International Union of Basic and Clinical Pharmacology CXIII: Nuclear Receptor Superfamily-Update 2023.

The NR superfamily comprises 48 transcription factors in humans that control a plethora of gene network programs involved in a wide range of physiologic processes. This review will summarize and discuss recent progress in NR biology and drug development derived from integrating various approaches, including biophysical techniques, structural studies, and translational investigation. We also highlight how defective NR signaling results in various diseases and disorders and how NRs can be targeted for therapeutic intervention via modulation via binding to synthetic lipophilic ligands. Furthermore, we also review recent studies that improved our understanding of NR structure and signaling. SIGNIFICANCE STATEMENT: Nuclear receptors (NRs) are ligand-regulated transcription factors that are critical regulators of myriad physiological processes. NRs serve as receptors for an array of drugs, and in this review, we provide an update on recent research into the roles of these drug targets.

A single N6-methyladenosine site regulates lncRNA HOTAIR function in breast cancer cells.

N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A "reader" YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes.

Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment.

BACKGROUND: Alpha-2-glycoprotein 1, zinc-binding (ZAG), a secreted protein encoded by the AZGP1 gene, is structurally similar to HLA class I. Despite its presumed immunological function, little is known about its role in tumor immunity. In this study, we thus aimed to determine the relationship between the expression of AZGP1/ZAG and the immunological profiles of breast cancer tissues at both the gene and protein level. METHODS: Using a publicly available gene expression dataset from a large-scale breast cancer cohort, we conducted gene set enrichment analysis (GSEA) to screen the biological processes associated with AZGP1. We analyzed the correlation between AZGP1 expression and immune cell composition in breast cancer tissues, estimated using CIBERSORTx. Previously, we evaluated the infiltration of 11 types of immune cells for 45 breast cancer tissues using flow cytometry (FCM). ZAG expression was evaluated by immunohistochemistry on these specimens and analyzed for its relationship with immune cell infiltration. The action of ZAG in M1/M2 polarization models using primary cultures of human peripheral blood mononuclear cells (PBMC)-derived macrophage (Mφ) was analyzed based on the expression of M1/M2 markers (CD86, CD80/CD163, MRC1) and HLA class I/II by FCM. RESULTS: AZGP1 expression was negatively correlated with multiple immunological processes and specific immune cell infiltration including Mφ M1 using GSEA and CIBERSORTx. ZAG expression was associated with decreased infiltration of monocytes/macrophages, non-classical monocytes, and myeloid-derived suppressor cells in tumor tissues assessed using FCM. In in vitro analyses, ZAG decreased the expression of CD80, CD163, MRC1, and HLA classes I/II in the M1 polarization model and the expression of CD163 and MRC1 in the M2 polarization model. CONCLUSION: ZAG is suggested to be a novel immunoregulatory factor affecting the Mφ phenotype in breast cancer tissues.

Breast cancers co-opt normal mechanisms of tolerance to promote immune evasion and metastasis.

Normal developmental processes, such as those seen during embryonic development and postpartum mammary gland involution, can be reactivated by cancer cells to promote immune suppression, tumor growth, and metastatic spread. In mammalian embryos, paternal-derived antigens are at risk of being recognized as foreign by the maternal immune system. Suppression of the maternal immune response toward the fetus, which is mediated in part by the trophoblast, is critical to ensure embryonic survival and development. The postpartum mammary microenvironment also exhibits immunosuppressive mechanisms accompanying the massive cell death and tissue remodeling that occurs during mammary gland involution. These normal immunosuppressive mechanisms are paralleled during malignant transformation, where tumors can develop neoantigens that may be recognized as foreign by the immune system. To circumvent this, tumors can dedifferentiate and co-opt immune-suppressive mechanisms normally utilized during fetal tolerance and postpartum mammary involution. In this review, we discuss those similarities and how they can inform our understanding of cancer progression and metastasis.

The underappreciated role of resident epithelial cell populations in metastatic progression: contributions of the lung alveolar epithelium.

