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Cancer remains one of the main causes of death in the world due to its increasing incidence and treatment difficulties. Although significant progress has been made in this field, innovative approaches are needed to reduce tumor incidence, progression, and spread. In particular, the development of cancer vaccines is currently ongoing as both a preventive and therapeutic strategy. This concept is not new, but few vaccines have been approved in oncology. Antigen-based vaccination emerges as a promising strategy, leveraging specific tumor antigens to activate the immune system response. However, challenges persist in finding suitable delivery systems and antigen preparation methods. Exosomes (EXs) are highly heterogeneous bilayer vesicles that carry several molecule types in the extracellular space. The peculiarity is that they may be released from different cells and may be able to induce direct or indirect stimulation of the immune system. In particular, EX-based vaccines may cause an anti-tumor immune attack or produce memory cells recognizing cancer antigens and inhibiting disease development. This review delves into EX composition, biogenesis, and immune-modulating properties, exploring their role as a tool for prevention and therapy in solid tumors. Finally, we describe future research directions to optimize vaccine efficacy and realize the full potential of EX-based cancer immunotherapy.
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BACKGROUND: Lung cancer (LC), including both non-small (NSCLC) and small (SCLC) subtypes, is currently treated with a combination of chemo- and immunotherapy. However, predictive biomarkers to identify high-risk patients are needed. Here, we explore the role of peripheral blood mononuclear cells (PBMCs) as a tool for novel biomarkers searching. METHODS: We analyzed the expression of the cGAS-STING pathway, a key DNA sensor that activates during chemotherapy, in PBMCs from LC patients divided into best responders (BR), responders (R) and non-responders (NR). The PBMCs were whole exome sequenced (WES). RESULTS: PBMCs from BR and R patients of LC cohorts showed the highest levels of STING (p < 0.0001) and CXCL10 (p < 0.0001). From WES, each subject had at least 1 germline/somatic alteration in a DDR gene and the presence of more DDR gene mutations correlated with clinical responses, suggesting novel biomarker implications. Thus, we tested the effect of the pharmacological DDR inhibitor (DDRi) in PBMCs and in three-dimensional spheroid co-culture of PBMCs and LC cell lines; we found that DDRi strongly increased cGAS-STING expression and tumor infiltration ability of immune cells in NR and R patients. Furthermore, we performed FACS analysis of PBMCs derived from LC patients from the BR, R and NR cohorts and we found that cytotoxic T cell subpopulations displayed the highest STING expression. CONCLUSIONS: cGAS-STING signaling activation in PBMCs may be a novel potential predictive biomarker for the response to immunotherapy and high levels are correlated with a better response to treatment along with an overall increased antitumor immune injury.
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Osimertinib is a third-generation tyrosine kinase inhibitor clinically approved for first-line treatment of EGFR-mutant non-small cell lung cancer (NSCLC) patients. Although an impressive drug response is initially observed, in most of tumors, resistance occurs after different time and an alternative therapeutic strategy to induce regression disease is currently lacking. The hyperactivation of MEK/MAPKs, is one the most common event identified in osimertinib-resistant (OR) NSCLC cells. However, in response to selective drug pressure, the occurrence of multiple mechanisms of resistance may contribute to treatment failure. In particular, the epithelial-to-mesenchymal transition (EMT) and the impaired DNA damage repair (DDR) pathways are recognized as additional cause of resistance in NSCLC thus promoting tumor progression. Here we showed that concurrent upregulation of ITGB1 and DDR family proteins may be associated with an increase of EMT pathways and linked to both osimertinib and MEK inhibitor resistance to cell death. Furthermore, this study demonstrated the existence of an interplay between ITGB1 and DDR and highlighted, for the first time, that combined treatment of MEK inhibitor with DDRi may be relevant to downregulate ITGB1 levels and increase cell death in OR NSCLC cells.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Receptores ErbB/metabolismo , Resistencia a Antineoplásicos/genética , Mutación , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Línea Celular TumoralRESUMEN
