Breast Cancer Therapeutic Resistance Research Articles

Abstract PO5-24-05: The Metabolic and Epigenetic Contributions of Fibroblast Growth Factor Receptor-Mediated Breast Cancer Progression

Abstract Despite recent improvements in treatment options, breast cancer remains the second leading cause of cancer-related deaths among women, with metastasis of the primary tumor accounting for most of these deaths. Signaling by fibroblast growth factor receptors (FGFRs) is active in 85% of breast cancers and results in enhanced proliferation, migration, invasion, and therapeutic resistance of tumor cells. A common feature of breast cancer is altered cholesterol metabolism including an increase in cholesterol stored intracellularly as cholesteryl esters (CEs). In breast cancer cells, high levels of CEs are associated with aggressive disease and CEs are essential for growth and migration of cancer cells. Furthermore, there is mounting evidence that modifications to metabolic pathways impact epigenetic processes including DNA methylation and histone post-translational modifications. These epigenetic changes support tumor progression and metastasis through oncogene activation and silencing of tumor suppressors. While there have been numerous studies focused on the role of metabolic and epigenetic changes in breast cancer, none have attempted to connect these altered pathways to upstream growth factor receptor pathways, such as the FGFR pathway, in cancer cells. We hypothesize that FGFR signaling promotes changes to cholesterol metabolism and epigenetic regulation which lead to breast cancer progression. To study the proposed roles of FGFR signaling, we utilized murine mammary cells of differing metastatic abilities with inducible, oncogenic FGFR1 signaling. We also included the FGFR-dependent murine mammary tumor lines 4T1 and 4T07 which demonstrate differing metastatic capabilities. Through gene expression studies, we found an FGFR-mediated increase in cholesterol metabolic processes including cholesterol storage, uptake, and synthesis. Along with this, we found an FGFR-mediated increase in CE content in our models. Interestingly, these changes were exclusive to our highly metastatic models. Additionally, we have identified FGFR-mediated changes in the expression of histone modifying enzymes in our cell lines. Many of the enzymes modified by FGFR have been previously reported to play a role in breast cancer metastasis. To further elucidate the role of enhanced cholesterol storage in breast cancer progression, we treated cells with pharmacological inhibitors of cholesterol storage. Using in vitro assays, we demonstrate that cell survival, migration, and invasion all decrease when cholesterol storage is inhibited. Again, we observe that our highly metastatic cell line models are more sensitive to this inhibition. Taken together, these data suggest a role for FGFR signaling in promoting breast cancer progression and metastasis. With further studies, this connection may inform the development of novel therapeutic strategies to combat therapeutic resistance in breast cancer. Citation Format: Jennifer Tuokkola, Lyndsay Reese, Kaylee Schwertfeger. The Metabolic and Epigenetic Contributions of Fibroblast Growth Factor Receptor-Mediated Breast Cancer Progression [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 PO5-24-05.

Analysis of miRNAs: Biomarkers for HER2-Targeted Breast Cancer Therapy

Breast cancer remains a leading lethal cancer in women globally, with the HER2+ positive subtype associated with a higher chance of therapeutic resistance. Treatments tailored specifically to this subtype, such as targeted therapies, have significantly developed over the past decade. However, the issue of drug resistance to targeted drugs underlines the necessity of better treatment approaches. Recent studies have focused on microRNAs (miRNAs), single-stranded non-coding RNAs, as critical regulators in drug resistance mechanisms. These miRNAs, capable of influencing various cellular processes, have emerged as critical modulators in numerous diseases, including different cancer types, particularly breast cancer. This article reviews current methodologies in the study of miRNAs within the context of HER2-positive breast cancer, from the selection of study models and sample extraction to comprehensive analysis methods. Our objective is to highlight the potential that miRNAs hold as biomarkers capable of regulating drug resistance in this specific cancer subtype. Moreover, we also discuss the importance of integrating advanced study models alongside the latest bioinformatics tools to enrich this research domain. Furthermore, this article evaluates the use of clinical samples and cell line models for studying miRNAs in this field, outlining the advantages and limitations of each method and proposing a refined approach to research design. The main contribution of this work is the establishment of a detailed taxonomy of research strategies that address current challenges while also outlining promising future directions, particularly focused on elucidating the regulatory mechanisms of miRNAs in therapeutic resistance in breast cancer. In addition, by underscoring the necessity of employing a diverse array of study models and capitalizing on bioinformatics advancements, this article seeks to uncover the complex interactions between miRNAs and drug resistance mechanisms in breast cancer. Ultimately, our goal is to pave the way toward overcoming therapeutic resistance, thereby improving the prognosis for patients afflicted with HER2-positive breast cancer.

