Defining Mutagenesis Pathways in Breast Cancer Evolution

Institution: Scripps Research Institute
Investigator(s): Ewa Lis, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11GB-0004 Award: $67,520
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2005)
Breast cancer is a genetic disease that begins with the mutation of DNA. Traditionally mutation, and therefore cancer, has been attributed to failure of the DNA replication and repair systems. More recently, researchers have come to appreciate that the cell must play an active role, and has the potential to induce mutations in response to stress. The capacity of cells to mutate themselves presents a novel approach to cancer prevention/treatment namely through the inhibition of proteins required for mutation.

Our approach is to study mutation processes in yeast, then translate these finding to human breast cancer. Yeast has proven to be an excellent model organism to dissect out evolutionarily conserved cellular pathways, such as cell cycle and DNA repair pathways. For example, the three known mutagenesis genes Rev1, Rev3 and Rev7 were first identified in yeast. These genes, when deleted, render cells essentially immutable. Our approach involves systematic screening of large yeast deletion strain libraries. Thus, the first objective of this project is to generate a set of genes involved in mutagenesis using multiple screens with a variety of selection systems. The second objective is to characterize that set of genes in terms of their relationship to the REV genes (both genetic as well as any physical protein interactions). The last objective is to take a selected set of evolutionarily conserved mutagenesis genes in humans and characterize them in breast cancer cell lines with respect to mutation rates and development of drug resistance. For these experiments, human homologs from the selected yeast gene set will be deleted in breast cancer cell lines and tested for their ability to reduce mutation rates and prevent the development of drug resistance. We will collaborate with Dr. Brunhilde Felding-Haberman, a CBCRP-funded researcher, to accomplish the breast cancer specific portion of these studies.

All stages of breast cancer show genetic instability whether it is the early stages when point mutations take place or the later stages when gross chromosomal abnormalities are observed. The majority of drug resistance is acquired through mutations in receptors and pumps in the cancer cells. The long term goal is to find an evolutionarily conserved mutation gene target in humans and develop a drug that would inhibit mutagenesis and prevent the emergence of drug resistance. Our novel approach of inhibiting mutagenesis has an immense potential of success not only because it fights cancer at its source, but also because it would be applicable to all patients with breast cancer undergoing therapy.


Final Report (2007)
The greatest difficulty in fighting cancer is that all cancers are different and hence require different treatments. However all cancers do begin the same way, with the mutation of DNA. Mutation also plays a central role in the evolution of drug resistance during chemotherapy. Traditionally, mutation has been attributed to the failure of DNA polymerases to maintain ultimate fidelity, and the failure of the repair systems to detect and/or correct the rare polymerase errors. We now know that this is not correct; cells must induce proteins that comprise a biochemical pathway whose function is to induce mutation.

Mutation in response to most types of DNA damage is thought to be mediated by the error-prone sub-branch of post-replication repair and the associated “translesion synthesis polymerases.” To further understand the mutagenic response to DNA damage, we screened 4,848 Saccharomyces cerevisiae (yeast) gene deletion strains to identify genes involved in damage induced mutation of the CAN1 gene. Extensive quantitative validation of the strains identified by the screen in different genetic backgrounds and with different mutation assays led to the identification of ten genes. Among the identified genes were those functioning in error-prone post replication repair as well as two additional genes, FYV6 and RNR4. Genetic characterization of FYV6 and RNR4 demonstrate that they are epistatic (i.e., epistasis takes place when the action of one gene is modified by one or several genes, sometimes called modifier genes) with respect to induced mutation, and that they function independently of post-replication repair, although FYV6 plays a smaller role. This novel pathway of induced mutation appears to be mediated by an increase in dNTP (DNA building blocks) levels that facilitates lesion bypass by the replicative polymerase Po16, and is as important as error-prone post-replication repair in the case of UV- and MMS (methylmethane sulfonate)-induced mutation, but solely responsible for EMS (ethylmethane sulfonate)-induced mutation. We propose that Rnr4/PolS and mutagenic post-replication repair constitute the two dominant pathways by which yeast induce mutation in response to DNA damage. Finally, we show that Rnr4/Po1S-induced mutation is efficiently inhibited by a small molecule inhibitor of ribonucleotide reductase, suggesting that if similar pathways exist in human cells, intervention in some forms of mutation may be possible.

Pathways for replication of damaged DNA are highly conserved from yeast to human cells. Thus, the aim of this research is to explore processes by which DNA replication can occur despite the presence of DNA mutations. Yeast strains that differ in their ability to replicate in the presence of UV- or chemically-induced mutations might be used to identify relevant genes in human cells. If we could develop new strategies to block replication of breast cancer cells when DNA mutations are present, then tumor initiation and progression might be blocked. Finally, the notion that cells mutate themselves in response to stress is not simply of academic interest, but rather, this idea identifies a new, potentially revolutionary, approach to cancer prevention and therapy – namely, the inhibition of proteins that are required for mutation. The overall goal of the proposed research was to identify the proteins of the induced mutation system in eukaryotes and validate them as drug targets in an effort to develop novel therapies for breast cancer.


Symposium Abstract (2007)
Breast cancer is a genetic disease that begins with the mutation of DNA. Traditionally mutation, and therefore cancer, has been attributed to failure of the DNA replication and repair systems. More recently, researchers have come to appreciate that the cell must play an active role with the potential to induce mutations in response to stress. The capacity of cells to mutate themselves presents a novel approach to cancer prevention/treatment namely through the inhibition of proteins required for mutation. Our approach is to study mutation processes in yeast, then translate these finding to human breast cancer. Yeast has proven to be an excellent model organism to dissect out evolutionarily conserved cellular pathways, such as cell cycle and DNA repair pathways.

Mutation in response to most types of DNA damage is thought to be mediated by the error-prone sub-branch of post-replication repair and the associated translesion synthesis polymerases. To further understand the mutagenic response to DNA damage, we screened 4,847 yeast deletion strains to identify genes involved in damage induced mutation of the CAN1 gene. We identified each of the known components of error-prone post-replication repair as well as two additional genes, FYV6 and RNR4. Genetic characterization of FYV6 and RNR4 demonstrate that they act in the same DNA repair pathway, and that this pathway is distinct from that of the conventional translesion polymerases. This novel pathway appears to be mediated by an increase in dNTP (nucleotide) levels that facilitates lesion bypass by the replicative polymerase, called PolD, and is as important as error-prone post-replication repair in the case of UV- and MMS-induced mutation, but solely responsible for EMS-induced mutation. We propose that Rnr4/Pold and mutagenic post-replication repair constitute the two dominant pathways by which yeast induce mutation in response to DNA damage. Finally, we show that Rnr4/PolD-induced mutation is efficiently inhibited by a small molecule inhibitor of ribonucleotide reductase, suggesting that if similar pathways exist in human cells, intervention in some forms of mutation may be possible.