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1.
Cancer Res Commun ; 2024 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-39177280

RESUMEN

Small cell lung cancer (SCLC) is the most lethal type of lung cancer. Paradoxically, this tumor displays an initial exquisite response to chemotherapy; however, at relapse, the tumor is highly resistant to subsequent available therapies. Here, we report that the expression of three prime repair exonuclease 1 (TREX1) is strongly induced in chemoresistant SCLCs. ATAC-seq and ChIP-seq revealed a significant increase in chromatin accessibility and transcriptional activity of TREX1 gene locus in chemoresistant SCLC. Analyses of human SCLC tumors and patient-derived xenografts (PDXs) also showed increase in TREX1 expression in post-chemotherapy samples. TREX1 depletion caused the activation of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway due to cytoplasmic accumulation of damage-associated double-stranded DNA, inducing immunogenicity and enhancing the sensitivity of drug resistant cells to chemotherapy. These findings suggest TREX1 upregulation may partially contribute to the survival of resistant cells, and its inhibition may represent a promising therapeutic strategy to enhance antitumor immunity and potentiate the efficacy of chemotherapy and/or immunotherapy in chemoresistant SCLCs.

2.
Commun Biol ; 6(1): 65, 2023 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-36653474

RESUMEN

Human cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DEAD-box helicase 4 (DDX4), a germline factor and RNA helicase conserved in all multicellular organisms, contributes to increased cell motility and cisplatin-mediated drug resistance in small cell lung cancer (SCLC) cells. Proteomic analysis suggests that DDX4 expression upregulates proteins related to DNA repair and immune/inflammatory response. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Further, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of immune/inflammatory response markers. Taken together, we propose that DDX4 increases SCLC cell survival, by increasing the DNA damage and immune response pathways, especially under challenging conditions such as cisplatin treatment.


Asunto(s)
Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Ratones , Animales , Humanos , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/genética , Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , Ratones Desnudos , Proteómica , Células Germinativas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo
3.
Cancer Cell ; 40(10): 1128-1144.e8, 2022 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-36150391

RESUMEN

KRAS-LKB1 (KL) mutant lung cancers silence STING owing to intrinsic mitochondrial dysfunction, resulting in T cell exclusion and resistance to programmed cell death (ligand) 1 (PD-[L]1) blockade. Here we discover that KL cells also minimize intracellular accumulation of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) to further avoid downstream STING and STAT1 activation. An unbiased screen to co-opt this vulnerability reveals that transient MPS1 inhibition (MPS1i) potently re-engages this pathway in KL cells via micronuclei generation. This effect is markedly amplified by epigenetic de-repression of STING and only requires pulse MPS1i treatment, creating a therapeutic window compared with non-dividing cells. A single course of decitabine treatment followed by pulse MPS1i therapy restores T cell infiltration in vivo, enhances anti-PD-1 efficacy, and results in a durable response without evidence of significant toxicity.


Asunto(s)
Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Decitabina , Genes ras , Humanos , Ligandos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo
4.
Cancer Res ; 82(21): 4079-4092, 2022 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-36066413

RESUMEN

Immunotherapy has shown limited efficacy in patients with EGFR-mutated lung cancer. Efforts to enhance the immunogenicity of EGFR-mutated lung cancer have been unsuccessful to date. Here, we discover that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (1). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI-resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of FOSL1. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI-resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI-resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8+ T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant EGFR-mutated lung cancers and promote immunogenicity. SIGNIFICANCE: MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Receptores ErbB/metabolismo , Amplificación de Genes , Neoplasias Pulmonares/patología , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/metabolismo , 5'-Nucleotidasa/metabolismo
5.
Cancers (Basel) ; 14(15)2022 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-35892819

RESUMEN

Cell therapies, including adoptive immune cell therapies and genetically engineered chimeric antigen receptor (CAR) T or NK cells, have shown promise in treating hematologic malignancies. Yet, immune cell infiltration and expansion has proven challenging in solid tumors due to immune cell exclusion and exhaustion and the presence of vascular barriers. Testing next-generation immune therapies remains challenging in animals, motivating sophisticated ex vivo models of human tumor biology and prognostic assays to predict treatment response in real-time while comprehensively recapitulating the human tumor immune microenvironment (TIME). This review examines current strategies for testing cell-based cancer immunotherapies using ex vivo microphysiological systems and microfluidic technologies. Insights into the multicellular interactions of the TIME will identify novel therapeutic strategies to help patients whose tumors are refractory or resistant to current immunotherapies. Altogether, these microphysiological systems (MPS) have the capability to predict therapeutic vulnerabilities and biological barriers while studying immune cell infiltration and killing in a more physiologically relevant context, thereby providing important insights into fundamental biologic mechanisms to expand our understanding of and treatments for currently incurable malignancies.

