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    Awarded Grants
    Targeting DNA polymerase ß and Base Excision Repair in Breast Cancer: Characterization of a Novel p53-Independent Anti-Tumor Response

    Scientific Abstract:
    Targeting DNA polymerase ß and Base Excision Repair in Breast Cancer: Characterization of a Novel p53-Independent Anti-Tumor Response. Background: Many chemotherapeutic agents mediate an anti-tumor response via DNA damage and subsequent induction of apoptosis. The hallmark response to therapeutically induced DNA damage is the activation of the tumor-suppressor gene p53 and the subsequent induction of the p53 network and activation of apoptosis. However, p53 is mutated in 50% of all cancers, in 20% of breast cancers and 100% of medullary breast carcinomas. It is therefore not surprising that p53 mutations in breast cancer correlate with a worse clinical outcome and indicates a need for chemotherapeutic targets that mediate an anti-tumor response independent of p53 status. In preliminary experiments, we have demonstrated that inhibition of the Base Excision DNA Repair (BER) pathway by loss or inhibition of DNA polymerase ß (ß-pol) expression leads to accumulation of the DNA repair intermediate 5’dRP. This intermediate induces a rapid cytotoxic response by a p53-independent mechanism. Most importantly, we have demonstrated that this toxic, p53-independent repair intermediate can be induced by administration of the orally available chemotherapeutic agent Temozolomide (TMZ). In preliminary cell culture studies, we have shown that deregulation of the BER pathway elicits a 50-fold increase in TMZ efficacy at doses with little or no cytotoxic effect on wild-type cells. Objective/Hypothesis: Our central hypothesis is that a majority of the anti-tumor action of TMZ is due to the formation of the BER intermediate 5’dRP and is therefore dependent on specific BER gene expression and activity. By deregulating the BER pathway in breast cancer cells, TMZ treatment will induce DNA base damage that will lead to the formation of 5’dRP and induce a cytotoxic response in a p53-independent manner. It is our objective to demonstrate that altering the BER pathway will initiate a robust anti-tumor response in p53-mutant breast cancer cells and xenograft models. Specific Aims: (1) To define the expression and activity profile of each member of the BER pathway in p53 (+) and p53 (-) breast cancer cell lines and correlate this activity profile to the cytotoxic response to TMZ. (2) To inhibit the BER pathway in p53 (+) and p53 (-) breast cancer cell lines (MCF-7 and MDA-MB-231) by siRNA- mediated knockdown of ß-pol mRNA and determine the increase in TMZ efficacy following loss of ß-pol expression. (3) To determine the tumor potential of ß-pol deficient breast cancer cell lines in nude mice, to determine efficacy of TMZ on these BER inhibited xenografts and to determine if BER intermediates produce anti-tumor effects in a p53-independent manner. Study Design: Aim 1 will use quantitative techniques to measure BER gene expression (mRNA and protein): alkyladenine DNA glycosylase (Aag), AP endonuclease, ß-pol, FEN-1 and PARP. Further, in this aim we will use our BER activity assays to quantitatively measure the alkylation damage repair capacity of these human breast cancer cell lines and correlate expression and activity to TMZ sensitivity. In Aim 2, we will use our siRNA expressing retroviruses to mediate Dicer-induced degradation of ß-pol mRNA and mediate the loss of ß-pol protein expression. In Aim 3, we will then determine the tumor potential of the ß-pol-deregulated breast cancer cell lines and measure the rate of tumor growth following administration of TMZ to determine the anti-tumor effect of the BER intermediate 5’dRP. Finally, we will introduce a second genetic alteration so as to increase 5’dRP production in vivo by the over-expression of Aag. Potential Outcomes and Benefits of the Research: The BER pathway is an unexplored therapeutic target in cancer chemotherapy. This project is designed to explore the possibility of tumor cell destruction via induction of the cytotoxic BER intermediate 5’dRP. The project outlined here will (i) aid in our understanding of the potential value of the BER pathway as a therapeutic target (ii) aid in identifying a major mechanism of efficacy of TMZ and (iii) ultimately direct the design of new therapeutics to both initiate BER and inhibit the ß-pol step of the pathway so as to specifically induce the toxicity of tumor cells in concert with pharmacological agents such as TMZ. It is anticipated that a more thorough understanding of the molecular mechanism of 5’dRP mediated replication arrest will aid in future studies to facilitate an even greater therapeutic efficacy of drugs such as TMZ.

    Lay Abstract:
    Targeting DNA polymerase ß and Base Excision Repair in Breast Cancer: Characterization of a Novel p53-Independent Anti-Tumor Response. Temozolomide (TMZ) has emerged as a novel treatment for many cancers. It is currently in clinical trials, is 100% bioavailable, is administered orally and exhibits very limited toxicity, with less than 5% of patients experiencing myelosuppression. As with many chemotherapeutic agents, the effectiveness of TMZ depends on the induction of genomic DNA damage. Tumor cells with a damaged genome are then unable to multiply and may initiate a form of cellular suicide, a process that, in most cases, requires a gene called p53. Unfortunately, the p53 gene is mutated in 50% of all cancers, in 20% of breast cancers and 100% of medullary breast carcinomas. It is not surprising that p53 mutations in breast cancer correlate with a worse clinical outcome. Therefore, there is a real need for chemotherapeutic targets that mediate an anti-tumor response independent of p53 status. To induce cell death and destroy tumors, TMZ causes 3 types of damage to DNA: O6MeG, N7MeG and N3MeA. Most studies have focused on one type of DNA damage (O6MeG) to explain how TMZ destroys tumor cells. Our preliminary results point to the significance of the other two major types of DNA damage (N7MeG and N3MeA); accounting for 80% of the genomic damage caused by TMZ. Our preliminary studies suggest that most (80%) of the DNA damage induced by TMZ is repaired by the base excision repair (BER) pathway, a repair system specific for genomic DNA base damage. Recently, we have discovered a dramatic biological impact mediated by a specific DNA intermediate that arises during the process of BER. This intermediate, 5’dRP, is formed during repair of many common DNA base lesions but is immediately removed by the repair protein DNA polymerase ß (ß-pol). We have found that this intermediate (5’dRP), if left un-repaired, can trigger cell death by an as-yet unknown mechanism that is independent of the p53 gene. In preliminary cell culture studies, we have shown that deregulation of the BER pathway elicits a 50-fold increase in TMZ efficacy at doses with little or no effect on wild-type cells. Our central hypothesis is that a majority of the anti-tumor action of TMZ is due to the formation and accumulation of the BER intermediate 5’dRP and is therefore dependent on specific BER gene expression and activity. By deregulating the BER pathway in breast cancer cells, we propose that TMZ treatment will induce DNA base damage that will lead to the accumulation of 5’dRP and induce a cell death response in a p53-independent manner. It is our objective to demonstrate that altering the BER pathway will initiate a robust anti-tumor response in p53-mutant breast cancer cells and xenograft models. The base excision repair pathway is a relatively unexplored therapeutic target in cancer chemotherapy. The project outlined here will (i) aid in our understanding of the potential value of the base excision repair pathway as a therapeutic target (ii) aid in identifying a major mechanism of efficacy of the chemotherapeutic agent Temozolomide (TMZ) and (iii) ultimately direct the design of new therapeutics to both initiate base excision repair and inhibit the ß-pol step of the BER pathway so as to specifically induce the toxicity of tumor cells either alone or in concert with pharmacological agents such as TMZ. It is anticipated that a more thorough understanding of the molecular mechanism of 5’dRP induced tumor cell death will aid in future studies to facilitate an even greater therapeutic efficacy of drugs such as TMZ.