RESUMEN
AIM: Deregulated signaling pathways are a hallmark feature of oncogenesis and driver of tumor progression. Dual specificity protein phosphatase 4 (DUSP4) is a critical negative regulator of the mitogen-activated protein kinase (MAPK) pathway and is often deleted or epigenetically silenced in tumors. DUSP4 alterations lead to hyperactivation of MAPK signaling in many cancers, including breast cancer, which often harbor mutations in cell cycle checkpoint genes, particularly in TP53. METHODS: Using a genetically engineered mouse model, we generated mammary-specific Dusp4-deleted primary epithelial cells to investigate the necessary conditions in which DUSP4 loss may drive breast cancer oncogenesis. RESULTS: We found that Dusp4 loss alone is insufficient in mediating tumorigenesis, but alternatively converges with loss in Trp53 and MYC amplification to induce tumorigenesis primarily through chromosome 5 amplification, which specifically upregulates Dbf4, a cell cycle gene that promotes cellular replication by mediating cell cycle checkpoint escape. CONCLUSIONS: This study identifies a novel mechanism for breast tumorigenesis implicating Dusp4 loss and p53 mutations in cellular acquisition of Dbf4 upregulation as a driver of cellular replication and cell cycle checkpoint escape.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Proteína p53 Supresora de Tumor , Animales , Ciclo Celular/genética , Transformación Celular Neoplásica/genética , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/metabolismo , Ratones , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
PURPOSE: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME). EXPERIMENTAL DESIGN: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1-high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood. RESULTS: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden. CONCLUSIONS: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.
Asunto(s)
Albúminas/administración & dosificación , Antígeno B7-H1/genética , Paclitaxel/administración & dosificación , Receptor de Muerte Celular Programada 1/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Adulto , Anciano , Albúminas/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Persona de Mediana Edad , Terapia Neoadyuvante/efectos adversos , Proteínas de Neoplasias/genética , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Paclitaxel/efectos adversos , Pronóstico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Supervivencia sin Progresión , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Clinical trials have demonstrated the efficacy of combining phosphoinositide 3-kinase (PI3K) inhibitors with endocrine therapies in hormone therapy-refractory breast cancer. However, biomarkers of PI3K pathway dependence in ER+ breast cancer have not been fully established. Hotspot mutations in the alpha isoform of PI3K (PIK3CA) are frequent in ER+ disease and may identify tumors that respond to PI3K inhibitors. It is unclear whether PIK3CA mutations are the only biomarker to suggest pathway dependence and response to therapy. We performed correlative molecular characterization of primary and metastatic tissue from patients enrolled in a phase Ib study combining buparlisib (NVP-BKM-120), a pan-PI3K inhibitor, with letrozole in ER+, human epidermal growth factor-2 (HER2)-negative, metastatic breast cancer. Activating mutations in PIK3CA and inactivating MAP3K1 mutations marked tumors from patients with clinical benefit (≥6 months of stable disease). Patients harboring mutations in both genes exhibited the greatest likelihood of clinical benefit. In ER+ breast cancer cell lines, siRNA-mediated knockdown of MAP3K1 did not affect the response to buparlisib. In a subset of patients treated with buparlisib or the PI3Kα inhibitor alpelisib each with letrozole where PAM50 analysis was performed, nearly all tumors from patients with clinical benefit had a luminal A subtype. Mutations in MAP3K1 in ER+ breast cancer may be associated with clinical benefit from combined inhibition of PI3K and ER, but we could not ascribe direct biological function therein, suggesting they may be a surrogate for luminal A status. We posit that luminal A tumors may be a target population for this therapeutic combination.
