Somatic mutations and copy number alterations (as a result of deletion or amplification of large portions of a chromosome) are major drivers of human lung cancers. Detailed analysis of lung cancer-associated chromosomal amplifications could identify novel oncogenes. By performing an integrative cytogenetic and gene expression analysis of non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) cell lines and tumors, we report here the identification of a frequently recurring amplification at chromosome 11 band p13. Within this region, only TNF receptor-associated factor 6 (TRAF6) exhibited concomitant mRNA overexpression and gene amplification in lung cancers. Inhibition of TRAF6 in human lung cancer cell lines suppressed NF-κB activation, anchorage-independent growth, and tumor formation. In these lung cancer cell lines, RAS required TRAF6 for its oncogenic capabilities. Furthermore, TRAF6 overexpression in NIH3T3 cells resulted in NF-κB activation, anchorage-independent growth, and tumor formation. Our findings show that TRAF6 is an oncogene that is important for RAS-mediated oncogenesis and provide a mechanistic explanation for the previously apparent importance of constitutive NF-κB activation in RAS-driven lung cancers.

Muscle cell differentiation, or myogenesis, is a well-characterized process and involves the expression of specific sets of genes in an orderly manner. A prerequisite for myogenesis is the exit from the cell cycle, which is associated with the up-regulation of the tumor suppressor Rb. In this study, we set to investigate the regulatory mechanism of the Rb promoter that allows adequate up-regulation in differentiating myoblasts. We report that Rb expression is regulated by the transcription factors GABP, HCF-1 and YY1. Before induction of differentiation, Rb is expressed at a low level and GABP and YY1 are both present on the promoter. YY1, which exerts an inhibitory effect on Rb expression, is removed from the promoter as cells advance through myogenesis and translocates from the nucleus to the cytoplasm. On the other hand, upon induction of differentiation, the GABP cofactor HCF-1 is recruited to and coactivates the promoter with GABP. RNAi-mediated knock-down of HCF-1 results in inhibition of Rb up-regulation as well as myotube formation. These results indicate that the Rb promoter is subject to regulation by positive and negative factors and that this intricate activation mechanism is critical to allow the accurate Rb gene up-regulation observed during myogenesis.

The transcription factor hGABP/E4TF1 is a heterotetrameric complex composed of two DNA-binding subunits (hGABP␣/E4TF1-60) and two transactivating subunits (hGABP␤/E4TF1-53). In order to understand the molecular mechanism of transcriptional regulation by hGABP, we searched for proteins that interact with the non-DNA-binding subunit, hGABP␤, using yeast two-hybrid screening. We identified a human cDNA encoding a protein related to YAF-2 (YY1-associated factor 2), which was previously isolated as an interacting partner of the Ying-Yang-1 (YY1) transcription factor. Reflecting this similarity, both YAF-2 and this novel protein (named YEAF1 for YY1-and E4TF1/hGABP-associated factor-1) interacted with hGABP␤ and YY1 in vitro and in vivo, indicating that YEAF1 and YAF-2 constitute a cofactor family for these two structurally distinct transcription factors. By using yeast three-hybrid assay, we demonstrated that hGABP␤ and YY1 formed a complex only in the presence of YEAF1, indicating that YEAF1 is a bridging factor of these two transcription factors. These cofactors are functionally different in that YAF-2 positively regulates the transcriptional activity of hGABP but YEAF1 negatively regulates this activity. Also, YAF-2 mRNA is highly expressed in skeletal muscle, whereas YEAF1 mRNA is highly expressed in placenta. We speculate that the transcriptional activity of hGABP is in part regulated by the expression levels of these tissue-specific cofactors. These results provide a novel mechanism of transcriptional regulation by functionally distinct cofactor family members.

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