RESUMO
Defects in vesicular trafficking underlie a wide variety of human diseases. Genetic disruption of leucine-rich repeat kinase 2 (LRRK2) in rodents results in epithelial vesicular trafficking errors that can also be induced by treatment of animals with LRRK2 kinase inhibitors. Here we demonstrate that defects in human renal cells lacking LRRK2 phenocopy those seen in the kidneys of Lrrk2 knockout mice, characterized by accumulation of intracellular waste vesicles and fragmentation of the Golgi apparatus. This phenotype can be recapitulated by knockdown of N-ethylmaleimide-sensitive factor, which physically associates with LRRK2 in renal cells. Deficiency in either protein leads to a defect in trans-Golgi to lysosome protein trafficking, which compromises the capacity of lysosomes to degrade endocytic and autophagic cargo. In contrast, neither bulk endocytosis nor autophagic flux are impaired when LRRK2 is acutely knocked down in normal immortalized human kidney (HK2) cells. These data collectively suggest that the primary renal defect caused by LRRK2 deficiency is in protein trafficking between the Golgi apparatus and late endosome/lysosome, which leads to progressive impairments in lysosomal function.
Assuntos
Endocitose , Células Epiteliais/enzimologia , Complexo de Golgi/enzimologia , Túbulos Renais Proximais/enzimologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/deficiência , Lisossomos/enzimologia , Autofagia , Linhagem Celular , Proliferação de Células , Células Epiteliais/patologia , Técnicas de Silenciamento de Genes , Genótipo , Complexo de Golgi/patologia , Humanos , Túbulos Renais Proximais/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Lisossomos/patologia , Proteínas Sensíveis a N-Etilmaleimida/genética , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Fenótipo , Transporte Proteico , ProteóliseRESUMO
Autophagy, the mechanism by which cells deliver material to the lysosome, has been associated with resistance to anticancer drugs, leading autophagy inhibition to be widely studied as a potential chemosensitization strategy for cancer cells. This strategy is based on the idea that inhibition of autophagy will increase drug sensitivity and kill more cancer cells. Here we report an unintended negative effect of this strategy. When modeling the effect of drug resistance in a heterogeneous cancer cell population, we found that autophagy inhibition in drug-sensitive tumor cells causes increased growth of drug-resistant cells in the population through a mechanism involving caspase activation and prostaglandin E2 signaling. These results emphasize the importance of understanding how autophagy manipulation in a tumor cell can have both cell-autonomous and nonautonomous effects and suggest that attempts to chemosensitize by inhibiting autophagy could be enhanced by adopting methods aimed at reducing tumor repopulation.
Assuntos
Autofagia/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/patologia , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Toxina Diftérica/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Kinase inhibitors are effective cancer therapies, but tumors frequently develop resistance. Current strategies to circumvent resistance target the same or parallel pathways. We report here that targeting a completely different process, autophagy, can overcome multiple BRAF inhibitor resistance mechanisms in brain tumors. BRAFV600Emutations occur in many pediatric brain tumors. We previously reported that these tumors are autophagy-dependent and a patient was successfully treated with the autophagy inhibitor chloroquine after failure of the BRAFV600E inhibitor vemurafenib, suggesting autophagy inhibition overcame the kinase inhibitor resistance. We tested this hypothesis in vemurafenib-resistant brain tumors. Genetic and pharmacological autophagy inhibition overcame molecularly distinct resistance mechanisms, inhibited tumor cell growth, and increased cell death. Patients with resistance had favorable clinical responses when chloroquine was added to vemurafenib. This provides a fundamentally different strategy to circumvent multiple mechanisms of kinase inhibitor resistance that could be rapidly tested in clinical trials in patients with BRAFV600E brain tumors.
Assuntos
Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Cloroquina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Indóis/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sulfonamidas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Resultado do Tratamento , VemurafenibRESUMO
Although autophagy controls cell death and survival, underlying mechanisms are poorly understood, and it is unknown whether autophagy affects only whether or not cells die or also controls other aspects of programmed cell death. MAP3K7 is a tumor suppressor gene associated with poor disease-free survival in prostate cancer. Here, we report that Map3k7 deletion in mouse prostate cells sensitizes to cell death by TRAIL (TNF-related apoptosis-inducing ligand). Surprisingly, this death occurs primarily through necroptosis, not apoptosis, due to assembly of the necrosome in association with the autophagy machinery, mediated by p62/SQSTM1 recruitment of RIPK1. The mechanism of cell death switches to apoptosis if p62-dependent recruitment of the necrosome to the autophagy machinery is blocked. These data show that the autophagy machinery can control the mechanism of programmed cell death by serving as a scaffold rather than by degrading cargo.