6.
Proc Natl Acad Sci U S A ; 119(28): e2111003119, 2022 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-35787058

RESUMEN

Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The stimulator of interferon genes (STING) DNA sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties. Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils, and natural killer (NK) populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, chimeric antigen receptor T cells, NK therapies, and immune checkpoint blockade.


Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Células Asesinas Naturales , Animales , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Humanos , Inmunidad , Inmunoterapia , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Microambiente Tumoral
7.
Cancer Immunol Res ; 10(8): 947-961, 2022 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-35678717

RESUMEN

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist-induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.


Asunto(s)
Inmunoterapia Adoptiva , Células Asesinas Naturales , Proteínas de la Membrana , Mesotelioma Maligno , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Proteínas de la Membrana/agonistas , Receptores Quiméricos de Antígenos
8.
Cancer Res ; 82(2): 248-263, 2022 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-34810201

RESUMEN

Neuroendocrine to nonneuroendocrine plasticity supports small cell lung cancer (SCLC) tumorigenesis and promotes immunogenicity. Approximately 20% to 25% of SCLCs harbor loss-of-function (LOF) NOTCH mutations. Previous studies demonstrated that NOTCH functions as a SCLC tumor suppressor, but can also drive nonneuroendocrine plasticity to support SCLC growth. Given the dual functionality of NOTCH, it is not understood why SCLCs select for LOF NOTCH mutations and how these mutations affect SCLC tumorigenesis. In a CRISPR-based genetically engineered mouse model of SCLC, genetic loss of Notch1 or Notch2 modestly accelerated SCLC tumorigenesis. Interestingly, Notch-mutant SCLCs still formed nonneuroendocrine subpopulations, and these Notch-independent, nonneuroendocrine subpopulations were driven by Runx2-mediated regulation of Rest. Notch2-mutant nonneuroendocrine cells highly express innate immune signaling genes including stimulator of interferon genes (STING) and were sensitive to STING agonists. This work identifies a Notch-independent mechanism to promote nonneuroendocrine plasticity and suggests that therapeutic approaches to activate STING could be selectively beneficial for SCLCs with NOTCH2 mutations. SIGNIFICANCE: A genetically engineered mouse model of NOTCH-mutant SCLC reveals that nonneuroendocrine plasticity persists in the absence of NOTCH, driven by a RUNX2-REST-dependent pathway and innate immune signaling.


Asunto(s)
Plasticidad de la Célula/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Neoplasias Pulmonares/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Transducción de Señal/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Animales , Sistemas CRISPR-Cas , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Mutación con Pérdida de Función , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Receptor Notch1/genética , Receptor Notch2/genética , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Transfección
9.
Front Oncol ; 11: 662943, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34026641

RESUMEN

INTRODUCTION: Brain metastases are the most common brain tumors in adults, whose management remains nuanced. Improved understanding of risk factors for surgical complications and mortality may guide treatment decisions. METHODS: A nationwide, multicenter analysis was conducted with a retrospective cohort. Adult patients in the 2012-2015 American College of Surgeons National Surgical Quality Improvement Project (ACS NSQIP) databases who received a craniotomy for resection of brain metastasis were included. RESULTS: 3500 cases were analyzed, of which 17% were considered frail and 24% were infratentorial. The most common 30-day medical complications were venous thromboembolism (3%, median time-to-event [TTE] 4.5 days), pneumonia (4%, median TTE 6 days), and urinary tract infections (2%, median TTE 5 days). Reoperation and unplanned readmission occurred in 5% and 12% of patients, respectively. Infratentorial approach and frailty were associated with reoperation before discharge (OR 2.0 for both; p=0.01 and p=0.03 respectively), but not after discharge. Infratentorial approaches conferred heightened risk for readmission for hydrocephalus (OR 5.1, p=0.02) and reoperation for cerebrospinal fluid diversion (OR 7.1, p<0.001).Overall 30-day mortality was 4%, with nearly three-quarters occurring after discharge. Pre-frailty and frailty were associated with increased odds for post-discharge mortality (OR 1.7 and 2.7, p<0.05), but not pre-discharge mortality. We developed a model to identify pre-/peri-operative variables associated with death, including frailty, thrombocytopenia, and high American Society of Anesthesiologists score (AUROC 0.75). CONCLUSIONS: Optimization of metrics contributing to patient frailty and heightened surveillance in patients with infratentorial metastases may be considered in the peri-operative period.