Single-cell morphological and transcriptome analysis unveil inhibitors of polyploid giant breast cancer cells in vitro

Considerable evidence suggests that breast cancer therapeutic resistance and relapse can be driven by polyploid giant cancer cells (PGCCs). The number of PGCCs increases with the stages of disease and therapeutic stress. Given the importance of PGCCs, it remains challenging to eradicate them. To discover effective anti-PGCC compounds, there is an unmet need to rapidly distinguish compounds that kill non-PGCCs, PGCCs, or both. Here, we establish a single-cell morphological analysis pipeline with a high throughput and great precision to characterize dynamics of individual cells. In this manner, we screen a library to identify promising compounds that inhibit all cancer cells or only PGCCs (e.g., regulators of HDAC, proteasome, and ferroptosis). Additionally, we perform scRNA-Seq to reveal altered cell cycle, metabolism, and ferroptosis sensitivity in breast PGCCs. The combination of single-cell morphological and molecular investigation reveals promising anti-PGCC strategies for breast cancer treatment and other malignancies.

Downregulation of TAB182 promotes cancer stem-like cell properties and therapeutic resistance in triple-negative breast cancer cells

TAB182 participates in DNA damage repair and radio-/chemosensitivity regulation in various tumors, but its role in tumorigenesis and therapeutic resistance in breast cancer remains unclear. In the current paper, we observed that triple-negative Breast Cancer (TNBC), a highly aggressive type of breast cancer, exhibits a lower expression of TAB182. TAB182 knockdown stimulates the proliferation, migration, and invasion of TNBC cells. Our study first obtained RNA-seq data to explore the cellular functions mediated by TAB182 at the genome level in TNBC cells. A transcriptome analysis and in vitro experiments enabled us to identify that TAB182 downregulation drives the enhanced properties of cancer stem-like cells (CSCs) in TNBC cells. Furthermore, TAB182 deletion contributes to the resistance of cells to olaparib or cisplatin, which can be rescued by silencing GLI2, a gene downstream of cancer stemness-related signaling pathways. Our results reveal a novel function of TAB182 as a potential negative regulator of cancer stem-like properties and drug sensitivity in TNBC cells, suggesting that TAB182 may be a tumor suppressor gene and is associated with increased therapeutic benefits for TNBC patients.

A novel signature based on cancer-associated fibroblast genes to predict prognosis, immune feature, and therapeutic response in breast cancer.

Breast cancer (BC) ranks first in the incidence of tumors in women and remains the most prevalent malignancy in women worldwide. Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) profoundly influence the progression, recurrence, and therapeutic resistance in BC. Here, we intended to establish a risk signature based on screened CAF-associated genes in BC (BCCGs) for patient stratification. Initially, BCCGs were screened by a combination of several CAF gene sets. The identified BCGGs were found to differ significantly in the overall survival (OS) of BC patients. Accordingly, we constructed a prognostic prediction signature of 5 BCCGs, which were independent prognostic factors associated with BC based on univariate and multivariate Cox regression. The risk model divided patients into low- and high-risk groups, accompanied by different OS, clinical features, and immune infiltration characteristics. Receiver operating characteristic (ROC) curves and a nomogram further validated the predictive performance of the prognostic model. Notably, 21 anticancer agents targeting these BCCGs possessed better sensitivity in BC patients. Meanwhile, the elevated expression of the majority of immune checkpoint genes suggested that the high-risk group may benefit more from immune checkpoint inhibitors (ICIs) therapy. Taken together, our well-established model is a robust instrument to precisely and comprehensively predict the prognosis, immune features, and drug sensitivity in BC patients, for combating BC.

The Role of Mesenchymal Stem Cells in Modulating the Breast Cancer Microenvironment.