BACKGROUND: ATM is a multifunctional serine/threonine kinase that in addition to its well-established role in DNA repair mechanisms is involved in a number of signaling pathways including regulation of oxidative stress response and metabolic diversion of glucose through the pentose phosphate pathway. Oncogene-driven tumorigenesis often implies the metabolic switch from oxidative phosphorylation to glycolysis which provides metabolic intermediates to sustain cell proliferation. The aim of our study is to elucidate the role of ATM in the regulation of glucose metabolism in oncogene-driven cancer cells and to test whether ATM may be a suitable target for anticancer therapy. METHODS: Two oncogene-driven NSCLC cell lines, namely H1975 and H1993 cells, were treated with ATM inhibitor, KU55933, alone or in combination with oncogene driver inhibitors, WZ4002 or crizotinib. Key glycolytic enzymes, mitochondrial complex subunits (OXPHOS), cyclin D1, and apoptotic markers were analyzed by Western blotting. Drug-induced toxicity was assessed by MTS assay using stand-alone or combined treatment with KU55933 and driver inhibitors. Glucose consumption, pyruvate, citrate, and succinate levels were also analyzed in response to KU55933 treatment. Both cell lines were transfected with ATM-targeted siRNA or non-targeting siRNA and then exposed to treatment with driver inhibitors. RESULTS: ATM inhibition deregulates and inhibits glucose metabolism by reducing HKII, p-PKM2Tyr105, p-PKM2Ser37, E1α subunit of pyruvate dehydrogenase complex, and all subunits of mitochondrial complexes except ATP synthase. Accordingly, glucose uptake and pyruvate concentrations were reduced in response to ATM inhibition, whereas citrate and succinate levels were increased in both cell lines indicating the supply of alternative metabolic substrates. Silencing of ATM resulted in similar changes in glycolytic cascade and OXPHOS levels. Furthermore, the driver inhibitors amplified the effects of ATM downregulation on glucose metabolism, and the combined treatment with ATM inhibitors enhanced the cytotoxic effect of driver inhibitors alone by increasing the apoptotic response. CONCLUSIONS: Inhibition of ATM reduced both glycolytic enzymes and OXPHOS levels in oncogene-driven cancer cells and enhanced apoptosis induced by driver inhibitors thus highlighting the possibility to use ATM and the driver inhibitors in combined regimens of anticancer therapy in vivo.
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Here, we tested whether co-targeting of glucose metabolism and oncogene drivers may enhance tumor response to tyrosine kinase inhibitors (TKIs) in NSCLC. To this end, pyruvate dehydrogenase kinase 1 (PDK1) was stably downregulated in oncogene-driven NSCLC cell lines exposed or not to TKIs. H1993 and H1975 cells were stably transfected with scrambled (shCTRL) or PDK1-targeted (shPDK1) shRNA and then treated with MET inhibitor crizotinib (1 µM), double mutant EGFRL858R/T790M inhibitor WZ4002 (1 µM) or vehicle for 48 h. The effects of PDK1 knockdown on glucose metabolism and apoptosis were evaluated in untreated and TKI-treated cells. PDK1 knockdown alone did not cause significant changes in glycolytic cascade, ATP production and glucose consumption, but it enhanced maximal respiration in shPDK1 cells when compared to controls. When combined with TKI treatment, PDK1 downregulation caused a strong enhancement of OXPHOS and a marked reduction in key glycolytic enzymes. Furthermore, increased levels of apoptotic markers were found in shPDK1 cells as compared to shCTRL cells after treatment with TKIs. Co-immunoprecipitation studies showed that PDK1 interacts with PKM2, Bcl-2 and Bcl-xL, forming macromolecular complexes at the ER-mitochondria interface. Our findings showed that downregulation of PDK1 is able to potentiate the effects of TKIs through the disruption of macromolecular complexes involving PKM2, Bcl-2 and Bcl-xL.
