RESUMO
MAPK inhibitors (MAPKi) remain an important component of the standard of care for metastatic melanoma. However, acquired resistance to these drugs limits their therapeutic benefit. Tumor cells can become refractory to MAPKi by reactivation of ERK. When this happens, tumors often become sensitive to drug withdrawal. This drug addiction phenotype results from the hyperactivation of the oncogenic pathway, a phenomenon commonly referred to as oncogene overdose. Several feedback mechanisms are involved in regulating ERK signaling. However, the genes that serve as gatekeepers of oncogene overdose in mutant melanoma remain unknown. Here, we demonstrate that depletion of the ERK phosphatase, DUSP4, leads to toxic levels of MAPK activation in both drug-naive and drug-resistant mutant melanoma cells. Importantly, ERK hyperactivation is associated with down-regulation of lineage-defining genes including MITF Our results offer an alternative therapeutic strategy to treat mutant melanoma patients with acquired MAPKi resistance and those unable to tolerate MAPKi.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Fosfatases de Especificidade Dupla/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Melanoma/genética , Melanoma/patologia , Proteínas de Membrana/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Oncogenes , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Lung cancer classification has been radically transformed in recent years as genomic profiling has identified multiple novel therapeutic targets including MET exon 14 (METex14) alterations and MET amplification. Utilizing targeted therapies in patients with molecularly-defined NSCLC leads to remarkable objective response rates and improved progression-free survival. However, acquired resistance is inevitable. Several recent phase II trials have confirmed that METex14 NSCLC can be treated effectively with MET kinase inhibitors, such as crizotinib, capmatinib, tepotinib, and savolitinib. However, response rates for many MET TKIs are modest relative to the activity of targeted therapy in other oncogene-driven lung cancers, where ORRs are more consistently greater than 60%. In spite of significant gains in the field of MET inhibition in NSCLC, challenges remain: the landscape of resistance mechanisms to MET TKIs is not yet well characterized, and there may be intrinsic and acquired resistance mechanisms that require further characterization to enable increased MET TKI activity. In this review, we overview MET pathway dysregulation in lung cancer, methods of detection in the clinic, recent clinical trial data, and discuss current mechanisms of TKI resistance, exploring emerging strategies to overcome resistance.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/genéticaRESUMO
The serine/threonine kinase AKT is a critical effector of the phosphoinositide 3-kinase (PI3K) signaling cascade and has a pivotal role in cell growth, proliferation, survival, and metabolism. AKT is one of the most commonly activated pathways in human cancer and dysregulation of AKT-dependent pathways is associated with the development and maintenance of a range of solid tumors. There are multiple small-molecule inhibitors targeting different components of the PI3K/AKT pathway currently at various stages of clinical development, in addition to new combination strategies aiming to boost the therapeutic efficacy of these drugs. Correlative and translational studies have been undertaken in the context of clinical trials investigating AKT inhibitors, however the identification of predictive biomarkers of response and resistance to AKT inhibition remains an unmet need. In this review, we discuss the biological function and activation of AKT, discuss its contribution to tumor development and progression, and review the efficacy and toxicity data from clinical trials, including both AKT inhibitor monotherapy and combination strategies with other agents. We also discuss the promise and challenges associated with the development of AKT inhibitors and associated predictive biomarkers of response and resistance.
RESUMO
OBJECTIVE: Epidermal growth factor receptor (EGFR) inhibition may be effective in biomarker-selected populations of advanced gastro-oesophageal adenocarcinoma (aGEA) patients. Here, we tested the association between outcome and EGFR copy number (CN) in pretreatment tissue and plasma cell-free DNA (cfDNA) of patients enrolled in a randomised first-line phase III clinical trial of chemotherapy or chemotherapy plus the anti-EGFR monoclonal antibody panitumumab in aGEA (NCT00824785). DESIGN: EGFR CN by either fluorescence in situ hybridisation (n=114) or digital-droplet PCR in tissues (n=250) and plasma cfDNAs (n=354) was available for 474 (86%) patients in the intention-to-treat (ITT) population. Tissue and plasma low-pass whole-genome sequencing was used to screen for coamplifications in receptor tyrosine kinases. Interaction between chemotherapy and EGFR inhibitors was modelled in patient-derived organoids (PDOs) from aGEA patients. RESULTS: EGFR amplification in cfDNA correlated with poor survival in the ITT population and similar trends were observed when the analysis was conducted in tissue and plasma by treatment arm. EGFR inhibition in combination with chemotherapy did not correlate with improved survival, even in patients with significant EGFR CN gains. Addition of anti-EGFR inhibitors to the chemotherapy agent epirubicin in PDOs, resulted in a paradoxical increase in viability and accelerated progression through the cell cycle, associated with p21 and cyclin B1 downregulation and cyclin E1 upregulation, selectively in organoids from EGFR-amplified aGEA. CONCLUSION: EGFR CN can be accurately measured in tissue and liquid biopsies and may be used for the selection of aGEA patients. EGFR inhibitors may antagonise the antitumour effect of anthracyclines with important implications for the design of future combinatorial trials.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Epirubicina/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Neoplasias Esofágicas/tratamento farmacológico , Panitumumabe/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Adenocarcinoma/química , Idoso , Antibióticos Antineoplásicos/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica , Biomarcadores Tumorais/análise , Epirubicina/administração & dosagem , Receptores ErbB/análise , Neoplasias Esofágicas/química , Humanos , Masculino , Pessoa de Meia-Idade , Panitumumabe/administração & dosagem , Neoplasias Gástricas/químicaRESUMO
The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.
