RESUMO
To identify novel therapeutic targets for non-small cell lung cancer (NSCLC), we conducted an integrative study in the following 3 stages: (i) identification of potential target gene(s) through shRNA functional screens in 2 independent NSCLC cell lines; (ii) validation of the clinical relevance of identified gene(s) using public databases; and (iii) investigation of therapeutic potential of targeting the identified gene(s) in vitro. A semi-genome-wide shRNA screen was performed in NCI-H358 cells, and was integrated with data from our previous screen in NCI-H460 cells. Among genes identified in shRNA screens, 24 were present in both NCI-H358 and NCI-H460 cells and were considered potential targets. Among the genes, we focused on eIF2ß, which is a subunit of heterotrimeric G protein EIF2 and functions as a transcription initiation factor. The eIF2ß protein is highly expressed in lung cancer cell lines compared with normal bronchial epithelial cells, and gene copy number analyses revealed that eIF2ß is amplified in a subset of NSCLC cell lines. Gene expression analysis using The Cancer Genome Atlas (TCGA) dataset revealed that eIF2ß expression is significantly upregulated in lung cancer tissues compared with corresponding normal lung tissues. Furthermore, high eIF2ß expression was correlated with poor survival in patients with lung adenocarcinoma, as shown in other cohorts using publicly available online tools. RNAi-mediated depletion of eIF2ß suppresses growth of lung cancer cells independently of p53 mutation status, in part through G1 cell cycle arrest. Our data suggest that eIF2ß is a therapeutic target for lung cancer.
Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Fator de Iniciação 2 em Eucariotos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Células A549 , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Idoso , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNARESUMO
To identify potential therapeutic targets for lung cancer, we performed semi-genome-wide shRNA screening combined with the utilization of genome-wide expression and copy number data. shRNA screening targeting 5043 genes in NCI-H460 identified 51 genes as candidates. Pathway analysis revealed that the 51 genes were enriched for the five pathways, including ribosome, proteasome, RNA polymerase, pyrimidine metabolism and spliceosome pathways. We focused on the proteasome pathway that involved six candidate genes because its activation has been demonstrated in diverse human malignancies, including lung cancer. Microarray expression and array CGH data showed that PSMA6, a proteasomal subunit of a 20S catalytic core complex, was highly expressed in lung cancer cell lines, with recurrent gene amplifications in some cases. Therefore, we further examined the roles of PSMA6 in lung cancer. Silencing of PSMA6 induced apoptosis or G2/M cell cycle arrest in cancer cell lines but not in an immortalized normal lung cell line. These results suggested that PSMA6 serves as an attractive target with a high therapeutic index for lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Domínio Catalítico/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Complexo de Endopeptidases do Proteassoma/genética , Células A549 , Idoso , Apoptose/genética , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Amplificação de Genes , Perfilação da Expressão Gênica/métodos , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Terapia de Alvo Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
Both pro- and anti-oncogenic roles of miR-221 and miR-222 microRNAs are reported in several types of human cancers. A previous study suggested their oncogenic role in invasiveness in lung cancer, albeit only one cell line (H460) was used. To further evaluate involvement of miR-221 and miR-222 in lung cancer, we investigated the effects of miR-221 and miR-222 overexpression on six lung cancer cell lines, including H460, as well as one immortalized normal human bronchial epithelial cell line, HBEC4. miR-221 and miR-222 induced epithelial-to-mesenchymal transition (EMT)-like changes in a minority of HBEC4 cells but, unexpectedly, both the microRNAs rather suppressed their invasiveness. Consistent with the prior report, miR-221 and miR-222 promoted growth in H460; however, miR-221 suppressed growth in four other cell lines with no effects in one, and miR-222 suppressed growth in three cell lines but promoted growth in two. These are the first results to show tumor-suppressive effects of miR-221 and miR-222 in lung cancer cells, and we focused on clarifying the mechanisms. Cell cycle and apoptosis analyses revealed that growth suppression by miR-221 and miR-222 occurred through intra-S-phase arrest and/or apoptosis. Finally, lung cancer cell lines transfected with miR-221 or miR-222 became more sensitive to the S-phase targeting drugs, possibly due to an increased S-phase population. In conclusion, our data are the first to show tumor-suppressive effects of miR-221 and miR-222 on lung cancer, warranting testing their potential as therapeutics for the disease.