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Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated to asbestos exposure. One of the most frequent genetic alteration in MPM patients is CDKN2A/ARF loss, leading to aberrant activation of the Rb pathway. In MPM cells, we previously demonstrated the therapeutic efficacy of targeting this signaling with the CDK4/6 inhibitor palbociclib in combination with PI3K/mTOR inhibitors. Here, we investigated whether such combination may have an impact on cell energy metabolism. Methods: The study was performed in MPM cells of different histotypes; metabolic analyses were conducted by measuring GLUT-1 expression and glucose uptake/consumption, and by SeaHorse technologies. Results: MPM cell models differed for their ability to adapt to metabolic stress conditions, such as glucose starvation and hypoxia. Independently of these differences, combined treatments with palbociclib and PI3K/mTOR inhibitors inhibited cell proliferation more efficaciously than single agents. The drugs alone reduced glucose uptake/consumption as well as glycolysis, and their combination further enhanced these effects under both normoxic and hypoxic conditions. Moreover, the drug combinations significantly impaired mitochondrial respiration as compared with individual treatments. These metabolic effects were mediated by the concomitant inhibition of Rb/E2F/c-myc and PI3K/AKT/mTOR signaling. Conclusions: Dual blockade of glycolysis and respiration contributes to the anti-tumor efficacy of palbociclib-PI3K/mTOR inhibitors combination.
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Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Metabolismo Energético/efeitos dos fármacos , Mesotelioma Maligno/metabolismo , Neoplasias Pleurais/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Linhagem Celular Tumoral , Glicólise/efeitos dos fármacos , Humanos , Mesotelioma Maligno/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Piperazinas/farmacologia , Neoplasias Pleurais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Prostaglandin E2 (PGE2), a key mediator of immunity, inflammation, and cancer, acts through 4 G-protein-coupled E-prostanoid receptors (EPs 1-4). Crosstalk between EPs and receptor tyrosine kinases also occurs. Colony-stimulating factor-1 receptor (CSF-1R) is an RTK that sustains the survival, proliferation, and motility of monocytes/macrophages, which are an essential component of innate immunity and cancer development. The aim of this study was to investigate on a possible crosstalk between EP and CSF-1R. In BAC1.2F5 and RAW264.7 murine macrophages, CSF-1 (EC50 = 18.1 and 10.2 ng/ml, respectively) and PGE2 (EC50 = 1.5 and 5.5 nM, respectively) promoted migration. PGE2 induced rapid CSF-1R phosphorylation that was dependent on Src family kinases (SFKs). CSF-1R inhibition reduced PGE2-elicited ERK1/2 phosphorylation and macrophage migration, indicating that CSF-1R plays a role in PGE2-mediated immunoregulation. EP4 appeared responsible for functional PGE2/CSF-1R crosstalk. Furthermore, PGE2 synergized with CSF-1 in inducing ERK1/2 phosphorylation and macrophage migration. ERK1/2 inhibition completely blocked migration induced by the combination CSF-1/PGE2. CSF-1/PGE2 functional interaction with respect to migration also occurred in bone marrow-derived murine macrophages (EC50 CSF-1, 6.7 ng/ml; EC50 PGE2, 16.7 nM). These results indicated that PGE2 transactivates CSF-1R and synergizes with its signaling at ERK1/2 level in promoting macrophage migration.
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Movimento Celular/efeitos dos fármacos , Dinoprostona/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Immunoblotting , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Osimertinib, a tyrosine kinase inhibitor targeting mutant EGFR, has received approval for initial treatment in patients with Non-Small Cell Lung Cancer (NSCLC). While effective in both first- and second-line treatments, patients eventually develop acquired resistance. Metabolic reprogramming represents a strategy through which cancer cells may resist and adapt to the selective pressure exerted by the drug. In the current study, we investigated the metabolic adaptations associated with osimertinib-resistance in NSCLC cells under low glucose culture conditions. We demonstrated that, unlike osimertinib-sensitive cells, osimertinib-resistant cells were able to survive under low glucose conditions by increasing the rate of glucose and glutamine uptake and by shifting towards mitochondrial metabolism. Inhibiting glucose/pyruvate contribution to mitochondrial respiration, glutamine deamination to glutamate, and oxidative phosphorylation decreased the proliferation and survival abilities of osimertinib-resistant cells to glucose starvation. Our findings underscore the remarkable adaptability of osimertinib-resistant NSCLC cells in a low glucose environment and highlight the pivotal role of mitochondrial metabolism in mediating this adaptation. Targeting the metabolic adaptive responses triggered by glucose shortage emerges as a promising strategy, effectively inhibiting cell proliferation and promoting cell death in osimertinib-resistant cells.