RESUMEN
Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , ADN Tumoral Circulante/genética , Resistencia a Antineoplásicos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Aminopiridinas/administración & dosificación , Aminopiridinas/farmacología , Animales , Antineoplásicos Hormonales/administración & dosificación , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Fulvestrant/administración & dosificación , Fulvestrant/farmacología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células MCF-7 , Ratones , Mutación , Naftalenos/farmacología , Piperazinas/farmacología , Supervivencia sin Progresión , Modelos de Riesgos Proporcionales , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Purinas/administración & dosificación , Purinas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Quinolinas/farmacología , Quinoxalinas/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The phase III ExteNET trial showed improved invasive disease-free survival in patients with HER2+ breast cancer treated with neratinib versus placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in patients with ER+/HER2+ tumors. We thus sought to discover mechanisms that may explain the benefit from extended adjuvant therapy with neratinib.Experimental Design: Mice with established ER+/HER2+ MDA-MB-361 tumors were treated with paclitaxel plus trastuzumab ± pertuzumab for 4 weeks, and then randomized to fulvestrant ± neratinib treatment. The benefit from neratinib was evaluated by performing gene expression analysis for 196 ER targets, ER transcriptional reporter assays, and cell-cycle analyses. RESULTS: Mice receiving "extended adjuvant" therapy with fulvestrant/neratinib maintained a complete response, whereas those treated with fulvestrant relapsed rapidly. In three ER+/HER2+ cell lines (MDA-MB-361, BT-474, UACC-893) but not in ER+/HER2- MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity, whereas treatment with fulvestrant resulted in increased HER2 and EGFR phosphorylation, suggesting compensatory reciprocal crosstalk between the ER and ERBB RTK pathways. ER transcriptional reporter assays, gene expression, and immunoblot analyses showed that treatment with neratinib/fulvestrant, but not fulvestrant, potently inhibited growth and downregulated ER reporter activity, P-AKT, P-ERK, and cyclin D1 levels. Finally, similar to neratinib, genetic and pharmacologic inactivation of cyclin D1 enhanced fulvestrant action against ER+/HER2+ breast cancer cells. CONCLUSIONS: These data suggest that ER blockade leads to reactivation of ERBB RTKs and thus extended ERBB blockade is necessary to achieve durable clinical outcomes in patients with ER+/HER2+ breast cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Biomarcadores de Tumor , Neoplasias de la Mama/patología , Línea Celular Tumoral , Quimioterapia Adyuvante , Modelos Animales de Enfermedad , Femenino , Fulvestrant/administración & dosificación , Humanos , Inmunohistoquímica , Ratones , Quinolinas/administración & dosificación , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Efferocytosis is the process by which apoptotic cells are cleared from tissue by phagocytic cells. The removal of apoptotic cells prevents them from undergoing secondary necrosis and releasing their inflammation-inducing intracellular contents. Efferocytosis also limits tissue damage by increasing immunosuppressive cytokines and leukocytes and maintains tissue homeostasis by promoting tolerance to antigens derived from apoptotic cells. Thus, tumor cell efferocytosis following cytotoxic cancer treatment could impart tolerance to tumor cells evading treatment-induced apoptosis with deleterious consequences in tumor residual disease. We report here that efferocytosis cleared apoptotic tumor cells in residual disease of lapatinib-treated HER2+ mammary tumors in MMTV-Neu mice, increased immunosuppressive cytokines, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg). Blockade of efferocytosis induced secondary necrosis of apoptotic cells, but failed to prevent increased tumor MDSCs, Treg, and immunosuppressive cytokines. We found that efferocytosis stimulated expression of IFN-γ, which stimulated the expression of indoleamine-2,3-dioxegenase (IDO) 1, an immune regulator known for driving maternal-fetal antigen tolerance. Combined inhibition of efferocytosis and IDO1 in tumor residual disease decreased apoptotic cell- and necrotic cell-induced immunosuppressive phenotypes, blocked tumor metastasis, and caused tumor regression in 60% of MMTV-Neu mice. This suggests that apoptotic and necrotic tumor cells, via efferocytosis and IDO1, respectively, promote tumor 'homeostasis' and progression. SIGNIFICANCE: These findings show in a model of HER2+ breast cancer that necrosis secondary to impaired efferocytosis activates IDO1 to drive immunosuppression and tumor progression.
Asunto(s)
Apoptosis , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Mamarias Experimentales/patología , Necrosis , Linfocitos T Reguladores/patología , Tirosina Quinasa c-Mer/metabolismo , Animales , Antineoplásicos/farmacología , Femenino , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Lapatinib/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Fagocitosis , Receptor ErbB-2/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Microambiente Tumoral/inmunología , Tirosina Quinasa c-Mer/genéticaRESUMEN