The role of mesenchymal stem cells (MSCs) in the breast tumor microenvironment (TME) is significant and multifaceted. MSCs are recruited to breast tumor sites through molecular signals released by tumor sites. Once in the TME, MSCs undergo polarization and interact with various cell populations, including immune cells, cancer-associated fibroblasts (CAFs), cancer stem cells (CSCs), and breast cancer cells. In most cases, MSCs play roles in breast cancer therapeutic resistance, but there is also evidence that indicates their abilities to sensitize cancer cells to chemotherapy and radiotherapy. MSCs possess inherent regenerative and homing properties, making them attractive candidates for cell-based therapies. Therefore, MSCs can be engineered to express therapeutic molecules or deliver anti-cancer agents directly to tumor sites. Unraveling the intricate relationship between MSCs and the breast TME has the potential to uncover novel therapeutic targets and advance our understanding of breast cancer biology.

Metabolomic and Mitochondrial Fingerprinting of the Epithelial-to-Mesenchymal Transition (EMT) in Non-Tumorigenic and Tumorigenic Human Breast Cells.

Epithelial-to-mesenchymal transition (EMT) is key to tumor aggressiveness, therapy resistance, and immune escape in breast cancer. Because metabolic traits might be involved along the EMT continuum, we investigated whether human breast epithelial cells engineered to stably acquire a mesenchymal phenotype in non-tumorigenic and H-RasV12-driven tumorigenic backgrounds possess unique metabolic fingerprints. We profiled mitochondrial-cytosolic bioenergetic and one-carbon (1C) metabolites by metabolomic analysis, and then questioned the utilization of different mitochondrial substrates by EMT mitochondria and their sensitivity to mitochondria-centered inhibitors. "Upper" and "lower" glycolysis were the preferred glucose fluxes activated by EMT in non-tumorigenic and tumorigenic backgrounds, respectively. EMT in non-tumorigenic and tumorigenic backgrounds could be distinguished by the differential contribution of the homocysteine-methionine 1C cycle to the transsulfuration pathway. Both non-tumorigenic and tumorigenic EMT-activated cells showed elevated mitochondrial utilization of glycolysis end-products such as lactic acid, β-oxidation substrates including palmitoyl-carnitine, and tricarboxylic acid pathway substrates such as succinic acid. Notably, mitochondria in tumorigenic EMT cells distinctively exhibited a significant alteration in the electron flow intensity from succinate to mitochondrial complex III as they were highly refractory to the inhibitory effects of antimycin A and myxothiazol. Our results show that the bioenergetic/1C metabolic signature, the utilization rates of preferred mitochondrial substrates, and sensitivity to mitochondrial drugs significantly differs upon execution of EMT in non-tumorigenic and tumorigenic backgrounds, which could help to resolve the relationship between EMT, malignancy, and therapeutic resistance in breast cancer.

PSME2 identifies immune-hot tumors in breast cancer and associates with well therapeutic response to immunotherapy.

Breast cancer (BrCa) is a heterogeneous disease, which leads to unsatisfactory prognosis in females worldwide. Previous studies have proved that tumor immune microenvironment (TIME) plays crucial roles in oncogenesis, progression, and therapeutic resistance in Breast cancer. However, biomarkers related to TIME features have not been fully discovered. Proteasome activator complex subunit 2 (PSME2) is a member of proteasome activator subunit gene family, which is critical to protein degradation mediated by the proteasome. In the current research, we comprehensively analyzed the expression and immuno-correlations of Proteasome activator complex subunit 2 in Breast cancer. Proteasome activator complex subunit 2 was significantly upregulated in tumor tissues but associated with well prognosis. In addition, Proteasome activator complex subunit 2 was overexpressed in HER2-positive Breast cancer but not related to other clinicopathological features. Interestingly, Proteasome activator complex subunit 2 was positively related to immune-related processes and identified immuno-hot TIME in Breast cancer. Specifically, Proteasome activator complex subunit 2 was positively correlated with immunomodulators, tumor-infiltrating immune cells (TIICs), immune checkpoints, and tumor mutation burden (TMB) levels. Moreover, the positive correlation between Proteasome activator complex subunit 2 and PD-L1 expression was confirmed in a tissue microarray (TMA) cohort. Furthermore, in an immunotherapy cohort of Breast cancer, patients with pathological complete response (pCR) expressed higher Proteasome activator complex subunit 2 compared with those with non-pathological complete response. In conclusion, Proteasome activator complex subunit 2 is upregulated in tumor tissues and correlated with the immuno-hot tumor immune microenvironment, which can be a novel biomarker for the recognition of tumor immune microenvironment features and immunotherapeutic response in Breast cancer.