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Notch1 plays a key role in epithelial-mesenchymal transition (EMT) and in the maintenance of cancer stem cells. In the present study we tested whether high levels of activated Notch1 in oncogene-driven NSCLC can induce a reversible shift of driver dependence from EGFR to Notch1, and thus causing resistance to EGFR inhibitors. Adherent cells (parental) and tumor spheres (TS) from NSCLC H1975 cells and patient-derived CD133-positive cells were tested for EGFR and Notch1 signaling cascade. The Notch1-dependent modulation of EGFR, NCID, Hes1, p53, and Sp1 were then analyzed in parental cells by binding assays with a Notch1 agonist, DLL4. TS were more resistant than parental cells to EGFR inhibitors. A strong upregulation of Notch1 and a concomitant downregulation of EGFR were observed in TS compared to parental cells. Parental cell exposure to DLL4 showed a dose-dependent decrease of EGFR and a simultaneous increase of NCID, Hes1, p53, and Sp1, along with the dislocation of Sp1 from the EGFR promoter. Furthermore, an enhanced interaction between p53 and Sp1 was observed in TS. In NSCLC cells, high levels of active Notch1 can promote a reversible shift of driver dependence from EGFR to Notch1, leading to resistance to EGFR inhibitors.
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The occurrence of phenotype switch from an epithelial to a mesenchymal cell state during the activation of the epithelial mesenchymal transition (EMT) program in cancer cells has been closely associated with the generation of invasive tumor cells that contribute to metastatic dissemination and treatment failure. Liquid biopsy represents an emergent non-invasive tool that may improve our understanding of the molecular events leading to cancer progression and initiating the metastatic cascade through the dynamic analysis of tumor-derived components isolated from body fluids. The present review will primarily focus on the applications of liquid biopsy in lung cancer patients for identifying EMT signature, elucidating molecular mechanisms underlying the acquisition of an invasive phenotype and detecting new targets for therapy.
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Among peritumoral cells, cancer-associated fibroblasts (CAFs) are major facilitators of tumor progression. This study describes the effects of two urokinase-derived, novel decapeptides, denoted as Pep 1 and its cyclic derivative Pep 2. In a mouse model of tumor dissemination, using HT1080 fibrosarcoma cells, Pep 2 reduced the number and size of lung metastases. Specific binding of fluoresceinated Pep 2 to HT1080 and telomerase immortalised fibroblasts (TIF) cell surfaces was enhanced by αv overexpression or abolished by excess vitronectin, anti-αv antibodies or silencing of ITGAV αv gene, identifying αv-integrin as the Pep 2 molecular target. In 3D-organotypic assays, peptide-exposed TIFs and primary CAFs from breast carcinoma patients both exhibited a markedly reduced pro-invasive ability of either HT1080 fibrosarcoma or MDA-MB-231 mammary carcinoma cells, respectively. Furthermore, TIFs, either exposed to Pep 2, or silenced for αv integrin, were impaired in their ability to chemoattract cancer cells and to contract collagen matrices, exhibiting reduced α-smooth muscle actin (α-SMA) levels. Finally, peptide exposure of αv-expressing primary CAFs led to the downregulation of α-SMA protein and to a dramatic reduction of their pro-invasive capability. In conclusion, the ability of the novel decapeptides to interfere with tumor cell invasion directly and through the down-modulation of CAF phenotype suggests their use as lead compounds for co-targeting anti-cancer strategies.
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Novel anticancer immunotherapy strategies such as immune checkpoint blockade have been successfully employed in patients with a variety of cancers and became a therapeutic option in the treatment of several malignancies. However, long-term durable responses to immune checkpoint inhibitors are currently limited to a fraction of patients raising the need of an accurate selection of potentially responding patients. Although several biomarkers have been proposed for patient selection and prediction of response to immune checkpoint blockade, none can be considered as an absolute selection criterion. Whole-body imaging with tracers recognizing targets for immunotherapy by allowing visualization of target expression in all tumor and extratumoral sites at baseline and during disease evolution may provide reliable predictive imaging biomarkers. Here we will provide an overview of preclinical imaging studies aiming at the development and validation of tracers recognizing targets for immunotherapy that can be used for selection and monitoring of patients undergoing immunotherapy and for testing novel immunotherapeutic agents or strategies. Furthermore, we will focus on the selection of animal models based on the main purpose of the study and implications for clinical transfer of the results.
