Therapy-resistant Breast Cancer Research Articles

Combined analysis of PTEN, HER2, and hormone receptors status: remodeling breast cancer risk profiling

BackgroundPhosphatase and tensin homolog (PTEN) loss is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker in these patients is controversial. We sought to determine whether the benefit of traditional biomarkers testing is improved by the analysis of PTEN status for the identification of high-risk breast cancer.MethodsA cohort of 608 patients with breast cancer was included in this study. Based on the expression on the neoplastic cells compared to the normal internal controls by immunohistochemistry (IHC), cases were classified as PTEN-low (PTEN-L) or PTEN-retained (PTEN-WT). The former constituted the study group, while the latter the control group. Analysis of gene expression was performed on publicly available genomic data and included 4265 patients from the METABRIC and MSK cohorts retrieved from cBioPortal. The Shapiro-Wilk test was used to analyze the normal distributions of continuous variables. Relationships between PTEN status and the clinicopathologic and molecular features of the patient population were assessed using Fisher’s exact test or Chi-squared/Wilcoxon rank-sum test. Survival curves were built according to the Kaplan-Meier method.ResultsAlteration in PTEN status was significantly different at protein and gene levels, where the reduced protein expression was observed in 280/608 cases (46.1%) from our group, while genetic aberrations in only 315/4265 (7.4%) cases of the METABRIC and MSK cohorts. PTEN-L tumors were significantly enriched for hormone receptors (HR) and HER2 negativity (n = 48, 17.1%) compared to PTEN-WT tumors (n = 22, 6.7%; p = 0.0008). Lack of HR with or without HER2 overexpression/amplification was significantly associated with worse overall survival (OS) in PTEN-L but not in PTEN-WT breast cancers (p < .0001). Moreover, PTEN-L protein expression but not gene alterations was related to the outcome, in terms of both OS and disease-free survival (p = 0.002).ConclusionsThe combined analysis of PTEN, HER2, and HR status offers relevant information for a more precise risk assessment of patients with breast cancer.

Prolactin and endocrine therapy resistance in breast cancer: The next potential hope for breast cancer treatment.

Breast cancer, a hormone‐dependent tumour, generally includes four molecular subtypes (luminal A, luminal B, HER2 enriched and triple‐negative) based on oestrogen receptor, progesterone receptor and human epidermal growth factor receptor‐2. Multiple hormones in the body regulate the development of breast cancer. Endocrine therapy is one of the primary treatments for hormone‐receptor‐positive breast cancer, but endocrine resistance is the primary clinical cause of treatment failure. Prolactin (PRL) is a protein hormone secreted by the pituitary gland, mainly promoting mammary gland growth, stimulating and maintaining lactation. Previous studies suggest that high PRL levels can increase the risk of invasive breast cancer in women. The expression levels of PRL and PRLR in breast cancer cells and breast cancer tissues are elevated in most ER+ and ER− tumours. PRL activates downstream signalling pathways and affects endocrine therapy resistance by combining with prolactin receptor (PRLR). In this review, we illustrated and summarized the correlations between endocrine therapy resistance in breast cancer and PRL, as well as the pathophysiological mechanisms and clinical practices. The study on PRL and its receptor would help explore reversing endocrine therapy‐resistance for breast cancer.

"γδT Cell-IL17A-Neutrophil" Axis Drives Immunosuppression and Confers Breast Cancer Resistance to High-Dose Anti-VEGFR2 Therapy.