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Many cytokines control tumor development by directly lowering cancer cell proliferation and inducing apoptotic cell death, or indirectly by activating the antitumoral activity of specific immune cells such as NK or CD8+ T-lymphocytes [...].
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Citocinas , Neoplasias , Humanos , Citocinas/metabolismo , Linfócitos T CD8-Positivos , Células Matadoras Naturais , Neoplasias/tratamento farmacológicoRESUMO
Hepatoblastoma (HB) and pediatric hepatocellular carcinoma (HCC) are rare primary malignant liver cancers in children and young adults. HB is the most common and accounts for about 70 % cases; it is usually diagnosed during the first 3 years of life. Instead, pediatric HCC is uncommon, and it is associated with a poor prognosis. Overall, the prognosis of pediatric HCC is dismal with 5-year event-free survival of <30 % as compared to >80 % for HB. Surgery approaches, either resection or transplant, remain the best chance for the cure of pediatric HCC. However, chemotherapy can be helpful as an adjuvant or neoadjuvant treatment. International groups have done trials in pediatric HCC with a chemotherapy regimen, based on cisplatin and doxorubicin (PLADO) as for HB, but the efficacy is limited. Sorafenib, a multi-kinase inhibitor, following positive results in adults and in a pilot study in children, is now tested in conjunction with chemotherapy in the PHITT phase III clinical trial. Some studies have been exploring the genetic profiles of patients to find biological hallmarks that determine the aggressiveness of pediatric HCC. Pathways involved in growth and differentiation are dysregulated and as demonstrated in HB and adult HCC, an important role of the Wnt/CTNNB1 pathway in the pathogenesis of pediatric HCC is also emerging. An extended molecular analysis of tumor samples could give information about pathways as possible targets of biological and immunotherapeutic agents bringing new pharmacological options for the treatment of pediatric HCC.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Hepatoblastoma , Neoplasias Hepáticas , Criança , Adulto Jovem , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Projetos Piloto , Hepatoblastoma/tratamento farmacológico , Hepatoblastoma/genética , Antineoplásicos/uso terapêuticoRESUMO
Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly devastating disease with rising incidence and poor prognosis. The lack of reliable prognostic biomarkers hampers the individual evaluation of the survival and recurrence potential. Methods: Here, we investigate the value of plasma levels of two potential key players in molecular mechanisms underlying PDAC aggressiveness and immune evasion, soluble TGF-beta (sTGF-beta) and sPD-L1, in both metastatic and radically-resected PDAC. To this aim we prospectively enrolled 38 PDAC patients and performed appropriate statistical analyses in order to evaluate their correlation, and role in the prediction of disease relapse/progression, and patients' outcome. Results: Metastatic patients showed lower levels of circulating sTGF-beta and higher levels of sPD-L1 compared to radically-resected patients. Moreover, a decrease in sTGF-beta levels (but not sPD-L1) was significantly associated with disease relapse in radically-resected patients. We also observed lower sTGF-beta at disease progression after first-line chemotherapy in metastatic patients, though this change was not statistically significant. We found a significant correlation between the levels of sTGF-beta and sPD-L1 before first-line chemotherapy. Conclusions: These findings support the possible interaction of TGF-beta and PD-L1 pathways and suggest that sTGF-beta and sPD-L1 might synergize and be new potential blood-based biomarkers.
