Exploring the Role of PARP Inhibitors in Breast Cancer

Institution: Stanford University
Investigator(s): Karlene Cimprich, Ph.D. -
Award Cycle: 2007 (Cycle 13) Grant #: 13IB-0029 Award: $157,516
Award Type: IDEA
Research Priorities
Etiology and Prevention>Etiology: the role of environment and lifestyle



Initial Award Abstract (2007)
Approximately 80% of breast cancers occur sporadically, while the remaining cases are inheritable. Mutations in the breast cancer associated genes BRCA1 and BRCA2 are responsible for a major portion of the hereditary cases. Importantly, the study of BRCA1/2’s role in normal cell chromosomal DNA repair pathways has provided great insights in how defects in these repair mechanisms underlie cancer progression. Notably, recent studies have shown that inhibitors of poly (ADP-ribose) polymerase (PARP), a key DNA repair enzyme, exhibit significantly enhanced toxicity to BRCA1- or 2-deficient cells, and clinical trials have been initiated in patients with BRCA deficiency. PARP is required for the repair of DNA single-stranded breaks, and inhibition of PARP leads to the conversion of single-strand breaks to double-strand breaks. In the absence of BRCA1 or 2, homologous recombination cannot occur and the double-strand breaks remain unrepaired leading to complex chromosomal alterations and apoptosis (programmed cell death).

Although BRCA1/2 mutations are only found in 5–10% of patients with breast cancer, studies have suggested that a significant portion of patients with sporadic breast cancer may have gene expression profiles similar to those in patients with hereditary BRCA1/2 mutations. This leads to two critical questions. First, which genes in the BRCA DNA repair pathway lead to breast cancer in a manner identical to BRCA mutations or deficiency? Second, will PARP inhibitors showing promise in treating patients with BRCA hereditary defects be applicable to a proportion of sporadic breast cancers?

We will employ a high-content genomic siRNA screen to identify genes for which loss of function increases the sensitivity of cells to PARP inhibitors. Next, the “hits” obtained from the siRNA screen will be validated, categorized and prioritized using a series of secondary assays. This will allow us to gain insight into the mechanism by which PARP inhibitors function and to choose those “hits” (i.e., DNA repair pathway genes of interest) most appropriate for further study. Our approach is to use immortalized breast epithelial cells to screen a large genomic library of siRNAs in the presence and absence of a PARP inhibitor. Using high-throughput techniques, we will analyze Rad51 focus formation and DNA content as markers for homologous recombination, cell cycle arrest and apoptosis to select our candidate genes in interest. We anticipate that the “hits” of highest interest are those which cause cell death (apoptosis) in the presence, but not absence, of the PARP inhibitor.

The use of PARP inhibitors is currently a promising avenue for therapy for BRCA-deficient patients due to a high therapeutic index, decreased toxicity of the treatment, and improved quality of life for breast cancer patients. We expect to identify additional genetic biomarkers to develop a novel “profile” of sporadic human breast cancers that would be sensitive to PARP inhibitors. Finally, these genes may also be important in the cell’s ability to maintain genome stability and as such are candidate breast cancer susceptibility genes.


Final Report (2008)
Approximately 80% of breast cancers are due to chance mutations in various and often unknown breast cancer genes. Among familial breast cancer cases, inactivation of the tumor suppressor genes BRCA1 and BRCA2 accounts for ~ 5-10% of the tumors while multiple unknown genes not related to BRCA1 and BRCA2 are implicated in the remaining cases of hereditary breast cancer. PARP is an enzyme needed to repair DNA following damaged produced by external stimuli and endogenous metabolites, and the BRCA genes are two of many genes involved in a different form of DNA repair. Drugs that inhibit PARP selectively kill breast cancer cells defective in the BRCA1 and BRCA2 genes and are currently in clinical trials. When PARP is inhibited, the BRCA genes help with a second, back-up form of repair for DNA damage, and cells are able to survive. When both the PARP and BRCA pathway are blocked, cells die because they are unable to repair damaged DNA. Importantly, studies in our lab and others suggested that other mutations in breast cancer cell lines might sensitize these cells to PARP inhibitors. Thus, we hypothesized that there might be other breast cancer patients that would be effectively treated with PARP inhibitors and that we might be able to identify them by identifying those genes that, when knocked down, sensitize cells to PARP inhibitors.

To do this, we planned to develop a high-throughput screen that could be used to screen the genome for those genes that might be needed to keep cells alive when PARP inhibitors were present. We also wanted to carry out this screen on a genome-wide scale. We were successful in developing a high-throughput and robust method for this screen, and we have implemented it on a small scale. We had some difficulties with our original readout that led us to redesign our assay. Although these difficulties did slow our overall progress, we think the modified assay is more robust, more sensitive, faster, and less expensive. We also believe it is now ready for a larger-scale screen, and plans are in progress to carry this screen out. Moreover, our small scale assay has already identified three genes that may predict sensitivity to PARP inhibitors. We plan now to work with our collaborator to determine if these genes might be mutated in breast cancer cells known to exhibit PARP inhibitor sensitivity, and to determine if they might be mutated in breast cancer.