Angiogenesis is an essential physiological process and hallmark of cancer. Currently, antiangiogenic therapy, mostly targeting the vascular endothelial growth factor (VEGF)/VEGFR2 signaling axis, is commonly used in the clinic for solid tumors. However, antiangiogenic therapies for breast cancer patients have produced limited survival benefits since cancer cells rapidly resistant to anti-VEGFR2 therapy. We applied the low-dose and high-dose VEGFR2 mAb or VEGFR2-tyrosine kinase inhibitor (TKI) agents in multiple breast cancer mouse models and found that low-dose VEGFR2 mAb or VEGFR2-TKI achieved good effects in controlling cancer progression, while high-dose treatment was not effective. To further investigate the mechanism involved in regulating the drug resistance, we found that high-dose anti-VEGFR2 treatment elicited IL17A expression in γδ T cells via VEGFR1-PI3K-AKT pathway activation and then promoted N2-like neutrophil polarization, thus inducing CD8+ T cell exhaustion to shape an immunosuppressive microenvironment. Combining anti-VEGFR2 therapy with immunotherapy such as IL17A, PD-1 or Ly-6G mAb therapy, which targeting the immunomodulatory axis of “γδT17 cells-N2 neutrophils” in vivo, showed promising therapeutic effects in breast cancer treatment. This study illustrates the potential mechanism of antiangiogenic therapy resistance in breast cancer and provides synergy treatment for cancer.

Induction of EnR stress by Melatonin enhances the cytotoxic effect of Lapatinib in HER2-positive breast cancer.

Despite HER2-targeted cancer treatments have provided considerable clinical benefits, resistance to HER2-targeted agents will inevitably develop. Targeting non-oncogene vulnerabilities including endoplasmic reticulum (EnR) stress has emerged as an attractive alternative approach to improve the efficacy of existing targeted cancer therapies. In the current study, we find that Melatonin sensitizes HER2-positive breast cancer cells to the dual tyrosine kinase inhibitor Lapatinib in vitro. Mechanistically, Melatonin enhances the cytotoxic effects of Lapatinib through promoting excessive EnR stress-induced unfolded protein response (UPR) and ROS overaccumulation. Consistently, the antioxidant N-acetylcysteine remarkably reverses the effects of the drug combination on ROS production, DNA damage and cytotoxicity. Furthermore, Melatonin significantly enhances the anti-tumor effect of Lapatinib in an HCC1954 xenograft model. Meanwhile, Lapatinib resistant HER2-positive breast cancer cells (LapR) display lower basal expression levels of UPR genes and enhanced tolerance to EnR stress with attenuated response to Brefeldin A and Tunicamycin. Importantly, Melatonin also increases the sensitivity of HCC1954 LapR cells to Lapatinib. Together, our findings highlight the potential utility of Melatonin as an adjuvant in the treatment of primary or therapy resistant HER2-positive breast cancer via EnR stress-mediated mechanisms.

Obesity and endocrine therapy resistance in breast cancer: Mechanistic insights and perspectives.

SummaryThe incidence of obesity, a recognized risk factor for various metabolic and chronic diseases, including numerous types of cancers, has risen dramatically over the recent decades worldwide. To date, convincing research in this area has painted a complex picture about the adverse impact of high body adiposity on breast cancer onset and progression. However, an emerging but overlooked issue of clinical significance is the limited efficacy of the conventional endocrine therapies with selective estrogen receptor modulators (SERMs) or degraders (SERDs) and aromatase inhibitors (AIs) in patients affected by breast cancer and obesity. The mechanisms behind the interplay between obesity and endocrine therapy resistance are likely to be multifactorial. Therefore, what have we actually learned during these years and which are the main challenges in the field? In this review, we will critically discuss the epidemiological evidence linking obesity to endocrine therapeutic responses and we will outline the molecular players involved in this harmful connection. Given the escalating global epidemic of obesity, advances in understanding this critical node will offer new precision medicine‐based therapeutic interventions and more appropriate dosing schedule for treating patients affected by obesity and with breast tumors resistant to endocrine therapies.

NP-ALT, a Liposomal:Peptide Drug, Blocks p27Kip1 Phosphorylation to Induce Oxidative Stress, Necroptosis, and Regression in Therapy-Resistant Breast Cancer Cells.

