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Combinations of immune checkpoint inhibitors (ICI, including anti-PD-1/PD-L1) and chemotherapy have been FDA approved for metastatic and early-stage triple-negative breast cancer (TNBC), but most patients do not benefit. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with anti-PD-L1 resistance. However, little is known about its regulation and effect on immune cell function in breast cancers. We assessed murine and human breast cancer cells to identify regulation mechanisms of B7-H4 in vitro. We used an immunocompetent anti-PD-L1-sensitive orthotopic mammary cancer model and induced ectopic expression of B7-H4. We assessed therapy response and transcriptional changes at baseline and under treatment with anti-PD-L1. We observed B7-H4 was highly associated with epithelial cell status and transcription factors and found to be regulated by PI3K activity. EMT6 tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. In addition, tumor-infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. Paradoxically, in human breast cancer, B7-H4 expression was associated with survival benefit for patients with metastatic TNBC treated with carboplatin plus anti-PD-L1 and was associated with no change in response or survival for patients with early breast cancer receiving chemotherapy plus anti-PD-1. While B7-H4 induces tumor resistance to anti-PD-L1 in murine models, there are alternative mechanisms of signaling and function in human cancers. In addition, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 independent of PD-L1. SIGNIFICANCE: This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target.
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Imunoterapia , Neoplasias de Mama Triplo Negativas , Inibidor 1 da Ativação de Células T com Domínio V-Set , Inibidor 1 da Ativação de Células T com Domínio V-Set/genética , Inibidor 1 da Ativação de Células T com Domínio V-Set/metabolismo , Animais , Humanos , Camundongos , Feminino , Linhagem Celular Tumoral , Imunoterapia/métodos , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/terapia , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias da Mama/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Despite the success of immune checkpoint inhibition (ICI) in treating cancer, patients with triple-negative breast cancer (TNBC) often develop resistance to therapy, and the underlying mechanisms are unclear. MHC-I expression is essential for antigen presentation and T-cell-directed immunotherapy responses. This study demonstrates that TNBC patients display intratumor heterogeneity in regional MHC-I expression. In murine models, loss of MHC-I negates antitumor immunity and ICI response, whereas intratumor MHC-I heterogeneity leads to increased infiltration of natural killer (NK) cells in an IFNγ-dependent manner. Using spatial technologies, MHC-I heterogeneity is associated with clinical resistance to anti-programmed death (PD) L1 therapy and increased NK:T-cell ratios in human breast tumors. MHC-I heterogeneous tumors require NKG2A to suppress NK-cell function. Combining anti-NKG2A and anti-PD-L1 therapies restores complete response in heterogeneous MHC-I murine models, dependent on the presence of activated, tumor-infiltrating NK and CD8+ T cells. These results suggest that similar strategies may enhance patient benefit in clinical trials. SIGNIFICANCE: Clinical resistance to immunotherapy is common in breast cancer, and many patients will likely require combination therapy to maximize immunotherapeutic benefit. This study demonstrates that heterogeneous MHC-I expression drives resistance to anti-PD-L1 therapy and exposes NKG2A on NK cells as a target to overcome resistance. This article is featured in Selected Articles from This Issue, p. 201.
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Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Imunoterapia/métodos , Células Matadoras Naturais , Linfócitos T CD8-Positivos , Antígeno B7-H1/metabolismoRESUMO
Rationale: Multiciliated cell (MCC) loss and/or dysfunction is common in the small airways of patients with chronic obstructive pulmonary disease (COPD), but it is unclear if this contributes to COPD lung pathology. Objectives: To determine if loss of p73 causes a COPD-like phenotype in mice and explore whether smoking or COPD impact p73 expression. Methods: p73floxE7-E9 mice were crossed with Shh-Cre mice to generate mice lacking MCCs in the airway epithelium. The resulting p73Δairway mice were analyzed using electron microscopy, flow cytometry, morphometry, forced oscillation technique, and single-cell RNA sequencing. Furthermore, the effects of cigarette smoke on p73 transcript and protein expression were examined using in vitro and in vivo models and in studies including airway epithelium from smokers and patients with COPD. Measurements and Main Results: Loss of functional p73 in the respiratory epithelium resulted in a near-complete absence of MCCs in p73Δairway mice. In adulthood, these mice spontaneously developed neutrophilic inflammation and emphysema-like lung remodeling and had progressive loss of secretory cells. Exposure of normal airway epithelium cells to cigarette smoke rapidly and durably suppressed p73 expression in vitro and in vivo. Furthermore, tumor protein 73 mRNA expression was reduced in the airways of current smokers (n = 82) compared with former smokers (n = 69), and p73-expressing MCCs were reduced in the small airways of patients with COPD (n = 11) compared with control subjects without COPD (n = 12). Conclusions: Loss of functional p73 in murine airway epithelium results in the absence of MCCs and promotes COPD-like lung pathology. In smokers and patients with COPD, loss of p73 may contribute to MCC loss or dysfunction.