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BACKGROUND: The loss of the CDKN2A/ARF (cyclin-dependent kinase inhibitor 2A/alternative reading frame) gene is the most common alteration in malignant pleural mesothelioma (MPM), with an incidence of about 70%, thus representing a novel target for mesothelioma treatment. In the present study, we evaluated the antitumor potential of combining the standard chemotherapy regimen used for unresectable MPM with the CDK4/6 (cyclin-dependent kinase 4 or 6) inhibitor abemaciclib. METHODS: Cell viability, cell death, senescence, and autophagy induction were evaluated in two MPM cell lines and in a primary MPM cell culture. RESULTS: The simultaneous treatment of abemaciclib with cisplatin and pemetrexed showed a greater antiproliferative effect than chemotherapy alone, both in MPM cell lines and in primary cells. This combined treatment induced cellular senescence or autophagic cell death, depending on the cell type. More in detail, the induction of cellular senescence was related to the increased expression of p21, whereas autophagy induction was due to the impairment of the AKT/mTOR signaling. Notably, the effect of the combination was irreversible and no resumption in tumor cell proliferation was observed after drug withdrawal. CONCLUSION: Our results demonstrated the therapeutic potential of CDK4/6 inhibitors in combination with chemotherapy for the treatment of MPM and are consistent with the recent positive results in the MiST2 arm in abemaciclib-treated patients.
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Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer with a poor prognosis and limited treatment options. Considering that alterations of the CDK4/6-cyclin D-Rb pathway occur frequently in HCC, we tested the efficacy of two CDK4/6 inhibitors, abemaciclib and ribociclib, in combination with lenvatinib, a multi-kinase inhibitor approved as first-line therapy for advanced HCC, in a panel of HCC Rb-expressing cell lines. The simultaneous drug combinations showed a superior anti-proliferative activity as compared with single agents or sequential schedules of treatment, either in short or in long-term experiments. In addition, the simultaneous combination of abemaciclib with lenvatinib reduced 3D cell growth, and impaired colony formation and cell migration. Mechanistically, these growth-inhibitory effects were associated with a stronger down-regulation of c-myc protein expression. Depending on the HCC cell model, reduced activation of MAPK, mTORC1/p70S6K or src/FAK signaling was also observed. Abemaciclib combined with lenvatinib arrested the cells in the G1 cell cycle phase, induced p21 accumulation, and promoted a stronger increase of cellular senescence, associated with elevation of ß-galactosidase activity and accumulation of ROS, as compared with single treatments. After drug withdrawal, the capacity of forming colonies was significantly impaired, suggesting that the anti-tumor efficacy of abemaciclib and lenvatinib combination was persistent. Our pre-clinical results demonstrate the effectiveness of the simultaneous combination of CDK4/6 inhibitors with lenvatinib in HCC cell models, suggesting that this combination may be worthy of further investigation as a therapeutic approach for the treatment of advanced HCC.
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Pancreatic Ductal Adenocarcinoma (PDAC) is one of the deadliest solid tumors and is estimated to become a leading cause of cancer-related death in coming years. Despite advances in surgical approaches and the emergence of new chemotherapy options, its poor prognosis has not improved in the last decades. The current treatment for PDAC is the combination of cytotoxic chemotherapy agents. However, PDAC shows resistance to many antineoplastic therapies with rapid progression. Although PDAC represents a heterogeneous disease, there are common alterations including oncogenic mutations of KRAS, and the frequent inactivation of different cell cycle regulators including the CDKN2A tumor suppressor gene. An emerging field of investigation focuses on inhibiting the function of proteins that suppress the immune checkpoint PD-1/PD-L1, with activation of the endogenous immune response. To date, all conventional immunotherapies have been less successful in patients with PDAC compared to other tumors. The need for new targets, associated with an extended molecular analysis of tumor samples could give new pharmacological options for the treatment of PDAC. It is, therefore, important to push for a broader molecular approach in PDAC research. Here, we provide a selected summary of emerging strategy options for targeting PDAC using CDK4/6 inhibitors, RAS inhibitors, and new drug combinations with immune checkpoint agents.
