IC90 Vandetanib for 48 hours, and the signatures of kinase activity were determined using a multiplex inhibitor bead library coupled with mass spectrometry. Differential gene (DGE) and protein (DPE) expression analyses were performed to determine enriched and depleted transcripts and kinase activity with Vandetanib treatment. Gene set enrichment analysis was performed to determine the shared and unique pathways regulated by Vandetanib for each model. A Vandetanib response signature was compiled using the top 20 downregulated genes and analyzed with METABRIC and SCAN-B datasets to assess effects on survival in ER+ breast cancer patients. Results: MCF7-TAM and MCF7-EXE demonstrated lower Tamoxifen IC50 when treated with Vandetanib compared to Tamoxifen alone, which showed synergistic effects. Data analysis of mRNA libraries showed reduced tumor progression and signaling of MAPK, PI3K/AKT, RAS, CCND1, MYC, and EGFR across all three lines, as well as increased ERBB2 signaling, which may denote a potential adaptive bypass mechanism to Vandetanib treatment. Gene sets showed increased estrogen response, ESR1 activity, and sensitivity to tamoxifen across all three lines. Notable key players in PI3K/AKT and MAPK signaling such as TYRO3 were significantly downregulated at both the gene and protein levels. Additionally, JAK2 demonstrated dual upregulation and has been implicated with favorable prognosis due to associations with immune infiltration. The downregulated Vandetanib gene set conferred significantly improved OS (HR=0.75, p<0.0001, METABRIC; HR=0.87, p=0.034, SCAN-B) and RFS (HR=0.60, p<0.0001, METABRIC) in ER+ breast cancer patients from both datasets. Multiple druggable kinases such as RET, EGFR, FGFR2/3, CDK6, and SRC were depleted by Vandetanib treatment. Upregulated druggable kinases identified with Vandetanib treatment include MTOR, FGFR4, ERBB2, and JAK1. Conclusions: Vandetanib overcomes antiestrogen resistance in MAPK-driven models of ER+ breast cancer by halting key signaling pathways such as MAPK, PI3K/AKT, MYC, and ERBB2, among others. Upregulated druggable kinases may serve as targets to overcome resistance to Vandetanib. Downregulated targets with Vandetanib could serve as biomarkers to select patients most likely to benefit from Vandetanib treatment. Citation Format: Rasha T Kakati, Austin Whitman, Hyunsoo Kim, Philip M Spanheimer. Vandetanib abrogates antiestrogen resistance in MAPK-driven ER+ breast cancer by halting key intracellular signaling pathways and multiple druggable kinases [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B007.

BCAR4 Expression as a Predictive Biomarker for Endocrine Therapy Resistance in Breast Cancer

BackgroundBreast cancer, particularly the estrogen receptor positive (ER+) subtype, remains a leading cause of cancer-related death among women. Endocrine therapy is the most effective treatment for ER+ breast cancer; however, the development of resistance presents a significant challenge. This study explored the role of the breast cancer antiestrogen resistance 4 (BCAR4) gene as a potential driver of resistance and a pivotal biomarker in breast cancer. Patients and MethodsThe researchers undertook a comprehensive analysis of 1743 patients spanning 6 independent cohorts. They examined the association of BCAR4 expression with patient outcomes across all breast cancer types and the PAM50 molecular subtypes. The relationship between elevated BCAR4 expression and resistance to endocrine therapy including AIs, the prevailing standard-of-care for endocrine therapy, was also investigated. ResultsThis meta-analysis corroborated the link between BCAR4 expression and adverse outcomes as well as resistance to endocrine therapy in breast cancer. Notably, BCAR4 expression is clinically significant in luminal A and B subtypes. Additionally, an association between BCAR4 expression and resistance to AI treatment was discerned. ConclusionThis study expands on previous findings by demonstrating that BCAR4 expression is associated with resistance to newer therapies. The identification of patients with intrinsic resistance to hormone therapy is crucial to avoid ineffective treatment strategies. These findings contribute to our understanding of endocrine therapy resistance in breast cancer and could potentially guide the development of more effective treatment strategies.

