RESUMO
Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Indóis/farmacologia , Neoplasias Pulmonares/metabolismo , Triazinas/farmacologia , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte , Linhagem Celular Tumoral , Proteína Coatomer/metabolismo , Feminino , Genes ras , Xenoenxertos , Humanos , Neoplasias Pulmonares/patologia , Lisossomos/metabolismo , Camundongos , Terapia de Alvo Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transplante de Neoplasias , Fosforilação OxidativaRESUMO
SW044248, identified through a screen for chemicals that are selectively toxic for non-small cell lung cancer (NSCLC) cell lines, was found to rapidly inhibit macromolecular synthesis in sensitive, but not in insensitive, cells. SW044248 killed approximately 15% of a panel of 74 NSCLC cell lines and was nontoxic to immortalized human bronchial cell lines. The acute transcriptional response to SW044248 in sensitive HCC4017 cells correlated significantly with inhibitors of topoisomerases and SW044248 inhibited topoisomerase 1 (Top1) but not topoisomerase 2. SW044248 inhibited Top1 differently from camptothecin and camptothecin did not show the same selective toxicity as SW044248. Elimination of Top1 by siRNA partially protected cells from SW044248, although removing Top1 was itself eventually toxic. Cells resistant to SW044248 responded to the compound by upregulating CDKN1A and siRNA to CDKN1A sensitized those cells to SW044248. Thus, at least part of the differential sensitivity of NSCLC cells to SW044248 is the ability to upregulate CDKN1A.
Assuntos
Antineoplásicos/farmacologia , Inibidores da Topoisomerase I/farmacologia , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Indóis/química , Indóis/farmacologia , Concentração Inibidora 50 , Neoplasias Pulmonares , Biossíntese de Proteínas/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Inibidores da Topoisomerase I/química , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Triazinas/química , Triazinas/farmacologia , Células Tumorais CultivadasRESUMO
In a previous genetic screen for Caenorhabditis elegans mutants that survive in the presence of an antimitotic drug, hemiasterlin, we identified eight strong mutants. Two of these were found to be resistant to multiple toxins, and in one of these we identified a missense mutation in phb-2, which encodes the mitochondrial protein prohibitin 2. Here we identify two additional mutations that confer drug resistance, spg-7 and har-1, also in genes encoding mitochondrial proteins. Other mitochondrial mutants, isp-1, eat-3, and clk-1, were also found to be drug-resistant. Respiratory complex inhibitors, FCCP and oligomycin, and a producer of reactive oxygen species (ROS), paraquat, all rescued wild-type worms from hemiasterlin toxicity. Worms lacking mitochondrial superoxide dismutase (MnSOD) were modestly drug-resistant, and elimination of MnSOD in the phb-2, har-1, and spg-7 mutants enhanced resistance. The antioxidant N-acetyl-l-cysteine prevented mitochondrial inhibitors from rescuing wild-type worms from hemiasterlin and sensitized mutants to the toxin, suggesting that a mechanism sensitive to ROS is necessary to trigger drug resistance in C. elegans. Using genetics, we show that this drug resistance requires pkc-1, the C. elegans ortholog of human PKCepsilon.
Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Resistência a Medicamentos/fisiologia , Mitocôndrias/metabolismo , Adenilato Quinase/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células HeLa , Herbicidas/farmacologia , Humanos , Metacrilatos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Paraquat/farmacologia , Proibitinas , Proteína Quinase C/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Tiazóis/farmacologia , Moduladores de Tubulina/farmacologiaRESUMO
Many cell lines derived from tumors as well as transformed cell lines are far more sensitive to V-ATPase inhibitors than normal counterparts. The molecular mechanisms underlying these differences in sensitivity are not known. Using global gene expression data, we show that the most sensitive responses to HeLa cells to low doses of V-ATPase inhibitors involve genes responsive to decreasing intracellular iron or decreasing cholesterol and that sensitivity to iron uptake is an important determinant of V-ATPase sensitivity in several cancer cell lines. One of the most sensitive cell lines, melanoma derived SK-Mel-5, over-expresses the iron efflux transporter ferroportin and has decreased expression of proteins involved in iron uptake, suggesting that it actively suppresses cytoplasmic iron. SK-Mel-5 cells have increased production of reactive oxygen species and may be seeking to limit additional production of ROS by iron.
Assuntos
Antinematódeos/uso terapêutico , Ferro/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Western Blotting , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Colesterol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Ferro/farmacologia , Macrolídeos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hemiasterlin is a potent antimitotic peptide that interferes with microtubule dynamics at picomolar concentrations in cell culture. The molecule largely eludes P glycoprotein-mediated drug efflux, and an analog is currently being evaluated in clinical trials as cancer chemotherapy. From a nonclonal genetic screen in Caenorhabditis elegans we isolated eight independent mutants resistant to a synthetic hemiasterlin analog. In one recessive mutant, phb-2(ad2154), a point mutation in prohibitin 2 (E130K) protects worms from drug-induced injury. Data indicate that direct binding of hemiasterlin to prohibitin 2 is unlikely. In fact, C. elegans phb-2(ad2154) was also found to be resistant to numerous other drugs that bind tubulin and to camptothecin, yet this mutant was sensitive to nocodazole and phalloidin. Thus, prohibitin 2 is implicated in a previously uncharacterized pathway of multidrug resistance.