Assuntos
Apoptose , Autofagia , Animais , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Proteína 5 Relacionada à Autofagia/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Necrose , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Morte Celular/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
UNLABELLED: Autophagy is a mechanism by which cells degrade cellular material to provide nutrients and energy for survival during stress. The autophagy is thought to be a critical process for cancer stem cell (CSC) or tumor-initiating cell maintenance but the mechanisms by which autophagy supports survival of CSCs remain poorly understood. In this study, inhibition of autophagy by knockdown of ATG7 or BECN1 modified the CD44(+)/CD24(low/-) population in breast cancer cells by regulating CD24 and IL6 secretion. In a breast cancer cell line that is independent of autophagy for survival, autophagy inhibition increased IL6 secretion to the media. On the other hand, in an autophagy-dependent cell line, autophagy inhibition decreased IL6 secretion, cell survival, and mammosphere formation. In these cells, IL6 treatment or conditioned media from autophagy-competent cells rescued the deficiency in mammosphere formation induced by autophagy inhibition. These results reveal that autophagy regulates breast CSC maintenance in autophagy-dependent breast cancer cells by modulating IL6 secretion implicating autophagy as a potential therapeutic target in breast cancer. IMPLICATIONS: Modulation of autophagy in breast cancer has different and even opposite effects, indicating the need for a selection strategy when trying to manipulate autophagy in the context of cancer therapy.
Assuntos
Autofagia , Neoplasias da Mama/patologia , Interleucina-6/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Neoplasias da Mama/imunologia , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Proteínas de Membrana/antagonistas & inibidores , Células-Tronco Neoplásicas/imunologia , Análise de Sequência de RNA , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidoresRESUMO
Autophagy is a conserved cellular pathway used to recycle nutrients through lysosomal breakdown basally and under times of stress (e.g., nutrient deprivation, chemotherapeutic treatment). Oncogenes are known to induce autophagy, which may be exploited by cancers for cell survival. To identify autophagy inhibitors with potential therapeutic value for cancer, we screened a panel of antimalarial agents and found that quinacrine (QN) had 60-fold higher potency of autophagy inhibition than chloroquine (CQ), a well-known autophagy inhibitor that functions by disrupting lysosomal activity. Despite desirable autophagy inhibiting properties, QN showed considerable cytotoxicity. Therefore, we designed and synthesized a novel series of QN analogs and investigated their effects on autophagy inhibition and cell viability. Notably, we found two compounds (33 and 34), bearing a backbone of 1,2,3,4-tetrahydroacridine, had limited cytotoxicity yet strong autophagy inhibition properties. In conclusion, these improved lysomotropic autophagy inhibitors may have use as anticancer agents in combination with conventional therapies.
Assuntos
Antineoplásicos/síntese química , Autofagia/efeitos dos fármacos , Quinacrina/análogos & derivados , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética , Cloroquina/química , Cloroquina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Lisossomos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Quinacrina/química , Relação Estrutura-AtividadeRESUMO
Autophagy is a dynamic cell survival mechanism by which a double-membrane vesicle, or autophagosome, sequesters portions of the cytosol for delivery to the lysosome for recycling. This process can be inhibited using the antimalarial agent chloroquine (CQ), which impairs lysosomal function and prevents autophagosome turnover. Despite its activity, CQ is a relatively inadequate inhibitor that requires high concentrations to disrupt autophagy, highlighting the need for improved small molecules. To address this, we screened a panel of antimalarial agents for autophagy inhibition and chemically synthesized a novel series of acridine and tetrahydroacridine derivatives. Structure-activity relationship studies of the acridine ring led to the discovery of VATG-027 as a potent autophagy inhibitor with a high cytotoxicity profile. In contrast, the tetrahydroacridine VATG-032 showed remarkably little cytotoxicity while still maintaining autophagy inhibition activity, suggesting that both compounds act as autophagy inhibitors with differential effects on cell viability. Further, knockdown of autophagy-related genes showed no effect on cell viability, demonstrating that the ability to inhibit autophagy is separate from the compound cytotoxicity profiles. Next, we determined that both inhibitors function through lysosomal deacidification mechanisms and ultimately disrupt autophagosome turnover. To evaluate the genetic context in which these lysosomotropic inhibitors may be effective, they were tested in patient-derived melanoma cell lines driven by oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B). We discovered that both inhibitors sensitized melanoma cells to the BRAF V600E inhibitor vemurafenib. Overall, these autophagy inhibitors provide a means to effectively block autophagy and have the potential to sensitize mutant BRAF melanomas to first-line therapies.