MYSM1 acts as a novel co-activator of ERα to confer antiestrogen resistance in breast cancer

Endocrine resistance is a crucial challenge in estrogen receptor alpha (ERα)-positive breast cancer (BCa). Aberrant alteration in modulation of E2/ERα signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Herein, we demonstrate that MYSM1 as a deubiquitinase participates in modulating ERα action via histone and non-histone deubiquitination. MYSM1 is involved in maintenance of ERα stability via ERα deubiquitination. MYSM1 regulates relevant histone modifications on cis regulatory elements of ERα-regulated genes, facilitating chromatin decondensation. MYSM1 is highly expressed in clinical BCa samples. MYSM1 depletion attenuates BCa-derived cell growth in xenograft models and increases the sensitivity of antiestrogen agents in BCa cells. A virtual screen shows that the small molecule Imatinib could potentially interact with catalytic MPN domain of MYSM1 to inhibit BCa cell growth via MYSM1-ERα axis. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ERα action and provide a potential therapeutic target for endocrine resistance in BCa.

Small molecule inhibition of TFF3 overcomes tamoxifen resistance and enhances taxane efficacy in ER+ mammary carcinoma

Even though tamoxifen has significantly improved the survival of estrogen receptor positive (ER+) mammary carcinoma (MC) patients, the development of drug resistance with consequent disease recurrence has limited its therapeutic efficacy. Trefoil factor-3 (TFF3) has been previously reported to mediate anti-estrogen resistance in ER+MC. Herein, the efficacy of a small molecule inhibitor of TFF3 (AMPC) in enhancing sensitivity and mitigating acquired resistance to tamoxifen in ER+MC cells was investigated. AMPC induced apoptosis of tamoxifen-sensitive and resistant ER+MC cells and significantly reduced cell survival in 2D and 3D culture in vitro. In addition, AMPC reduced cancer stem cell (CSC)-like behavior in ER+MC cells in a BCL2-dependent manner. Synergistic effects of AMPC and tamoxifen were demonstrated in ER+MC cells and AMPC was observed to improve tamoxifen efficacy in tamoxifen-sensitive cells and to re-sensitize cells to tamoxifen in tamoxifen-resistant ER+MC in vitro and in vivo. Additionally, tamoxifen-resistant ER+MC cells were concomitantly resistant to anthracycline, platinum and fluoropyrimidine drugs, but not to Taxanes. Taxane treatment of tamoxifen-sensitive and resistant ER+MC cells increased TFF3 expression indicating a combination vulnerability for tamoxifen-resistant ER+MC cells. Taxanes increased CSC-like behavior of tamoxifen-sensitive and resistant ER+MC cells which was reduced by AMPC treatment. Taxanes synergized with AMPC to promote apoptosis and reduce CSC-like behavior in vitro and in vivo. Hence, AMPC restored the sensitivity of tamoxifen and enhanced the efficacy of Taxanes in tamoxifen-resistant ER+MC. In conclusion, pharmacological inhibition of TFF3 may serve as an effective combinatorial therapeutic strategy for the treatment of tamoxifen-resistant ER+MC.

THU552 Understanding Radiation Therapy Sensitivity In Invasive Lobular Carcinoma Of The Breast

Abstract Disclosure: M.R. Boysen: None. M.J. Sikora: None. M. Shackleford: None. J. Sottnik: None. Invasive lobular carcinoma of the breast (ILC) affects ∼40,000 US women annually and is a top-ten most common cancer affecting women. Despite most ILC having biomarkers of “low risk” disease (e.g. ∼95% are estrogen receptor α /ER-positive), ILC is associated with poorer long-term outcomes and anti estrogen resistance. ILC are also notoriously non-responsive to chemotherapy, critically limiting treatment options for advanced or metastatic ILC. However, ILC are highly responsive to radiation therapy (XRT), which suppresses ILC recurrence and improves survival. We hypothesize that this sensitivity may be due to a dysfunction in DNA damage repair unique to ILC cells. We examined formation and resolution of DNA damage repair foci (namely RAD51, γH2AX and 53BP1 foci) by immunofluorescence with ILC and IDC cell lines treated with ionizing radiation +/- anti-estrogen therapy as surrogates for the initiation and resolution of DNA repair. We also assessed cell growth via 7-day growth assays with ILC and IDC cell lines treated with ionizing radiation. Imaging with immunofluorescence suggested a deficiency in γH2AX foci formation in ILC cells compared to IDC cells following treatment with XRT, despite robust formation of 53BP1 foci. This suggests that a deficiency in performing DNA damage repair in response to XRT is likely present in ILC cells. Additionally, our growth assays demonstrated an inability in ILC cells to recover from ionizing radiation compared to IDC cells. Taken together, the potential dysfunction in γH2AX foci formation in ILC cells suggests that ILC cells may not form the appropriate DNA repair structures to initiate the DNA damage response, suggesting that a form of DNA repair deficiency underlies radiation sensitivity in ILC cells. Presentation: Thursday, June 15, 2023

