RESUMO
Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation, how ULK1 promotes autophagy remains poorly understood. Here, we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered 15 phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.
Assuntos
Autofagia/fisiologia , Benzamidas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/farmacologia , Sequência de Aminoácidos , Animais , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Benzamidas/química , Domínio Catalítico/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Sequência Consenso , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Dados de Sequência Molecular , Fosforilação , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Pirimidinas/química , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
We used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to profile the transcriptome of pancreatic neuroendocrine tumors (PNET) that were either non-invasive or highly invasive, seeking to identify pro- and anti-invasive molecules. Expression of multiple components of desmosomes, structures that help maintain cellular adhesion, was significantly reduced in invasive carcinomas. Genetic deletion of one of these desmosomal components, desmoplakin, resulted in increased local tumor invasion without affecting tumor growth parameters in RT2 PNETs. Expression of cadherin 1, a component of the adherens junction adhesion complex, was maintained in these tumors despite the genetic deletion of desmoplakin. Our results demonstrate that loss of desmoplakin expression and resultant disruption of desmosomal adhesion can promote increased local tumor invasion independent of adherens junction status.
Assuntos
Desmoplaquinas/genética , Desmossomos/genética , Deleção de Genes , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Junções Aderentes/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Desmoplaquinas/metabolismo , Desmossomos/patologia , Modelos Animais de Doenças , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos/genéticaRESUMO
Cancer is a disease subject to both genetic and environmental influences. In this study, we used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to identify a genetic locus that influences tumor progression to an invasive growth state. RT2 mice inbred into the C57BL/6 (B6) background develop both noninvasive pancreatic neuroendocrine tumors (PNET) and invasive carcinomas with varying degrees of aggressiveness. In contrast, RT2 mice inbred into the C3HeB/Fe (C3H) background are comparatively resistant to the development of invasive tumors, as are RT2 C3HB6(F1) hybrid mice. Using linkage analysis, we identified a 13-Mb locus on mouse chromosome 17 with significant linkage to the development of highly invasive PNETs. A gene residing in this locus, the anaplastic lymphoma kinase (Alk), was expressed at significantly lower levels in PNETs from invasion-resistant C3H mice compared with invasion-susceptible B6 mice, and pharmacological inhibition of Alk led to reduced tumor invasiveness in RT2 B6 mice. Collectively, our results demonstrate that tumor invasion is subject to polymorphic genetic control and identify Alk as a genetic modifier of invasive tumor growth.
Assuntos
Adenoma de Células das Ilhotas Pancreáticas/genética , Adenoma de Células das Ilhotas Pancreáticas/patologia , Transformação Celular Neoplásica/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Polimorfismo Genético , Adenoma de Células das Ilhotas Pancreáticas/tratamento farmacológico , Quinase do Linfoma Anaplásico , Animais , Cromossomos de Mamíferos , Ligação Genética , Loci Gênicos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/uso terapêutico , Receptores Proteína Tirosina QuinasesRESUMO
The LKB1 tumor suppressor is often mutationally inactivated in non-small cell lung cancer (NSCLC). LKB1 phosphorylates and activates members of the AMPK family of Ser/Thr kinases. Within this family, the salt-inducible kinases (SIKs) modulate gene expression in part via the inhibitory phosphorylation of the CRTCs, coactivators for CREB (cAMP response element-binding protein). The loss of LKB1 causes SIK inactivation and the induction of the CRTCs, leading to the up-regulation of CREB target genes. We identified CRTC2 as a critical factor in LKB1-deficient NSCLC. CRTC2 is unphosphorylated and therefore constitutively activated in LKB1-mutant NSCLC, where it promotes tumor growth, in part via the induction of the inhibitor of DNA binding 1 (ID1), a bona fide CREB target gene. As ID1 expression is up-regulated and confers poor prognosis in LKB1-deficient NSCLC, our results suggest that small molecules that inhibit CRTC2 and ID1 activity may provide therapeutic benefit to individuals with NSCLC.