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Advanced hepatocarcinoma (HCC) is an aggressive malignancy with poor prognosis and limited treatment options. Alterations of the cyclin D-CDK4/6-Rb pathway occur frequently in HCC, providing the rationale for its targeting at least in a molecular subset of HCC. In a panel of HCC cell lines, we investigated whether the CDK4/6 inhibitor palbociclib might improve the efficacy of regorafenib, a powerful multi-kinase inhibitor approved as second-line treatment for advanced HCC after sorafenib failure and currently under clinical investigation as first-line therapy in combination with immunotherapy. In Rb-proficient cells, the simultaneous drug combination, but not the sequential schedules, inhibited cell proliferation, either in short or in long-term experiments, and induced cell death more strongly than individual treatments. Moreover, the combination significantly reduced spheroid cell growth and inhibited cell migration/invasion. The superior efficacy of palbociclib plus regorafenib emerged also under hypoxia and was associated with a significant down-regulation of CDK4/6-Rb-myc and mTORC1/p70S6K signaling. Moreover, regorafenib suppressed palbociclib-induced expression of cyclin D1 contributing to the cytotoxic effects of the combination. Besides these inhibitory effects on cell viability/proliferation, palbociclib and regorafenib reduced glucose uptake, although this effect was dependent on the cell model and on the oxygen availability (normoxia or hypoxia). Palbociclib and regorafenib combination impaired glucose uptake and utilization, down-regulating basal and hypoxia-induced expression of HIF-1α, HIF-2α, GLUT-1, and MCT4 proteins as well as the activity/expression of glycolytic enzymes (HK2, PFKP, aldolase A, PKM2). In addition, regorafenib alone reduced mitochondrial respiration. The combined treatment impaired glucose metabolism and respiration without enhancing the effects of the single agents. Our findings provide pre-clinical evidence for the effectiveness of palbociclib and regorafenib combination in HCC cell models.
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Abemaciclib is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6 that inhibits the transition from the G1 to the S phase of the cell cycle by blocking downstream CDK4/6-mediated phosphorylation of Rb. The effects of abemaciclib alone or combined with the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib were examined in a panel of PC9 and HCC827 osimertinib-resistant non-small cell lung cancer (NSCLC) cell lines carrying EGFR-dependent or -independent mechanisms of intrinsic or acquired resistance. Differently from sensitive cells, all the resistant cell lines analyzed maintained p-Rb, which may be considered as a biomarker of osimertinib resistance and a potential target for therapeutic intervention. In these models, abemaciclib inhibited cell growth, spheroid formation, colony formation, and induced senescence, and its efficacy was not enhanced in the presence of osimertinib. Interestingly, in osimertinib sensitive PC9, PC9T790M, and H1975 cells the combination of abemaciclib with osimertinib significantly inhibited the onset of resistance in long-term experiments. Our findings provide a preclinical support for using abemaciclib to treat resistance in EGFR mutated NSCLC patients progressed to osimertinib either as single treatment or combined with osimertinib, and suggest the combination of osimertinib with abemaciclib as a potential approach to prevent or delay osimertinib resistance in first-line treatment.
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Immunotherapy has significantly changed the treatment landscape for advanced non-small-cell lung cancer (NSCLC) with the introduction of drugs targeting programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1). In particular, the addition of the anti-PD-1 antibody pembrolizumab to platinum-pemetrexed chemotherapy resulted in a significantly improved overall survival in patients with non-squamous NSCLC, regardless of PD-L1 expression. In this preclinical study, we investigated whether chemotherapy can modulate PD-L1 expression in non-squamous NSCLC cell lines, thus potentially affecting immunotherapy efficacy. Among different chemotherapeutic agents tested, only pemetrexed increased PD-L1 levels by activating both mTOR/P70S6K and STAT3 pathways. Moreover, it also induced the secretion of cytokines, such as IFN-γ and IL-2, by activated peripheral blood mononuclear cells PBMCs that further stimulated the expression of PD-L1 on tumor cells, as demonstrated in a co-culture system. The anti-PD-1/PD-L1 therapy enhanced T cell-mediated cytotoxicity of NSCLC cells treated with pemetrexed and expressing high levels of PD-L1 in comparison with untreated cells. These data may explain the positive results obtained with pemetrexed-based chemotherapy combined with pembrolizumab in PD-L1-negative NSCLC and can support pemetrexed as one of the preferable chemotherapy partners for immunochemotherapy combination regimens.