THU477 Drivers Of Estrogen Induced Invasive Lobular Carcinoma

Abstract Disclosure: C. Ufondu: None. L. Clem: None. M. Musick: None. M.J. Sikora: None. J.H. Ostrander: None. Invasive lobular carcinoma (ILC) is an estrogen-receptor (ER)-positive histologic subtype of breast cancer typically treated with anti-estrogens (i.e., tamoxifen and aromatase inhibitors). Anti-estrogen resistance and disease progression occur in about 34% of patients with ILC, but mechanisms of resistance have been underexplored. Additionally, estrogen-only hormone replacement therapy increases the risk of ILC, but not the more common invasive ductal carcinoma (IDC), highlighting a potentially unique role for estrogen in ILC initiation and progression. To identify mediators of estrogen-induced ILC progression we are using the the mouse mammary intraductal (MIND) model, which better recapitulates the progression of ILC and response to estrogen in vivo. MM134, MM330, and SUM44PE ILC cell lines were injected intraductally into the #4 mammary glands of NSG mice. Mice were monitored for up to 20 weeks with or without estrogen supplemented in the drinking water. Our results show that MIND supports an in situ-like to invasive disease progression for ILC cell lines, and that estrogen accelerated tumor growth and invasion in vivo. Current studies are aimed at identifying mediators that drive estrogen-induced tumor growth and invasion in vivo, focusing on E-cadherin (CDH1) loss as the genetic hallmark of ILC occurring in ∼95% of cases. We aim to identify how loss of E-cadherin in normal human mammary epithelial cells (HMECs) may lead to reprogramming of ER and promote the development of ILC. We are developing a CRISPR/Cas9 approach for knocking out CDH1 in HMECs. We will study estrogen response in the CDH1-knockout versus control HMECs in vitro to examine whether loss contributes to ILC-specific estrogen activity. Using the MIND model, we will engraft the control and knockout cells into the ducts of adult female NSG mice and monitor tumor growth through bioluminescence imaging. ER reprogramming will be assessed in the engrafted cells using RNAseq and CUT&RUN (Cleavage Under Target & Release Using Nuclease). We predict that E-cadherin loss will reprogram ER DNA binding, dysregulate transcription factors linked to E-cadherin, such as TCF/LEF, and confirm critical downstream regulators that mediate estrogen-driven tumorigenesis. Understanding the unique contributions of ER in ILC could lead to new therapeutic strategies for the prevention and treatment of ILC. Presentation: Thursday, June 15, 2023

RETooling the RET Inhibitor Pralsetinib for ESR1 Fusion-Positive Breast Cancer and Beyond.

Transcriptionally active fusions of ESR1 (ESR1-TAF) and somatic mutations in the estrogen receptor alpha (ERα) ligand-binding domain (LBD) cause endocrine therapy resistance in breast cancer. In searching for therapeutic target kinase(s) in these breast cancers, Gou and colleagues identified FLT4, RET, JAK1, and IGF1R as the top upregulated kinases induced by ESR1-TAFs and ERα LBD mutants in breast cancer cells. Among them, inhibition of RET by pralsetinib suppressed ESR1-TAF-driven and ERα LBD mutant-driven cell proliferation and patient-derived xenograft growth. Pralsetinib is an inhibitor of the RET protein tyrosine kinase that is approved for treating oncogenic RET mutation-positive and RET fusion-positive thyroid cancers and non-small cell lung cancer. The work by Gou and colleagues reinforces the knowledge of RET as an ESR1 target gene and highlights that RET interacts with ERα to promote breast cancer tumorigenesis and antiestrogen resistance. It also raises the prospect of repositioning pralsetinib to target wildtype RET in ER-positive breast cancer. See related article by Gou et al., p. 3237.

