Characterization of COP1 in Gastrointestinal Stromal Tumor (GIST) Pathogenesis

Gastrointestinal stromal tumors (GISTs) are defined by gain of function mutations in either the KIT or PDGFRA receptor tyrosine kinases. Secondary mutations in KIT or PDGFRA have led to kinase-inhibitor drug-resistance, presenting a need for alternative molecular targets for advanced GISTs. We have recently discovered that an ETS family transcription factor, ETV1, is highly expressed in GISTs and is required for the growth and development of GIST cells. We have previously observed that the protein level of ETV1 is stablized by active KIT and its downstream MAP kinase signaling. Regulatory mechanisms of ETV1 KIT/MAPK-dependent protein stability were investigated by isolating ETV1-containing nuclear complexes; in which mass-spectrometry and western blot identified COP1 as a member of the core-complex. COP1 is an E3 ubiquitin ligase that has been characterized as a tumor suppressor that regulates ETV1 protein stability in prostate cancers harboring ETV1 translocations. To further understand how the signaling-dependent ETV1 protein stability is regulated in GIST cells, we isolated and biochemically characterized the endogenous COP1 core complex (eCOP1) by performing nuclear extraction, column fractionation, and immunopurification. Preliminary mass-spectrometry identified ETV1 as one of the core-complex members, consistent with our previous biochemical characterization of the ETV1 endogenous complex. The rest of the core-complex members are currently being identified by mass spectrometry and will be validated by western blot, gel filtration chromatography, and in vitro binding-assays with recombinant proteins. These studies are crucial to exploring alternative treatment options for advanced GISTs by identifying potential novel therapeutics that may destabilize ETV1.