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    Monitoring the Effectiveness of NSAIDs Chemoprevention in Breast Cancer Using the CD13/APN Cell Surface Peptidase

    Scientific Abstract:
    Monitoring the effectiveness of NSAIDs chemoprevention in breast cancer using the CD13/APN cell surface peptidase Background: We have shown that the cell surface peptidase, CD13/Aminopeptidase N (CD13/APN), is a critical regulator of endothelial cell invasion and capillary network formation and is required for angiogenesis. During endothelial cell activation, CD13/APN expression is induced by tumor-derived growth factors via the Ras-MAPK and PI-3 kinase pathways converging on ERK/2. These signal transduction pathways transmit signals to the nucleus where they enhance the binding of a potentially novel transcription factor to the CD13/APN promoter that is critical for its upregulation and, therefore, its function. This Ras/MAPK association prompted our investigation of the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the angiogenic induction of CD13/APN in endothelial cells. We found that treatment of cells with NSAIDs inhibits the induction of CD13/APN by interfering with the binding of the inducible transcription complex. We are confident that the ability of NSAIDS to modulate expression of the CD13/APN cell surface peptidase represents a fundamental aspect of their documented antitumor activity. Therefore, this mechanistic relationship between a chemopreventive treatment, transcriptional modulation, and therapeutic effect warrants further investigation to exploit this link and allow us to predict treatment efficacy and design more effective adjuvant therapies. Objective/Hypothesis: The fact that NSAIDs directly affect CD13/APN expression suggests that CD13/APN can serve as a surrogate for NSAIDs-modulated tumor neovascularization and may serve as a marker of treatment efficacy. Our working hypothesis is that the efficacy of NSAIDS chemoprevention of at-risk breast carcinoma patients can be monitored by assessment of CD13/APN expression either in serum or biopsy specimens. In addition, we propose that inhibition of CD13/APN is a factor in NSAIDs anti-tumor effects. Specific Aims: In this proposal, we plan to investigate the validity of CD13/APN as a surrogate biomarker for breast cancer chemoprevention. Since the accuracy and strength of the mechanistic link between the treatment, its specific effect at the site of action, and the desired therapeutic outcome dictates the legitimacy and prognostic potential of predictive biomarkers, we will first strengthen this link by determining which NSAIDs are most effective at inhibiting transcription factor binding and CD13/APN expression in vitro (Specific Aim 1). We will then test the effects of the two lead drugs on CD13/APN levels (Specific Aim 2) and tumorigenesis (Specific Aim 3) in vivo in a murine model of breast cancer to measure the efficacy of CD13/APN in tissue and serum as a predictor of NSAIDs therapeutic efficacy. Finally, animals treated in parallel with a well characterized peptidase inhibitor will clarify the contribution of CD13/APN inhibition to NSAIDs efficacy. Study Design: Early results of NSAIDs treatment of murine tumor models clearly show that this class of drugs is effective in the prevention of breast cancer. Development of this therapy would be furthered by the identification of a biomarker that allows convenient assessment of treatment efficacy. We will use the MMTV-Her2/neu transgenic model of breast cancer to evaluate CD13/APN expression as a surrogate marker of NSAIDs treatment. Cohorts of transgenic mice will be treated in preventive protocols with a panel of NSAIDs that have been tested to block CD13/APN transcription or a well characterized CD13/APN inhibitor and evaluated at progressive time points. The results of treatment on tumor parameters such as size, vascularity, incidence, and multiplicity will be assessed in relation to CD13/APN levels in biopsy and serum samples to determine if CD13/APN levels accurately reflect the effects of NSAIDs on tumorigenesis as well as the relative contribution of CD13/APN downregulation to NSAIDs anti-tumor effects. Further studies will test the dose dependence and rebound effects of the drug that we find to be most effective. Potential Outcomes and Benefits of the Research: The experimental plan outlined in this proposal will characterize in detail the features of CD13/APN as an NSAIDS modulatable surrogate marker for the chemoprevention of breast carcinoma and strengthen the link between its modulation and NSAIDs mechanistic effects. Surrogate biomarkers are essential tools in the development and evaluation of potential clinical therapies since they often can predict the eventual success or failure of a strategy or can significantly aid in comparing one strategy to another in the early stages of drug or therapy development. Such information can be extremely valuable considering the tremendous cost of drug development and validation. Furthermore, the recent success of therapies combining anti-angiogenic drugs with conventional chemotherapeutic agents will undoubtedly stimulate similar trials of combination therapies requiring the assessment of potential therapeutic efficacy with biologically relevant surrogate markers.