MRI-based breast cancer radiogenomics using RNA profiling: association with subtypes in a single-center prospective study

BackgroundThere are few prospective studies on the correlations between MRI features and whole RNA-sequencing data in breast cancer according to molecular subtypes. The purpose of our study was to explore the association between genetic profiles and MRI phenotypes of breast cancer and to identify imaging markers that influences the prognosis and treatment according to subtypes.MethodsFrom June 2017 to August 2018, MRIs of 95 women with invasive breast cancer were prospectively analyzed, using the breast imaging-reporting and data system and texture analysis. Whole RNA obtained from surgical specimens was analyzed using next-generation sequencing. The association between MRI features and gene expression profiles was analyzed in the entire tumor and subtypes. Gene networks, enriched functions, and canonical pathways were analyzed using Ingenuity Pathway Analysis. The P value for differential expression was obtained using a parametric F test comparing nested linear models and adjusted for multiple testing by reporting Q value.ResultsIn 95 participants (mean age, 53 years ± 11 [standard deviation]), mass lesion type was associated with upregulation of CCL3L1 (sevenfold) and irregular mass shape was associated with downregulation of MIR421 (sixfold). In estrogen receptor-positive cancer with mass lesion type, CCL3L1 (21-fold), SNHG12 (11-fold), and MIR206 (sevenfold) were upregulated, and MIR597 (265-fold), MIR126 (12-fold), and SOX17 (fivefold) were downregulated. In triple-negative breast cancer with increased standard deviation of texture analysis on precontrast T1-weighted imaging, CLEC3A (23-fold), SRGN (13-fold), HSPG2 (sevenfold), KMT2D (fivefold), and VMP1 (fivefold) were upregulated, and IGLC2 (73-fold) and PRDX4 (sevenfold) were downregulated (all, P < 0.05 and Q < 0.1). Gene network and functional analysis showed that mass type estrogen receptor-positive cancers were associated with cell growth, anti-estrogen resistance, and poor survival.ConclusionMRI characteristics are associated with the different expressions of genes related to metastasis, anti-drug resistance, and prognosis, depending on the molecular subtypes of breast cancer.

Abstract 3890: TMPRSS2 serin protease is a novel biomarker for ER+ breast cancer patient prognosis and survival and mediates resistance to anti-estrogen treatment in ER+ breast cancer

Abstract Breast cancer (BC) is the most commonly diagnosed cancer in women and the second leading cause of cancer related deaths in the US. About 70-80% of patients have ER+ BC and initially respond to anti-estrogen (ie, Tamoxifen) or endocrine/homonal treatments. However, about 60% of the patients inevitably develops resistance to anti-estrogen therapies (due to intrinsic (15-20%) and acquire resistance (30-40%), representing a major clinical problem, resulting in relapses and metastasis, leading to significant patient mortality. Therefore, understanding the underlying mechanisms causing resitance to hormonal or anti-estrogen treatments is needed the for developing highly effective threapies and improving patient survival. Cell surface proteases, including serine proteases, are proteolytic modifiers of particular targets, including growth factors and protease-activated receptors, which are critical for the activation of oncogenic signaling pathways. TMPRSS2, a member of type II transmembrane serine protease (TTSP) family, has been shown to be highly expressed in prostate cancer cells and its role in carcinogenesis. We found that TMPRSS2 is highly upregulated well established tamoxifene resistant cell lines derivative of MCF7 (LCC2 and LCC9) compared to MCF7 cells. Performing Kaplan-Meier survival analyses in TCGA-BC database we also found that TMPRSS2 expression is associated with poor prognosis and significantly shorter patient survival in ER+ BC patients (p=0.015, n=221 patients). In addition, we found that TMPRSS2 is regulated by Estrogen/Estrogen Receptor-alpha (ERα) signaling as in vitro inhibition of estrogen/ERα signaling by ERα siRNA, tamoxifen or faslodex reduced TMPRSS2 protein expression ER+ MCF7 BC cells. Moreover, we found that inhibition of TMPRSS2 by 3 different siRNA molecules suppressed cell proliferation of MCF7 cells and reduced expression critical proteins for cell cycle and proliferation, including cyclin D1, c-Myc and pERK/MAPK signaling, suggesting that TMPRSS2 promotes the oncogenic signaling pathways in ER+ BC cells. Currently, we are targeting TMPRSS2 in in vitro and in vivo Tamox-resistant LCC2 and LCC9 models in mice by siRNA alone and in combination with Tamoxifen or faslodex to demostrate inhibition of TMPRSS2 sensitize tumors to anti-estrogen treatments. In conclusion, TMPRSS2 is a potential biomarker for poor patient survival and anti-estrogen resistance, and targeting TMPRSS2 alone as a monothreapy and in combination with anti- Tamoxifen or faslodex may be a promising approach for overcoming resistance to estrogen thereapies in ER+ BC patients. Citation Format: Rumeysa Ozyurt, Nermin Kahraman, Pinar Atalay Dundar, Bulent Ozpolat. TMPRSS2 serin protease is a novel biomarker for ER+ breast cancer patient prognosis and survival and mediates resistance to anti-estrogen treatment in ER+ breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3890.

