Abstract
Background: The application of a multidimensional integrated analysis of recurring genomic and expression alterations can identify new insights into the molecular mechanisms involved in the pathogenesis of NSCLC. Distinguishing the key mechanisms and causal events driving tumorigenesis will lead not only to a better understanding of lung cancer phenotypes and biology, but also new molecular markers and therapeutic targets. Using this approach, we identified the chromosomal region at 12q13-15, and more specifically Glioma amplified sequence 41 (GAS41) to be frequently amplified and overexpressed in NSCLC. A putative transcription factor, amplification of GAS41 has been reported in dedifferentiated liposarcomas and in the earliest stages of glioma and astrocytoma. While the oncogene MDM2 has long been believed to be the driver of this amplicon, we hypothesize GAS41 is an oncogene capable of driving selection of the 12q15 amplicon, and not merely a passenger event.
Methods: An integrative genomics approach, examining 261 NSCLC tumors (169 adenocarcinomas (AC) and 92 squamous cell carcinomas (SqCC)) profiled for copy number and gene expression alterations was used to identify novel candidate oncogenes in NSCLC. Recurrent DNA amplifications were identified using the GISTIC algorithm and integrated with gene expression data to identify genes frequently amplified and overexpressed. Genes were classified as overexpressed if the fold change between tumor and matched non-malignant tissues was greater than 2 fold. The functional significance of GAS41 was assessed by lentiviral knockdown and ectopic overexpression in lung cancer cell lines with and without GAS41 amplification, and human bronchial epithelial cells respectively. In vitro assays measuring proliferation, anchorage independent growth, senescence and apoptosis were used to assess the phenotypic effect of gene dosage manipulation. Survival analysis was performed using the Cox regression model for multiple independent cohorts.
Results: GAS41 is gained or amplified in over 20% of NSCLC tumors, with similar frequencies of amplification in both AC (26%) and SqCC (24%). Although frequently co-amplified with MDM2, amplification of GAS41 was observed to occur in the absence of MDM2 amplification. Overexpression of GAS41 in human bronchial epithelial cells abrogated senescence, whereas knockdown reduced cell proliferation, impaired colony formation and induced cellular senescence only in lung cancer cell lines with amplification. Western blotting revealed increased p21, cleaved PARP and reduced levels of phospho-p53 in knockdown lines as compared to empty vector controls, suggesting GAS41 is implicated in the regulation of the p21-p53 pathway. Consistent with in vitro results, patients expressing high levels of GAS41 displayed poorer outcomes compared to those with lower levels of GAS41.
Conclusions: Our findings reveal GAS41 as a candidate oncogene frequently amplified and overexpressed in NSCLC, both in the presence and absence of MDM2 amplification. Gene dosage manipulation resulted in distinct phenotypic changes characteristic of oncogenes, and thus implicate amplification of GAS41 as a novel mechanism of NSCLC tumorigenesis.
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