Resistance to cyclin D-CDK4/6 inhibitors (CDK4/6i) represents an unmet clinical need and is frequently caused by compensatory CDK2 activity. Here we describe a novel strategy to prevent CDK4i resistance by using a therapeutic liposomal:peptide formulation, NP-ALT, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. We find that NP-ALT blocks proliferation in HR+ breast cancer cells, as well as CDK4i-resistant cell types, including triple negative breast cancer (TNBC). The peptide ALT is not as stable in primary mammary epithelium, suggesting that NP-ALT has little effect in nontumor tissues. In HR+ breast cancer cells specifically, NP-ALT treatment induces ROS and RIPK1-dependent necroptosis. Estrogen signaling and ERα appear required. Significantly, NP-ALT induces necroptosis in MCF7 ESRY537S cells, which contain an ER gain of function mutation frequently detected in metastatic patients, which renders them resistant to endocrine therapy. Here we show that NP-ALT causes necroptosis and tumor regression in treatment naïve, palbociclib-resistant, and endocrine-resistant BC cells and xenograft models, demonstrating that p27 is a viable therapeutic target to combat drug resistance. IMPLICATIONS: This study reveals that blocking p27 tyrosine phosphorylation inhibits CDK4 and CDK2 activity and induces ROS-dependent necroptosis, suggesting a novel therapeutic option for endocrine and CDK4 inhibitor-resistant HR+ tumors.

Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in estrogen receptor-positive advanced breast cancer. However, most patients will eventually experience disease progression on this combination, underscoring the need for effective subsequent treatments or better initial therapies. Here, we show that triple inhibition with fulvestrant, CDK4/6i and AKT inhibitor (AKTi) durably impairs growth of breast cancer cells, prevents progression and reduces metastasis of tumor xenografts resistant to CDK4/6i-fulvestrant combination or fulvestrant alone. Importantly, switching from combined fulvestrant and CDK4/6i upon resistance to dual combination with AKTi and fulvestrant does not prevent tumor progression. Furthermore, triple combination with AKTi significantly inhibits growth of patient-derived xenografts resistant to combined CDK4/6i and fulvestrant. Finally, high phospho-AKT levels in metastasis of breast cancer patients treated with a combination of CDK4/6i and endocrine therapy correlates with shorter progression-free survival. Our findings support the clinical development of ER, CDK4/6 and AKT co-targeting strategies following progression on CDK4/6i and endocrine therapy combination, and in tumors exhibiting high phospho-AKT levels, which are associated with worse clinical outcome.

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.

CDK4/6 signaling pathway and its targeted therapeutic agents in cancer therapy: a review

The development and progression of most cancers have been well recognized as the result of highly activated cell cycle. Cyclin dependent kinase 4/6 plays important roles not only in mitosis, but also in multiple biological processes that contribute to cancer development, such as aging, apoptosis and histone modification. Three FDA approved CDK4/6 inhibitors, Palbociclib, Ribociclib and Abemaciclib, have been used as targeted cancer therapeutic agents to benefit patients with endocrine therapy-resistant breast cancer and other types of cancer, prolonging their survival. However, the clinical application of these inhibitors also leads to acquired drug resistance and other problems. This paper reviews the regulatory roles of CDK4/6, the application of CDK4/6 inhibitors in cancer and the challenge of drug resistance.

Growth Inhibitory Efficacy of Tabebuia Avellanedae in a Model for Triple Negative Breast Cancer