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The recent approval of immune checkpoint inhibitors drastically changed the standard treatments in many advanced cancer patients, but molecular changes within the tumor can prevent the activity of immunotherapy drugs. Thus, the introduction of the inhibitors of the immune checkpoint programmed death-1/programmed death ligand-1 (PD-1/PD-L1), should prompt deeper studies on resistance mechanisms, which can be caused by oncogenic mutations detected in cancer cells. PTEN, a tumor suppressor gene, dephosphorylates the lipid signaling intermediate PIP3 with inhibition of AKT activity, one of the main effectors of the PI3K signaling axis. As a consequence of genetic or epigenetic aberrations, PTEN expression is often altered, with increased activation of PI3K axis. Interestingly, some data confirmed that loss of PTEN expression modified the pattern of cytokine secretion creating an immune-suppressive microenvironment with increase of immune cell populations that can promote tumor progression. Moreover, PTEN loss may be ascribed to reduction of tumor infiltrating lymphocytes (TILs), which can explain the absence of activity of immune checkpoint inhibitors. This review describes the role of PTEN loss as a mechanism responsible for resistance to anti PD-1/PD-L1 treatment. Moreover, combinatorial strategies between PD-1/PD-L1 inhibitors and PI3K/AKT targeting drugs are proposed as a new strategy to overcome resistance to immune checkpoint inhibition.
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BACKGROUND: Osimertinib is a new third-generation, epidermal growth factor receptor-tyrosine kinase inhibitor highly selective for the epidermal growth factor receptor with both activating and T790M mutations. A recent phase III trial showed a statistically significant progression-free survival benefit with osimertinib vs. gefitinib or erlotinib as first-line treatment for EGFR-mutated non-small cell lung cancer, and preliminary data are available on resistance mechanisms to first-line osimertinib therapy. OBJECTIVE: The objective of this study was to examine potential in vitro mechanisms of acquired resistance to osimertinib in a cell model carrying an EGFR exon 19 deletion. METHODS: PC9 cells were cultured in the presence of increasing concentrations of osimertinib (ranging from 10 to 500 nM) to generate resistant cells. Three clones resistant to osimertinib (half maximal inhibitory concentration > 1 µM) were isolated, genotyped by next-generation sequencing and tested for drug sensitivity. Cell proliferation and migration, cell death, and signaling transduction pathways were analyzed. RESULTS: Our study revealed that all the three resistant clones developed acquired resistance via the BRAF G469A mutation maintaining a constitutive activation of the ERK pathway. Stable transfection of PC9 and HCC827 cells with a plasmid containing BRAF G469A rendered the cells resistant to osimertinib. Treatment with selumetinib and trametinib, but not dabrafenib, restored the sensitivity to osimertinib and enhanced cell death in the resistant clones with the BRAF G469A mutation. CONCLUSIONS: Our in vitro studies revealed the BRAF G469A-activating mutation as a potential mechanism of acquired resistance to first-line osimertinib treatment, and provide a strategy of intervention to overcome this mechanism of resistance.
