2008 Scientific Report

VARI | 2008
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
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.
35