Background: Tabebuia avellanedae (TA) is a tree that is indigenous to the Amazon rainforest. The experiments in the present study were designed to examine the inhibitory effects of TA, and to identify mechanistic targets for its efficacy in the estrogen-α receptor (ER-α), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) negative MDA-MB-231 model for triple negative breast cancer (TNBC). Methods: Non-fractionated aqueous extract from the inner bark of TA was used in the experiments. Anchorage dependent growth, anchorage independent (AI) colony formation, cell cycle progression, and expressions of relevant regulatory proteins represented quantitative end points. Results: Long-term treatment for 21 days with the maximum cytostatic concentration of 2.5% TA resulted in a 90% inhibition (P=0.014) in AI colony number. Short-term treatment for 2 days with 1.0% TA (IC50) resulted in about a 1.3 fold increase (P=0.014) in G1: S+G2/M ratio, about a 1.48 fold increase (P=0.010) in the sub G0 (apoptotic) cells and about a 3.2 fold increase (P=0.014) in the pro-apoptotic caspase 3/7 activity. Mechanistically, the short-term treatment with 2.5% TA decreased Cyclin D1 expression by about 83.3%, and pRB expression by about 73.3%. Conclusion: TNBC represents an aggressive cancer notable for its resistance to conventional and targeted therapy. Non-toxic natural substances may represent testable alternatives. This study identifies potential mechanistic leads for TA as a novel naturally occurring testable alternative for secondary prevention/therapy of TNBC, and validates a novel mechanistic approach to evaluate efficacious non-toxic phytochemicals and herbs as testable alternatives against therapy resistant breast cancer.

The synergy between miR-486–5p and tamoxifen causes profound cell death of tamoxifen-resistant breast cancer cells

Breast cancer (BC) is the most common type of malignancy in women. A subset of breast cancers show resistance to endocrine-based therapies. The estrogen receptor (ER) plays a critical role in developing hormone-dependent BC. Loss of ER contributes to resistance to tamoxifen therapy and may contribute to mortality. Thus, it is crucial to overcome this problem. Here, using luciferase reporter assays, qRT-PCR, and Western blot analyses, we demonstrate that the microRNA miR-486–5p targets HMGA1 mRNA, decreasing its mRNA and protein levels in ER-positive (ER+) BC cells. Consistently, miR-486–5p is significantly downregulated, whereas HMGA1 is considerably upregulated in ER+ BC samples. Remarkably, while both miR-486–5p and tamoxifen individually cause G2/M cell cycle arrest, combination treatment synergistically causes profound cell death, specifically in tamoxifen-resistant ER+ cells but not in tamoxifen-sensitive ER+ cells. Combined treatment with miR-486–5p and tamoxifen also additively reduces cell migration, invasion, colony formation, mammary spheroid formation and a CD24-CD44+ cell population, representing decreased cancer stemness. However, these phenomena are independent of the tamoxifen responsiveness of the ER+ BC cells. Thus, miR-486–5p and tamoxifen exhibit additive and synergistic tumor-suppressive effects, most importantly causing profound cell death specifically in tamoxifen-resistant BC cells. Therefore, our work suggests that combining miR-486–5p replacement therapy with tamoxifen treatment is a promising strategy to treat endocrine therapy-resistant BC.

Abstract 743: Altered ER and PR transcription factor activity associated with endocrine therapy resistance in breast cancer

Abstract Half of estrogen receptor (ERα)-positive breast cancer patients treated with endocrine therapies display intrinsic or acquired therapy resistance. One-third of patients with acquired resistance present with metastatic tumors containing ERα-Y537S mutations. This mutation results in constitutive activity of ERα and altered ERα-associated gene expression. Previous research suggests that ERα and the progesterone receptor (PR) engage in complex interactions involving reciprocal regulation of ERα and PR transcription factor activity known as ERα/PR crosstalk. My central hypothesis is that ERα-Y537S alters the transcription factor activity of ERα and PR, causing dysregulation of gene expression in SERM-resistant breast cancer. While previous research has assessed the impact of the ERα-Y537S mutation on ERα-associated chromatin binding and gene expression, here I assess the effect of the resistance-associated ERα-Y537S mutation on ERα and PR activity and crosstalk. Simultaneous analysis of transcription factor binding activity (ChIP-seq), gene expression (RNA-seq), and proximity-based interaction (NanoBRET) indicates that active PR binding across the genome is increased in the context of ERα-Y537S, suggesting that ERα/PR crosstalk is significantly altered. Importantly, PR chromatin binding in the context of ERα-Y537S appears to be elevated regardless of the stimulation of PR activity with the agonist R5020. These findings, along with identification of candidate genes of potential therapeutic value, will be applied using patient-derived tumor organoids to determine the efficacy of simultaneously targeting ERα and PR to overcome endocrine therapy resistance in breast cancer. Citation Format: Rosemary J. Huggins. Altered ER and PR transcription factor activity associated with endocrine therapy resistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 743.

