Research Interests
The primary focus of the Systems Biology laboratory is identifying and understanding the genes and signaling pathways that, when mutated, contribute to the pathophysiology of cancer. We take advantage of RNA interference (RNAi) and novel proteomic approaches to identify the enzymes that control cell growth, proliferation, and survival. For example, after screening the human genome for more than 600 kinases and 200 phosphatases—called the “kinome” and “phosphatome”, respectively—that act with chemotherapeutic agents in controlling apoptosis, we identified several essential kinases and phosphatases whose roles in cell survival were previously unrecognized. We are asking several questions. How are these survival enzymes regulated at the molecular level? What signaling pathway(s) do they regulate? Does changing the number of enzyme molecules present inhibit waves of compensatory changes at the cellular level (system-level changes)? What are the system-level changes after reduction or loss of each gene?
Novel Modulators of Chemotherapeutic Sensitization
Kinases and phosphatases play an integral role in balancing the survival and apoptotic signals within a cell. In an attempt to define proteins with a major role in these processes, we tested an RNAi library against all known kinases and phosphatases in the human genome and assayed various phenotypes, including sensitization to apoptosis and chemoresistance. A group of apoptosis sensitizers was identified whose siRNA knock-out conferred a marked increase in cell survival as well as a striking chemoresistant phenotype (Figure 1). One of these proteins, MK-STYX, resembles the dual-specificity phosphatases implicated in MAP kinase signaling, but it is catalytically inactive due to a cysteine-to-serine mutation at its active site. When MK-STYX is knocked down via RNAi, the cells display a profound decrease in apoptosis; MK-STYX-overexpressing cells, on the other hand, are sensitized to apoptotic signals. We propose that MK-STYX could function as a dead phosphatase, sequestering potential phosphoproteins that promote survival. Through further experiments, we plan to characterize MK-STYX and elucidate its mechanism of apoptotic sensitization; these studies may identify a survival signal that would constitute a novel target for chemotherapy.
 |
Figure 1. Human kinase and phosphatase siRNA library screen. HeLa cells were transfected with siRNAs directed against all known and putative human phosphatases and kinases. Cells were incubated for 72 h to allow target knockdown, and apoptosis was measured by a DNA-fragmentation ELISA. The graph shows relative apoptosis for 600 kinase and 200 phosphatase siRNA targets. |
Monitoring Cellular Signaling
Phosphatidylinositol-3-kinase (PI3K) phosphorylates the 3´ ring position of phosphatidylinositol to generate lipid products important for signal transduction, membrane trafficking, and other cellular processes. The identification of PI3Ks as key players in cellular functions ranging from vesicular trafficking to cell survival merits further study to identify factors acting immediately up and downstream of these lipid kinases, as well as characterizing the phosphatase regulating these molecular pathways. Roles for PI3K isoforms in amino acid sensing and in signaling through the mTOR pathway, as well as in autophagy, have also recently emerged. Note that these functions of PI3Ks might not merely rely on their lipid kinase activity, since they are large enzymes that could also serve as platforms for the assembly of protein complexes. Understanding is needed of the mechanisms of PI3K signaling involved in these various cellular functions.
Parkinson Disease–Associated Genes in Cancer
Renal cell carcinoma (RCC) is an aggressive cancer that is highly metastatic and refractory to all forms of systemic cancer therapy. Using bioinformatic analysis and over 150 RCC tumor samples, we have identified Parkinson disease–associated (PD) kinases as a novel molecular constituent of the renal tubule epithelium whose expression is specifically down-regulated during the progression of papillary RCC (Figure 2). These PD kinases are highly expressed in the brain and kidney and have been previously linked to familial Parkinson disease. Activating mutations in these genes sensitize cells to oxidative stress and lead to increased death of neural cells, implying that these genes may function as a sensor of oxidative stress in the renal epithelium and induce cell death pathways in response to toxic levels of reactive oxygen species. Selective loss of each gene in more aggressive and metastatic RCC tumors suggests that this protein may also be a tumor suppressor. The goal of this project is to define the relationship between oxidative stress management and malignant tumor progression in the kidney, with a particular emphasis on the role of kinase signaling. This project is a collaboration with VARI’s Kyle Furge and Bin Teh.
 |
Figure 2. PD genes are located within a conserved RCC amplicon on chromosome 12. Expression profiles from more than 150 normal and RCC tissue samples were obtained by microarray analysis with the Affymetrix HGU-133 Plus 2.0 chip. |
Colorectal Cancer
Chemoresistance is a therapeutic problem that severely limits successful treatment of most human cancers. This is particularly true of colorectal cancer, in which the development of resistance is common: most anti-cancer regimens are ineffective, with the five-year survival rates for late-stage colorectal cancer being only 8%. How colorectal cancer resistance develops is largely unknown, and the response to therapy varies based on individual patient tumors. With this in mind, how can we prevent cancer emergence or progression at the level of individual tumors? Recent studies have shown that a large percentage of colorectal tumors have mutations in a key gene, for class I PI3K. While mutations play an important causative role in colorectal cancer, it is currently unclear how these mutations can be exploited as drug targets and whether we can develop targeted cancer agents based on the gene. We have ongoing projects to determine the molecular pathways (and genes) that can be used to prevent progression of precancerous lesions to colorectal cancer. Further, we are defining each pathway activation in each patient’s tumor and comparing the pathways with a novel chemopreventive agent against PI3K/mTOR.
Graded MAPK Signaling and Switch-like c-Fos Induction
We also take a systems biology approach to understanding two key molecular pathways, Ras/MAPK and PI3K/mTOR. One project in the lab involves the question of whether the evolutionarily conserved pathways exhibit a switch-like or a graded response in mammalian cells. Ultrasensitive switch-like responses control cell-fate decisions in many biological settings, and the regulation of kinase activity is one way in which such behavior can be initiated. Signaling molecules switch between two discontinuous, stable states with no intermediate; this is referred to as a bistable response. Given the irreversible, all-or-none nature of many cell behaviors, including cell cycle control and apoptosis, significant effort has been focused on identifying the cellular mechanisms underlying bistability.