Assuntos
Adenosina/análogos & derivados , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Proteína Proto-Oncogênica N-Myc/biossíntese , Neuroblastoma/tratamento farmacológico , Temozolomida/farmacologia , Adenosina/farmacologia , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Elementos Facilitadores Genéticos , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: AKT, a critical effector of the phosphoinositide 3-kinase (PI3K) signalling cascade, is an intensely pursued therapeutic target in oncology. Two distinct classes of AKT inhibitors have been in clinical development, ATP-competitive and allosteric. Class-specific differences in drug activity are likely the result of differential structural and conformational requirements governing efficient target binding, which ultimately determine isoform-specific potency, selectivity profiles and activity against clinically relevant AKT mutant variants. METHODS: We have carried out a systematic evaluation of clinical AKT inhibitors using in vitro pharmacology, molecular profiling and biochemical assays together with structural modelling to better understand the context of drug-specific and drug-class-specific cell-killing activity. RESULTS: Our data demonstrate clear differences between ATP-competitive and allosteric AKT inhibitors, including differential effects on non-catalytic activity as measured by a novel functional readout. Surprisingly, we found that some mutations can cause drug resistance in an isoform-selective manner despite high structural conservation across AKT isoforms. Finally, we have derived drug-class-specific phosphoproteomic signatures and used them to identify effective drug combinations. CONCLUSIONS: These findings illustrate the utility of individual AKT inhibitors, both as drugs and as chemical probes, and the benefit of AKT inhibitor pharmacological diversity in providing a repertoire of context-specific therapeutic options.
Assuntos
Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Células HT29 , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Conformação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
Aberrant activation of the PI3K pathway is one of the commonest oncogenic events in human cancer. AKT is a key mediator of PI3K oncogenic function, and thus has been intensely pursued as a therapeutic target. Multiple AKT inhibitors, broadly classified as either ATP-competitive or allosteric, are currently in various stages of clinical development. Herein, we review the evidence for AKT dependence in human tumours and focus on its therapeutic targeting by the two drug classes. We highlight the future prospects for the development and implementation of more effective context-specific AKT inhibitors aided by our increasing knowledge of both its regulation and some previously unrecognised non-canonical functions.
Assuntos
Trifosfato de Adenosina/química , Sítio Alostérico , Ligação Competitiva , Regulação Neoplásica da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antineoplásicos/farmacologia , Catálise , Desenho de Fármacos , Genótipo , Humanos , Camundongos , Isoformas de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Transdução de Sinais/efeitos dos fármacosRESUMO
Human primary glioblastomas (GBM) often harbor mutations within the epidermal growth factor receptor (EGFR). Treatment of EGFR-mutant GBM cell lines with the EGFR/HER2 tyrosine kinase inhibitor lapatinib can effectively induce cell death in these models. However, EGFR inhibitors have shown little efficacy in the clinic, partly because of inappropriate dosing. Here, we developed a computational approach to model the in vitro cellular dynamics of the EGFR-mutant cell line SF268 in response to different lapatinib concentrations and dosing schedules. We then used this approach to identify an effective treatment strategy within the clinical toxicity limits of lapatinib, and developed a partial differential equation modeling approach to study the in vivo GBM treatment response by taking into account the heterogeneous and diffusive nature of the disease. Despite the inability of lapatinib to induce tumor regressions with a continuous daily schedule, our modeling approach consistently predicts that continuous dosing remains the best clinically feasible strategy for slowing down tumor growth and lowering overall tumor burden, compared to pulsatile schedules currently known to be tolerated, even when considering drug resistance, reduced lapatinib tumor concentrations due to the blood brain barrier, and the phenotypic switch from proliferative to migratory cell phenotypes that occurs in hypoxic microenvironments. Our mathematical modeling and statistical analysis platform provides a rational method for comparing treatment schedules in search for optimal dosing strategies for glioblastoma and other cancer types.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Quinazolinas/administração & dosagem , Antineoplásicos/farmacocinética , Barreira Hematoencefálica , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Esquema de Medicação , Receptores ErbB/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Lapatinib , Modelos Logísticos , Dose Máxima Tolerável , Modelos Biológicos , Mutação , Inibidores de Proteínas Quinases/farmacocinética , Quinazolinas/farmacocinéticaRESUMO
Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin ß family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.