The tumor microenvironment as driver of stemness and therapeutic resistance in breast cancer: New challenges and therapeutic opportunities

Breast cancer (BC), the second most common cause of cancer-related deaths, remains a significant threat to the health and wellness of women worldwide. The tumor microenvironment (TME), comprising cellular components, such as cancer-associated fibroblasts (CAFs), immune cells, endothelial cells and adipocytes, and noncellular components such as extracellular matrix (ECM), has been recognized as a critical contributor to the development and progression of BC. The interplay between TME components and cancer cells promotes phenotypic heterogeneity, cell plasticity and cancer cell stemness that impart tumor dormancy, enhanced invasion and metastasis, and the development of therapeutic resistance. While most previous studies have focused on targeting cancer cells with a dismal prognosis, novel therapies targeting stromal components are currently being evaluated in preclinical and clinical studies, and are already showing improved efficacies. As such, they may offer better means to eliminate the disease effectively. In this review, we focus on the evolving concept of the TME as a key player regulating tumor growth, metastasis, stemness, and the development of therapeutic resistance. Despite significant advances over the last decade, several clinical trials focusing on the TME have failed to demonstrate promising effectiveness in cancer patients. To expedite clinical efficacy of TME-directed therapies, a deeper understanding ofthe TME is of utmost importance. Secondly, the efficacy of TME-directed therapies when used alone or in combination with chemo- or radiotherapy, and the tumor stage needs to be studied. Likewise, identifying molecular signatures and biomarkers indicating the type of TME will helpin determining precise TME-directed therapies.

E2A Modulates Stemness, Metastasis, and Therapeutic Resistance of Breast Cancer.

Cancer stem cells (CSC) are considered responsible for tumor initiation, therapeutic resistance, and metastasis. A comprehensive knowledge of the mechanisms governing the acquisition and maintenance of cancer stemness is crucial for the development of new therapeutic approaches in oncology. E2A basic helix-loop-helix (bHLH) transcription factors are associated with epithelial-mesenchymal transition (EMT) and tumor progression, but knowledge of their functional contributions to cancer biology is still limited. Using a combination of in vivo and in vitro analyses in a novel PyMT-E2A conditional knockout mouse model and derived primary tumor cell lines, we report here an essential role of E2A in stemness, metastasis, and therapeutic resistance in breast cancer. Targeted deletion of E2A in the mammary gland impaired tumor-initiating ability and dedifferentiation potential and severely compromised metastatic competence of PyMT-driven mammary tumors. Mechanistic studies in PyMT-derived cell lines indicated that E2A actions are mediated by the upregulation of Snai1 transcription. Importantly, high E2A and SNAIL1 expression occurred in aggressive human basal-like breast carcinomas, highlighting the relevance of the E2A-Snail1 axis in metastatic breast cancer. In addition, E2A factors contributed to the maintenance of genomic integrity and resistance to PARP inhibitors in PyMT and human triple-negative breast cancer cells. Collectively, these results support the potential for E2A transcription factors as novel targets worthy of translational consideration in breast cancer. SIGNIFICANCE: These findings identify key functions of E2A factors in breast cancer cell stemness, metastasis, and drug resistance, supporting a therapeutic vulnerability to targeting E2A proteins in breast cancer.

Hormonal Regulation of Semaphorin 7a Promotes Therapeutic Resistance in Breast Cancer