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Diagnóstico por Imagen/métodos , Evasión Inmune , Neoplasias/diagnóstico por imagen , Neoplasias/inmunología , Animales , Humanos , Inmunoterapia , Trazadores RadiactivosRESUMEN
Preclinical imaging with radiolabeled probes can provide noninvasive tools to test the efficacy of targeted agents in tumors harboring specific genetic alterations and to identify imaging parameters that can be used as pharmacodynamics markers in cancer patients. The present review will primarily focus on preclinical imaging studies that can accelerate the clinical approval of targeted agents and promote the development of imaging biomarkers for clinical applications. Since only subgroups of patients may benefit from treatment with targeted anticancer agents, the identification of a patient population expressing the target is of primary importance for the success of clinical trials. Preclinical imaging studies tested the ability of new radiolabeled compounds to recognize mutant, amplified, or overexpressed targets and some of these tracers were transferred to the clinical setting. More common tracers such as 18F-Fluorothymidine and 18F-Fluorodeoxyglucose were employed in animal models to test the inhibition of the target and downstream pathways through the evaluation of early changes of proliferation and glucose metabolism allowing the identification of sensitive and resistant tumors. Furthermore, since the majority of patients treated with targeted anticancer agents will invariably develop resistance, preclinical imaging studies were performed to test the efficacy of reversal agents to overcome resistance. These studies provided consistent evidence that imaging with radiolabeled probes can monitor the reversal of drug resistance by newly designed alternative compounds. Finally, despite many difficulties and challenges, preclinical imaging studies targeting the expression of immune checkpoints proved the principle that it is feasible to select patients for immunotherapy based on imaging findings. In conclusion, preclinical imaging can be considered as an integral part of the complex translational process that moves a newly developed targeted agent from laboratory to clinical application intervening in all clinically relevant steps including patient selection, early monitoring of drug effects and reversal of drug resistance.
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Diagnóstico por Imagen/métodos , Neoplasias/diagnóstico por imagen , Neoplasias/radioterapia , Animales , Terapia Combinada , Resistencia a Antineoplásicos/efectos de la radiación , Terapia Molecular Dirigida , Resultado del TratamientoRESUMEN
Since many oncogenes, including BCR-ABL, may promote the acquisition and maintenance of the glycolytic phenotype, we tested whether treatment of BCR-ABL-driven human leukemia cells with imatinib, a selective BCR-ABL inhibitor, can modulate the expression of key glycolytic enzymes and mitochondrial complex subunits thus causing alterations of glucose metabolism. BCR-ABL-driven K562 and KCL-22 cells were incubated with increasing concentrations of imatinib to preliminarily test drug sensitivity. Then untreated and treated cells were analyzed for levels of BCR-ABL signaling mediators and key proteins of glycolytic cascade and oxidative phosphorylation. Effective inhibition of BCR-ABL caused a concomitant reduction of p-ERK1/2, p-AKT, phosphorylated form of STAT3 (at Tyr705 and Ser727), c-Myc and cyclin D1 along with an increase of cleaved PARP and caspase 3 at 48 h after treatment. Furthermore, a strong reduction of the hexokinase II (HKII), phosphorylated form of PKM2 (at Tyr105 and Ser37) and lactate dehydrogenase A (LDH-A) was observed in response to imatinib along with a strong upregulation of mitochondrial complexes (OXPHOS). According to these findings, a significant reduction of glucose consumption and lactate secretion along with an increase of intracellular ATP levels was observed in response to imatinib. Our findings indicate that imatinib treatment of BCR-ABL-driven human leukemia cells reactivates mitochondrial oxidative phosphorylation thus allowing potential co-targeting of BCR-ABL and OXPHOS.
