RESUMO
Hepatic steatosis is a major etiological factor in hepatocellular carcinoma (HCC), but factors causing lipid accumulation leading to HCC are not understood. We identify BNIP3 (a mitochondrial cargo receptor) as an HCC suppressor that mitigates against lipid accumulation to attenuate tumor cell growth. Targeted deletion of Bnip3 decreased tumor latency and increased tumor burden in a mouse model of HCC. This was associated with increased lipid in bnip3-/- HCC at early stages of disease, while lipid did not accumulate until later in tumorigenesis in wild-type mice, as Bnip3 expression was attenuated. Low BNIP3 expression in human HCC similarly correlated with increased lipid content and worse prognosis than HCC expressing high BNIP3. BNIP3 suppressed HCC cell growth by promoting lipid droplet turnover at the lysosome in a manner dependent on BNIP3 binding LC3. We have termed this process "mitolipophagy" because it involves the coordinated autophagic degradation of lipid droplets with mitochondria.
RESUMO
Mitophagy formed the basis of the original description of autophagy by Christian de Duve when he demonstrated that GCG (glucagon) induced macroautophagic/autophagic turnover of mitochondria in the liver. However, the molecular basis of liver-specific activation of mitophagy by GCG, or its significance for metabolic stress responses in the liver is not understood. Here we show that BNIP3 is required for GCG-induced mitophagy in the liver through interaction with processed LC3B; an interaction that is also necessary to localize LC3B out of the nucleus to cytosolic mitophagosomes in response to nutrient deprivation. Loss of BNIP3-dependent mitophagy caused excess mitochondria to accumulate in the liver, disrupting metabolic zonation within the liver parenchyma, with expansion of zone 1 metabolism at the expense of zone 3 metabolism. These results identify BNIP3 as a regulator of metabolic homeostasis in the liver through its effect on mitophagy and mitochondrial mass distribution.Abbreviations: ASS1, arginosuccinate synthetase; BNIP3, BCL2/adenovirus E1B interacting protein 3; CV, central vein; GCG - glucagon; GLUL, glutamate- ammonia ligase (glutamine synthetase); HCQ, hydroxychloroquine; LIR, LC3-interacting region; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 beta; mtDNA:nucDNA, ratio of mitochondrial DNA to nuclear DNA; PV, periportal vein; TOMM20, translocase of outer mitochondrial membrane protein 20.
Assuntos
Fígado/citologia , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Animais , Células Cultivadas , Citosol/metabolismo , Glucagon/metabolismo , Glucagon/farmacologia , Homeostase , Humanos , Fígado/efeitos dos fármacos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Mitofagia/efeitos dos fármacos , Mitofagia/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The reversible modification of cysteine residues by thioester formation with palmitate (S-palmitoylation) is an abundant lipid post-translational modification (PTM) in mammalian systems. S-palmitoylation has been observed on mitochondrial proteins, providing an intriguing potential connection between metabolic lipids and mitochondrial regulation. However, it is unknown whether and/or how mitochondrial S-palmitoylation is regulated. Here we report the development of mitoDPPs, targeted fluorescent probes that measure the activity levels of "erasers" of S-palmitoylation, acyl-protein thioesterases (APTs), within mitochondria of live cells. Using mitoDPPs, we discover active S-depalmitoylation in mitochondria, in part mediated by APT1, an S-depalmitoylase previously thought to reside in the cytosol and on the Golgi apparatus. We also find that perturbation of long-chain acyl-CoA cytoplasm and mitochondrial regulatory proteins, respectively, results in selective responses from cytosolic and mitochondrial S-depalmitoylases. Altogether, this work reveals that mitochondrial S-palmitoylation is actively regulated by "eraser" enzymes that respond to alterations in mitochondrial lipid homeostasis.