Abstract LB078: Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) inhibits hormone therapy resistant breast cancer cells, in part by Inhibiting CYP3A4 arachidonic acid epoxygenase activity

Abstract Introduction: Small molecule therapeutics of estrogen receptor-positive/HER2-negative breast cancer remains an area of active investigation where novel agents are greatly needed for treatment of hormone therapy resistant metastatic disease. The biguanide hexyl-benzyl-biguanide (HBB) is a potent inhibitor of CYP3A4 arachidonic acid (AA) epoxygenase activity and inhibits breast cancer cell proliferation and MCF-7 breast cancer tumor growth in nude mice. To explore the impact of bioisosteric substitution of the benzyl moiety of HBB with a cubane moiety, we synthesized hexyl-(cuban-1-yl-methyl)-biguanide (HCB) and tested its potency for the inhibition of the cognate CYP3A4 target AA epoxygenase activity as well as breast cancer cell proliferation of hormone therapy sensitive and resistant cell lines. Results: HCB selectively inhibited CYP3A4-mediated biosynthesis of (±)-14,15-EET with an IC50 of 4.7±0.2 uM vs. 64.8±6.5 uM for 8,9-EET and 26.5±1.9 uM for 11,12-EET. At 24 hours, HCB inhibited proliferation of MCF-7 (ER+HER2-), BT474 (ER+HER2+) and MDA-MB-231 (ER-HER2-) cells at IC50 of 8.4±1.2, 11±1.3 and 15±0.9 uM, respectively. At 48 hours, HCB inhibited proliferation of aromatase inhibitor and fulvestrant resistant (LR,FR), and cyclin dependent kinase inhibitor (CDKi) palbociclib resistant (LR,FR,PR) MCF-7 cell lines; LR,FR MCF-7AC1 (IC50 =1.34±0.1 uM) and LR,FR,PR MCF-7AC1 (IC50 =1.64±0.2 uM). Addition of 14,15-EET (1 uM) partially rescues MCF-7 cells from HCB-mediated inhibition of proliferation. OXPHOS is promoted, in part, by EETs. HCB is a potent OXPHOS inhibitor and rapidly inhibits O2 consumption of the MCF-7 and ZR75 (ER+HER2-) cells in a dose-dependent fashion (P&amp;lt;0.05). HCB treatment (10 uM) reduces mitochondrial membrane potential to 57.4±15.3% (P&amp;lt;0.001) of vehicle control in MCF-7 cells. Treatment with HCB at 20 uM for 0.5 hour also causes mitochondrial swelling in MCF-7 cells. HCB (10 uM) activates AMPK within 0.5 hour and increases the level of phosphorylation from 2.4±0.3 to 25.1±6.0 folds in a time dependent fashion in MCF-7 cells from 0.5-24 hours. Conclusion: These results show that HCB inhibits proliferation of ER+HER2- breast cancer cells, in part through inhibition of OXPHOS and suppression of the CYP product 14,15-EET. This inhibition is highly active in hormonal therapy and CDKi resistant ER+HER2- breast cancer cells. These results suggest that HCB is a novel and potent biguanide that has potential to be developed for inhibition of hormone therapy resistant and CDKi resistant breast cancer. Citation Format: Zhijun Guo, Jianxun Lei, Kwon Ho Hong, Beverly Norris, Craig M. Flory, Swaathi Jayaraman, Connor McDermott, Elizabeth Ambrose, Irina Sevrioukova, Tom Poulos, Ilia Denisov, Stephen Sliga, Robert J. Schumacher, Gunda I. Georg, John R. Hawse, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) inhibits hormone therapy resistant breast cancer cells, in part by Inhibiting CYP3A4 arachidonic acid epoxygenase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB078.