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Enfisema , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Animais , Humanos , Camundongos , Epitélio/metabolismo , Pulmão , Doença Pulmonar Obstrutiva Crônica/patologiaRESUMO
Importance: Agents targeting programmed death ligand 1 (PD-L1) have demonstrated efficacy in triple-negative breast cancer (TNBC) when combined with chemotherapy and are now the standard of care in patients with PD-L1-positive metastatic disease. In contrast to microtubule-targeting agents, the effect of combining platinum compounds with programmed cell death 1 (PD-1)/PD-L1 immunotherapy has not been extensively determined. Objective: To evaluate the efficacy of atezolizumab with carboplatin in patients with metastatic TNBC. Design, Setting, and Participants: This phase 2 randomized clinical trial was conducted in 6 centers from August 2017 to June 2021. Interventions: Patients with metastatic TNBC were randomized to receive carboplatin area under the curve (AUC) 6 alone or with atezolizumab, 1200 mg, every 3 weeks until disease progression or unacceptable toxic effects with a 3-year duration of follow-up. Main Outcome and Measures: The primary end point was investigator-assessed progression-free survival (PFS). Secondary end points included overall response rate (ORR), clinical benefit rate (CBR), and overall survival (OS). Other objectives included correlation of response with tumor PD-L1 levels, tumor-infiltrating lymphocytes (TILs), tumor DNA- and RNA-sequenced biomarkers, TNBC subtyping, and multiplex analyses of immune markers. Results: All 106 patients with metastatic TNBC who were enrolled were female with a mean (range) age of 55 (27-79) years, of which 12 (19%) identified as African American/Black, 1 (1%) as Asian, 73 (69%) as White, and 11 (10%) as unknown. Patients were randomized and received either carboplatin (n = 50) or carboplatin and atezolizumab (n = 56). The combination improved PFS (hazard ratio [HR], 0.66; 95% CI, 0.44-1.01; P = .05) from a median of 2.2 to 4.1 months, increased ORR from 8.0% (95% CI, 3.2%-18.8%) to 30.4% (95% CI, 19.9%-43.3%), increased CBR at 6 months from 18.0% (95% CI, 9.8%-30.1%) to 37.5% (95% CI, 26.0%-50.6%), and improved OS (HR, 0.60; 95% CI, 0.37-0.96; P = .03) from a median of 8.6 to 12.6 months. Subgroup analysis showed PD-L1-positive tumors did not benefit more from adding atezolizumab (HR, 0.62; 95% CI, 0.23-1.65; P = .35). Patients with high TILs (HR, 0.12; 95% CI, 0.30-0.50), high mutation burden (HR, 0.50; 95% CI, 0.23-1.06), and prior chemotherapy (HR, 0.59; 95% CI, 0.36-0.95) received greater benefit on the combination. Patients with obesity and patients with more than 125 mg/dL on-treatment blood glucose levels were associated with better PFS (HR, 0.35; 95% CI, 0.10-1.80) on the combination. TNBC subtypes benefited from adding atezolizumab, except the luminal androgen receptor subtype. Conclusions and Relevance: In this randomized clinical trial, the addition of atezolizumab to carboplatin significantly improved survival of patients with metastatic TNBC regardless of PD-L1 status. Further, lower risk of disease progression was associated with increased TILs, higher mutation burden, obesity, and uncontrolled blood glucose levels. Trial Registration: ClinicalTrials.gov Identifier: NCT03206203.
