RESUMO
KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention.
Assuntos
Oncogenes , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Adenilato Quinase/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry-based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.
Assuntos
Proteínas de Membrana/análise , Proteínas Mutantes/análise , Proteômica/métodos , Proteínas Proto-Oncogênicas p21(ras)/análise , Antígenos CD/análise , Antígenos de Neoplasias , Basigina/análise , Moléculas de Adesão Celular/análise , Linhagem Celular Tumoral , Movimento Celular , Biologia Computacional , Transição Epitelial-Mesenquimal , Glicoproteínas/classificação , Glicoproteínas/fisiologia , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Proteínas de Neoplasias/análiseRESUMO
The Kaposi sarcoma associated herpesvirus (KSHV) genome encodes more than 85 open reading frames (ORFs). Serological evaluation of KSHV infection now generally relies on reactivity to just one latent and/or one lytic protein (commonly ORF73 and K8.1). Most of the other polypeptides encoded by the virus have unknown antigenic profiles. We have systematically expressed and purified products from 72 KSHV ORFs in recombinant systems and analyzed seroreactivity in US patients with KSHV-associated malignancies, and US blood donors (low KSHV seroprevalence population). We identified several KSHV proteins (ORF38, ORF61, ORF59 and K5) that elicited significant responses in individuals with KSHV-associated diseases. In these patients, patterns of reactivity were heterogeneous; however, HIV infection appeared to be associated with breadth and intensity of serological responses. Improved antigenic characterization of additional ORFs may increase the sensitivity of serologic assays, lead to more rapid progresses in understanding immune responses to KSHV, and allow for better comprehension of the natural history of KSHV infection. To this end, we have developed a bead-based multiplex assay detecting antibodies to six KSHV antigens.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 8/imunologia , Técnicas Imunológicas , Proteoma/imunologia , Adulto , Idoso , Anticorpos Antivirais/sangue , Coinfecção/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Infecções por HIV/complicações , Infecções por HIV/imunologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Ribonucleotide reductase inhibitors enhance the anti-HIV-1 activities of a variety of nucleoside analogs, including those that act as chain terminators and those that increase the HIV-1 mutation rate. However the use of these ribonucleotide reductase inhibitors is limited by their associated toxicities. The hydroxylated phytostilbene resveratrol has activity in a host of systems including inhibition of ribonucleotide reductase and has minimal toxicity. Here we synthesized derivatives of resveratrol and examined them for anti-HIV-1 activity and their ability to enhance the antiviral activity of decitabine, a nucleoside analog that decreases viral replication by increasing the HIV-1 mutation rate. The data demonstrates that six of the derivatives have anti-HIV-1 activity greater than resveratrol. However, only resveratrol acted in synergy with decitabine to inhibit HIV-1 infectivity. These results reveal novel resveratrol derivatives with anti-HIV-1 activity that may have mechanisms of action that differ from the drugs currently used to treat HIV-1.