Abstract 676: Fulvestrant and radiation modify the tumor immune microenvironment in ER+ metastatic breast cancer and cooperate to enhance response to anti-PDL1 checkpoint blockade

Abstract Metastatic, hormone therapy-resistant breast cancers expressing estrogen receptor (ER+) account for most breast cancer deaths. A lack of tumor-infiltrating lymphocytes (TILs) and low tumor mutation burden render most ER+ breast cancers immunologically “cold” and unresponsive to immune checkpoint inhibitors (ICI). Radiation therapy (RT) can augment tumor immune susceptibility, but RT may also increase immunosuppressive cell recruitment to the tumor. Recent studies demonstrate that ER inhibition may antagonize the trafficking and activation of myeloid derived suppressive cells (MDSCs) in tumors. We hypothesized that combining RT and a selective ER degrader (SERD), fulvestrant, would cooperate to relieve immunosuppression, increase tumor immune susceptibility, and facilitate response to ICI in a syngeneic murine model of ER+ breast cancer. Using a cell line (TC11) developed in the Schuler laboratory, cells were transplanted to caudal mammary fat pads of female FVB/N mice. When tumors reached ~200mm3, mice were treated with combinations of vehicle, fulvestrant (250mg/kg sc, weekly), 8Gy RT on 3 consecutive days (8Gy x 3, days -1, 0, 1), and/or anti-PDL1 (200µg, IP, at days 3, 6, and 9). Age-matched controls that received identical treatments were included. Tumors from these mice were collected at day 28, and cytokines in tumor lysates were evaluated using a multiplex immunoassay. In a similar experiment, tumors were collected at day 10 for qPCR and flow cytometry analysis. Therapeutic intervention demonstrated that fulvestrant with RT slowed tumor growth compared to either treatment alone. 8Gy RT elicited a type-1 interferon response in TC11 tumors as measured by upregulated expression of Mx1, Pdl1, Oas2, Oas3, Trex1, and IfnB mRNAs peaking at 3-5 days post RT and persisting to day 10. Cytokine analysis from tumor lysate revealed significantly increased IP-10 in tumors from mice treated with both RT and fulvestrant compared to controls, and similar trends were seen for IL6, VEGF, and MCSF. With the addition of RT and fulvestrant to anti-PDL1, IL2, IL3, IL6, IL7, IL17, and IP-10 were significantly increased compared to anti-PDL1 alone. At day 28 RT and fulvestrant together primed a greater response to anti-PDL1. Combined RT and fulvestrant may reduce immunosuppression compared to RT alone and render this immunologically cold tumor model more responsive to anti-PDL1 blockade. RT increases tumor cell immune susceptibility by activation of a type I IFN response and combining fulvestrant with RT does not affect this IFN1 response. These results demonstrate a cooperative interaction between RT and fulvestrant in favorably modulating the TME and response to ICIs in an immunologically cold, hormone-therapy resistant, murine ER+ breast cancer model. Further experiments are needed to explore the mechanisms of these immune effects. Citation Format: Erin J. Nystuen, Amber M. Bates, Kathleen A. O'Leary, Sarah E. Emma, Elizabeth G. Sumiec, Linda A. Schuler, Zachary S. Morris. Fulvestrant and radiation modify the tumor immune microenvironment in ER+ metastatic breast cancer and cooperate to enhance response to anti-PDL1 checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 676.

Rac inhibition as a novel therapeutic strategy for EGFR/HER2 targeted therapy resistant breast cancer