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Antineoplásicos/farmacología , Glucólisis/efectos de los fármacos , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Ciclina D1/metabolismo , Humanos , Ácido Láctico/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Neutrophil extracellular traps (NETs), in addition to their function as a host defense mechanism, play a relevant role in thrombus formation and metastatic dissemination of cancer cells. Here we screened different cancer cell lines endogenously expressing a variety of integrins for their ability to bind to NETs. To this end, we used NETs isolated from neutrophil-like cells as a substrate for adhesion assays of HT1080, U-87 MG, H1975, DU 145, PC-3 and A-431 cells. Levels of α5, αIIb, αv, ß1, ß3 and ß5 chains were determined by western blot analysis in all cell lines and levels of whole integrins on the plasma membrane were assessed by fluorescence-activated cell sorting (FACS) analysis. We found that high levels of α5ß1, αvß3 and αvß5 enhance cell adhesion to NETs, whereas low expression of α5ß1 prevents cell attachment to NETs. Excess of cyclic RGD peptide inhibited cell adhesion to NETs by competing with fibronectin within NETs. The maximal reduction of such adhesion was similar to that obtained by DNase 1 treatment causing DNA degradation. Our findings indicate that NETs from neutrophil-like cells may be used as a substrate for large screening of the adhesion properties of cancer cells expressing a variety of RGD-binding integrins.
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Adhesión Celular/genética , Cadenas alfa de Integrinas/genética , Cadenas beta de Integrinas/genética , Transporte de Proteínas/genética , Membrana Celular , Trampas Extracelulares , Fibronectinas/genética , Citometría de Flujo , Humanos , Neutrófilos/citología , Neutrófilos/metabolismo , Células PC-3 , Péptidos/genética , Unión ProteicaRESUMEN
Purpose: Our aim was to test whether imaging with 18F-fluorothymidine (18F-FLT) PET/CT was able to detect the combined effects of EGFR and MET inhibitors in oncogene-driven non-small cell lung cancer (NSCLC) and to elucidate the mechanisms underlying the enhanced efficacy of drug combination.Experimental Design: NSCLC cells bearing MET amplification (H1993 and H820) were treated with EGFR and MET inhibitors either alone or in combination and then tested for cell viability and inhibition of signaling. Nude mice bearing H1993 tumors underwent 18F-FLT PET/CT scan before and after treatment with erlotinib and crizotinib alone or in combination (1:1 ratio) and posttreatment changes of 18F-FLT uptake in tumors were determined. The role of inositol trisphosphate receptor type 3 (IP3R3) in mediating the combined action of EGFR and MET inhibitors was tested by transfecting NSCLC cells with IP3R3-targeted siRNA.Results: Imaging studies showed a significant reduction of 18F-FLT uptake in response to combined treatment with EGFR and MET inhibitors that was higher than that obtained with single agents (ANOVA, F-ratio = 6.215, P = 0.001). Imaging findings were confirmed by analysis of surgically excised tumors. Levels of IP3R3 were significantly reduced in both cells and tumors after treatment with crizotinib, whereas EGFR inhibitors caused a reduction of IP3R3 interaction with K-Ras mainly through dephosphorylation of serine residues of K-Ras.Conclusions: Our findings indicate that 18F-FLT PET/CT is able to detect the enhanced efficacy of EGFR and MET inhibitors in oncogene-driven NSCLC and that such enhancement is mediated by IP3R3 through its interaction with K-Ras. Clin Cancer Res; 24(13); 3126-36. ©2018 AACR.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Didesoxinucleósidos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/genética , Ratones , Terapia Molecular Dirigida , Mutación , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neutrophil extracellular traps (NETs), originally recognized as a host defense mechanism, were reported to promote thrombosis and metastatic dissemination of cancer cells. Here we tested the role of integrins α5ß1 and ανß3 in the adhesion of cancer cells to NETs. Neutrophil-like cells stimulated with calcium ionophore (A23187) were used as a stable source of cell-free NETs-enriched suspensions. Using NETs as an adhesion substrate, two human K562 cell lines, differentially expressing α5ß1 and ανß3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5ß1 or ανß3, alone or in combination with DNAse 1, and Proteinase K. As expected DNAse 1 treatment strongly inhibited adhesion of both cell lines to NETs. An equivalent significant reduction of cell adhesion to NETs was obtained after treatment of cells with blocking antibodies against α5ß1 or ανß3 indicating that both integrins were able to mediate cell adhesion to NETs. Furthermore, the combination of DNAse 1 and anti-integrin antibody treatment almost completely blocked cell adhesion. Western blot analysis and immunoprecipitation experiments showed a dose-dependent increase of fibronectin levels in samples from stimulated neutrophil-like cells and a direct or indirect interaction of fibronectin with histone H3. Finally, co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. In conclusion, our study showed that α5ß1 and ανß3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin. Therefore, in addition to mechanical trapping and aspecific adsorption of different cell types driven by DNA/histone complexes, NETs may provide specific binding sites for integrin-mediated cell adhesion of neutrophils, platelets, endothelial and cancer cells thus promoting intimate interactions among these cells.