Abstract P1-13-13: Nuclear envelope Expression of the Oncogene Adipocyte-Associated Methionine Domain Containing Conveys Inferior Prognosis but Increased Sensitivity to Statins and Fluorouracil-Based Therapy

Abstract The sub-classification of invasive breast cancer into Integrated Clusters by a combined analysis of genomic change and expression profiling has revealed novel cancer drivers. The integrated Cluster 2 breast cancer sub-group represents a cohort with aggressive, largely estrogen receptor positive tumours with a high relapse rate. It is characterized by an amplification of chromosome 11 at the heart of which is a little studied gene which codes for the protein Adipocyte-Associated Methionine Domain Containing (AAMDC). Initial cell line and murine studies demonstrated oncogenic behaviours for AAMDC including increased proliferation and invasion, increased colony formation and anti-estrogen resistance. Downstream gene expression analysis showed the protein to modulate cholesterol biosynthesis, one carbon metabolism and mTOR signaling. To assess the clinical impact of differing levels and sub-localizations of AAMDC, immunohistochemistry for AAMDC was carried out using tissue microarrays from a cohort of 420 patients with invasive breast cancer. Expression was noted in a number of sub-cellular localizations including diffuse cytoplasm, nucleus and nuclear envelope. Using both dichotomous and continuous scoring, no significant association for any expression site with standard prognostic factors was identified including size, lymph node status, grade or receptor statuses. However, both cytoplasmic and nuclear envelope expressions correlated with significantly worse overall survival (p=0.04 and p=0.04 respectively) whereas nuclear expression showed a trend to better survival (p=0.06). Distant relapse and breast cancer deaths were lowest where there was nuclear expression but no nuclear envelope expression (4.7% and 4.7% respectively) but significantly higher for the reverse expression pattern (18.9%, p=0.02 and 17.6%, p=0.03 respectively). Considering the pre-clinical impact of AAMDC on genes involved in cholesterol biosynthesis, we studied the effects of statin prescription in the early disease setting in the context of AAMDC expression. Statins were found to be generally protective of relapse across the group. Only nuclear envelope AAMDC expression interacted, with a hazard ratio (HR) of 0.33 for distant relapse with high expressors, compared to a HR of 0.90 in low expression (p=0.02 for difference). Similarly, considering one carbon metabolism, we explored the impact of the anti-metabolite drug capecitabine compared to other chemotherapy treatments, largely taxane-based, in the metastatic setting. Again, only nuclear envelope expression interacted with median progression-free survivals on capecitabine of 2.0 v 12.2 months for low and high nuclear envelope expression respectively, p=0.03. In summary, AAMDC nuclear envelope expression correlates with poor prognosis which may be mitigated by statin administration in the early disease setting. This expression pattern also confers sensitivity to flurouracil-based metastatic treatment. Citation Format: Andrew D. Redfern, Indunil Weerasena, Lisa Spalding, Monique Ong, Emily Golden, Eleanor Woodward, Pilar Blancafort. Nuclear envelope Expression of the Oncogene Adipocyte-Associated Methionine Domain Containing Conveys Inferior Prognosis but Increased Sensitivity to Statins and Fluorouracil-Based Therapy [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 P1-13-13.

