Roles of DMP1 in Ras signaling and lung cancer

Wake Forest University Health Sciences, Winston-Salem NC

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Abstract

DESCRIPTION (provided by applicant): INK4a/ARF, located on chromosome 9p21, is one of the most commonly disrupted locus, in human cancer regardless of the patients' age or histology of tumors. The ARF tumor suppressor protein is a major mediator of the activation of p53 in response to oncogenic stress signaling and ARF-null mice die of malignancy within 15 months after birth. A number of transcription factors/nuclear proteins play a role in ARF regulation, either positive or negative. The positive regulators include E2F1, c-Myc, DMP1 and the negative regulators include Twist, Bmi-1, JunD, TBX, and p53 itself. Although hyperproliferative signaling stemming from oncogenic Ras has been shown to induce p53-dependent premature cell senescence by upregulating ARF, very little is known about the molecular mechanisms, especially at the levels of transcription. DMP1, cyclin D-interacting Myb-like Protein 1, is a novel transcription factor that had been isolated in a yeast two-hybrid screen with cyclin D2 bait. We have been accumulating evidence that suggests the importance of the DMP1 transcription factor in Ras-ARF signaling. For instance, 1) DMP1 directly binds and activates the ARF promoter and causes ARF-dependent cell cycle arrest. 2) DMP1-null murine embryonic fibroblasts are transformed by oncogenic Ras alone, bypassing the effects of immortalizing oncogenes such as c-Myc. 3) DMP1-knockout mice are hypersensitive to tumor formation, especially lung cancer, in response to dimethylbenzanthracene that causes Ha-Ras mutations. Furthermore, our preliminary data suggest that the DMP1 promoter is activated by oncogenic Ras in primary culture cells. Therefore, we hypothesize that oncogenic Ras signaling activates the ARF gene expression through transactivation of the DMP1 promoter. We will prove our hypothesis by reporter assays using ARF and DMP1 promoters, as well as by electrophoretic mobility shift assays, and by chromatin immunoprecipitation. We will crossbreed DMP1-knockout mice with activated K-Ras transgenic mice to observe the latency and spectra of tumor formation, in order to study the cooperativity of DMP1 inactivation and K-Ras mutation in living animals. We will search for aberrant expression and mutations of DMP1 in human lung cancer cells. Our studies will provide direct evidence that indicates that DMP1 plays a crucial role in conveying oncogenic Ras signaling to the ARF-p53 tumor suppressor pathway and will point to the role of DMP1 in human tumor suppression.