Background: The majority of all breast cancers (BC) are estrogen receptor positive (ER+). While ER-targeting endocrine therapies have improved patient survival, many of these tumors develop drug resistance and recur within 20 years. Therefore, novel targets are needed to predict for recurrence and to treat recurrent ER+BC. Previous reports describe a tumor-promotional role for Semaphorin 7A (SEMA7A) in ER- disease; yet, the role of SEMA7A in ER+ disease is poorly characterized. Hypothesis: SEMA7A promotes cell survival and drug resistance in ER+ BC. Methods: We overexpressed SEMA7A in ER+ BC cells, then used the ER-targeting agents tamoxifen and fulvestrant to test how SEMA7A-expressing cells respond to endocrine therapy. In vitro, we used proliferation and cell survival assays. In vivo, we implanted ER+ BC cells, then treated the animals with fulvestrant to measure how SEMA7A affects tumor growth and metastasis. We also utilized drug resistant cells, which have high endogenous SEMA7A levels, to measure markers of stemness and multi-drug resistance via flow cytometry. Results: We first found that SEMA7A expression correlates with decreased relapse free survival in patients with ER+BC who received endocrine therapy (Kmplotter; p=0.042). We also observe that SEMA7A is hormonally regulated in ER+BC, but its expression does not uniformly decrease with endocrine therapy agents. Instead, long term estrogen deprivation and ER-targeting drug treatments increase SEMA7A expression, likely through the action of other hormone receptors such as the androgen receptor, which also increases with long term estrogen deprivation. Further, in ER+ cell lines, overexpression of SEMA7A promotes in vitro growth in the face of estrogen-deprivation, tamoxifen, or fulvestrant treatments. In vivo, SEMA7A promotes fulvestrant resistance in the primary tumor and induces lung metastases. Finally, we report that pro-survival signaling is a therapeutic vulnerability of ER+SEMA7A+ tumors. Conclusion: These studies describe that SEMA7A promotes drug resistance in ER+ BC. We propose that targeting pro-survival signaling may prove efficacious for treating SEMA7A+ tumors, which are less likely to respond to endocrine therapies.

Advance in metabolism and target therapy in breast cancer stem cells.

Breast cancer, like many other cancers, is believed to be driven by a population of cells that display stem cell properties. Recent studies suggest that cancer stem cells (CSCs) are essential for tumor progression, and tumor relapse is thought to be caused by the presence of these cells. CSC-targeted therapies have also been proposed to overcome therapeutic resistance in breast cancer after the traditional therapies. Additionally, the metabolic properties of cancer cells differ markedly from those of normal cells. The efficacy of metabolic targeted therapy has been shown to enhance anti-cancer treatment or overcome therapeutic resistance of breast cancer cells. Metabolic targeting of breast CSCs (BCSCs) may be a very effective strategy for anti-cancer treatment of breast cancer cells. Thus, in this review, we focus on discussing the studies involving metabolism and targeted therapy in BCSCs.

Triple-negative breast cancer therapeutic resistance: Where is the Achilles' heel?

Triple-negative breast cancer (TNBC) shows a higher response rate to systemic therapy compared with other breast cancer subtypes. However, the tumor differentiation of TNBC is poorer, with an early tendency to metastasis and a higher recurrence rate. Relapsed and metastatic TNBCs usually progress more rapidly, showing strong resistance to chemotherapy and radiotherapy. Due to the lack of combinatorial targeted drugs, alternative treatments fail to improve these patient's prognosis and the quality of life. Finding the Achilles' heel of TNBC is critical for patients with advanced TNBC. Here, we summarize the latest advances in the mechanisms underlying TNBC therapeutic resistance, consider how these mechanisms may affect the development and utilization of TNBC targeted drugs, and discuss the rationale of relevant signals as therapeutic targets. Also, we review the clinical trials registered in ClinicalTrial.gov for TNBC patients, which comprehensively reveals current research and development of novel TNBC targeted drugs and future trends.

P-cadherin induces anoikis-resistance of matrix-detached breast cancer cells by promoting pentose phosphate pathway and decreasing oxidative stress