Assuntos
Acrilamidas/uso terapêutico , Compostos de Anilina/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , Deleção de Sequência/genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Receptores ErbB/genética , Éxons/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
Triple Negative Breast Cancer (TNBC) is a challenging disease due to the lack of druggable targets; therefore, chemotherapy remains the standard of care and the identification of new targets is a high clinical priority. Alterations in the components of the cell cycle machinery have been frequently reported in cancer; given the success obtained with the CDK4/6 inhibitor palbocicib in ER-positive BC, we explored the potential of combining this drug with chemotherapy in Rb-positive TNBC cell models. The simultaneous combination of palbociclib with paclitaxel exerted an antagonistic effect; by contrast, the sequential treatment inhibited cell proliferation and increased cell death more efficaciously than single treatments. By down-regulating the E2F target c-myc, palbociclib reduced HIF-1α and GLUT-1 expression, and hence glucose uptake and consumption both under normoxic and hypoxic conditions. Importantly, these inhibitory effects on glucose metabolism were enhanced by palbociclib/paclitaxel sequential combination; the superior efficacy of such combination was ascribed to the ability of paclitaxel to inhibit palbociclib-mediated induction of AKT and to further down-regulate the Rb/E2F/c-myc signaling. Our results suggest that the efficacy of standard chemotherapy can be significantly improved by a pre-treatment with palbociclib, thus offering a better therapeutic option for Rb-proficient TNBC.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Sinergismo Farmacológico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose , Proliferação de Células , Feminino , Humanos , Paclitaxel/administração & dosagem , Piperazinas/administração & dosagem , Piridinas/administração & dosagem , Células Tumorais CultivadasRESUMO
Second- and third-generation inhibitors of EGFR possess an acrylamide group which alkylates Cys797, allowing to overcome resistance due to insurgence of T790M mutation. Less reactive warheads, yet capable to bind the target cysteine, may be useful to design newer and safer inhibitors. In the present work, we synthesized a 2-chloro-N-(4-(phenylamino)quinazolin-6-yl)acetamide (8) derivative as a prototype of EGFR inhibitor potentially able to react with Cys797 by nucleophilic substitution. We then tuned the reactivity of the acetamide fragment by replacing the chlorine leaving group with (hetero)-aromatic thiols or carboxylate esters. Among the synthesized derivatives, the 2-((1H-imidazol-2-yl)thio)acetamide 16, while showing negligible reactivity with cysteine in solution, caused long-lasting inhibition of wild-type EGFR autophosphorylation in A549 cells, resulted able to bind recombinant EGFR L858R/T790M in a time-dependent manner, and inhibited both EGFR autophosphorylation and proliferation in gefitinib-resistant H1975 lung cancer cells (expressing EGFR L858R/T790M mutant) at low micromolar concentration.
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Antineoplásicos/farmacologia , Tioacetamida/farmacologia , Células A549 , Acetamidas/química , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisteína/metabolismo , Receptores ErbB/antagonistas & inibidores , Gefitinibe/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosforilação , Tioacetamida/análogos & derivados , Tioacetamida/síntese químicaRESUMO
BACKGROUND: The third generation Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) osimertinib has been initially approved for T790M positive Non-Small Cell Lung Cancer (NSCLC) and more recently for first-line treatment of EGFR-mutant T790M negative NSCLC patients. Similarly to previous generation TKIs, despite the high response rate, disease progression eventually occurs and current clinical research is focused on novel strategies to delay the emergence of osimertinib resistance. In this study we investigated the combination of osimertinib with pemetrexed or cisplatin in EGFR-mutated NSCLC cell lines and xenografts. METHODS: Tumor growth was evaluated in a PC9T790M xenograft model and tissue composition was morphometrically determined. PC9, PC9T790M and HCC827 cell lines were employed to test the efficacy of osimertinib and chemotherapy combination in vitro. Cell viability and cell death were evaluated by MTT assay and fluorescence microscopy. Protein expression and gene status were analysed by Western blotting, fluorescence in situ hybridization analysis, next-generation sequencing and digital droplet PCR. RESULTS: In xenograft models, osimertinib significantly inhibited tumor growth, however, as expected, in 50% of mice drug-resistance developed. A combination of osimertinib with pemetrexed or cisplatin prevented or at least delayed the onset of resistance. Interestingly, such combinations increased the fraction of fibrotic tissue and exerted a long-lasting activity after stopping therapy. In vitro studies demonstrated the stronger efficacy of the combination over the single treatments in inhibiting cell proliferation and inducing cell death in PC9T790M cells as well as in T790M negative PC9 and HCC827 cell lines, suggesting the potential role of this strategy also as first-line treatment. Finally, we demonstrated that osimertinib resistant clones, either derived from resistant tumors or generated in vitro, were less sensitive to pemetrexed prompting to use a chemotherapy regimen non-containing pemetrexed in patients after progression to osimertinib treatment. CONCLUSIONS: Our results identify a combination between osimertinib and pemetrexed or cisplatin potentially useful in the treatment of EGFR-mutated NSCLC patients, which might delay the appearance of osimertinib resistance with long-lasting effects.