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Anticorpos Monoclonais Humanizados , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Pessoa de Meia-Idade , Idoso , Masculino , Carboplatina/uso terapêutico , Neoplasias de Mama Triplo Negativas/patologia , Antígeno B7-H1/imunologia , Glicemia , Ligantes , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Biomarcadores , Progressão da Doença , Obesidade , ApoptoseRESUMO
Genomic and transcriptomic analysis has furthered our understanding of many tumors. Yet, thyroid cancer management is largely guided by staging and histology, with few molecular prognostic and treatment biomarkers. Here, we utilize a large cohort of 251 patients with 312 samples from two tertiary medical centers and perform DNA/RNA sequencing, spatial transcriptomics, and multiplex immunofluorescence to identify biomarkers of aggressive thyroid malignancy. We identify high-risk mutations and discover a unique molecular signature of aggressive disease, the Molecular Aggression and Prediction (MAP) score, which provides improved prognostication over high-risk mutations alone. The MAP score is enriched for genes involved in epithelial de-differentiation, cellular division, and the tumor microenvironment. The MAP score also identifies aggressive tumors with lymphocyte-rich stroma that may benefit from immunotherapy. Future clinical profiling of the stromal microenvironment of thyroid cancer could improve prognostication, inform immunotherapy, and support development of novel therapeutics for thyroid cancer and other stroma-rich tumors.
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Triple-negative breast cancer (TNBC) is a heterogeneous disease with varying responses to neoadjuvant chemotherapy (NAC). The identification of biomarkers to predict NAC response and inform personalized treatment strategies is essential. In this study, we conducted large-scale gene expression meta-analyses to identify genes associated with NAC response and survival outcomes. The results showed that immune, cell cycle/mitotic, and RNA splicing-related pathways were significantly associated with favorable clinical outcomes. Furthermore, we integrated and divided the gene association results from NAC response and survival outcomes into four quadrants, which provided more insights into potential NAC response mechanisms and biomarker discovery.
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Triple-negative breast cancer (TNBC) is a collection of biologically diverse cancers characterized by distinct transcriptional patterns, biology, and immune composition. TNBCs subtypes include two basal-like (BL1, BL2), a mesenchymal (M) and a luminal androgen receptor (LAR) subtype. Through a comprehensive analysis of mutation, copy number, transcriptomic, epigenetic, proteomic, and phospho-proteomic patterns we describe the genomic landscape of TNBC subtypes. Mesenchymal subtype tumors display high mutation loads, genomic instability, absence of immune cells, low PD-L1 expression, decreased global DNA methylation, and transcriptional repression of antigen presentation genes. We demonstrate that major histocompatibility complex I (MHC-I) is transcriptionally suppressed by H3K27me3 modifications by the polycomb repressor complex 2 (PRC2). Pharmacological inhibition of PRC2 subunits EZH2 or EED restores MHC-I expression and enhances chemotherapy efficacy in murine tumor models, providing a rationale for using PRC2 inhibitors in PD-L1 negative mesenchymal tumors. Subtype-specific differences in immune cell composition and differential genetic/pharmacological vulnerabilities suggest additional treatment strategies for TNBC.
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Antineoplásicos/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Animais , Metilação de DNA , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genômica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Proteínas do Grupo Polycomb/antagonistas & inibidores , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Proteogenômica , Proteômica , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Cell-cell interactions (CCIs) and cell-cell communication (CCC) are critical for maintaining complex biological systems. The availability of single-cell RNA sequencing (scRNA-seq) data opens new avenues for deciphering CCIs and CCCs through identifying ligand-receptor (LR) gene interactions between cells. However, most methods were developed to examine the LR interactions of individual pairs of genes. Here, we propose a novel approach named LR hunting which first uses random forests (RFs)-based data imputation technique to link the data between different cell types. To guarantee the robustness of the data imputation procedure, we repeat the computation procedures multiple times to generate aggregated imputed minimal depth index (IMDI). Next, we identify significant LR interactions among all combinations of LR pairs simultaneously using unsupervised RFs. We demonstrated LR hunting can recover biological meaningful CCIs using a mouse cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) dataset and a triple-negative breast cancer scRNA-seq dataset.
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p53 is mutated in over half of human cancers. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. To study mutant p53 GOF mechanisms and phenotypes, we genetically engineered non-transformed and tumor-derived WT p53 cell line models to express endogenous missense mutant p53 (R175H and R273H) or to be deficient for p53 protein (null). Characterization of the models, which initially differed only by TP53 genotype, revealed that aneuploidy frequently occurred in mutant p53-expressing cells. GOF phenotypes occurred clonally in vitro and in vivo, were independent of p53 alteration and correlated with increased aneuploidy. Further, analysis of outcome data revealed that individuals with aneuploid-high tumors displayed unfavorable prognoses, regardless of the TP53 genotype. Our results indicate that genetic variation resulting from aneuploidy accounts for the diversity of previously reported mutant p53 GOF phenotypes.