BackgroundEven though targeted therapies are available for cancers expressing oncogenic epidermal growth receptor (EGFR) and (or) human EGFR2 (HER2), acquired or intrinsic resistance often confounds therapy success. Common mechanisms of therapy resistance involve activating receptor point mutations and (or) upregulation of signaling downstream of EGFR/HER2 to Akt and (or) mitogen activated protein kinase (MAPK) pathways. However, additional pathways of resistance may exist thus, confounding successful therapy.MethodsTo determine novel mechanisms of EGFR/HER2 therapy resistance in breast cancer, gefitinib or lapatinib resistant variants were created from SKBR3 breast cancer cells. Syngenic therapy sensitive and resistant SKBR3 variants were characterized for mechanisms of resistance by mammosphere assays, viability assays, and western blotting for total and phospho proteins.ResultsGefitinib and lapatinib treatments reduced mammosphere formation in the sensitive cells, but not in the therapy resistant variants, indicating enhanced mesenchymal and cancer stem cell-like characteristics in therapy resistant cells. The therapy resistant variants did not show significant changes in known therapy resistant pathways of AKT and MAPK activities downstream of EGFR/HER2. However, these cells exhibited elevated expression and activation of the small GTPase Rac, which is a pivotal intermediate of GFR signaling in EMT and metastasis. Therefore, the potential of the Rac inhibitors EHop-016 and MBQ-167 to overcome therapy resistance was tested, and found to inhibit viability and induce apoptosis of therapy resistant cells.ConclusionsRac inhibition may represent a viable strategy for treatment of EGFR/HER2 targeted therapy resistant breast cancer.

Sodium/glucose cotransporter 1-dependent metabolic alterations induce tamoxifen resistance in breast cancer by promoting macrophage M2 polarization

Endocrine therapy is the standard treatment for estrogen receptor (ER)-positive breast cancer, but tumors eventually develop resistance. However, endocrine therapy resistance mechanisms mediated through interactions between breast cancer cells and tumor-associated macrophages (TAMs) are still unclear. Here, we characterized sodium/glucose cotransporter 1 (SGLT1) overexpression drives the highly glycolytic phenotype of tamoxifen-resistant breast cancer cells where enhanced lactic acid secretion promotes M2-like TAM polarization via the hypoxia-inducible factor-1α/signal transducer and activator of transcription-3 pathway. In turn, M2-like TAMs activate breast cancer cells through EGFR/PI3K/Akt signaling, providing feedback to upregulate SGLT1 and promote tamoxifen resistance and accelerate tumor growth in vitro and in vivo. Higher expression of SGLT1 and CD163+ TAMs was associated with endocrine-resistant ER-positive breast cancers. Our study identifies a novel vicious cycle of metabolic reprogramming, M2-like TAM polarization, and endocrine therapy resistance, which involves SGLT1, proposing SGLT1 as a therapeutic target to overcome endocrine therapy resistance in breast cancer.

Mechanisms of endocrine therapy resistance in breast cancer

The most commonly diagnosed breast cancer (BC) subtype is characterized by estrogen receptor (ER) expression. Treatment of this BC subtype typically involves modalities that either suppress the production of estrogen or impede the binding of estrgen to its receptors, constituting the basis for endocrine therapy. While many patients have benefitted from endocrine therapy with clear reduction in mortality and cancer recurrence, one of the clinical hurdles that remain involves overcoming intrinsic (de novo) or acquired resistance to endocrine therapy driven by diverse and complex changes occurring in the tumor microenvironment. Moreover, such resistance may persist even after progression through additional antiestrogen therapies thus demonstrating the importance of further investigation of mechanisms of ER modulation. Here, we discuss a number of advances that provide a better understanding of the complex mechanistic basis for resistance to endocrine therapy as well as future therapeutic maneuvers that may break this resistance.

Lasofoxifene as a potential treatment for therapy-resistant ER-positive metastatic breast cancer