Metastatic cancer is difficult to treat and is responsible for the majority of cancer-related deaths. After cancer cells initiate metastasis and successfully seed a distant site, resident cells in the tissue play a key role in determining how metastatic progression develops. The lung is the second most frequent site of metastatic spread, and the primary site of metastasis within the lung is alveoli. The most abundant cell type in the alveolar niche is the epithelium. This review will examine the potential contributions of the alveolar epithelium to metastatic progression. It will also provide insight into other ways in which alveolar epithelial cells, acting as immune sentinels within the lung, may influence metastatic progression through their various interactions with cells in the surrounding microenvironment.

Secreted indicators of androgen receptor activity in breast cancer pre-clinical models.

PURPOSE: Accumulating evidence has attracted attention to the androgen receptor (AR) as a biomarker and therapeutic target in breast cancer. We hypothesized that AR activity within the tumor has clinical implications and investigated whether androgen responsive serum factors might serve as a minimally invasive indicator of tumor AR activity. METHODS: Based on a comprehensive gene expression analysis of an AR-positive, triple negative breast cancer patient-derived xenograft (PDX) model, 163 dihydrotestosterone (DHT)-responsive genes were defined as an androgen responsive gene set. Among them, we focused on genes that were DHT-responsive that encode secreted proteins, namely KLK3, AZGP1 and PIP, that encode the secreted factors prostate specific antigen (PSA), zinc-alpha-2-glycoprotein (ZAG) and prolactin induced protein (PIP), respectively. Using AR-positive breast cancer cell lines representing all breast cancer subtypes, expression of candidate factors was assessed in response to agonist DHT and antagonist enzalutamide. Gene set enrichment analysis (GSEA) was performed on publically available gene expression datasets from breast cancer patients to analyze the relationship between genes encoding the secreted factors and other androgen responsive gene sets in each breast cancer subtype. RESULTS: Anti-androgen treatment decreased proliferation in all cell lines tested representing various tumor subtypes. Expression of the secreted factors was regulated by AR activation in the majority of breast cancer cell lines. In GSEA, the candidate genes were positively correlated with an androgen responsive gene set across breast cancer subtypes. CONCLUSION: KLK3, AZGP1 and PIP are AR regulated and reflect tumor AR activity. Further investigations are needed to examine the potential efficacy of these factors as serum biomarkers.

Constitutive expression of microRNA-150 in mammary epithelium suppresses secretory activation and impairs de novo lipogenesis.

Profiling of RNA from mouse mammary epithelial cells (MECs) isolated on pregnancy day (P)14 and lactation day (L)2 revealed that the majority of differentially expressed microRNA declined precipitously between late pregnancy and lactation. The decline in miR-150, which exhibited the greatest fold-decrease, was verified quantitatively and qualitatively. To test the hypothesis that the decline in miR-150 is crucial for lactation, MEC-specific constitutive miR-150 was achieved by crossing ROSA26-lox-STOP-lox-miR-150 mice with WAP-driven Cre recombinase mice. Both biological and foster pups nursed by bitransgenic dams exhibited a dramatic decrease in survival compared with offspring nursed by littermate control dams. Protein products of predicted miR-150 targets Fasn, Olah, Acaca, and Stat5B were significantly suppressed in MECs of bitransgenic mice with constitutive miR-150 expression as compared with control mice at L2. Lipid profiling revealed a significant reduction in fatty acids synthesized by the de novo pathway in L2 MECs of bitransgenic versus control mice. Collectively, these data support the hypothesis that a synchronized decrease in miRNAs, such as miR-150, at late pregnancy serves to allow translation of targets crucial for lactation.

The long non-coding RNA MALAT1 promotes ovarian cancer progression by regulating RBFOX2-mediated alternative splicing.