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Acrilamidas/administração & dosagem , Compostos de Anilina/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Pemetrexede/administração & dosagem , Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Camundongos , Mutação , Pemetrexede/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and ß1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.
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Movimento Celular , Fibronectinas/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Macrófagos/citologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Modelos Biológicos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Osimertinib is a third-generation EGFR-TKI with a high selective potency against T790M-mutant NSCLC patients. Considering that osimertinib can lead to enhanced HER-2 expression on cell surface and HER-2 overexpression is a mechanism of resistance to osimertinib, this study was addressed to investigate the potential of combining osimertinib with trastuzumab emtansine (T-DM1) in order to improve the efficacy of osimertinib and delay or overcome resistance in NSCLC cell lines with EGFR activating mutation and with T790M mutation or HER-2 amplification. METHODS: The effects of osimertinib combined with T-DM1 on cell proliferation, cell cycle, cell death, antibody-dependent cell-mediated cytotoxicity (ADCC), and acquisition of osimertinib resistance was investigated in PC9, PC9-T790M and H1975 cell lines. The potential of overcoming osimertinib resistance with T-DM1 was tested in a PC9/HER2c1 xenograft model. RESULTS: T-DM1 exerted an additive effect when combined with osimertinib in terms of inhibition of cell proliferation, cell death and ADCC induction in PC9, PC9-T790M and H1975 cell lines. Combining osimertinib and T-DM1 using different schedules in long-term growth experiments revealed that the appearance of osimertinib-resistance was prevented in PC9-T790M and delayed in H1975 cells when the two drugs were given together. By contrast, when osimertinib was followed by T-DM1 an antagonistic effect was observed on cell proliferation, cell death and resistance acquisition. In xenograft models, we demonstrated that HER-2 amplification was associated with osimertinib-resistance and that T-DM1 co-administration is a potential strategy to overcome this resistance. CONCLUSIONS: Our data suggest that concomitant treatment with osimertinib and T-DM1 may be a promising therapeutic strategy for EGFR-mutant NSCLC.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Maitansina/análogos & derivados , Inibidores de Proteínas Quinases/uso terapêutico , Trastuzumab/uso terapêutico , Ado-Trastuzumab Emtansina , Animais , Antineoplásicos Imunológicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/patologia , Maitansina/farmacologia , Maitansina/uso terapêutico , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Trastuzumab/farmacologiaRESUMO
Fibroblast Growth Factor Receptor (FGFR) signaling is a complex pathway which controls several processes, including cell proliferation, survival, migration, and metabolism. FGFR1 signaling is frequently deregulated via amplification/over-expression in NSCLC of squamous histotype (SQCLC), however its inhibition has not been successfully translated in clinical setting. We determined whether targeting downstream signaling implicated in FGFR1 effects on glucose metabolism potentiates the anti-tumor activity of FGFR1 inhibition in SQCLC. In FGFR1 amplified/over-expressing SQCLC cell lines, FGF2-mediated stimulation of FGFR1 under serum-deprivation activated both MAPK and AKT/mTOR pathways and increased glucose uptake, glycolysis, and lactate production, through AKT/mTOR-dependent HIF-1α accumulation and up-regulation of GLUT-1 glucose transporter. These effects were hindered by PD173074 and NVP-BGJ398, selective FGFR inhibitors, as well as by dovitinib, a multi-kinase inhibitor. Glucose metabolism was hampered by the FGFR inhibitors also under hypoxic conditions, with consequent inhibition of cell proliferation and viability. In presence of serum, glucose metabolism was impaired only in cell models in which FGFR1 inhibition was associated with AKT/mTOR down-regulation. When the activation of the AKT/mTOR pathway persisted despite FGFR1 down-regulation, the efficacy of NVP-BGJ398 could be significantly improved by the combination with NVP-BEZ235 or other inhibitors of this signaling cascade, both in vitro and in xenotransplanted nude mice. Collectively our results indicate that inhibition of FGFR1 signaling impacts on cancer cell growth also by affecting glucose energy metabolism. In addition, this study strongly suggests that the therapeutic efficacy of FGFR1 targeting molecules in SQCLC may be implemented by combined treatments tackling on glucose metabolism.