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Adhesión Celular , Trampas Extracelulares/metabolismo , Integrinas/metabolismo , Neutrófilos/fisiología , Biomarcadores , Fibronectinas/metabolismo , Histonas/metabolismo , Humanos , Peroxidasa/metabolismo , Unión Proteica , Transporte de ProteínasRESUMEN
Preclinical imaging with radiolabeled probes became an integral part of the complex translational process that moves a newly developed compound from laboratory to clinical application. Imaging studies in animal tumor models may be undertaken to test a newly synthesized tracer, a newly developed drug or to interrogate, in the living organism, specific molecular and biological processes underlying tumor growth and progression. The aim of the present review is to outline the current knowledge and future perspectives of preclinical imaging in oncology by providing examples from recent literature. Among the biological processes and molecular targets that can be visualized with radiolabeled probes in animal tumor models, we focused on proliferation, expression of targets suitable for therapy, glycolytic phenotype, metastatic dissemination, tumor angiogenesis and survival. The major contribution of preclinical imaging emerging from these studies is the development and validation of imaging biomarkers that can be translated into the clinical context for patient selection and evaluation of tumor response to molecularly targeted agents.
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Diagnóstico por Imagen/métodos , Neoplasias/diagnóstico por imagen , Animales , Proliferación Celular , Humanos , Inmunoterapia , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neoplasias/terapia , Neovascularización Patológica , Análisis de SupervivenciaRESUMEN
BACKGROUND: The two main mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) are the occurrence of T790M secondary mutation in the kinase domain of EGFR and MET amplification. The aim of the present study was to test whether early changes of 18F-fluorodeoxyglucose (18F-FDG) uptake in animal models bearing erlotinib-resistant NSCLC may have different imaging patterns of response to erlotinib depending on the molecular mechanisms underlying resistance. Animal tumor models were developed using NSCLC H1975 cells bearing the T790M mutation and H1993 cells with MET amplification. Nude mice bearing erlotinib-resistant H1975 and H1993 xenografts (four animals for each cell line and for each treatment) were subjected to 18F-FDG PET/CT scan before and immediately after treatment (50 mg/kg p.o. for 3 days) with erlotinib, WZ4002, crizotinib, or vehicle. A three-dimensional region of interest analysis was performed to determine the percent change of 18F-FDG uptake in response to treatment. At the end of the imaging studies, tumors were removed and analyzed for glycolytic and mitochondrial proteins as well as levels of cyclin D1. RESULTS: Imaging studies with 18F-FDG PET/CT in H1975 tumor-bearing mice showed a reduction of 18F-FDG uptake of 25.87 % ± 8.93 % after treatment with WZ4002 whereas an increase of 18F-FDG uptake up to 23.51 % ± 9.72 % was observed after treatment with erlotinib or vehicle. Conversely, H1993 tumors showed a reduction of 18F-FDG uptake after treatment with both crizotinib (14.70 % ± 1.30 %) and erlotinib (18.40 % ± 9.19 %) and an increase of tracer uptake in vehicle-treated (56.65 % ± 5.65 %) animals. The in vivo reduction of 18F-FDG uptake was always associated with downregulation of HKII and p-PKM2 Tyr105 glycolytic proteins and upregulation of mitochondrial complexes (subunits I-IV) in excised tumors. CONCLUSIONS: 18F-FDG uptake is a reliable imaging biomarker of T790M-mediated resistance and its reversal in NSCLC whereas it may not be accurate in the detection of MET-mediated resistance.