10.
Cancer Discov ; 11(8): 1952-1969, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33707236

RESUMEN

Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. SIGNIFICANCE: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection.This article is highlighted in the In This Issue feature, p. 1861.


Asunto(s)
Plasticidad de la Célula , Neoplasias Pulmonares/inmunología , Carcinoma Pulmonar de Células Pequeñas/inmunología , Animales , Estudios de Cohortes , Modelos Animales de Enfermedad , Registros Electrónicos de Salud , Humanos , Neoplasias Pulmonares/patología , Ratones , Carcinoma Pulmonar de Células Pequeñas/patología
11.
Expert Opin Ther Targets ; 25(3): 167-174, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33703985

RESUMEN

INTRODUCTION: KRAS mutations drive tumorigenesis by altering cell signaling and the tumor immune microenvironment. Recent studies have shown promise for KRAS-G12C covalent inhibitors, which are advancing rapidly through clinical trials. The sequencing and combination of these agents with other therapies including immune checkpoint blockade (ICB) will benefit from strategies that also address the immune microenvironment to improve durability of response. AREAS COVERED: This paper reviews KRAS signaling and discusses downstream effects on cytokine production and the tumor immune microenvironment. RAS targeted therapy is introduced and perspectives on therapeutic targeting of KRAS-G12C and its immunosuppressive tumor microenvironment are offered. EXPERT OPINION: The availability of KRAS-G12C covalent inhibitors raises hopes for targeting this pervasive oncogene and designing better therapeutic combinations to promote anti-tumor immunity. A comprehensive mechanistic understanding of KRAS immunosuppression is required in order to prioritize agents for clinical trials.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Diseño de Fármacos , Humanos , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunidad Innata , Terapia Molecular Dirigida , Mutación , Neoplasias/genética , Neoplasias/inmunología , Proteínas Proto-Oncogénicas p21(ras)/genética , Microambiente Tumoral/inmunología
12.
Cancers (Basel) ; 13(4)2021 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-33578789

RESUMEN

Despite recent advances in first-line treatment for small-cell lung cancer (SCLC), durable responses remain rare. The DNA repair enzyme poly-(ADP)-ribose polymerase (PARP) was identified as a therapeutic target in SCLC using unbiased preclinical screens and confirmed in human and mouse models. Early trials of PARP inhibitors, either alone or in combination with chemotherapy, showed promising but limited responses, suggesting that selecting patient subsets and treatment combinations will prove critical to further clinical development. Expression of SLFN11 and other components of the DNA damage response (DDR) pathway appears to select for improved responses. Combining PARP inhibitors with agents that damage DNA and inhibit DDR appears particularly effective in preclinical and early trial data, as well as strategies that enhance antitumor immunity downstream of DNA damage. A robust understanding of the mechanisms of DDR in SCLC, which exhibits intrinsic replication stress, will improve selection of agents and predictive biomarkers. The most effective combinations will target multiple nodes in the DNA damage/DDR/immune activation cascade to minimize toxicity from synthetic lethality.

13.
Front Immunol ; 11: 2090, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33013881

RESUMEN

Intratumoral recruitment of immune cells following innate immune activation is critical for anti-tumor immunity and involves cytosolic dsDNA sensing by the cGAS/STING pathway. We have previously shown that KRAS-LKB1 (KL) mutant lung cancer, which is resistant to PD-1 blockade, exhibits silencing of STING, impaired tumor cell production of immune chemoattractants, and T cell exclusion. Since the vasculature is also a critical gatekeeper of immune cell infiltration into tumors, we developed a novel microfluidic model to study KL tumor-vascular interactions. Notably, dsDNA priming of LKB1-reconstituted tumor cells activates the microvasculature, even when tumor cell STING is deleted. cGAS-driven extracellular export of 2'3' cGAMP by cancer cells activates STING signaling in endothelial cells and cooperates with type 1 interferon to increase vascular permeability and expression of E selectin, VCAM-1, and ICAM-1 and T cell adhesion to the endothelium. Thus, tumor cell cGAS-STING signaling not only produces T cell chemoattractants, but also primes tumor vasculature for immune cell escape.


