Although initiated by genetic mutation, the unchecked proliferation, aberrant differentiation, and altered motility of cancer cells depends upon the integrity and activation state of specific signal transduction pathways. Our laboratory is interested in understanding how alterations in these signaling pathways contribute to human cancer, and whether exploitation of that understanding can aid in the development of new diagnostics, prognostics and therapeutic intervention strategies. To this end, we employ a global “systems level” integrative discovery platform, one that has as a foundation mass spectrometry-based proteomic interaction networks. More specifically, through LC/MS/MS, we define the physical interaction network for a signaling pathway of oncogenic interest. By small molecule and functional genomic screening, we then annotate the human genome for functional contribution to the pathway of interest. Integration of these data with cancer-associated mutation data and cancer-associated gene expression data yields a powerful tool for oncogenic discovery—a cancer annotated physical/functional map for a specific signaling pathway of interest. The models and hypotheses produced though integrative screening are challenged through mechanistic studies employing cultured human cancer cells, zebrafish, mice and in vitro biochemical systems. With this general approach, we are currently pursuing the following projects:
Wnt/β-catenin Signal Transduction
Of the relatively small number of signaling pathways that function as master regulators of development, adult tissue homeostasis and cancer, the β-catenin dependent Wnt pathway (Wnt/β-catenin) figures prominently; it regulates the growth and fate of neoplastic cells in tissues of diverse origin, notably the colon, kidney, breast and skin. Integrative molecular analysis of Wnt/β-catenin signaling in kidney and colon cancer models yielded numerous discoveries of mechanistic and clinical importance. Work in my lab is now focusing on an integrative analysis of Wnt signaling in pancreatic adenocarcinoma and lung cancer. For both cancer models, independent functional genomic screens have been completed and have identified numerous novel regulators, some of which are mutated in human cancer. In addition, we are looking more closely at a family of proteins I discovered to regulate Wnt signaling in Wilms tumor. Specifically, we are working to understand the mechanism(s) by which members of the WTX gene family regulate Wnt signaling and oncogenesis.
Keap1/Nrf2 Signal Transduction
Keap1 is an E3 ubiquitin ligase important for cellular defense against genotoxic stress, and in that context contributes fundamentally to aging and a myriad of human cancers, most notably lung cancer. Keap1 functions by ubiquitinating the Nrf2 transcription factor, which ultimately results in the proteosomal degradation of Nrf2. In an effort to better understand Keap1 in an oncogenic contect, I completed quantitative proteomic analysis of the Keap1 protein complex as well as functional genomic screen of the Keap1/Nrf2 pathway. The resulting integrative map has identified numerous novel proteins which both physically associate with keap1 as well as functional regulate Keap1/Nrf2 signaling. Ongoing work in the lab is focused on understanding the mechanisms by which these proteins control Keap1 function as well as uncovering new cancer connections.
E3 Ubiquitin Ligase Substrate Identification
E3 ubiquitin ligase complexes provide specificity and catalysis for the transfer of ubiquitin to target proteins, a post-translational modification that results in proteosome-mediated degradation, altered subcellular localization or changes in protein interaction. As such, E3 ubiquitin ligases regulate every facet of cell biology, and importantly, are frequently perturbed in disease states such as cancer. While expanding my protein-protein interaction network for the Wnt/β-catenin pathway, I performed tandem affinity purification and mass spectrometry on βTrCP, the E3 ubiquitin ligase responsible for β-catenin degration. As the E3 complex is catalytic in action, many of the known βTrCP substrates were not identified by LC/MS/MS. As such I designed and implemented a novel approach which stabilizes the E3-substrate interaction. Using this strategy, I have identified novel substrates for both the βTrCp and Keap1 E3 ubiquitin ligases. Work in my laboratory is exploiting this system to identify substrates for uncharacterized E3 ubiquitin ligases, specifically those with established connections to human disease. Follow-up experiments require biochemical purification of target substrate following gain-of-function and/or loss-of-function of the E3, in vitro analysis of the ubiquitination reaction, and histological assessment of substrate expression in patient tissue samples. Of great importance, and in fact the greatest challenge, is the demonstration of biological significance for the E3-substrate relationship. To address this, my lab implements an array of technologies encompassing cDNA microarray analysis, proteomics and whole organism studies.
Genetic Screening Platforms
In addition to this integrative analysis of signaling, we are also actively working to develop a human somatic cell forward-genetic screening platform. Eukaryotic cells sense and interpret extracellular cues through a highly integrated network of intracellular signaling pathways. The identification of these pathways and their constituent proteins can be largely attributed to genetic screens in yeast, Drosophila, c-elegans and zebrafish, each of which permits random mutagenesis screening and clonal isolation in a homozygous state. RNA interference provides analogous loss-of-function technologies in mammalian systems, and by doing so has revolutionized the significance and speed of our discoveries. However, RNA interference-based screens are expensive and fraught with artifact, owing in part to off-target effects and incomplete silencing. An exciting and ongoing effort in my lab is to realize somatic cell genetic screens in human cells. We are pursuing this goal through retroviral insertional mutagenesis of stable near-haploid human cell lines. Leveraging our existing expertise in proteomic and functional genomic analysis of signal transduction, we are employing this haploid screening platform to comprehensively identify proteins and microRNAs required for the following signaling pathways, each of which functions as a master regulator of human disease and human development: TGFβ, Wnt/β-catenin, retinoic acid, NF-κB and hedgehog. A systems-level integration of the resulting functional data with protein-protein interaction networks, genome-wide association data and transcriptional signatures will reveal targets of diagnostic, prognostic and therapeutic value. Considering only the haploid screening approach, success promises rapid, unbiased and inexpensive complete loss-of-function phenotypic screening of human cells, and therefore has the potential to transform the experimental strategies taken in both basic and applied sciences.