Assuntos
Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Glioma/genética , beta Carioferinas/genética , Proteína ran de Ligação ao GTP/genética , Transporte Ativo do Núcleo Celular/genética , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Doxorrubicina/farmacologia , Receptores ErbB/metabolismo , Retroalimentação Fisiológica , Feminino , Técnicas de Silenciamento de Genes , Glioma/tratamento farmacológico , Glioma/metabolismo , Células HEK293 , Humanos , Camundongos Knockout , Camundongos SCID , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42-JNK pathway.
Assuntos
Claudina-2/metabolismo , Claudina-4/metabolismo , Junções Íntimas/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Claudina-2/genética , Claudina-4/genética , Cães , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Células Madin Darby de Rim Canino , Junções Íntimas/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
The serine-threonine kinase AKT regulates proliferation and survival by phosphorylating a network of protein substrates. In this study, we describe a kinase-independent function of AKT. In cancer cells harboring gain-of-function alterations in MET, HER2, or Phosphatidyl-Inositol-3-Kinase (PI3K), catalytically inactive AKT (K179M) protected from drug induced cell death in a PH-domain dependent manner. An AKT kinase domain mutant found in human melanoma (G161V) lacked enzymatic activity in vitro and in AKT1/AKT2 double knockout cells, but promoted growth factor independent survival of primary human melanocytes. ATP-competitive AKT inhibitors failed to block the kinase-independent function of AKT, a liability that limits their effectiveness compared to allosteric AKT inhibitors. Our results broaden the current view of AKT function and have important implications for the development of AKT inhibitors for cancer.
Assuntos
Sobrevivência Celular , Melanoma/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Humanos , Melanoma/enzimologiaRESUMO
The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra-physiological levels of phospho-tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry-based phospho-proteomics, we show that glucose withdrawal initiates a unique signature of phospho-tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal-induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS-mediated cell death. Taken together, these findings illustrate the systems-level cross-talk between metabolism and signaling in the maintenance of cancer cell homeostasis.
Assuntos
Glucose/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patologia , Fosfotirosina/metabolismo , Transdução de Sinais/fisiologia , Morte Celular , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Adesões Focais , Humanos , Espectrometria de Massas , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfotirosina/análise , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
UNLABELLED: Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors that bind to the inactive EGFR conformation, however, potently inhibit EGFR EC mutants and induce cell death in EGFR-mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these 2 cancer types. SIGNIFICANCE: Approximately 40% of human glioblastomas harbor oncogenic EGFR alterations, but attempts to therapeutically target EGFR with first-generation EGFR kinase inhibitors have failed. Here, we demonstrate selective sensitivity of glioma-specific EGFR mutants to ATP-site competitive EGFR kinase inhibitors that target the inactive conformation of the catalytic domain.