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Aneuploidia , Mutação com Ganho de Função , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação com Perda de Função , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
p73 and p63 are members of the p53 family that exhibit overlapping and distinct functions in development and homeostasis. The evaluation of p73 and p63 isoform expression across human tissue can provide greater insight to the functional interactions between family members. We determined the mRNA isoform expression patterns of TP73 and TP63 across a panel of 36 human tissues and protein expression within the highest-expressing tissues. TP73 and TP63 expression significantly correlated across tissues. In tissues with concurrent mRNA expression, nuclear co-expression of both proteins was observed in a majority of cells. Using GTEx data, we quantified p73 and p63 isoform expression in human tissue and identified that the α-isoforms of TP73 and TP63 were the predominant isoform expressed in nearly all tissues. Further, we identified a previously unreported p73 mRNA product encoded by exons 4 to 14. In sum, these data provide the most comprehensive tissue-specific atlas of p73 and p63 protein and mRNA expression patterns in human and murine samples, indicating coordinate expression of these transcription factors in the majority of tissues in which they are expressed.
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Regulação da Expressão Gênica , Especificidade de Órgãos/genética , Fatores de Transcrição/genética , Proteína Tumoral p73/genética , Proteínas Supressoras de Tumor/genética , Processamento Alternativo/genética , Animais , Epitélio/metabolismo , Éxons/genética , Humanos , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Proteína Tumoral p73/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Triple-negative breast cancer (TNBC) has a poor outcome compared to other breast cancer subtypes, and new therapies that target the molecular alterations driving tumor progression are needed. Annexin A1 is an abundant multi-functional Ca2+ binding and membrane-associated protein. Reported roles of Annexin A1 in breast cancer progression and metastasis are contradictory. Here, we sought to clarify the functions of Annexin A1 in the development and progression of TNBC. The association of Annexin A1 expression with patient prognosis in subtypes of TNBC was examined. Annexin A1 was stably knocked down in a panel of human and murine TNBC cell lines with high endogenous Annexin A1 expression that were then evaluated for orthotopic growth and spontaneous metastasis in vivo and for alterations in cell morphology in vitro. The impact of Annexin A1 knockdown on the expression of genes involved in mammary epithelial cell differentia tion and epithelial to mesenchymal transition was also determined. Annexin A1 mRNA levels correlated with poor patient prognosis in basal-like breast tumors and also in the basal-like 2 subset of TNBCs. Unexpectedly, loss of Annexin A1 expression had no effect on either primary tumor growth or spontaneous metastasis of MDA-MB-231_HM xenografts, but abrogated the growth rate of SUM149 orthotopic tumors. In an MMTV-PyMT driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation in both immuno-competent and immuno-deficient mice and induced epithelial to mesenchymal transition and upregulation of basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1- population containing putative tumor initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in a model of human breast cancer and suggest that some basal-like TNBCs may require high endogenous tumor cell Annexin A1 expression for continued growth.
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Actively growing tumors are often histologically associated with Ki67 positivity, while the detection of invasiveness relies on non-quantitative pathologic evaluation of mostly advanced tumors. We recently reported that reduced expression of the Ca2+-dependent membrane-binding annexin A6 (AnxA6) is associated with increased expression of the Ca2+ activated RasGRF2 (GRF2), and that the expression status of these proteins inversely influence the growth and motility of triple negative breast cancer (TNBC) cells. Here, we establish that the reciprocal expression of AnxA6 and GRF2 is at least in part, dependent on inhibition of non-selective Ca2+ channels in AnxA6-low but not AnxA6-high TNBC cells. Immunohistochemical staining of breast cancer tissues revealed that compared to non-TNBC tumors, TNBC tumors express lower levels of AnxA6 and higher Ki67 expression. GRF2 expression levels strongly correlated with high Ki67 in pretreatment biopsies from patients with residual disease and with residual tumor size following chemotherapy. Elevated AnxA6 expression more reliably identified patients who responded to chemotherapy, while low AnxA6 levels were significantly associated with shorter distant relapse-free survival. Finally, the reciprocal expression of AnxA6 and GRF2 can delineate GRF2-low/AnxA6-high invasive from GRF2-high/AnxA6-low rapidly growing TNBCs. These data suggest that AnxA6 may be a reliable biomarker for distant relapse-free survival and response of TNBC patients to chemotherapy, and that the reciprocal expression of AnxA6 and GRF2 can reliably delineate TNBCs into rapidly growing and invasive subsets which may be more relevant for subset-specific therapeutic interventions.