Assuntos
Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Tioléster Hidrolases/metabolismo , Células A549 , Acil Coenzima A/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinética , Lipoilação , Células MCF-7 , Microscopia Confocal , Interferência de RNA , Tioléster Hidrolases/genéticaRESUMO
Mitophagy is a selective mode of autophagy in which mitochondria are specifically targeted for degradation at the autophagolysosome. Mitophagy is activated by stresses such as hypoxia, nutrient deprivation, DNA damage, inflammation and mitochondrial membrane depolarization and plays a role in maintaining mitochondrial integrity and function. Defects in mitophagy lead to mitochondrial dysfunction that can affect metabolic reprogramming in response to stress, alter cell fate determination and differentiation, which in turn affects disease incidence and etiology, including cancer. Here, we discuss how different mitophagy adaptors and modulators, including Parkin, BNIP3, BNIP3L, p62/SQSTM1 and OPTN, are regulated in response to physiological stresses and deregulated in cancers. Additionally, we explore how these different mitophagy control pathways coordinate with each other. Finally, we review new developments in understanding how mitophagy affects stemness, cell fate determination, inflammation and DNA damage responses that are relevant to understanding the role of mitophagy in cancer.
Assuntos
Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitofagia , Neoplasias/genética , Neoplasias/metabolismo , Adaptação Biológica , Animais , Autofagia , Dano ao DNA , Metabolismo Energético , Humanos , Inflamação/genética , Inflamação/metabolismo , Transdução de Sinais , Estresse FisiológicoRESUMO
Autophagy is a conserved catabolic process that plays a housekeeping role in eliminating protein aggregates and organelles and is activated during nutrient deprivation to generate metabolites and energy. Autophagy plays a significant role in tumorigenesis, although opposing context-dependent functions of autophagy in cancer have complicated efforts to target autophagy for therapeutic purposes. We demonstrate that autophagy inhibition reduces tumor cell migration and invasion in vitro and attenuates metastasis in vivo. Numerous abnormally large focal adhesions (FAs) accumulate in autophagy-deficient tumor cells, reflecting a role for autophagy in FA disassembly through targeted degradation of paxillin. We demonstrate that paxillin interacts with processed LC3 through a conserved LIR motif in the amino-terminal end of paxillin and that this interaction is regulated by oncogenic SRC activity. Together, these data establish a function for autophagy in FA turnover, tumor cell motility, and metastasis.
Assuntos
Autofagia , Movimento Celular , Adesões Focais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/patologia , Paxilina/metabolismo , Quinases da Família src/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Paxilina/química , Ligação Proteica , Estabilidade Proteica , Transporte ProteicoRESUMO
BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesistwo processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunctioncaused by defects in mitophagycan promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.
Assuntos
Neoplasias Mamárias Experimentais/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitofagia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Biomarcadores Tumorais/análise , Progressão da Doença , Feminino , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Proteínas de Membrana/deficiência , Camundongos , Proteínas Mitocondriais/deficiência , Metástase Neoplásica , Neovascularização Patológica/metabolismo , Prognóstico , Espécies Reativas de Oxigênio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Mutação , Neuroblastoma/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular Tumoral , Crizotinibe , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Amplificação de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/enzimologia , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Interferência de RNA , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proteína S6 Ribossômica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent reports indicate that inactivation of the RB, TP53 or PTEN tumour suppressor genes is detected in tumour stroma of oropharyngeal, breast and other human cancers. Mouse models have validated the tumour-promoting effects of deleting Rb, Pten or p53 in fibroblasts that converts them from normal fibroblasts to carcinoma associated fibroblasts (CAFs). The tumour-promoting activity of CAFs in these contexts was associated with increased paracrine signaling to tumour cells through production of specific growth factors, chemokines and MMPs by CAFs. The conversion of NOFs into CAFs through acquisition of specific mutations, such as loss of tumour suppressors, or deregulated expression of microRNAs or key epigenetic events, can clearly occur independently of genetic and epigenetic changes in tumour cells but an alternative source of CAFs that is being reconsidered is that CAFs derive from the tumour cells by EMT. Recent mouse models employing lineage-tracing techniques have suggested that this can take place in vivo and the extent to which this is relevant more broadly is discussed.