Quantitative proteomic analysis of proteins important in cancer

In this seminar, I will talk about the ICAT (isotope-coded affinity tags) quantitative proteomics technology and its applications to 1) the analysis of Myc network function, 2) the identification of the substrates of ubiquitin ligases, and 3) the analysis of protein secretion.

Our initial ICAT proteomic comparisons of myc-expressing and myc-non-expressing fibroblasts revealed a novel cytoskeletal function for Myc (EMBO J. 21: 5088-96, 2002), and a critical role for a mitochondrial chloride channel, mtCLIC, in Myc-induced apoptosis (J. Biol. Chem., 281: 2750-6, 2005)), which had escaped detection in all mRNA expression analyses to date. In the process of these screens, we also extended the analysis of Myc-regulated proteins to nuclear regulatory factors by developing a method to identify and quantify chromatin-associated proteins using a combination of chromatin isolation and ICAT analysis (J. Am. Soc. Mass Spec. 14: 696-703, 2003). We have also developed a method to systematically identify the components of multi-protein complexes using ICAT and applied this method to the analysis of proteins binding to SAP25, a novel transcriptional co-repressor discovered by the PI (Mol. Cell. Biol. 26: 1386-97, 2006).

We have now extended these studies to address regulation at the level of protein ubiquitination and degradation through proteomic identification of the substrates of cancer-associated ubiquitin ligases. A number of ubiquitin ligases play key roles in tumorigenesis, but many of their critical substrates are yet to be identified. The problem stems from that there is no established method to identify ubiquitin ligase substrates. We devised two proteomic approaches to identify ubiquitin ligase substrates and identified a novel substrate for VHL tumor suppressor, which may mediate apoptosis of tumor cells, and a novel substrate for BRCA1 tumor suppressor, which may be a missing link between BRCA1 and breast/ovary-specific tumor suppression

Cancer cells secrete many proteins which can be detected in cancer patient serum and could be used as cancer protein biomarkers. However, direct proteomic analysis of whole serum proteins is technically difficult because of the complexity of the serum proteome. To identify candidate biomarkers without directly analyzing patient serum, we are interested in the quantitative proteomic analysis of proteins secreted from cultured cancer cells. We have developed an ICAT-based method to systematically identify and quantify proteins secreted in culture supernatant and using this method, we conducted a pilot proteomic analysis of the secretory pattern associated with replicative senescence of human fibroblasts. Some of the proteins displaying increased secretion from senescent fibroblasts have biological activities that can affect the function of other cells and support the hypothesis that protein secretion from senescent cells affects the physiology of neighboring tissues including tumor growth.