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Anexina A6/metabolismo , Canais de Cálcio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , Animais , Anexina A6/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Metástase Neoplásica/genética , Prognóstico , Transplante Heterólogo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade , Fatores ras de Troca de Nucleotídeo Guanina/genéticaRESUMO
Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that does not respond to endocrine therapy or human epidermal growth factor receptor 2 (HER2)-targeted therapies. Individuals with TNBC experience higher rates of relapse and shorter overall survival compared to patients with receptor-positive breast cancer subtypes. Preclinical discoveries are needed to identify, develop, and advance new drug targets to improve outcomes for patients with TNBC. Here, we report that MYCN, an oncogene typically overexpressed in tumors of the nervous system or with neuroendocrine features, is heterogeneously expressed within a substantial fraction of primary and recurrent TNBC and is expressed in an even higher fraction of TNBCs that do not display a pathological complete response after neoadjuvant chemotherapy. We performed high-throughput chemical screens on TNBC cell lines with varying amounts of MYCN expression and determined that cells with higher expression of MYCN were more sensitive to bromodomain and extraterminal motif (BET) inhibitors. Combined BET and MEK inhibition resulted in a synergistic decrease in viability, both in vitro and in vivo, using cell lines and patient-derived xenograft (PDX) models. Our preclinical data provide a rationale to advance a combination of BET and MEK inhibitors to clinical investigation for patients with advanced MYCN-expressing TNBC.
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Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Recidiva Local de Neoplasia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Preclinical data demonstrating androgen receptor (AR)-positive (AR+) triple-negative breast cancer (TNBC) cells are sensitive to AR antagonists, and PI3K inhibition catalyzed an investigator-initiated, multi-institutional phase Ib/II study TBCRC032. The trial investigated the safety and efficacy of the AR-antagonist enzalutamide alone or in combination with the PI3K inhibitor taselisib in patients with metastatic AR+ (≥10%) breast cancer. PATIENTS AND METHODS: Phase Ib patients [estrogen receptor positive (ER+) or TNBC] with AR+ breast cancer received 160 mg enzalutamide in combination with taselisib to determine dose-limiting toxicities and the maximum tolerated dose (MTD). Phase II TNBC patients were randomized to receive either enzalutamide alone or in combination with 4 mg taselisib until disease progression. Primary endpoint was clinical benefit rate (CBR) at 16 weeks. RESULTS: The combination was tolerated, and the MTD was not reached. The adverse events were hyperglycemia and skin rash. Overall, CBR for evaluable patients receiving the combination was 35.7%, and median progression-free survival (PFS) was 3.4 months. Luminal AR (LAR) TNBC subtype patients trended toward better response compared with non-LAR (75.0% vs. 12.5%, P = 0.06), and increased PFS (4.6 vs. 2.0 months, P = 0.082). Genomic analyses revealed subtype-specific treatment response, and novel FGFR2 fusions and AR splice variants. CONCLUSIONS: The combination of enzalutamide and taselisib increased CBR in TNBC patients with AR+ tumors. Correlative analyses suggest AR protein expression alone is insufficient for identifying patients with AR-dependent tumors and knowledge of tumor LAR subtype and AR splice variants may identify patients more or less likely to benefit from AR antagonists.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Receptores Androgênicos/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antagonistas de Receptores de Andrógenos/administração & dosagem , Antagonistas de Receptores de Andrógenos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Benzamidas , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Humanos , Imidazóis/administração & dosagem , Imidazóis/efeitos adversos , Pessoa de Meia-Idade , Metástase Neoplásica , Nitrilas , Oxazepinas/administração & dosagem , Oxazepinas/efeitos adversos , Feniltioidantoína/administração & dosagem , Feniltioidantoína/efeitos adversos , Feniltioidantoína/análogos & derivados , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Taxa de Sobrevida , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Melanomas are characterized by driver