BackgroundEndocrine therapy remains the mainstay of treatment for estrogen receptor-positive (ER+) breast cancer. Constitutively active mutations in the ligand binding domain of ERα render tumors resistant to endocrine agents. Breast cancers with the two most common ERα mutations, Y537S and D538G, have low sensitivity to fulvestrant inhibition, a typical second-line endocrine therapy. Lasofoxifene is a selective estrogen receptor modulator with benefits on bone health and breast cancer prevention potential. This study investigated the anti-tumor activity of lasofoxifene in breast cancer xenografts expressing Y537S and D538G ERα mutants. The combination of lasofoxifene with palbociclib, a CDK4/6 inhibitor, was also evaluated.MethodsLuciferase-GFP tagged MCF7 cells bearing wild-type, Y537S, or D538G ERα were injected into the mammary ducts of NSG mice (MIND model), which were subsequently treated with lasofoxifene or fulvestrant as single agents or in combination with palbociclib. Tumor growth and metastasis were monitored with in vivo and ex vivo luminescence imaging, terminal tumor weight measurements, and histological analysis.ResultsAs a monotherapy, lasofoxifene was more effective than fulvestrant at inhibiting primary tumor growth and reducing metastases. Adding palbociclib improved the effectiveness of both lasofoxifene and fulvestrant for tumor suppression and metastasis prevention at four distal sites (lung, liver, bone, and brain), with the combination of lasofoxifene/palbociclib being generally more potent than that of fulvestrant/palbociclib. X-ray crystallography of the ERα ligand binding domain (LBD) shows that lasofoxifene stabilizes an antagonist conformation of both wild-type and Y537S LBD. The ability of lasofoxifene to promote an antagonist conformation of Y537S, combined with its long half-life and bioavailability, likely contributes to the observed potent inhibition of primary tumor growth and metastasis of MCF7 Y537S cells.ConclusionsWe report for the first time the anti-tumor activity of lasofoxifene in mouse models of endocrine therapy-resistant breast cancer. The results demonstrate the potential of using lasofoxifene as an effective therapy for women with advanced or metastatic ER+ breast cancers expressing the most common constitutively active ERα mutations.

'γδT Cell-IL-17A-Neutrophil' Axis Drives Immunosuppression and Confers Breast Cancer Resistance to High-Dose Anti-VEGFR2 Therapy

Background: Angiogenesis is an essential physiological process and hallmark of cancer. Currently, antiangiogenic therapy, mostly targeting the VEGF/VEGFR2 signaling axis, is commonly used in the clinic for solid tumors. However, antiangiogenic therapies for breast cancer patients have produced limited survival benefits since cancer cells rapidly acquire resistance to anti-VEGFR2 therapy. Methods: We tested the low-dose and high-dose VEGFR2 mAb or VEGFR2-tyrosine kinase inhibitor (TKI) agents in 4T1-, EMT6-tumor bearing and MMTV-PyMT breast cancer mouse models. Flow cytometric, western blot and tissue immunofluorescence stain were used to elucidate the role and regulatory mechanism of immune cells under the anti-VEGFR2 therapy. Findings: Low-dose VEGFR2 mAb or VEGFR2-TKI achieved good effects in controlling cancer progression in multiple breast cancer mouse models, while high dose treatment was not effective. To further investigate the mechanism involved in regulating this resistance, we found that high-dose anti-VEGFR2 treatment elicited IL-17A expression in γδ T cells via VEGFR1-PI3K-AKT pathway activation and then promoted N2-like neutrophil polarization, thus inducing CD8+ T cell exhaustion to shape an immunosuppressive microenvironment. Combining anti-VEGFR2 therapy with IL-17, PD-1 and Ly-6G mAb targeting the immunomodulatory axis of “γδT17 cells-N2 neutrophils” in vivo showed promising therapeutic effects in breast cancer treatment. Interpretation: Taken together, this study illustrates the potential mechanism of antiangiogenic therapy resistance in breast cancer and provides combinational treatment options for cancer therapy. Funding Information: This work was supported by grants from the National Natural Science Foundation of China (81930079,81872317,81902981,81902626). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: All mouse protocols and procedures were reviewed and approved by the Ethics Review Committee of the Second Affiliated Hospital of the Zhejiang University School of Medicine.

10P The clinical actionability of PTEN protein and gene expression analysis in HR- and HER2+ breast cancers

Phosphatase and tensin homologue (PTEN) loss is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker requires further studies. We seek to identify clinically relevant subtypes of breast cancer based on PTEN status and other clinicopathologic features.