Abstract MS1-1: Oncogene signal transduction and antiestrogen resistance

Abstract Background: Recent studies suggest that enhancer reprogramming underlies heterogeneity and disease progression in estrogen receptor-positive (ER+) BC. Cell-type/state specific transcription is governed by high-order assemblies of master transcription factors (TFs) and epigenetically defined regulatory regions including super-enhancers (SEs). We previously showed that aberrant activation of the pioneer TF FOXA1 promotes enhancer and transcriptional reprogramming in endocrine-resistant BC, involving the ER and the AP-1 FRA1 and c-JUN TFs. As SEs maintain a robust cell-type/state specific core transcriptional regulatory circuitry (CRC) in developmental and tumorigenic processes, we sought to identify key additional TFs in SE/FOXA1-driven CRCs in endocrine resistance, which could serve as attractive therapeutic targets. Methods: TF binding motif at the shared SEs (mapped by H3K27ac ChIP-seq) between MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE) and the endogenous FOXA1-amplifed tamoxifen-resistant (TamR) cells was analyzed by HOMER. ER-bound SEs distinguishing TamR vs. P cells were defined by integrating the SEs with our prior ER ChIP-seq data (PMID 28507152). KLF4 motif within these ER-bound SEs was scanned using FIMO and linked to nearby genes by intersection with the previously defined promoter-tethered regions (PTRs) (PMID 24141950). Differential gene expression in MCF7-TamR cells upon KLF4 knockdown (KD) by 3 unique siRNAs was analyzed using limma from edgeR. The biological and clinical significance of the KLF4-dependent genes was analyzed using Gene Ontology and survival modeling with METABRIC and the ER+ metastatic BC cohort ( SABCS19-GS2-02 ). Cell migration was assessed by the wound-healing assay. Citation Format: Rachel Schiff. Oncogene signal transduction and antiestrogen 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 MS1-1.

Upregulation of the EGFR/MEK1/MAPK1/2 signaling axis as a mechanism of resistance to antiestrogen‑induced BimEL dependent apoptosis in ER+ breast cancer cells.

The epidermal growth factor receptor (EGFR) is commonly upregulated in multiple cancer types, including breast cancer. In the present study, evidence is provided in support of the premise that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitates the escape of breast cancer cells from BimEL‑dependent apoptosis, conferring resistance to therapy. This conclusion is based on the findings that ectopic BimEL cDNA overexpression and confocal imaging studies confirm the pro‑apoptotic role of BimEL in ERα expressing breast cancer cells and that upregulated EGFR/MEK1/MAPK1/2 signaling blocks BimEL pro‑apoptotic action in an antiestrogen‑resistant breast cancer cell model. In addition, the present study identified a pro‑survival role for autophagy in antiestrogen resistance while EGFR inhibitor studies demonstrated that a significant percentage of antiestrogen‑resistant breast cancer cells survive EGFR targeting by pro‑survival autophagy. These pre‑clinical studies establish the possibility that targeting both the MEK1/MAPK1/2 signaling axis and pro‑survival autophagy may be required to eradicate breast cancer cell survival and prevent the development of antiestrogen resistance following hormone treatments. The present study uniquely identified EGFR upregulation as one of the mechanisms breast cancer cells utilize to evade the cytotoxic effects of antiestrogens mediated through BimEL‑dependent apoptosis.

OR07-2 Endocrine Response And Resistance Is Driven By Novel Transcriptional Co-Regulator Activity In Invasive Lobular Carcinoma Of The Breast