and loss-of-function mutations that promote mitogen-activated protein kinase (MAPK) signaling. MEK inhibitors are approved for use in BRAF-mutated melanoma; however, early-phase clinical trials show occasional responses in driver-negative melanoma, suggesting other alterations conferring MAPK/ERK dependency. To identify additional structural alterations in melanoma, we evaluated RNA-Seq from a set of known MAPK/ERK regulators using a novel population-based algorithm in The Cancer Genome Atlas (TCGA). We identified recurrent MAP3K8 rearrangements in 1.7% of melanomas in TCGA, occurring in more than 15% of tumors without known driver mutations (BRAF, NRAS, KIT, GNAQ, GNA11, and NF1). Using an independent tumor set, we validated a similar rearrangement frequency by FISH. MAP3K8-rearranged melanomas exhibit a low mutational burden and absence of typical UV-mutational patterns. We identified two melanoma cell lines that harbor endogenous truncating MAP3K8 rearrangements that demonstrate exquisite dependency. Rearrangement and amplification of the MAP3K8 locus in melanoma cells result in increased levels of a truncated, active MAP3K8 protein; oncogenic dependency on the aberrant MAP3K8; and a concomitant resistance to BRAF inhibition and sensitivity to MEK or ERK1/2 inhibition. Our findings reveal and biochemically characterize targetable oncogenic MAP3K8 truncating rearrangements in driver mutation-negative melanoma, and provide insight to therapeutic approaches for patients with these tumors. These data provide rationale for using MEK or ERK inhibitors in a subset of driver-negative, MAPK/ERK-dependent melanomas harboring truncating MAP3K8 rearrangements. IMPLICATIONS: This is the first mechanistic study and therapeutic implications of truncating MAP3K8 rearrangements in driver-negative melanoma.
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MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Melanoma/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Análise de Sequência de RNA/métodos , Deleção de Sequência , Algoritmos , Linhagem Celular Tumoral , Bases de Dados Genéticas , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Análise de Sobrevida , Translocação Genética , Regulação para CimaRESUMO
The role of AnxA6 in breast cancer and in particular, the mechanisms underlying its contribution to tumor cell growth and/or motility remain poorly understood. In this study, we established the tumor suppressor function of AnxA6 in triple negative breast cancer (TNBC) cells by showing that loss of AnxA6 is associated with early onset and rapid growth of xenograft TNBC tumors in mice. We also identified the Ca2+ activated RasGRF2 as an effector of AnxA6 mediated TNBC cell growth and motility. Activation of Ca2+ mobilizing oncogenic receptors such as epidermal growth factor receptor (EGFR) in TNBC cells or pharmacological stimulation of Ca2+ influx led to activation, subsequent degradation and altered effector functions of RasGRF2. Inhibition of Ca2+ influx or overexpression of AnxA6 blocked the activation/degradation of RasGRF2. We also show that AnxA6 acts as a scaffold for RasGRF2 and Ras proteins and that its interaction with RasGRF2 is modulated by GTP and/or activation of Ras proteins. Meanwhile, down-regulation of RasGRF2 and treatment of cells with the EGFR-targeted tyrosine kinase inhibitor (TKI) lapatinib strongly attenuated the growth of otherwise EGFR-TKI resistant AnxA6 high TNBC cells. These data not only suggest that AnxA6 modulated Ca2+ influx and effector functions of RasGRF2 underlie at least in part, the AnxA6 mediated TNBC cell growth and/or motility, but also provide a rationale to target Ras-driven TNBC with EGFR targeted therapies in combination with inhibition of RasGRF2.
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Recently, Georgia State University's Centennial Hall was the premier location for the 2017 International Conference on Triple Negative Breast Cancer (TNBC): Illuminating Actionable Biology, which was held from Sept. 18 to 20, 2017, in Atlanta, USA. The conference featured a stellar line-up of domestic and international speakers and diverse participants including TNBC survivors, luminaries in breast cancer research, medical students and fellows, clinicians, translational researchers, epidemiologists, biostatisticians, bioinformaticians, and representatives from the industry. This report distills the burning questions that spiked the event and summarizes key themes, findings, unique opportunities and future directions that emerged from this confluence of thought leaders.