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PURPOSE: One of the hallmarks of cancer cells is the excessive conversion of glucose to lactate under normoxic conditions, also known as the Warburg effect. Here, we tested whether the targeted inhibition of EGFR may revert this effect and reactivate mitochondrial oxidative phosphorylation in non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Sensitive (HCC827) and resistant (H1975 and H1993) NSCLC cells were treated with a panel of EGFR or MET inhibitors, and then tested for changes of EGFR signaling, glycolytic cascade, and mitochondrial function. Silencing of key glycolytic enzymes was then performed with targeted siRNAs. Furthermore, tumor-bearing nude mice treated with EGFR inhibitors were evaluated with (18)F-FDG PET/CT and tumors were analyzed for glycolytic and mitochondrial proteins. RESULTS: Effective inhibition of EGFR signaling in NSCLC cells induced a dramatic reduction of hexokinase II (HKII) and phospho-pyruvate kinase M2 (p-PKM2, Tyr105) levels as well as an upregulation of mitochondrial complexes subunits (OXPHOS). Accordingly, a decreased lactate secretion and increased intracellular ATP levels were also observed in response to EGFR inhibitors. Downregulation of HKII and PKM2 by targeted siRNA transfection did not cause upregulation of OXPHOS but enhanced the effects of EGFR TKIs. Conversely, selective inhibition of AKT and ERK1/2 caused OXPHOS upregulation and glycolysis inhibition, respectively. Similar findings were obtained in tumors from animals treated with appropriate EGFR inhibitors. CONCLUSIONS: Our findings indicate that EGFR inhibitors may reactivate oxidative phosphorylation of cancer cells and provide a mechanistic clue for the rational combination of agents targeting EGFR-dependent proliferation and glucose metabolism in cancer therapy.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Receptores ErbB/genética , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas c-met/genética , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Glucosa/metabolismo , Humanos , Ácido Láctico/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Fosforilación Oxidativa/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: MET amplification is one of the mechanisms underlying acquired resistance to EGFR tyrosine kinase inhibitors (TKI) in non-small cell lung cancer (NSCLC). Here, we tested whether 3'-deoxy-3'-[(18)F]-fluorothymidine ([(18)F]FLT) positron emission tomography/computerized tomography (PET/CT) can detect MET-mediated resistance to EGFR TKIs and monitor the effects of MET inhibitors in NSCLC. EXPERIMENTAL DESIGN: H1993 and H820 NSCLC cells with high and low levels of MET amplification, respectively, and HCC827-expressing MET, but without gene amplification, were tested for the effects of MET inhibitors on the EGFR pathway and proliferation both in vitro and in vivo. Nude mice bearing NSCLCs with and without MET amplification were subjected to [(18)F]FLT PET/CT before and after treatment with crizotinib or erlotinib (50 mg/kg and 100 mg/kg p.o. for 3 days). RESULTS: H1993 cells showed high responsiveness to MET inhibitors and were resistant to erlotinib. Conversely, HCC827 cells showed high sensitivity to erlotinib and were resistant to MET inhibitors. Accordingly, H1993 tumors bearing MET amplification showed a mean reduction in [(18)F]FLT uptake of 28% and 41% after low- and high-dose treatment with crizotinib for 3 days, whereas no posttherapy changes of [(18)F]FLT uptake were observed in HCC827 tumors lacking MET amplification. Furthermore, a persistently high [(18)F]FLT uptake was observed in H1993 tumors after treatment with erlotinib, whereas HCC827 tumors showed up to 39% reduction of [(18)F]FLT uptake following erlotinib treatment. Imaging findings were confirmed by Ki67 immunostaining of tumor sections. CONCLUSIONS: [(18)F]FLT PET/CT can detect MET-mediated resistance to EGFR TKIs and its reversal by MET inhibitors in NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico por imagen , Resistencia a Antineoplásicos/genética , Radioisótopos de Flúor , Neoplasias Pulmonares/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Radiofármacos , Animales , Antineoplásicos/farmacología , Western Blotting , Línea Celular Tumoral , Crizotinib , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/genética , Pirazoles/farmacología , Piridinas/farmacología , Quinazolinas/farmacología , Interferencia de ARN , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: Src tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. To