Asunto(s)
Células Endoteliales/metabolismo , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica , Nucleótidos Cíclicos/metabolismo , Transducción de Señal , Línea Celular Tumoral , Técnicas de Cocultivo , Células Endoteliales/patología , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/genética , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Nucleótidos Cíclicos/genética
14.
Thromb Haemost ; 120(7): 1137-1141, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32483771
15.
Cancer Cell ; 37(1): 104-122.e12, 2020 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-31935369

RESUMEN

Eradicating tumor dormancy that develops following epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment of EGFR-mutant non-small cell lung cancer, is an attractive therapeutic strategy but the mechanisms governing this process are poorly understood. Blockade of ERK1/2 reactivation following EGFR TKI treatment by combined EGFR/MEK inhibition uncovers cells that survive by entering a senescence-like dormant state characterized by high YAP/TEAD activity. YAP/TEAD engage the epithelial-to-mesenchymal transition transcription factor SLUG to directly repress pro-apoptotic BMF, limiting drug-induced apoptosis. Pharmacological co-inhibition of YAP and TEAD, or genetic deletion of YAP1, all deplete dormant cells by enhancing EGFR/MEK inhibition-induced apoptosis. Enhancing the initial efficacy of targeted therapies could ultimately lead to prolonged treatment responses in cancer patients.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Senescencia Celular , Receptores ErbB/metabolismo , Femenino , Eliminación de Gen , Humanos , Neoplasias Pulmonares/patología , MAP Quinasa Quinasa 1/metabolismo , Masculino , Ratones , Ratones Noqueados , Mutación , Transducción de Señal , Transcripción Genética , Proteínas Señalizadoras YAP
17.
Cancer Res ; 80(1): 44-56, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31662325

RESUMEN

Triple-negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here, we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TANK-binding kinase 1 (TBK1)/IκB kinase ε (IKKε) on the same serine-72 (S72) site. An unbiased search for novel TBK1/IKKε substrates using stable isotope labeling with amino acids in cell culture phosphoproteomic analysis identified Rab7-S72 as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated stimulator of interferon genes (STING) degradation. Thus, convergence of PTEN loss and TBK1/IKKε activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFNß. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyperresponsive to STING agonists. Together, these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKε in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic. SIGNIFICANCE: These findings identify Rab7 as a substrate for TBK1 for regulation of innate immune signaling, thereby providing important insight for strategies aimed at manipulating the immune response to enhance therapeutic efficacy in TNBC.


Asunto(s)
Quinasa I-kappa B/metabolismo , Inmunidad Innata , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias de la Mama Triple Negativas/inmunología , Proteínas de Unión al GTP rab/metabolismo , Mama/inmunología , Mama/patología , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosforilación/genética , Fosforilación/inmunología , Proteolisis , Serina/genética , Serina/metabolismo , Transducción de Señal/inmunología , Neoplasias de la Mama Triple Negativas/patología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión a GTP rab7
19.
FASEB J ; 31(5): 1903-1915, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28174207

RESUMEN

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.


Asunto(s)
Diferenciación Celular , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Neuroblastoma/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Receptores ErbB/metabolismo , Proteoglicanos de Heparán Sulfato/farmacología , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Humanos , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Regulación hacia Arriba/efectos de los fármacos
20.
J Clin Invest ; 124(7): 3016-31, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24937430

RESUMEN

Neuroblastoma prognosis is dependent on both the differentiation state and stromal content of the tumor. Neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here, we identified critical growth-limiting components of the differentiating stroma secretome and designed a potential therapeutic strategy based on their central mechanism of action. We demonstrated that expression of heparan sulfate proteoglycans (HSPGs), including TßRIII, GPC1, GPC3, SDC3, and SDC4, is low in neuroblasts and high in the Schwannian stroma. Evaluation of neuroblastoma patient microarray data revealed an association between TGFBR3, GPC1, and SDC3 expression and improved prognosis. Treatment of neuroblastoma cell lines with soluble HSPGs promoted neuroblast differentiation via FGFR1 and ERK phosphorylation, leading to upregulation of the transcription factor inhibitor of DNA binding 1 (ID1). HSPGs also enhanced FGF2-dependent differentiation, and the anticoagulant heparin had a similar effect, leading to decreased neuroblast proliferation. Dissection of individual sulfation sites identified 2-O, 3-O-desulfated heparin (ODSH) as a differentiating agent, and treatment of orthotopic xenograft models with ODSH suppressed tumor growth and metastasis without anticoagulation. These studies support heparan sulfate signaling intermediates as prognostic and therapeutic neuroblastoma biomarkers and demonstrate that tumor stroma biology can inform the design of targeted molecular therapeutics.


Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Proteoglicanos de Heparán Sulfato/farmacología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparina/análogos & derivados , Heparina/química , Heparina/metabolismo , Heparina/farmacología , Humanos , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Ratones , Ratones SCID , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Neuroblastoma/patología , Pronóstico , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Células del Estroma/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
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