Assuntos
Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/terapia , Animais , Biomarcadores Tumorais , Atenção à Saúde , Feminino , Disparidades em Assistência à Saúde , Humanos , Terapia de Alvo Molecular , Fatores de Risco , Neoplasias de Mama Triplo Negativas/epidemiologia , Neoplasias de Mama Triplo Negativas/etiologiaRESUMO
Triple-negative breast cancer (TNBC) is an operational term for breast cancers lacking targetable estrogen receptor expression and HER2 amplifications. TNBC is, therefore, inherently heterogeneous, and is associated with worse prognosis, greater rates of metastasis, and earlier onset. TNBC displays mutational and transcriptional diversity, and distinct mRNA transcriptional subtypes exhibiting unique biology. High-throughput sequencing has extended cancer research far beyond protein coding regions that include non-coding small RNAs, such as miRNA, isomiR, tRNA, snoRNAs, snRNA, yRNA, 7SL, and 7SK. In this study, we performed small RNA profiling of 26 TNBC cell lines, and compared the abundance of non-coding RNAs among the transcriptional subtypes of triple negative breast cancer. We also examined their co-expression pattern with corresponding mRNAs. This study provides a detailed description of small RNA expression in triple-negative breast cancer cell lines that can aid in the development of future biomarker and novel targeted therapies.
RESUMO
We hypothesize that the relative mitochondria copy number (MTCN) can be estimated by comparing the abundance of mitochondrial DNA to nuclear DNA reads using high throughput sequencing data. To test this hypothesis, we examined relative MTCN across 13 breast cancer cell lines using the RT-PCR based NovaQUANT Human Mitochondrial to Nuclear DNA Ratio Kit as the gold standard. Six distinct computational approaches were used to estimate the relative MTCN in order to compare to the RT-PCR measurements. The results demonstrate that relative MTCN correlates well with the RT-PCR measurements using exome sequencing data, but not RNA-seq data. Through analysis of copy number variants (CNVs) in The Cancer Genome Atlas, we show that the two nuclear genes used in the NovaQUANT assay to represent the nuclear genome often experience CNVs in tumor cells, questioning the accuracy of this gold-standard method when it is applied to tumor cells.
Assuntos
Neoplasias da Mama/genética , Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Linhagem Celular Tumoral , Biologia Computacional/normas , Mineração de Dados , Bases de Dados Genéticas , Feminino , Genes Essenciais , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Mitocôndrias/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Análise de Sequência de DNA/normas , Análise de Sequência de RNA/normas , Sequenciamento do Exoma/normasRESUMO
BACKGROUND: Triple negative breast cancer (TNBC) is a heterogeneous disease that lacks unifying molecular alterations that can guide therapy decisions. We previously identified distinct molecular subtypes of TNBC (TNBCtype) using gene expression data generated on a microarray platform using frozen tumor specimens. Tumors and cell lines representing the identified subtypes have distinct enrichment in biologically relevant transcripts with differing sensitivity to standard chemotherapies and targeted agents. Since our initial discoveries, RNA-sequencing (RNA-seq) has evolved as a sensitive and quantitative tool to measure transcript abundance. METHODS: To demonstrate that TNBC subtypes were similar between platforms, we compared gene expression from matched specimens profiled by both microarray and RNA-seq from The Cancer Genome Atlas (TCGA). In the clinical care of patients with TNBC, tumor specimens collected for diagnostic purposes are processed by formalin fixation and paraffin-embedding (FFPE). Thus, for TNBCtype to eventually have broad and practical clinical utility we performed RNA-seq gene expression and molecular classification comparison between fresh-frozen (FF) and FFPE tumor specimens. RESULTS: Analysis of TCGA showed consistent subtype calls between 91% of evaluable samples demonstrating conservation of TNBC subtypes across microarray and RNA-seq platforms. We compared RNA-seq performed on 21-paired FF and FFPE TNBC specimens and evaluated genome alignment, transcript coverage, differential transcript enrichment and concordance of TNBC molecular subtype calls. We demonstrate that subtype accuracy between matched FF and FFPE samples increases with sequencing depth and correlation strength to an individual TNBC subtype. CONCLUSIONS: TNBC subtypes were reliably identified from FFPE samples, with highest accuracy if the samples were less than 4 years old and reproducible subtyping increased with sequencing depth. To reproducibly subtype tumors using gene expression, it is critical to select genes that do not vary due to platform type, tissue processing or RNA isolation method. The majority of differentially expressed transcripts between matched FF and FFPE samples could be attributed to transcripts selected for by RNA enrichment method. While differentially expressed transcripts did not impact TNBC subtyping, they will provide guidance on determining which transcripts to avoid when implementing a gene set size reduction strategy. TRIAL REGISTRATION: NCT00930930 07/01/2009.