    Lay Abstract:
    Monitoring the effectiveness of NSAIDs chemoprevention in breast cancer using the CD13/APN cell surface peptidase Clinical studies in women at risk for breast cancer have clearly shown that chemoprevention can substantially lower this risk. Chemoprevention approaches are based upon the premise that interfering with early cancer causing events will avert subsequent steps leading to disease progression. Since the goal of chemoprevention is to stop tumor formation, monitoring a treatment’s effectiveness must rely on indicators other than overt disease that will accurately reflect tumor progression. Therefore, identifying and verifying such indicators, or surrogate biomarkers, capable of gauging the malignant potential of a tumor would prove extremely useful for predicting the success of a therapeutic intervention. In particular, markers that are directly modulated by the chemopreventive drug are especially useful in clinical trials designed to determine optimal dose and to assess relative risk, toxicity, and treatment efficacy. The CD13/aminopeptidase N (CD13/APN) cell surface peptidase is a potent regulator of new blood vessel formation (angiogenesis). Its expression is much higher in the new blood vessels of tumors due to factors that are unique to the tumor and its surroundings. Clinical studies indicate that solid tumors, including breast cancers, must build new blood vessels in order to invade, grow, and metastasize. Important to prevention strategies, these new blood vessels form very early in tumor development and can control the rate of disease progression. Pertinent to this proposal, CD13/APN is found in the new vessels of mouse models of human breast carcinoma, and treatment of tumor bearing mice with CD13/APN inhibitors blocks tumor growth, consistent with its inhibition of tumor-induced angiogenesis. In addition, in breast cancer patients high CD13/APN levels in the blood correlate with poor outcome suggesting that it is readily measurable and reflects disease severity. Non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and celebrex, have been shown to be effective chemoprevention agents in both human studies and murine models of breast carcinoma, where they work in part by impeding angiogenesis. We have shown that treatment of vascular endothelial cells with NSAIDs significantly inhibits CD13/APN expression, likely contributing to the anti-angiogenic effects of these drugs. Therefore, CD13/APN offers a unique opportunity for monitoring NSAID chemoprevention strategies in breast cancer: it is both a modulatable biomarker (its levels are regulated by NSAIDs) as well as a therapeutic target (NSAIDs-induced loss of CD13/APN is anti-angiogenic), suggesting that its expression level may be directly indicative of treatment efficacy. We propose that during breast cancer tumorigenesis CD13/APN promotes formation of new vessels necessary for sustaining tumor progression by its expression on tumor-associated blood vessels and that the level of CD13/APN expression may directly correlate with breast cancer angiogenesis and tumor progression. Our working hypothesis is that the efficacy of NSAIDS chemoprevention of at-risk breast carcinoma patients can be monitored by assessment of CD13/APN expression in serum or biopsy specimens. For example, if the NSAIDs treatment is inhibiting tumor progression, CD13/APN expression will remain low or be reduced, consequently affecting the ability of tumors to grow and form new blood vessels. However, an increase in CD13/APN expression during treatment would suggest that the tumor is resistant and continues to progress. Therefore the existing treatment regimen is not effective and must be modified or supplemented. In this way, chemoprevention can be monitored and tailored on a case by case basis. Finally, our demonstration that NSAIDS treatment specifically interferes with a molecule necessary for angiogenesis provides a mechanistic basis for NSAIDs effectiveness and strengthens the link between marker and therapy. The long range goal of this study is to characterize the utility of CD13/APN as an NSAIDS-modulatable surrogate marker for the chemoprevention of breast carcinoma. The objective of the current proposal is to validate CD13/APN as a biomarker of chemoprevention by correlating its expression with tumor formation following NSAIDs treatment in a murine breast cancer model. Identifying surrogate markers is critically important in the development of therapy and the monitoring of effective treatment, where obtaining critical information about efficacy in early stages can be critical to therapeutic outcome and patient survival.