Novel Inhibitors of Rad51-DNA Repair in Breast Cancer

Institution: Pangene Corporation
Investigator(s): Anne Vallerga, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7WB-0153 Award: $130,332
Award Type: STEP Award
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2001)
Surgery, radiation and chemotherapy are the most commonly used therapies for breast cancer. Both radiation and chemotherapy kill cells by damaging DNA, not only in tumor cells but also in normal cells, leading to unwanted side effects and toxicity. Moreover, breast tumor and other tumor cells contain elevated levels of DNA repair enzymes that repair the damage caused by radiation and chemotherapy, resulting in resistance to these treatments. Recent research from our laboratory and our collaborator, suggest that breast tumor tissues contain high levels of special DNA repair protein, called Rad51. We know much about how Rad51 works, because very similar proteins from bacteria (e.g., E. Coil RecA) have been well characterized. Rad51 is associated with a number of key breast cancer genes, including BRCA1, 2 and the tumor suppressor, p53. The activity of Rad51 is regulated by a direct protein:protein interactions with p53. In cases where p53 is absent or mutated, Rad51 has increased DNA repair activity. The net effect of increased Rad51 activity and reduced p53 function is to allow cancer cells to effectively repair DNA damage to neutralize radiation and chemotherapy treatments. Our hypothesis is that we can modulate Rad51 DNA repair by using synthetic peptide fragments of p53.

Thus, we are developing Rad51 inhibitors that may serve to improve conventional breast cancer therapy by reducing the tumor resistance and, perhaps, resulting in novel therapeutics. The inhibitors are based on the peptide regions of p53 which bind to Rad51. Our methodology uses a special biosensor. This approach allows us to pass p53 peptides through a cell of the biosensor that contains Rad51 on its surface. Unbound peptides are removed and potentially active peptides that bind to Rad51 will be released further study. Promising Rad51-binding peptides will be analyzed by mass spectroscopy for their amino acid content and their structure will be inferred by molecular modeling. We propose to determine the smallest fragments or peptides of p53 protein that regulates the function of Rad51. Finally, these p53 peptides will be introduced into breast cancer cells and tested for their ability to neutralize the function of Rad51. We hope to demonstrate that p53 peptides can prevent the translocation Rad51 to the nucleus, and that the peptides enhance the effectiveness of chemotherapeutics and radiation for killing tumor cells

It is becoming obvious that treating cancer effectively will involve an increased understanding of the underlying biology of resistance to current and future therapeutics. Our approach is novel, because we propose to inhibit the enzymes that repair the DNA damage sustained during radiation or chemotherapy.


Final Report (2002)
Note: Anne K. Vallerga, Ph.D., replaced Dr. Reddy as PI during the final 3 months of the grant.

Rad51 protein catalyses repair of double-strand breaks in the DNA of higher organisms and is over-expressed in direct response to double-strand (ds) DNA damage either by dsDNA damaging chemicals, including many types of cancer chemotherapeutic drugs or by radiation therapy. We discovered that Rad51 is highly over-expressed in breast tumor cell lines and primary breast tumor patient samples. Furthermore, Rad51 over-expression appears to be an important diagnostic marker for breast tumor grading and potentially for therapy resistant tumor progression or tumor recurrence. In contrast, Rad51 protein is expressed at relatively low levels in normal breast epithelial cells.

We found that inhibitors of Rad51 DNA repair enzyme promote apoptosis in breast tumor cells and inhibit repair of dsDNA breaks, thus, sensitizing apoptosis resistant breast tumor cells to dsDNA-breaking chemotherapeutic anti-cancer drugs. We also discovered p53-derived oligopeptide inhibitors of Rad51 enzyme, which were characterized and enabled us to identify the p53 binding domain on Rad51 protein. We found that this site is distinct from the nucleoside binding domain or the DNA binding domains on Rad51. The p53 oligopeptide binding site on Rad51 protein was specifically competed by p53 polypeptide. We systematically tested Rad51 antisense oligonucleotide inhibitors of Rad51 RNA and Rad51 protein expression. Novel Rad51 antisense oligonucleotides that targeted either coding or non-coding regions of the Rad51 gene were designed and measured for their ability to downregulate Rad51 RNA and Rad51 protein expression. Importantly, these inhibitors served to sensitize cells to the dsDNA damaging cancer chemotherapeutics, doxorubucin, etoposide or cisplatin.

In similar experiments, we found that Rad51 antisense oligonucleotides specifically inhibited Rad51 RNA and Rad51 repair enzyme in MDA-MB-231 and MCF7 human breast tumor cell lines. Rad51 enzyme downregulation by Rad51 antisense oligonucleotides in breast tumor cells (MDA-MB-231) selectively sensitized, by approximately 11- and 12-fold, the breast tumor cells but not normal mammary epithelial cells to lower doxorubicin and cisplatin drug concentrations, respectively. MDA-MB-231 cells treated with control sense or scrambled non-sense oligonucleotides were not sensitized.

Thus, Rad51 specific inhibition in breast tumor cells appears to remove the block to apoptosis and significantly enhances cell killing by dsDNA damaging chemotherapeutic drugs, including cisplatin and the DNA topoisomerase II inhibitors, doxorubicin and etoposide. These results show that Rad51 antisense inhibitors may be used in combination therapy with dsDNA damaging breast cancer therapeutics, including cisplatin and doxorubicin, for treating radioresistant and chemoresistant human breast tumor cells.