The role of MAP3K8 in lung tumorigenesis.

摘要

The MAP3K8 (Cot/Tpl-2) protooncogene is a serine/threonine kinase that participates in the MAP kinase cascades (MEK-1, SEK-1, and MKK6), NFAT activation, the NF-kB signalsome, and Caspase-9 induced apoptosis. Activation of MAP3K8 is believed to contribute to cellular transformation and tumorigenesis. MAP3K8 was examined in lung cancer cells to determine if the gene played a role in lung tumorigenesis. Three areas related to tumorigenesis were examined: mutational analysis of the MAP3K8 gene in human lung cancer cells, expression analysis of MAP3K8 in lung cancer cell lines, and alterations of non-transformed lung cells upon transfection of MAP3K8. PCR-based techniques (RT-PCR, SSCP, RACE, and Realtime PCR) analyzed mutational and expressional alterations of the gene. This was complemented with primer extension analysis to identify the promoter and Western blot analysis to confirm protein expression. Additional assays characterized alterations of lung cell activity including transfection, FACS analysis, Caspase activity analysis, and two different protein arrays. In this thesis, the first mutation of MAP3K8 occurring in a primary human tumor was identified in a lung adenocarcinoma resulting from a rearrangement of the 3' end of the RNA. Additional mutations were not found in either the 3' end or open reading frame of the gene. Expression analysis demonstrated increased levels of MAP3K8 transcript in a large fraction of NSCLC cell lines, contrasting decreased levels of transcript in the majority of SCLC cell lines. These levels, however, did not correlate with protein expression in the cell lines examined. The transfection of an immortalized bronchial epithelial cell line (9HTE) with wildtype or mutant MAP3K8 plasmids slowed proliferation of the cells in a non-apoptotic manner compared to cells transfected with an empty vector. Examination of the activated pathways in the transfected cells identified altered activity of multiple transcription factors including NFAT, c-Myb, and Brn-3; and implicated a role for MAP3K8 in the mTOR and PKC signaling pathways. These data demonstrate that MAP3K8 is rarely mutated in lung tumors, but suggest that both altered transcriptional and translational regulation are associated with lung tumorigenesis. The transfection of MAP3K8 in non-transformed lung cells slowed proliferation, indicating that other molecular alterations are necessary to complement an oncogenic role for MAP3K8 in lung cancer.