Abstract Invasive lobular carcinoma of the breast (ILC) affects ∼40,000 US women annually and is a top ten most diagnosed cancer in women. ∼95% of ILC express estrogen receptor alpha (ER), but ILC is associated with poor long-term outcomes and anti-estrogen resistance, suggesting ER function is distinct in ILC cells. We reported that MDC1 (mediator of DNA damage checkpoint 1) is an ILC-specific ER co-regulator required for ER-driven proliferation, and that MDC1 knockdown dysregulates the ER transcriptome in ILC. Mechanisms of MDC1 co-regulator activity are unknown, but must be defined to understand endocrine response and resistance in ILC. To understand MDC1 co-regulator functions, we profiled ER/MDC1 genomic binding (ie. "cistrome") using CUT&amp;RUN in ER+ ILC cell line MDA MB 134VI (MM134). We identified ∼2500 binding sites each for ER and MDC1, but only ∼8% of sites were bound by both factors. However, using BETA to integrate cistrome and transcriptome data predicted that both ER and MDC1 activate estrogen-regulated genes with over half of direct ER target genes also direct MDC1 targets (p=9.8×10-375). Among shared ER: MDC1 direct target genes (n=760), 58% had MDC1 binding in the promoter region with ER bound at nearby enhancers, while only 15% were linked to shared ER: MDC1 binding sites, suggesting MDC1 primarily facilitates promoter access for ER-bound enhancers. Further, MDC1 knockdown reduced ER binding at enhancer sites at ER: MDC1 target genes, implicating MDC1 in regulating chromatin access. Importantly, MDC1 does not have known enzymatic activity to directly regulate chromatin structure. We profiled MDC1-interacting proteins in ILC cells by RIME (Rapid immunoprecipitation mass spectrometry of endogenous proteins), and determined whether knockdown of putative ER: MDC1 partners disrupted ER function. This identified the SWI/SNF complex protein BRG1 (SMARCA4) as a critical ER: MDC1 partner. BRG1 was required for ILC cell growth, and BRG1 knockdown specifically suppressed ER regulation of MDC1-dependent ER target genes. Taken together, our data suggest that in ILC cells, MDC1 facilitates ER access to and activity at target gene promoters. The BRG1 SWI/SNF complex is a putative ER: MDC1 partner and mediator of MDC1 co-regulator functions. Ongoing research is focused on understanding how MDC1 regulates ILC-specific ER activity by licensing access to ILC-specific target genes, and the role of the SWI/SNF complex in mediating genomic ER: MDC1 activity. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.

HAS2-Ezrin-ER axis plays a role in acquired antiestrogen resistance of ER-positive breast cancer.

The development of endocrine resistance is a major clinical problem in estrogen receptor-positive (ER+) breast cancer (BrCa) treatment, in which how cancer cells acquire resistance remains obscure. Hyaluronan synthase 2 (HAS2) is the most critical synthase in producing hyaluronan and is well known for its involvement in cancer growth, metabolism and metastasis. Recent evidence has proved that HAS2 is involved in cellular acquired resistance to drug therapy in BrCa. In this work, we first observed that HAS2 expression was decreased in the endocrine-resistant ER+ BrCa cells. Further knocking-out experiments confirmed that the loss of HAS2 in parental ER+ BrCa cells resulted in a following antiestrogen resistance. Next, we found that the HAS2-loss could induce an upregulation of Ezrin, a member of the membrane cytoskeletal protein family who plays key roles in cellular signal transduction. Notably, we identified that the increase of Ezrin induced by HAS2-loss could inhibit the ERα expression and augment antiestrogen resistance, suggesting that a HAS2-Ezrin-ER axis may be associated with the acquirement of endocrine resistance in ER+ BrCa cells. Finally, knockdown or inhibition of Ezrin could restore the sensitivity of endocrine-resistant cells to antiestrogens treatment by activating ERα signaling. Taken together, our findings unraveled a novel HAS2-Ezrin-ER route in regulating the sensitivity of ER+ BrCa cells to antiestrogens, in which Ezrin may be a potential target in endocrine therapy.

Molecular Mechanisms of Anti-Estrogen Therapy Resistance and Novel Targeted Therapies.

Breast cancer (BC) is the most commonly diagnosed cancer in women, constituting one-third of all cancers in women, and it is the second leading cause of cancer-related deaths in the United States. Anti-estrogen therapies, such as selective estrogen receptor modulators, significantly improve survival in estrogen receptor-positive (ER+) BC patients, which represents about 70% of cases. However, about 60% of patients inevitably experience intrinsic or acquired resistance to anti-estrogen therapies, representing a major clinical problem that leads to relapse, metastasis, and patient deaths. The resistance mechanisms involve mutations of the direct targets of anti-estrogen therapies, compensatory survival pathways, as well as alterations in the expression of non-coding RNAs (e.g., microRNA) that regulate the activity of survival and signaling pathways. Although cyclin-dependent kinase 4/6 and phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) inhibitors have significantly improved survival, the efficacy of these therapies alone and in combination with anti-estrogen therapy for advanced ER+ BC, are not curative in advanced and metastatic disease. Therefore, understanding the molecular mechanisms causing treatment resistance is critical for developing highly effective therapies and improving patient survival. This review focuses on the key mechanisms that contribute to anti-estrogen therapy resistance and potential new treatment strategies alone and in combination with anti-estrogen drugs to improve the survival of BC patients.