Immunotherapies targeting the PD-1 pathway produce durable responses in many cancers, but the tumor-intrinsic factors governing response and resistance are largely unknown. MHC-II expression on tumor cells can predict response to anti-PD-1 therapy. We therefore sought to determine how MHC-II expression by tumor cells promotes PD-1 dependency. Using transcriptional profiling of anti-PD-1-treated patients, we identified unique patterns of immune activation in MHC-II+ tumors. In patients and preclinical models, MHC-II+ tumors recruited CD4+ T cells and developed dependency on PD-1 as well as Lag-3 (an MHC-II inhibitory receptor), which was upregulated in MHC-II+ tumors at acquired resistance to anti-PD-1. Finally, we identify enhanced expression of FCRL6, another MHC-II receptor expressed on NK and T cells, in the microenvironment of MHC-II+ tumors. We ascribe this to what we believe to be a novel inhibitory function of FCRL6 engagement, identifying it as an immunotherapy target. These data suggest a MHC-II-mediated context-dependent mechanism of adaptive resistance to PD-1-targeting immunotherapy.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunoterapia , Receptores de Superficie Celular/metabolismo , Inmunidad Adaptativa , Animales , Anticuerpos Neutralizantes , Neoplasias de la Mama/metabolismo , Linfocitos T CD4-Positivos , Línea Celular Tumoral , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T , Linfocitos T/inmunología , Microambiente Tumoral , Proteína del Gen 3 de Activación de LinfocitosAsunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/terapia , Inmunoterapia , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/metabolismo , Neoplasias de la Mama/patología , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/metabolismo , Femenino , Humanos , Ipilimumab/uso terapéutico , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/terapiaRESUMEN
Potentiating anti-tumor immunity by inducing tumor inflammation and T cell-mediated responses are a promising area of cancer therapy. Immunomodulatory agents that promote these effects function via a wide variety of mechanisms, including upregulation of antigen presentation pathways. Here, we show that major histocompatibility class-I (MHC-I) genes are methylated in human breast cancers, suppressing their expression. Treatment of breast cancer cell lines with a next-generation hypomethylating agent, guadecitabine, upregulates MHC-I expression in response to interferon-γ. In murine tumor models of breast cancer, guadecitabine upregulates MHC-I in tumor cells promoting recruitment of CD8+ T cells to the microenvironment. Finally, we show that MHC-I genes are upregulated in breast cancer patients treated with hypomethylating agents. Thus, the immunomodulatory effects of hypomethylating agents likely involve upregulation of class-I antigen presentation to potentiate CD8+ T cell responses. These strategies may be useful to potentiate anti-tumor immunity and responses to checkpoint inhibition in immune-refractory breast cancers.
Asunto(s)
Azacitidina/análogos & derivados , Neoplasias de la Mama/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes MHC Clase I/fisiología , Linfocitos T Citotóxicos/fisiología , Animales , Antineoplásicos/farmacología , Azacitidina/farmacología , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes MHC Clase I/genética , Humanos , Neoplasias Mamarias Experimentales , Ratones , Regiones Promotoras GenéticasRESUMEN
Most patients with advanced triple-negative breast cancer (TNBC) develop drug resistance. MYC and MCL1 are frequently co-amplified in drug-resistant TNBC after neoadjuvant chemotherapy. Herein, we demonstrate that MYC and MCL1 cooperate in the maintenance of chemotherapy-resistant cancer stem cells (CSCs) in TNBC. MYC and MCL1 increased mitochondrial oxidative phosphorylation (mtOXPHOS) and the generation of reactive oxygen species (ROS), processes involved in maintenance of CSCs. A mutant of MCL1 that cannot localize in mitochondria reduced mtOXPHOS, ROS levels, and drug-resistant CSCs without affecting the anti-apoptotic function of MCL1. Increased levels of ROS, a by-product of activated mtOXPHOS, led to the accumulation of HIF-1α. Pharmacological inhibition of HIF-1α attenuated CSC enrichment and tumor initiation in vivo. These data suggest that (1) MYC and MCL1 confer resistance to chemotherapy by expanding CSCs via mtOXPHOS and (2) targeting mitochondrial respiration and HIF-1α may reverse chemotherapy resistance in TNBC.
Asunto(s)
Resistencia a Antineoplásicos , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fosforilación Oxidativa , Proteínas Proto-Oncogénicas c-myc/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosforilación Oxidativa/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Inhibition of proliferation in estrogen receptor-positive (ER+) breast cancers after short-term antiestrogen therapy correlates with long-term patient outcome. We profiled 155 ER+/human epidermal growth factor receptor 2-negative (HER2-) early breast cancers from 143 patients treated with the aromatase inhibitor letrozole for 10 to 21 days before surgery. Twenty-one percent of tumors remained highly proliferative, suggesting that these tumors harbor alterations associated with intrinsic endocrine therapy resistance. Whole-exome sequencing revealed a correlation between 8p11-12 and 11q13 gene amplifications, including FGFR1 and CCND1, respectively, and high Ki67. We corroborated these findings in a separate cohort of serial pretreatment, postneoadjuvant chemotherapy, and recurrent ER+ tumors. Combined inhibition of FGFR1 and CDK4/6 reversed antiestrogen resistance in ER+FGFR1/CCND1 coamplified CAMA1 breast cancer cells. RNA sequencing of letrozole-treated tumors revealed the existence of intrachromosomal ESR1 fusion transcripts and increased expression of gene signatures indicative of enhanced E2F-mediated transcription and cell cycle processes in cancers with high Ki67. These data suggest that short-term preoperative estrogen deprivation followed by genomic profiling can be used to identify druggable alterations that may cause intrinsic endocrine therapy resistance.