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Genetic Control of Cell Proliferation, Cancer, and Genomic Integrity

Research Summary

Stephen Elledge is interested in understanding cell cycle control, the cellular response to DNA damage and how the proteome is remodeled by ubiquitin-mediated proteolysis. He also develops genetic technologies to aid in gene and drug discovery. He developed new methods to identify targets of autoimmunity. Recently he has applied genetic and computational strategies to understand how aneuploidy drives tumorigenesis and how genetics can uncover vulnerabilities in cancer cells.

The etiology of cancer is complex and involves multiple events. However, among these changes must be disruption of normal control of cell proliferation and genomic stability. In mammals, cell proliferation is primarily controlled at the point of cell cycle entry, the G1- to S-phase transition. A large number of genes exist that can influence cell proliferation. They normally function to control cell division spatially and temporally during embryonic development. Many of these genes, when activated in an inappropriate tissue or at an abnormal time, can cause cells that are not dividing to enter the cell cycle and begin to proliferate. They do so by interfacing with the basic cell cycle–regulatory machinery to activate cell cycle entry. As tumor cells evolve, they lose their ability to prevent mutations and maintain control of genomic stability. This allows mutations in growth regulatory genes to accumulate and tumors to form. We hope that by understanding the basic regulatory machinery responsible for catalyzing cell cycle entry and understanding how cells maintain genomic stability, we will uncover mechanisms that may be points through which general growth-inhibitory therapies may be targeted to attack cancer.

Cellular Responses to DNA Damage
Cancer occurs by the accumulation of mutations in growth-controlling genes. Conditions under which increased frequencies of mutations occur often lead to cancer, and there are several examples of human diseases that cause higher mutation rates and result in a predisposition to cancer. Enhanced mutation frequencies can result from environmental factors, such as increased exposure to DNA-damaging agents, or genetic factors that decrease the efficiency of normal DNA repair processes. Organisms have evolved elaborate sensory networks to detect and repair DNA damage to prevent alterations in their genetic material. Cellular responses to DNA damage and to blocks in DNA replication are similar: cells arrest progression through the cell cycle at distinct transitions and induce the transcription of genes whose products facilitate DNA repair.

The capacity to detect and respond to DNA damage is central to an organism's ability to avoid damage to its genetic material. To investigate how eukaryotic cells accomplish this task, we have undertaken genetic approaches to identify genes responsible for this in yeast and humans. We have uncovered a signal transduction pathway that senses and responds to DNA damage. Near the top of this pathway is a pair of PIK-related protein kinases, ATM and ATR. Of these, ATR is the most important, as it, together with its regulatory partner, ATRIP (ATR-interacting protein), responds to problems in DNA replication, the major source of genomic instability in cells. We recently discovered that the ATR-ATRIP complex senses DNA damage by binding to single-stranded DNA (ssDNA) coated with the binding protein RPA (replication protein A). This RPA-ssDNA complex, which is the signal for DNA damage and stalled replication forks, initiates the checkpoint response.

A second sensor of DNA damage is the Rad17/Rfc2–5 complex. We discovered that this complex also recognizes RPA-ssDNA complexes and when it does, it loads a PCNA (proliferating cell nuclear antigen)-related trimeric complex called 9-1-1 onto sites of DNA damage. Rad17 and 9-1-1 interact with the ATR-ATRIP complex on DNA to initiate a solid-state signaling apparatus at the site of damage and replication stress.

A key aspect of this pathway is the identification of the substrates for the ATM and ATR kinases in response to DNA damage. Through proteomic analysis, we have identified more than 1,000 proteins phosphorylated in response to DNA damage in vivo. These substrates implicate a large number of novel regulatory pathways in the DNA damage response and establish the framework for understanding this response. It is clear that the presence of DNA damage profoundly alters cellular physiology. Using this substrate list, we have identified several new proteins involved in the DNA damage response, including two new human disease genes, the Fanconi anemia I (FANCI) gene required for cross-link repair and the SMARCAL1 protein defective in Schimke immunoosseous dysplasia (SIOD). We have also discovered a new Brca1 complex, the A complex, which includes Brca1, Bard1, and three new proteins, Abraxas, Nba1, and Rap80.

Genetic Screens in Mammals
The tremendous power of genetics to unravel the mechanisms underlying multiple pathways in model organisms has transformed our understanding of biology. Until recently, large-scale genetic screens have not been possible in mammals. Recently, however, sophisticated tools have emerged that allow us to perform both gain-of-function and loss-of-function genetic screens in mammals.

Identification of genes that restrain or promote cell proliferation.

Using the power of RNA interference to silence gene expression, we are now undertaking loss-of-function screens in mammalian cells to investigate cell cycle control, DNA damage signaling, tumorigenesis, and transcriptional control of important regulators of cell proliferation. We discovered a collection of human genes whose overexpression or loss confers cancer-like phenotypes on cells. We have coupled this with an analysis of genes mutated in cancer and uncovered the mechanism through which aneuploidy acts to drive tumorigenesis.

We also have the ability to identify genes upon which cancer cells depend for survival. We have identified a number of genes whose depletion is lethal to cells bearing Kras mutations, and we identified a large mitotic network of genes Kras depends on for survival.

Control of Protein Stability
We discovered Skp1, F-box proteins and Rbx1, which together with the Cullen scaffold form the SCF ubiquitin ligase machine (now known as Cullin-Ring Ligases, CRL) that couples signal transduction to control of protein stability.  There are over 200 different CRLs controlling 1000s of substrates. To identify ubiquitination substrates, we developed Global Protein Stability (GPS) profiling. GPS allows us to measure the half-lives of thousands of proteins simultaneously and to discover proteins whose stabilities are altered in response to stimuli such as drugs or pathogen invasion. Using GPS, we have identified several hundred substrates of the Cul1–5 ubiquitin ligase family. We plan to use these proteins to understand how the proteome is remodeled after translation in the same way that microRNAs remodel the transcriptome after transcription and how this changes in response to stimuli. Thus we are taking a systems biology approach to investigate the plasticity of the proteome.

A grant from the National Institutes of Health provided support for much of the DNA damage signaling work in my lab.

As of February 26, 2014

Scientist Profile

Investigator
Brigham and Women's Hospital
Genetics, Molecular Biology