Successful metastatic spreading relies on cancer cells with stem-like properties, glycolytic metabolism and increased antioxidant protection, allowing them to escape anoikis and to survive in circulation. The expression of P-cadherin, a poor prognostic factor in breast cancer, is associated with hypoxic, glycolytic and acidosis biomarkers. In agreement, P-cadherin-enriched breast cancer cell populations presents a glycolytic and an acid-resistance phenotype.Our aim was to evaluate whether P-cadherin expression controls the glycolytic and oxidative phosphorylation fluxes of matrix-detached breast cancer cells, acting as an antioxidant and enhancing their survival in anchorage-independent conditions. By using matrix-detached breast cancer cells, we concluded that P-cadherin increases glucose-6-phosphate dehydrogenase expression, up-regulating the carbon flux through the pentose phosphate pathway, while inhibiting pyruvate oxidation to acetyl-coA via pyruvate dehydrogenase kinase-4 (PDK-4) activation. Accordingly, P-cadherin expression conferred increased sensitivity to dichloroacetate (DCA), a PDK inhibitor. P-cadherin expression also regulates oxidative stress in matrix-detached breast cancer cells, through the control of antioxidant systems, such as catalase and superoxide dismutases (SOD)1 and 2, providing these cells with an increased resistance to doxorubicin-induced anoikis. Importantly, this association was validated in primary invasive breast carcinomas, where an enrichment of SOD2 was found in P-cadherin-overexpressing breast carcinomas.In conclusion, we propose that P-cadherin up-regulates carbon flux through the pentose phosphate pathway and decreases oxidative stress in matrix-detached breast cancer cells. These metabolic remodeling and antioxidant roles of P-cadherin can promote the survival of breast cancer cells in circulation and in metastatic sites, being a possible player in breast cancer therapeutic resistance to pro-oxidant-based interventions.

Abstract 5467: Outlier analysis to identify determinants of therapeutic resistance in breast cancer

Abstract Tumors are typically characterized by variable responses to targeted therapy driven by the heterogeneity of drivers among tumors. Inter-tumor heterogeneity also results in different resistance determinants to the same therapeutic agent. Outlier analysis has been applied extensively to identify drivers of cancer when comparing a heterogenous population of tumor samples to a relatively homogenous population of normal samples. However, the application of outlier analysis has rarely been applied to identify intrinsic determinants of resistance to targeted cancer therapies. We recently applied outlier analysis to baseline proteomics data collected from HER2/ERBB2 positive breast cancer patients treated with anti-HER2 therapy, comparing protein levels in each patient that did not show pathological complete response (pCR) to the population of patients that did. Three of the five non-pCR patients were outliers for low expression of the ERBB2 protein relative to the pCR patients, suggesting that lack of protein expression of the direct therapeutic target drives resistance in some cases whereas other mechanisms contribute to resistance in other cases. Here, we systematically evaluate the ability of multiple outlier analysis methods, including the method we implemented for the HER2 study (outlieR), to identify molecular genes associated with poor prognosis in estrogen receptor (ESR1) positive patients receiving hormone therapy (targeting ESR1) using a large-scale publicly available dataset which contains baseline gene expression and survival data for ~2000 breast cancer patients (METABRIC). Specifically, we compared expression data from tumors of 72 patients that showed poor prognosis (died of disease within 5 years) to data from 226 tumors from patients with good prognosis (did not die of disease, with at least five years of follow-up time). For this evaluation, we focused on the direct target of hormone therapy, ESR1, and growth factor receptors associated with endocrine therapy resistance. Scores from outlier analysis methods consistently ranked ESR1, EGFR, and genes associated with the ERBB2 locus (amplification of the locus drives ERBB2 gene expression in HER2 positive breast cancer) more highly than the the T-test metric did, with the outlieR method outperforming another established method, Outlier Sums (OS), in most cases. Furthermore, four targets of FDA-approved drugs, including two genes in the MAPK pathway, were amongst the top 200 genes identified by the outlieR method, whereas TOP2A was the only approved target in the top 200 genes identified by the T-test, a target of cytotoxic chemotherapy treatments. Finally, the outlieR method generates outlier scores for each gene in each non-responder relative to the set of non-responders, allowing for the evaluation of genes associated with resistance on a patient-by-patient basis. These observations suggest that outlier analysis can be used to prioritize molecular features as potential mechanisms of resistance and alternative drug targets by accounting for heterogeneity between resistant tumors. Citation Format: Eric James Jaehnig, Meenakshi Anurag, Jonathan T. Lei, Bing Zhang. Outlier analysis to identify determinants of therapeutic resistance 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 5467.

Managing therapeutic resistance in breast cancer: from the lncRNAs perspective.