identify the mechanism by which Src overexpression sustains this resistance, we tested a panel of breast cancer cell lines either sensitive or resistant to lapatinib. METHODS: To determine the role of Src in lapatinib resistance, we evaluated the effects of Src inhibition/silencing in vitro on survival, migration, and invasion of lapatinib-resistant cells. In vivo experiments were performed in JIMT-1 lapatinib-resistant cells orthotopically implanted in nude mice. We used artificial metastasis assays to evaluate the effect of Src inhibition on the invasiveness of lapatinib-resistant cells. Src-dependent signal transduction was investigated with Western blot and ELISA analyses. RESULTS: Src activation was higher in lapatinib-resistant than in lapatinib-sensitive cells. The selective small-molecule Src inhibitor saracatinib combined with lapatinib synergistically inhibited the proliferation, migration, and invasion of lapatinib-resistant cells. Saracatinib combined with lapatinib significantly prolonged survival of JIMT-1-xenografted mice compared with saracatinib alone, and impaired the formation of lung metastases. Unexpectedly, in lapatinib-resistant cells, Src preferentially interacted with epidermal growth factor receptor (EGFR) rather than with HER2. Moreover, EGFR targeting and lapatinib synergistically inhibited survival, migration, and invasion of resistant cells, thereby counteracting Src-mediated resistance. These findings demonstrate that Src activation in lapatinib-resistant cells depends on EGFR-dependent rather than on HER2-dependent signaling. CONCLUSIONS: Complete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Familia-src Quinasas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzodioxoles/farmacología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Receptores ErbB/metabolismo , Femenino , Humanos , Lapatinib , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica/genética , Trasplante de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Receptor ErbB-2/metabolismo , Transducción de Señal/genética , Trasplante Heterólogo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
UNLABELLED: Epidermal growth factor receptor (EGFR) mutational status, activation of downstream signaling, and effective apoptotic cascade are all factors that may affect the tumor response to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Here we test whether 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET/CT can provide clues for the selection of patients with NSCLC as candidates for treatment with reversible and irreversible EGFR TKIs or combination treatment with Bcl-x(L) inhibitors. METHODS: HCC827, H1975, and H1650 NSCLC cells were subcutaneously injected into flanks of nude mice. Tumor-bearing animals were treated daily for 3 d by oral gavage with erlotinib at 50 and 150 mg/kg, CL-387,785 (an irreversible EGFR TKI) at 50 mg/kg, WZ4002 (a more potent irreversible EGFR TKI) at 25 and 50 mg/kg, ABT-263 (a Bcl-x(L) inhibitor) at 6.25 mg/kg, and a combination of erlotinib (50 mg/kg) and ABT-263 (6.25 mg/kg). Imaging studies were performed before and after 3 d of treatment by intravenous injection of 7.4 MBq of (18)F-FLT and small-animal PET/CT of animals at 1 h after injection. Quantitative analysis of reconstructed images of baseline and posttreatment scans was performed, and the percentage change in (18)F-FLT uptake in each animal was determined. Tumor sections were tested for Ki67 immunostaining and the percentage of apoptotic cells. RESULTS: Sensitive tumors (HCC827) showed mean decreases in (18)F-FLT uptake of 45% and 28% with high- and low-dose regimens of erlotinib, respectively. Resistant NSCLC cells bearing a T790M mutation (H1975) showed mean increases in (18)F-FLT uptake of 27% and 33% with high and low doses of erlotinib, respectively. Treatment with CL-387,785, low-dose WZ4002, and high-dose WZ4002 caused mean decreases in tracer uptake of 21%, 26%, and 36%, respectively. NSCLC cells that were resistant because of dysregulation of Bcl-2 family members (H1650) showed mean reductions in (18)F-FLT uptake of 49% and 23% with high and low doses of erlotinib, respectively, whereas the addition of ABT-263 did not affect tracer uptake but significantly increased the percentage of apoptotic cells in tumor sections. CONCLUSION: PET/CT with (18)F-FLT may contribute to the selection of patients who may benefit from treatment with reversible and irreversible EGFR TKIs and may provide clues about which patients with NSCLC may be candidates for combination treatment with erlotinib and Bcl-x(L) inhibitors.