Ovarian cancer metastasizes via direct seeding, whereby cancer cells shed from the primary site, resist cell death in the peritoneal cavity, then metastasize to peritoneal organs. We sought to identify molecular mechanisms that facilitate ovarian cancer cell anchorage independent survival. Gene expression profiling was performed on ovarian cancer cells grown in attached or forced suspension culture and confirmed by RT-qPCR. Anoikis was measured by Caspase 3/7 assay. Since the long non-coding RNA Metastasis Associated Lung Adenocarcinoma transcript 1 (MALAT1) was among the transcripts most highly increased in forced suspension culture, modified anti-sense oligonucleotides (ASO) were used to inhibit its expression. Knockdown of RBFOX2 and KIF1B was performed using shRNAs. Publically available datasets were analyzed for association of MALAT1 gene expression with clinicopathological variables. In multiple anoikis-resistant ovarian cancer cell lines MALAT1 expression increased after 24 and 48 h in forced suspension culture compared to attached culture. High MALAT1 is associated with increased stage, recurrence, and reduced survival in ovarian cancer, and in a small percentage of ovarian cancers MALAT1 is amplified. MALAT1 knockdown resulted in decreased proliferation, invasion, anchorage-independent growth, and increased anoikis. Suppression of MALAT1 also resulted in decreased expression of RBFOX2, and alternative processing of the pro-apoptotic tumor suppressor gene KIF1B. RBFOX2 suppression resulted in preferential splicing of the pro-apoptotic isoform of KIF1B (KIFB1B-beta) and increased anoikis. The lncRNA MALAT1 facilitates a pro-metastatic phenotype in ovarian cancer by promoting alternative RNA processing and differential expression of anti-apoptosis and epithelial to mesenchymal transition (EMT)-related genes.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Targeting Tryptophan Catabolism in Ovarian Cancer to Attenuate Macrophage Infiltration and PD-L1 Expression.

High-grade serous carcinoma (HGSC) of the fallopian tube, ovary, and peritoneum is the most common type of ovarian cancer and is predicted to be immunogenic because the presence of tumor-infiltrating lymphocytes conveys a better prognosis. However, the efficacy of immunotherapies has been limited because of the immune-suppressed tumor microenvironment (TME). Tumor metabolism and immune-suppressive metabolites directly affect immune cell function through the depletion of nutrients and activation of immune-suppressive transcriptional programs. Tryptophan (TRP) catabolism is a contributor to HGSC disease progression. Two structurally distinct rate-limiting TRP catabolizing enzymes, indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2), evolved separately to catabolize TRP. IDO1/TDO2 are aberrantly expressed in carcinomas and metabolize TRP into the immune-suppressive metabolite kynurenine (KYN), which can engage the aryl hydrocarbon receptor to drive immunosuppressive transcriptional programs. To date, IDO inhibitors tested in clinical trials have had limited efficacy, but those inhibitors did not target TDO2, and we find that HGSC cell lines and clinical outcomes are more dependent on TDO2 than IDO1. To identify inflammatory HGSC cancers with poor prognosis, we stratified patient ascites samples by IL6 status, which correlates with poor prognosis. Metabolomics revealed that IL6-high patient samples had enriched KYN. TDO2 knockdown significantly inhibited HGSC growth and TRP catabolism. The orally available dual IDO1/TDO2 inhibitor, AT-0174, significantly inhibited tumor progression, reduced tumor-associated macrophages, and reduced expression of immune-suppressive proteins on immune and tumor cells. These studies demonstrate the importance of TDO2 and the therapeutic potential of AT-0174 to overcome an immune-suppressed TME. SIGNIFICANCE: Developing strategies to improve response to chemotherapy is essential to extending disease-free intervals for patients with HGSC of the fallopian tube, ovary, and peritoneum. In this article, we demonstrate that targeting TRP catabolism, particularly with dual inhibition of TDO2 and IDO1, attenuates the immune-suppressive microenvironment and, when combined with chemotherapy, extends survival compared with chemotherapy alone.

The spatial structure of the tumor immune microenvironment can explain and predict patient response in high-grade serous carcinoma.