Breast cancer (BC) is the most common female malignancy and the second leading cause of cancer-related death worldwide. In spite of significant advances in clinical management, the mortality of BC continues to increase due to the frequent occurrence of treatment resistance. Intensive studies have been conducted to elucidate the molecular mechanisms underlying BC therapeutic resistance, including increased drug efflux, altered drug targets, activated bypass signaling pathways, maintenance of cancer stemness, and deregulated immune response. Emerging evidence suggests that long noncoding RNAs (lncRNAs) are intimately involved in BC therapy resistance through multiple modes of action. Therefore, an in-depth understanding of the implication of lncRNAs in resistance to clinical therapies may improve the clinical outcome of BC patients. Here, we highlight the role and underlying mechanisms of lncRNAs in regulating BC treatment resistance with an emphasis on lncRNAs-mediated resistance in different clinical scenarios, and discuss the potential of lncRNAs as novel biomarkers or therapeutic targets to improve BC therapy response.

Abstract 120: Paracrine TGF-β1 induces tamoxifen resistance in cancer-associated fibroblsts by activation of p44/42 MAPK signaling pathway

Abstract Introduction: Biochemical crosstalk between tumor and stroma by several secretory factors has evolved as a significant contributor for the development of therapeutic resistance in breast cancer, where cancer-associated fibroblasts (CAFs) in the tumor stroma play a critical role. However, the role of secretory factors of cancer cells in assisting CAF chemoresistance is least apparent. TGF-β is the major cytokine secreted by the cancer cells to the tumor stroma that further imparts resistance in CAFs to the chemotherapy. Here, we hypothesize that tamoxifen-resistant breast cancer cells stimulate surrounding CAFs towards chemoresistant phenotype through TGF-β1 activated p44/42 MAPK signaling pathway. Methodology: The tamoxifen-resistant (TAM-R) clones of parental MCF-7 cell line were established by treating with sub-lethal doses over several cycles and characterized by MTT assay. Western blot (WB) was performed to quantify the TGF-β1 expression in the parental MCF-7 and the resistant MCF-7 (MCF-7/TAM-R) cells. ELISA was carried out to quantify TGF-β1 in the conditioned media (CM) from MCF-7 and MCF-7/TAM-R. CM from both the MCF-7 and MCF-7/TAM-R was collected and treated to the fibroblasts in dose and time-dependent manner. Initial screening for the expression of CAF markers α-SMA and caveolin-1 was carried out by qPCR and confirmed by WB. Cell cycle analysis was performed by flow cytometry and WB. Scratch assay was performed to evaluate migration and proliferation activity of CAFs. Downstream signaling pathway analysis was carried out by WB. Expression of TGF-β1 and p44/42 was downregulated by appropriate inhibitors. Results: Tamoxifen treatment to the MCF-7 cells showed a marked increase in TGF- β1 expression both in time and dose-dependent manner. α-SMA was upregulated, and Caveolin-1 was downregulated in CAFs after treatment of CM from MCF-7/ TAM-R cells. MTT result showed a higher IC50 value of TAM in CAFs treated with CM compared to untreated one. WB analysis confirmed the elevated anti-apoptotic and reduced pro-apoptotic protein expressions in CAFs post-treatment. Flow cytometry analysis depicts less number of cells in the sub G0/G1 phage after TAM treatment in CM treated CAFs compared to untreated one. WB results showed increased cyclin D1, and cyclin E in CM treated CAFs. Further, inhibiting TGF- β1 in MCF-7/TAM-R cells and p44/42 MAPK signaling cascade in CM treated CAFs found a strong correlation between the elevated expressions of TGF- β1 in MCF-7/TAM-R cells and p44/42 signaling pathway in CM treated CAFs. Conclusion: Here we report that chemoresistant breast cancer cells with increased TGF- β1 expression exhibit a greater potential in inducing TAM resistance in CAFs compared to parental cells, with an up-regulated p44/42 MAPK signaling cascade. The mechanism underscores the p44/42 MAPK signaling pathway as an important therapeutic target in tumor stroma. Note: This abstract was not presented at the meeting. Citation Format: Bikash Chandra Jena, Chandan Kanta Das, Deblina Bharadwaj, Subhayan Das, Indranil Banerjee, Santosh Gupta, Mahitosh Mandal. Paracrine TGF-β1 induces tamoxifen resistance in cancer-associated fibroblsts by activation of p44/42 MAPK signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 120.

