Project Summary/Abstract Even though Inducible cAMP Early Repressor (ICER) has the functional characteristics of a tumor suppressor, there is no genetic evidence to demonstrate that ICER is a bona fide tumor suppressor gene product. Thus, altered post-translational events might be the cause of the observed abnormalities of ICER protein expression in cancer cells. On this basis it is hypothesized that in cancer cells, ICER is deregulated by ubiquitination resulting in constitutive proteasomal degradation and/or abnormal subcellular localization. Finding alternatives to rescue endogenous ICER nuclear expression in malignant cells could lead to the development of novel cancer treatment modalities. Through this project, we will study the mechanisms and physiological consequences of ICER ubiquitination and subcellular localization. We will use melanoma as a paradigm for the study. This study will focus on two specific aims. Aim 1. Determine the functional and physiological consequences of ICER ubiquitination by characterizing the specific ubiquitination sites on ICER and ubiquitin E3 ligases using biochemistry, chromatin binding, and siRNA techniques. Aim 2. Examine the effect on tumorgenesis upon expression of ?ubiquitinatable?-deficient ICER using mouse xenograft models and an established zebrafish model for melanoma. The overarching goal of this project is to determine the functional consequences of ICER post- translational modifications in cancer. The experiments described in this proposal will provide the basis for more extensive analyses of multi-component complexes associated with the regulation of ICER in cancer cells. Subsequent research will: 1) Use the zebrafish melanoma model to further characterize the molecular mechanisms of ICER ubiquitination during melanoma genesis; 2) Establish collaborative efforts to develop small-molecule compounds and small ICER-related peptides as specifics Ub-ligases inhibitors to test specific approaches to restore endogenous ICER nuclear expression in cancer cells; and 3) Study the effect of these Ub-ligase inhibitors in animal models for cancer.