Despite ovarian cancer being the deadliest gynecological malignancy, there has been little change to therapeutic options and mortality rates over the last three decades. Recent studies indicate that the composition of the tumor immune microenvironment (TIME) influences patient outcomes but are limited by a lack of spatial understanding. We performed multiplexed ion beam imaging (MIBI) on 83 human high-grade serous carcinoma tumors - one of the largest protein-based, spatially-intact, single-cell resolution tumor datasets assembled - and used statistical and machine learning approaches to connect features of the TIME spatial organization to patient outcomes. Along with traditional clinical/immunohistochemical attributes and indicators of TIME composition, we found that several features of TIME spatial organization had significant univariate correlations and/or high relative importance in high-dimensional predictive models. The top performing predictive model for patient progression-free survival (PFS) used a combination of TIME composition and spatial features. Results demonstrate the importance of spatial structure in understanding how the TIME contributes to treatment outcomes. Furthermore, the present study provides a generalizable roadmap for spatial analyses of the TIME in ovarian cancer research.

The miR-200 and miR-221/222 microRNA families: opposing effects on epithelial identity.

Carcinogenesis is a complex process during which cells undergo genetic and epigenetic alterations. These changes can lead tumor cells to acquire characteristics that enable movement from the primary site of origin when conditions become unfavorable. Such characteristics include gain of front-rear polarity, increased migration/invasion, and resistance to anoikis, which facilitate tumor survival during metastasis. An epithelial to mesenchymal transition (EMT) constitutes one way that cancer cells can gain traits that promote tumor progression and metastasis. Two microRNA (miRNA) families, the miR-200 and miR-221 families, play crucial opposing roles that affect the differentiation state of breast cancers. These two families are differentially expressed between the luminal A subtype of breast cancer as compared to the less well-differentiated triple negative breast cancers (TNBCs) that exhibit markers indicative of an EMT. The miR-200 family promotes a well-differentiated epithelial phenotype, while high miR-221/222 results in a poorly differentiated, mesenchymal-like phenotype. This review focuses on the mechanisms (specific proven targets) by which these two miRNA families exert opposing effects on cellular plasticity during breast tumorigenesis and metastasis.

RUNX1 Is Regulated by Androgen Receptor to Promote Cancer Stem Markers and Chemotherapy Resistance in Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which no effective targeted therapies are available. Growing evidence suggests that chemotherapy-resistant cancer cells with stem-like properties (CSC) may repopulate the tumor. The androgen receptor (AR) is expressed in up to 50% of TNBCs, and AR inhibition decreases CSC and tumor initiation. Runt-related transcription factor 1 (RUNX1) correlates with poor prognosis in TNBC and is regulated by the AR in prostate cancer. Our group has shown that RUNX1 promotes TNBC cell migration and regulates tumor gene expression. We hypothesized that RUNX1 is regulated by the AR and that both may work together in TNBC CSC to promote disease recurrence following chemotherapy. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments in MDA-MB-453 revealed AR binding to RUNX1 regulatory regions. RUNX1 expression is upregulated by dihydrotestosterone (DHT) in MDA-MB-453 and in an AR+-TNBC HCI-009 patient-derived xenograft (PDX) tumors (p < 0.05). RUNX1 is increased in a CSC-like experimental model in MDA-MB-453 and SUM-159PT cells (p < 0.05). Inhibition of RUNX1 transcriptional activity reduced the expression of CSC markers. Interestingly, RUNX1 inhibition reduced cell viability and enhanced paclitaxel and enzalutamide sensitivity. Targeting RUNX1 may be an attractive strategy to potentiate the anti-tumor effects of AR inhibition, specifically in the slow-growing CSC-like populations that resist chemotherapy which lead to metastatic disease.

Cell State and Cell Type: Deconvoluting Circulating Tumor Cell Populations in Liquid Biopsies by Multi-Omics.