JAK/STAT inhibition in macrophages promotes therapeutic resistance by inducing expression of protumorigenic factors

Tumor-associated macrophages contribute to tumor progression and therapeutic resistance in breast cancer. Within the tumor microenvironment, tumor-derived factors activate pathways that modulate macrophage function. Using in vitro and in vivo models, we find that tumor-derived factors induce activation of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in macrophages. We also demonstrate that loss of STAT3 in myeloid cells leads to enhanced mammary tumorigenesis. Further studies show that macrophages contribute to resistance of mammary tumors to the JAK/STAT inhibitor ruxolitinib in vivo and that ruxolitinib-treated macrophages produce soluble factors that promote resistance of tumor cells to JAK inhibition in vitro. Finally, we demonstrate that STAT3 deletion and JAK/STAT inhibition in macrophages increases expression of the protumorigenic factor cyclooxygenase-2 (COX-2), and that COX-2 inhibition enhances responsiveness of tumors to ruxolitinib. These findings define a mechanism through which macrophages promote therapeutic resistance and highlight the importance of understanding the impact of targeted therapies on the tumor microenvironment.

3300 Progesterone receptor alters lipid biology in luminal breast cancer

OBJECTIVES/SPECIFIC AIMS: These studies seek to evaluate hormonal regulation of luminal breast cancer lipid metabolism and to identify targetable progesterone-mediated changes in lipid biology that contribute to therapeutic resistance in breast cancer. METHODS/STUDY POPULATION: Established and patient-derived luminal breast cancer cell lines, which express ER and PR, were used for this study. RNA transcript and protein expression levels were evaluated by qRT-PCR and immunoblot, respectively. Broad scale lipidomics of progesterone-treated cells was conducted via ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) through the UCD Skaggs School of Pharmacy Mass Spectrometry Core. RESULTS/ANTICIPATED RESULTS: Data mining of previously published microarray data of CK5+ and CK5− syngeneic cancer sublines revealed that CK5+ cells have increased expression of lipid processing genes, including LPL and PPARG. As progestin treatment induces a subpopulation of cells to turn on CK5 expression in luminal breast cancers, UHPLC-MS-based lipidomics analysis will expose whether modulation of the lipid landscape occurs in all cells with progesterone treatment, or whether this phenomenon is heightened specifically in CK5+ cells. I also expect that ER+ breast cancers with progestin induced-altered lipid content, such as lipid droplet formation, will evade therapy-induced death. DISCUSSION/SIGNIFICANCE OF IMPACT: There are numerous approved and developmental therapeutics targeting lipid biology. By determining if progestins alter lipid metabolic genes specifically in CK5+ CSCs, which are endocrine resistant, strategies may be devised to target these resistant cells using combination therapy in conjunction with existing therapies to prevent tumor recurrence.

LSD1 activation promotes inducible EMT programs and modulates the tumour microenvironment in breast cancer

Complex regulatory networks control epithelial-to-mesenchymal transition (EMT) but the underlying epigenetic control is poorly understood. Lysine-specific demethylase 1 (LSD1) is a key histone demethylase that alters the epigenetic landscape. Here we explored the role of LSD1 in global epigenetic regulation of EMT, cancer stem cells (CSCs), the tumour microenvironment, and therapeutic resistance in breast cancer. LSD1 induced pan-genomic gene expression in networks implicated in EMT and selectively elicits gene expression programs in CSCs whilst repressing non-CSC programs. LSD1 phosphorylation at serine-111 (LSD1-s111p) by chromatin anchored protein kinase C-theta (PKC-θ), is critical for its demethylase and EMT promoting activity and LSD1-s111p is enriched in chemoresistant cells in vivo. LSD1 couples to PKC-θ on the mesenchymal gene epigenetic template promotes LSD1-mediated gene induction. In vivo, chemotherapy reduced tumour volume, and when combined with an LSD1 inhibitor, abrogated the mesenchymal signature and promoted an innate, M1 macrophage-like tumouricidal immune response. Circulating tumour cells (CTCs) from metastatic breast cancer (MBC) patients were enriched with LSD1 and pharmacological blockade of LSD1 suppressed the mesenchymal and stem-like signature in these patient-derived CTCs. Overall, LSD1 inhibition may serve as a promising epigenetic adjuvant therapy to subvert its pleiotropic roles in breast cancer progression and treatment resistance.