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Mechanisms of cisplatin resistance in ovarian cancer

1. Summary of project

Ovarian cancer is the 5th leading cause of cancer deaths for U.S. women. Approximately half the women diagnosed with ovarian cancer die from chemoresistant metastatic disease. Many cytotoxic chemotherapeutics such as cisplatin work by causing DNA damage. A major contributor to acquired chemoresistance is increased DNA repair in tumor cells. The level of DNA damage tolerated by tumor cells is directly related to DNA repair capacity in the tumor cells. Chemoresistance in ovarian cancer correlates with increased expression of DNA repair genes including XPA, ERCC1, ERCC6 and ERCC3. Thus, tumor cells with elevated constitutive DNA repair capacity or the ability to induce their DNA repair capacity in response to cytotoxic therapies will be more resistant than those with lower DNA repair. We have been investigating DNA repair gene induction in an in vitro model for cisplatin resistant ovarian cancer. The essential nucleotide excision repair protein XPA is induced by cisplatin treatment in resistant cells under conditions that also induce ERCC1. Neither gene is induced in sensitive cells. This result suggests that XPA and ERCC1 may be coordinately regulated in cisplatin resistant cells in response to cisplatin treatment. We have shown that XPA protein induction is paralleled by induction of the XPA promoter using reporter assays. We localized the cisplatin response element to a 253 bp fragment of the XPA promoter. Several nuclear protein factors bind this fragment. One of these factors is present in sensitive cells but lacking in resistant cells. These results suggest the hypothesis that the transcription factor present in cisplatin sensitive ovarian carcinoma cells that is lost on conversion to cisplatin resistance is a negative acting factor preventing induction of the DNA repair gene XPA and that loss of this factor allows inducibility of XPA. Ultimately, loss of this factor is part of the conversion to chemoresistant status. We propose to continue these studies by identifying this factor and showing that it is a negative acting factor that prevents XPA induction. Identification and purification of the factor will allow the production of antibodies that can be used for probing cells and tissues for presence of the factor. The association of loss of this negative factor with cisplatin resistance will be investigated in other cisplatin sensitive/resistant paired cancer cell lines. Future studies will determine whether presence of the factor is a useful biomarker of sensitivity to cisplatin therapy by examining primary ovarian cancer tissues and correlating presence of the factor with subsequent responsiveness to platinum based therapy. These studies may also be extended into investigations of chemoresistance in other types of cancer as well.

2. Description of project

2. A.    Specific Aims

Ovarian cancer is the fifth leading cause of cancer deaths in women in the U.S. Approximately half the women diagnosed with ovarian cancer will die from chemoresistant metastatic disease. Understanding the mechanisms of chemoresistance will aid in developing new treatment strategies and in identifying those patients that will respond to current strategies. Prognostication of responsiveness to current strategies is important in maximizing quality of life and chances of remission.

We have been investigating the role of DNA repair gene expression in resistance to cisplatin based therapies using an in vitro model of acquired cisplatin resistance in ovarian carcinoma. The model consists of a pair of ovarian carcinoma cell lines: a cisplatin sensitive cell line (A2780) and a derivative resistant cell line (A2780/CP70, "CP70"). DNA repair gene expression is inducible in CP70 cells but not A2780 cells. Induction is mediated at the transcriptional level. We have localized the region of transcriptional control in the promoter of XPA (an essential DNA repair gene) and have identified several transcription factors which bind to this region. In addition, we have observed a correlation between the presence of an unknown XPA promoter binding factor and non-inducibility of XPA promoter activity in A2780 cells. CP70 cells appear to have lost this factor upon conversion to cisplatin resistance and XPA is inducible by cisplatin treatment. Thus, DNA repair gene inducibility and chemoresistance correlate with loss of this unknown factor. The long term goal of this research is to understand the mechanisms regulating DNA repair induction in chemoresistant cancer in order to develop approaches to overcome this major obstacle to successful treatment.

The hypothesis to be tested is that the transcription factor present in cisplatin sensitive ovarian carcinoma cells that is lost on conversion to cisplatin resistance is a negative acting factor preventing the induction of the DNA repair gene XPA and that loss of this factor allows inducibility of XPA. Ultimately, loss of this factor is part of the conversion to chemoresistant status.

In order to test the above hypothesis, the following specific aims will be accomplished:

  1. Map the binding site of the putative negative factor by mutation of the XPA promoter derived 30-mer oligonucleotide that binds the factor and assay for factor binding by electrophoretic mobility shift analysis (EMSA). Mutation of the sequence within the binding site will eliminate binding and mobility shift. Mapping the binding site will assist in identification of the binding factor. A match to a known factor and availability of antibodies will prompt super-shift analyses to confirm the identity of the factor.
  2. Identify the binding factor by proteomic approaches. Even if a match is obtained by binding site mapping, it is possible that a complex with another factor(s) is acting at the site. Biotinylated oligonucleotides will be used to isolate protein(s) which bind the native sequence but not a mutant sequence identified in aim 1. Isolated proteins will be resolved by SDS-PAGE and their identities determined using mass spectrometry and bioinformatics. If a match to a known factor is obtained and antibodies are available, the identity of the factor will be confirmed by super-shift analysis. If the factor is novel, then further characterization by database mining and molecular cloning of the cDNA will be pursued.
  3. Test the ability of mutations in the factor binding site that eliminate EMSA to confer inducibility on a reporter construct in cisplatin sensitive ovarian carcinoma cells. The mutations are a proxy for loss of the factor and will be engineered into the reporter construct. Inducibility by cisplatin of the mutant reporter construct(s) will be assayed in cisplatin sensitive cells that cannot induce expression of the normal construct. These experiments will confirm the negative function of the factor.

Characterization of the factor that is lost upon conversion of cancer cells from cisplatin sensitivity to cisplatin resistance will allow for development of antibodies if the factor is novel. The antibodies will then be available for probing other models of chemoresistance (both ovarian and non-ovarian cancer cells) and tumors for the presence of the factor to determine the generality of the correlation between loss of the factor and development of chemoresistance. Thus, the identification and characterization of this factor will be important for predicting the responsiveness of a tumor to platin-based therapies. Tumors that are likely to be resistant to platin-based therapies can be subjected to other modalities sooner and patients need not be subjected to quality of life destroying therapies needlessly. Those patients with tumors that are likely to be responsive can be identified and their disease treated aggressively to obtain a cure. Ultimately, understanding the mechanisms of resistance will provide the framework for design of alternative treatments that circumvent these resistance mechanisms.

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