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    Functional Significance of HtrA1 Downregulation in Breast Cancer: Implications for Platinum Resistance

    Scientific Abstract:
    Shridhar V PDF0403257 SCIENTIFIC ABSTRACT Functional Significance of HtrA1 Downregulation in Breast Cancer: Implications for Platinum Resistance Background: Breast cancer is one of the leading causes of death among women in the United States with over 203,000 cases diagnosed each year and 40,000 resulting in mortality. If the disease is diagnosed in its early stages both surgery and radiotherapy treatment result in better cure rate. However, patients with advanced or more aggressive tumors often experience chemoresistance and recurrence leading to eventual death. Although the molecular mechanisms of chemoresistance vary from classical drug metabolisms, such as drug efflux pumps and upregulation of glutathione-S-transferase, to novel extracellular matrix –mediated drug resistance, one of the underlying factors that influences chemoresistance is the regulation of apoptosis by cancer cells. When a cell sustains damage due to chemotherapy, it can either (i) attempt to repair the damage or (ii) if the damage is extreme, self-destruct through the suicide (or apoptosis) pathway. Cancer cells, in their evolution, lose components of this suicide machinery as a mechanism to survive drug-induced cell death. Our laboratory has identified a pro-apoptotic serine protease, HtrA1, as a down regulated gene in several types of cancer. Our preliminary results indicate that 1) HtrA1 is expressed in ductal epithelial cells of breast tissue. 2) HtrA1 expression is markedly reduced or lost in 3 of 9 breast cancer cell lines. 3) HtrA1 co-localizes with microtubules. 4) More importantly relevant to this proposal we have shown that platinum agent, cisplatin, upregulates HtrA1 expression in MCF-7 cells within 1 hour of exposure. 5) Forced expression of HtrA1 leads to destruction of microtubules, severe cytoplasmic reduction, cell rounding, and apoptosis. These results suggest the possibility that HtrA1 may modulate platinum sensitivity by mediating platinum-induced apoptosis. Hypothesis: Based on these novel observations, we propose that HtrA1 is a mediator of platinum-induced cell death and that loss of HtrA1 contributes to platinum resistance. Specific Aims: To further evaluate the functional significance of HtrA1 down-regulation and platinum resistance, we propose the following three specific aims: AIM 1: To evaluate the effect of loss of HtrA1 on platinum chemoresistance in vitro and in vivio. AIM 2: To examine the mechanisms of HtrA1 inactivation in breast cancer. AIM 3: To determine HtrA1 expression in breast tumors and correlate HtrA1 expression with clinical outcome. Study Design: To address the first aim, HtrA1 expression in MCF-7 will be inhibited by siRNA, and platinum chemosensitivity will be determined by assessing nuclear morphology with Hoechst’ stain following platinum treatment at various times and concentration points. MCF-7 clonal lines stably expressing HtrA1 siRNA (in pRetroSuper) will be established, and chemosensitivity will be determined by clonogenic assay in vitro and by evaluating tumor burden in nude mice in vivo following platinum treatment. To address aim 2, loss of heterozygosity, methylation, and mutational analyses will be performed in both breast cancer cell lines and primary tumors. To address aim 3, HtrA1 expression will be analyzed in 40 each of node negative and node positive breast tumors on tissue microarrays by immunohistochemistry. Correlation between HtrA1 expression and clinical outcome will be determined. Potential Outcomes and Benefits of the Research: This proposal addresses one of the basic problems in cancer, namely the development of chemoresistant disease, and proposes to identify the mechanistic basis of this phenomenon in the context of how the loss of HtrA1 contributes to drug resistant phenotype. The basic goal of this proposal is to determine the role of HtrA1 in apoptosis and chemosensitivity and to explore the potential use of HtrA1 as a prognostic marker. While the specific aims complement each other, each specific aim is unique and independent of each other. We believe the functional characterization of HtrA1 in conferring chemosensitivity to breast cancer cells will provide valuable insights into how breast cancer cells become resistant to commonly used drugs in the treatment of this cancer and provide novel therapeutic approaches in the treatment of this malignancy.

    Lay Abstract:
    Shridhar V PDF0403257 LAY ABSTRACT Functional Significance of HtrA1 Downregulation in Breast Cancer: Implications for Platinum Resistance Breast cancer is one of the leading causes of death among women in the United States with over 203,000 cases diagnosed each year and 40,000 resulting in mortality. If the disease is diagnosed in its early stages both surgery and radiotherapy treatment result in better cure rate. However, patients with advanced or more aggressive tumors often experience chemoresistance and recurrence leading to eventual death. Although multiple mechanisms could account for chemoresistance, one of the underlying factors that influences chemoresistance is the regulation of programmed cell death by cancer cells. Normal cells are programmed to undergo cell death when cellular integrity becomes inconsistent with normal functional parameters of the cells--such as unchecked cell growth, injury to the cell, or DNA damage induced by chemotherapeutic drugs. Cancer cells, however, could continue to survive under these circumstances because they often lose the ability to undergo programmed cell death. One of the mechanisms by which cells may avoid programmed cell death is by losing expression of proteins involved in cell death. When the cell loses these proteins, it becomes more aggressive and resistant to chemotherapy. Therefore, proteins involved in cell death machinery play an important role in cancer development and chemoresistance. We have recently identified HtrA1 as a protein involved in programmed cell death. HtrA1 expression increases following platinum treatment, and overexpression of HtrA1 induces cell death. These data suggest that HtrA1 may participate in platinum-induced cell death and that presence of HtrA1 may make cancer cells more sensitive to platinum agents. Unfortunately, HtrA1 is absent or present at very low levels in more than 30% of breast cancer cell lines. We believe that this condition could make cancer cells more resistant to platinum-base chemotherapy because these cells no longer possess a mechanism by which platinum induces cell death. To determine if our hypothesis is correct, we will 1) test whether absence of HtrA1 make cancer cells resistant to platinum agents; 2) analyze the expression of HtrA1 in breast tumors, and correlate the expression with clinical outcome and aggressive invasive phenotype of breast tumors; and 3) determine the cause leading to loss of HtrA1 in breast cancer. At the conclusion of this study, we hope to 1) define how loss of HtrA1 contributes to the development of breast cancer and chemoresistance, 2) provide evidence that HtrA1 expression can be used to predict clinical outcome, aggressive nature of the disease, or chemoresponse, and 3) determine the cause leading to loss of HtrA1 in breast cancer. Collectively, the outcome of the proposed studies will contribute to the understanding of the biological basis of chemoresistance, and provide innovative approaches to the development of novel therapeutic targets for breast cancer.