Assuntos
Neoplasias Encefálicas/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Glioma/genética , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Glioma/metabolismo , Humanos , Lapatinib , Neoplasias Pulmonares/metabolismo , Camundongos , Mutação , Quinazolinas/farmacologiaRESUMO
PURPOSE OF REVIEW: Inhibition of the epidermal growth factor receptor (EGFR) by small molecules or antibodies has been pursued as a paradigm to treat human cancers for over two decades. It is now clear that these agents can induce tumor regressions in a variety of human cancers, proving the critical role of EGFR signals for tumor maintenance in subsets of patients with these cancers. Clinically meaningful responses, however, are only transient and further refinement of EGFR-targeted therapies is urgently needed. RECENT FINDINGS: Several studies provide new insights into the molecular basis of EGFR kinase inhibitor resistance, including co-activation of the MET growth factor receptor, loss of the phosphatase and tensin homolog (PTEN) tumor suppressor, and KRAS mutation. Potential strategies are emerging to overcome acquired EGFR kinase inhibitor resistance associated with the T790 M EGFR mutation, including a new compound identified in a chemical library screen and a combination regimen of an anti-EGFR antibody plus a small molecule EGFR kinase inhibitor. Lastly, pulsatile dosing schedules are being pursued to accomplish more complete target inhibition. SUMMARY: Current data point toward a strong association between EGFR genotype and EGFR kinase inhibitor response, similar to the findings with other oncogenic kinases (BCR-ABL, HER2, KIT, PDGFRA, BRAF). This relationship is less obvious for antibodies targeting EGFR. More complete inhibition of EGFR in tumor cells and more focused clinical drug development remain important goals toward further success with this class of anticancer agents.
Assuntos
Receptores ErbB/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Receptores ErbB/genética , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
The phosphatase and tensin homolog (PTEN) is a tumor suppressor that is inactivated in many human cancers. PTEN loss has been associated with resistance to inhibitors of the epidermal growth factor receptor (EGFR), but the molecular basis of this resistance is unclear. It is believed that unopposed phosphatidylinositol-3-kinase (PI3K) activation through multiple receptor tyrosine kinases (RTKs) can relieve PTEN-deficient cancers from their "dependence" on EGFR or any other single RTK for survival. Here we report a distinct resistance mechanism whereby PTEN inactivation specifically raises EGFR activity by impairing the ligand-induced ubiquitylation and degradation of the activated receptor through destabilization of newly formed ubiquitin ligase Cbl complexes. PTEN-associated resistance to EGFR kinase inhibitors is phenocopied by expression of dominant negative Cbl and can be overcome by more complete EGFR kinase inhibition. PTEN inactivation does not confer resistance to inhibitors of the MET or PDGFRA kinase. Our study identifies a critical role for PTEN in EGFR signal termination and suggests that more potent EGFR inhibition should overcome resistance caused by PI3K pathway activation.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose , Linhagem Celular , Ativação Enzimática , Humanos , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , UbiquitinaçãoRESUMO
Cellular signalling pathways are critical in regulating the balance between latency and lytic replication of herpesviruses. Here, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway on replication of two gamma-2 herpesviruses, murine gammaherpesvirus-68 (MHV-68) and human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV). We found that de novo infection of MHV-68 induced PI3K-dependent Akt activation and the lytic replication of MHV-68 was enhanced by inhibiting the PI3K-Akt pathway with both chemical inhibitors and RNA interference technology. Inhibiting the activity of Akt using Akt inhibitor VIII also facilitated the reactivation of KSHV from latency. Both lytic replication and latency depend on the activity of viral transactivator RTA and we further show that the activity of RTA is increased by reducing Akt1 expression. The data suggest that the PI3K-Akt pathway suppresses the activity of RTA and thereby contributes to the maintenance of viral latency and promotes tumorigenesis.
Assuntos
Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Viral , Latência Viral , Replicação Viral , Células 3T3 , Animais , Linhagem Celular , Gammaherpesvirinae/genética , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Mutation of the gene PARK2, which encodes an E3 ubiquitin ligase, is the most common cause of early-onset Parkinson's disease. In a search for multisite tumor suppressors, we identified PARK2 as a frequently targeted gene on chromosome 6q25.2-q27 in cancer. Here we describe inactivating somatic mutations and frequent intragenic deletions of PARK2 in human malignancies. The PARK2 mutations in cancer occur in the same domains, and sometimes at the same residues, as the germline mutations causing familial Parkinson's disease. Cancer-specific mutations abrogate the growth-suppressive effects of the PARK2 protein. PARK2 mutations in cancer decrease PARK2's E3 ligase activity, compromising its ability to ubiquitinate cyclin E and resulting in mitotic instability. These data strongly point to PARK2 as a tumor suppressor on 6q25.2-q27. Thus, PARK2, a gene that causes neuronal dysfunction when mutated in the germline, may instead contribute to oncogenesis when altered in non-neuronal somatic cells.