Bi-directional crosstalk between the tumor and the tumor microenvironment (TME) has been shown to increase the rate of tumor evolution and to play a key role in neoplastic progression, therapeutic resistance, and a patient's overall survival. Here, we set out to use a comprehensive liquid-biopsy analysis to study cancer and specific TME cells in circulation and their association with disease status. Cytokeratin+, CD45- circulating rare cells (CRCs) from nine breast and four prostate cancer patients were characterized through morphometrics, single-cell copy number analysis, and targeted multiplexed proteomics to delineate cancer cell lineage from other rare cells originating in the TME. We show that we can detect epithelial circulating tumor cells (EPI.CTC), CTCs undergoing epithelial-to-mesenchymal transition (EMT.CTC) and circulating endothelial cells (CECs) using a universal rare event detection platform (HDSCA). Longitudinal analysis of an index patient finds that CTCs are present at the time of disease progression, while CECs are predominately present at the time of stable disease. In a small cohort of prostate and breast cancer patients, we find high inter-patient and temporal intra-patient variability in the expression of tissue specific markers such as ER, HER2, AR, PSA and PSMA and EpCAM. Our study stresses the importance of the multi-omic characterization of circulating rare cells in patients with breast and prostate carcinomas, specifically highlighting overlapping and cell type defining proteo-genomic characteristics of CTCs and CECs.

Phase II trial of fulvestrant plus enzalutamide in ER+/HER2- advanced breast cancer.

This clinical trial combined fulvestrant with the anti-androgen enzalutamide in women with metastatic ER+/HER2- breast cancer (BC). Eligible patients were women with ECOG 0-2, ER+/HER2- measurable or evaluable metastatic BC. Prior fulvestrant was allowed. Fulvestrant was administered at 500 mg IM on days 1, 15, 29, and every 4 weeks thereafter. Enzalutamide was given at 160 mg po daily. Fresh tumor biopsies were required at study entry and after 4 weeks of treatment. The primary efficacy endpoint of the trial was the clinical benefit rate at 24 weeks (CBR24). The median age was 61 years (46-87); PS 1 (0-1); median of 4 prior non-hormonal and 3 prior hormonal therapies for metastatic disease. Twelve had prior fulvestrant, and 91% had visceral disease. CBR24 was 25% (7/28 evaluable). Median progression-free survival (PFS) was 8 weeks (95% CI: 2-52). Adverse events were as expected for hormonal therapy. Significant (p < 0.1) univariate relationships existed between PFS and ER%, AR%, and PIK3CA and/or PTEN mutations. Baseline levels of phospho-proteins in the mTOR pathway were more highly expressed in biopsies of patients with shorter PFS. Fulvestrant plus enzalutamide had manageable side effects. The primary endpoint of CBR24 was 25% in heavily pretreated metastatic ER+/HER2- BC. Short PFS was associated with activation of the mTOR pathway, and PIK3CA and/or PTEN mutations were associated with an increased hazard of progression. Thus, a combination of fulvestrant or other SERD plus AKT/PI3K/mTOR inhibitor with or without AR inhibition warrants investigation in second-line endocrine therapy of metastatic ER+ BC.

Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

Mutual exclusivity of ESR1 and TP53 mutations in endocrine resistant metastatic breast cancer.

Both TP53 and ESR1 mutations occur frequently in estrogen receptor positive (ER+) metastatic breast cancers (MBC) and their distinct roles in breast cancer tumorigenesis and progression are well appreciated. Recent clinical studies discovered mutual exclusivity between TP53 and ESR1 mutations in metastatic breast cancers; however, mechanisms underlying this intriguing clinical observation remain largely understudied and unknown. Here, we explored the interplay between TP53 and ESR1 mutations using publicly available clinical and experimental data sets. We first confirmed the robust mutational exclusivity using six independent cohorts with 1,056 ER+ MBC samples and found that the exclusivity broadly applies to all ER+ breast tumors regardless of their clinical and distinct mutational features. ESR1 mutant tumors do not exhibit differential p53 pathway activity, whereas we identified attenuated ER activity and expression in TP53 mutant tumors, driven by a p53-associated E2 response gene signature. Further, 81% of these p53-associated E2 response genes are either direct targets of wild-type (WT) p53-regulated transactivation or are mutant p53-associated microRNAs, representing bimodal mechanisms of ER suppression. Lastly, we analyzed the very rare cases with co-occurrences of TP53 and ESR1 mutations and found that their simultaneous presence was also associated with reduced ER activity. In addition, tumors with dual mutations showed higher levels of total and PD-L1 positive macrophages. In summary, our study utilized multiple publicly available sources to explore the mechanism underlying the mutual exclusivity between ESR1 and TP53 mutations, providing further insights and testable hypotheses of the molecular interplay between these two pivotal genes in ER+ MBC.

