Scientific Overview: Systems Biology

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, cell proliferation, and cell 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 73 kinases and 72 phosphatases whose roles in cell survival were previously unrecognized. We are asking several questions. How are these novel 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?

Identification of kinases that regulate cell survival

We have performed RNAi screens in the presence of apoptosis-inducing chemotherapeutic agents (Taxol, cisplatin, and etoposide) and identified a group of kinases whose loss of function sensitizes cells to undergo cell death, the most interesting of these being PINK1 (PTEN-induced kinase 1). PINK1 was originally shown to be up-regulated by the tumor suppressor PTEN. Although PINK1 does not fall into a particular kinase subfamily, it has a known role in maintaining mitochondrial membrane potential. Other work has recently shown inherited mutations at chromosomal location 1p36 in familial Parkinson disease, and the two mutated genes that map to this region are PINK1 (PARK6) and DJ-1 (PARK7). Both genes are responsible for early-onset autosomal recessive parkinsonism. We had previously noted that DJ-1 is overexpressed in non–small cell lung carcinoma and that its down-regulation enhances apoptosis. Further, Parkinson disease–causing mutations in LRRK2 (PARK8), which are dominantly inherited gain-of-function mutations, sensitize neurons to cell death, and a significant fraction of the LRRK2 population is associated with the mitochondria. We are currently investigating whether the molecular mechanisms of PINK1 and LRRK2 in cancer and in Parkinson disease are linked.

Identification of phosphatases that regulate chemoresistance

Our research has shown that a large percentage of phosphatases and their regulatory subunits contribute to cell survival. This is a previously unrecognized general role for phosphatases as negative regulators of apoptosis, and it is important because phosphatases may no longer be simply viewed as enzymes that oppose the action of kinases. This research also identified a number of phosphatases whose loss of function results in chemoresistance, implicating these proteins as potential tumor suppressors. In our RNAi study, 5% of all phosphatases were shown to act in this way; an example is MK-STYX. Down-regulation of MK-STYX resulted in dramatic cellular resistance to cisplatin-, Taxol- or etoposide-induced cell death, which is consistent with up-regulated survival signals in these cells (Fig. 1). Also, MK-STYX is located at 7q11.23, a chromosome region mutated in colon cancer. MK-STYX is similar to MKP-1, which inactivates MAPKs; MK-STYX, however, is predicted to be a catalytically inactive phosphatase. Our observations suggest that MK-STYX acts against cell survival by sequestering pro-survival signaling components in a way analogous to the “substrate-trapping” effects of catalytically inactive phosphatases.

Figure 1. Identification of MK-STYX as a potential tumor suppressor phosphatase. Cells were transfected with control siRNA or MK-STYX siRNA for 48 h and then were treated for an additional 24 h with solvent control (–) or 50 μM cisplatin (+). Cell viability was visualized by A) crystal violet stain and B) cleavage of full-length PARP measured by western blot analysis.

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. In the Ras/MAPK pathway, growth factors activate the small G protein Ras, which recruits Raf to the plasma membrane where it is activated and phosphorylates MEK1/2, which in turn phosphorylates ERK1/2-MAPKs. Activated ERK1/2 phosphorylates additional kinases (such as RSK) and specific transcription factors (such as c-Fos and Elk-1) that are important in cellular proliferation, differentiation, and survival.

One project in the lab involved the question of whether the evolutionarily conserved MAPK pathway exhibits 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 (Fig. 2, top panel). 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. Our research and that of others has provided solid evidence for graded MAPK signaling in mammalian cells (Fig. 2, lower panel); that is, as agonist concentration increases, single-cell kinase activity increases proportionally. Yet we have also found that the proliferative response to growth factor stimulation is switch-like, demonstrating that the ultrasensitive step in the MAPK pathway occurs at the level of MAPK nuclear concentration and switch-like c-Fos induction. Although c-Fos induction and cell cycle entry in mammalian cells is switch-like, graded MAPK activation could have an important role in cell survival, since many MAPK targets regulating cell survival are in the cytoplasm.

Figure 2. Total cell population MAPK measurements. Single cells exhibiting a bistable (all-or-none) response or graded response (linear).