Assuntos
Neoplasias do Colo/genética , Glioblastoma/genética , Mutação , Ubiquitina-Proteína Ligases/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Dosagem de Genes , Variação Genética , Genótipo , Glioblastoma/patologia , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Doença de Parkinson/genética , Estrutura Terciária de Proteína , Transplante Heterólogo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
EGFR is a major anticancer drug target in human epithelial tumors. One effective class of agents is the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. These drugs induce dramatic responses in individuals with lung adenocarcinomas characterized by mutations in exons encoding the EGFR tyrosine kinase domain, but disease progression invariably occurs. A major reason for such acquired resistance is the outgrowth of tumor cells with additional TKI-resistant EGFR mutations. Here we used relevant transgenic mouse lung tumor models to evaluate strategies to overcome the most common EGFR TKI resistance mutation, T790M. We treated mice bearing tumors harboring EGFR mutations with a variety of anticancer agents, including a new irreversible EGFR TKI that is under development (BIBW-2992) and the EGFR-specific antibody cetuximab. Surprisingly, we found that only the combination of both agents together induced dramatic shrinkage of erlotinib-resistant tumors harboring the T790M mutation, because together they efficiently depleted both phosphorylated and total EGFR. We suggest that these studies have immediate therapeutic implications for lung cancer patients, as dual targeting with cetuximab and a second-generation EGFR TKI may be an effective strategy to overcome T790M-mediated drug resistance. Moreover, this approach could serve as an important model for targeting other receptor tyrosine kinases activated in human cancers.
Assuntos
Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Quinazolinas/metabolismo , Afatinib , Anfirregulina , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/uso terapêutico , Cetuximab , Família de Proteínas EGF , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Epirregulina , Receptores ErbB/genética , Cloridrato de Erlotinib , Perfilação da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Transplante de Neoplasias , Paclitaxel/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
DNA damage checkpoints are critical for preventing tumorigenesis and regulating the response of cells to genotoxic agents. It is believed that the coordinated actions of a number of effectors underlie proper checkpoint function. The kinase Chk2, p21 and 14-3-3sigma have each been shown to be independent effectors of the G(2) DNA damage checkpoint. However, the relative roles of these proteins remain unclear. To help elucidate this question, we have perturbed each of these 3 genes in combination in human cells. We show that Chk2 depletion causes markedly increased sensitivity to DNA damage in p21(-/-), 14-3-3sigma(-/-) cells but not in cells lacking only one or none of these genes. This greater sensitivity was due to an increase in apoptosis following DNA damage and not due to exacerbation of G(2) checkpoint defects. Pharmacologic inhibition of Chk2 in p21(-/-), 14-3-3sigma(-/-) cells also resulted in greater sensitivity to DNA damage. Our data indicates that p21 and 14-3-3sigma synergize as molecular determinants of sensitivity to DNA damage following Chk2 inhibition, and Chk2 modulates the biological rheostat that determines whether a cancer cell undergoes arrest versus death after treatment with a chemotherapeutic agent. These findings have implications for the targeting of Chk2 in human cancers.
Assuntos
Biomarcadores Tumorais/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Exonucleases/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas 14-3-3 , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , Inibidor de Quinase Dependente de Ciclina p21/genética , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/farmacologia , Exonucleases/genética , Exorribonucleases , Fase G2 , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Tyrosine phosphorylation plays a critical role in regulating cellular function and is a central feature in signaling cascades involved in oncogenesis. The regulation of tyrosine phosphorylation is coordinately controlled by kinases and phosphatases (PTPs). Whereas activation of tyrosine kinases has been shown to play vital roles in tumor development, the role of PTPs is much less well defined. Here, we show that the receptor protein tyrosine phosphatase delta (PTPRD) is frequently inactivated in glioblastoma multiforme (GBM), a deadly primary neoplasm of the brain. PTPRD is a target of deletion in GBM, often via focal intragenic loss. In GBM tumors that do not possess deletions in PTPRD, the gene is frequently subject to cancer-specific epigenetic silencing via promoter CpG island hypermethylation (37%). Sequencing of the PTPRD gene in GBM and other primary human tumors revealed that the gene is mutated in 6% of GBMs, 13% of head and neck squamous cell carcinomas, and in 9% of lung cancers. These mutations were deleterious. In total, PTPRD inactivation occurs in >50% of GBM tumors, and loss of expression predicts for poor prognosis in glioma patients. Wild-type PTPRD inhibits the growth of GBM and other tumor cells, an effect not observed with PTPRD alleles harboring cancer-specific mutations. Human astrocytes lacking PTPRD exhibited increased growth. PTPRD was found to dephosphorylate the oncoprotein STAT3. These results implicate PTPRD as a tumor suppressor on chromosome 9p that is involved in the development of GBMs and multiple human cancers.