NPC1 Confers Metabolic Flexibility in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) often undergoes at least partial epithelial-to-mesenchymal transition (EMT) to facilitate metastasis. Identifying EMT-associated characteristics can reveal novel dependencies that may serve as therapeutic vulnerabilities in this aggressive breast cancer subtype. We found that NPC1, which encodes the lysosomal cholesterol transporter Niemann-Pick type C1 is highly expressed in TNBC as compared to estrogen receptor-positive (ER+) breast cancer, and is significantly elevated in high-grade disease. We demonstrated that NPC1 is directly targeted by microRNA-200c (miR-200c), a potent suppressor of EMT, providing a mechanism for its differential expression in breast cancer subtypes. The silencing of NPC1 in TNBC causes an accumulation of cholesterol-filled lysosomes, and drives decreased growth in soft agar and invasive capacity. Conversely, overexpression of NPC1 in an ER+ cell line increases invasion and growth in soft agar. We further identified TNBC cell lines as cholesterol auxotrophs, however, they do not solely depend on NPC1 for adequate cholesterol supply. The silencing of NPC1 in TNBC cell lines led to altered mitochondrial function and morphology, suppression of mTOR signaling, and accumulation of autophagosomes. A small molecule inhibitor of NPC1, U18666A, decreased TNBC proliferation and synergized with the chemotherapeutic drug, paclitaxel. This work suggests that NPC1 promotes aggressive characteristics in TNBC, and identifies NPC1 as a potential therapeutic target.

Antigens Expressed by Breast Cancer Cells Undergoing EMT Stimulate Cytotoxic CD8+ T Cell Immunity.

Antigenic differences formed by alterations in gene expression and alternative splicing are predicted in breast cancer cells undergoing epithelial to mesenchymal transition (EMT) and the reverse plasticity known as MET. How these antigenic differences impact immune interactions and the degree to which they can be exploited to enhance immune responses against mesenchymal cells is not fully understood. We utilized a master microRNA regulator of EMT to alter mesenchymal-like EO771 mammary carcinoma cells to a more epithelial phenotype. A computational approach was used to identify neoantigens derived from the resultant differentially expressed somatic variants (SNV) and alternative splicing events (neojunctions). Using whole cell vaccines and peptide-based vaccines, we find superior cytotoxicity against the more-epithelial cells and explore the potential of neojunction-derived antigens to elicit T cell responses through experiments designed to validate the computationally predicted neoantigens. Overall, results identify EMT-associated splicing factors common to both mouse and human breast cancer cells as well as immunogenic SNV- and neojunction-derived neoantigens in mammary carcinoma cells.

ESR1 mutant breast cancers show elevated basal cytokeratins and immune activation.

Estrogen receptor alpha (ER/ESR1) is frequently mutated in endocrine resistant ER-positive (ER+) breast cancer and linked to ligand-independent growth and metastasis. Despite the distinct clinical features of ESR1 mutations, their role in intrinsic subtype switching remains largely unknown. Here we find that ESR1 mutant cells and clinical samples show a significant enrichment of basal subtype markers, and six basal cytokeratins (BCKs) are the most enriched genes. Induction of BCKs is independent of ER binding and instead associated with chromatin reprogramming centered around a progesterone receptor-orchestrated insulated neighborhood. BCK-high ER+ primary breast tumors exhibit a number of enriched immune pathways, shared with ESR1 mutant tumors. S100A8 and S100A9 are among the most induced immune mediators and involve in tumor-stroma paracrine crosstalk inferred by single-cell RNA-seq from metastatic tumors. Collectively, these observations demonstrate that ESR1 mutant tumors gain basal features associated with increased immune activation, encouraging additional studies of immune therapeutic vulnerabilities.