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    Research Grants Awarded

    Tyrosine Phosphorylation And Functional Regulation Of Pcna In Breast Cancer

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    Although significant progress has been made in the past decade in developing targeted therapy for breast cancer, chemotherapy continues to be the first-line treatment option for which the major challenge is to enhance chemosensitivity to overcome the tumor-derived chemoresistance and the associated non-specific cytotoxicity of which accumulated dosage can result in increased risk of life-threatening side effect. A promising strategy of chemosensitization is to combine the conventional chemotherapy with treatment targeting other malignancy-promoting genes. The epithelial growth factor receptor EGFR, a receptor tyrosine kinase (RTK) of the ErbB family, plays a profound role in many adverse features associated with malignant phenotypes, such as elevated proliferation, promoted metastasis, and reduced apoptosis. It has been widely expected that the concurrent treatment of tumors by chemotherapeutic agents combined with inhibitors of EGFR can generate a significant impact in improving patient outcome. However, recent clinical studies implementing such approaches to treat breast cancer and other cancer types have not been successful in that there is no therapeutic advantage for patients receiving chemotherapy with concomitant anti-EGFR regimens. These unexpected and disappointing results cast a serious concern for further development of combination cancer therapy to target EGFR-associated etiology in breast cancer. Thus, there is an urgent need for critical understanding of the biological mechanisms of EGFR in regulating proliferation and response to cytotoxic stress in breast cancer cells and for the development of biomarkers to identify patients sensitive to the therapeutic strategies. Proliferating cell nuclear antigen (PCNA) is the indispensable molecular coordinator during DNA synthesis, the obligated process for cancer proliferation and the most vulnerable stage of cancer cells to genotoxic chemotherapeutic agents, such as doxorubicin, etoposide and cisplatin. Because of its critical role in cell proliferation and DNA replication, PCNA has been widely used as a prognostic marker in multiple tumor types, including breast cancer. Intriguingly, however, the correlation between PCNA expression and patient survival has been controversial as studies either challenging or supporting the correlation have been reported. We recently found that EGFR functions as a protein kinase to phosphorylate PCNA on tyrosine 211 (Y211). The phosphorylation is required to stabilize PCNA protein on the chromatin and enhances PCNA-mediated DNA synthesis activity in proliferating cancer cells. Y211 phosphorylation earmarks PCNA function in proliferation and is highly correlated with poor prognosis in a cohort of breast cancer patients. More importantly, EGFR-positive breast cancer cells expressing the wild-type PCNA are more sensitive to conventional chemotherapeutic agents than the cancer cells expressing the Y211F mutant PCNA. Pre-treatment of EGFR inhibitor resulted in de-sensitization to subsequent treatment with chemotherapeutic drugs. We propose that the EGFR-PCNA signaling pathway positively regulates cancer cell proliferation and subsequently sensitizes these proliferating cells to chemotherapy. To test our hypotheses, in Specific Aim 1, we will determine whether EGFR directly stimulates DNA replication through PCNA phosphorylation. The outcome of the study will have critical implication in future designing of genotoxic therapy against EGFR-positive breast cancer cells. The experiments of this Aim will also provide further evidence for the role of phospho-Y211 PCNA as a novel tumor marker in breast cancer. In Specific Aim 2, we will begin a series of experiments to study exactly how the EGFR-PCNA signaling responds to chemotherapeutic agents in cancer cells. This study will also test the potential interplay of key signaling events of PCNA in response to DNA damaging stresses. The resulted knowledge can be used to develop potential tumor markers to evaluate or predict chemoresponse of cancer cells. In Specific Aim 3, we will study the translational impact of manipulating the EGFR-PCNA signaling pathway for chemosensitization in cultured cancer cells and in orthotopic tumor model. We will determine whether the sequential combination therapy starting with genotoxic treatment followed by EGFR targeting can achieve improved therapeutic efficacy compared with the conventional regimens (pre- or concurrent inhibition of EGFR). The proposed studies are likely to unveil a critical insight of how cell proliferation, DNA damage repair, and the response to chemotherapy can be regulated through PCNA functions in breast cancer dictated by receptor tyrosine kinases, and to provide an opportunity of identifying novel therapeutic targets and prognostic markers of breast cancer. Deregulated EGFR function is frequently associated with breast cancer development and therefore has been an important target of cancer prevention and therapy. Furthermore, the similar concept of sequentially combined treatments may also be applied to therapies targeting other oncoproteins or employing other chemotherapeutic agents preferentially targeting proliferating cells. Thus, success of the proposed research is expected to benefit a large cohort of breast cancer patients. In addition, because all these reagents have been approved for clinical use, the knowledge resulted from the proposed study can yield significant translational value in the near future.

    Lay Abstract:
    Chemotherapy is an important component of the front-line approaches for breast cancer therapy. However cancer cells can evolve to become resistant to these chemotherapeutic drugs. Indeed, the majority of treatment failure can be attributed to drug resistance of the tumor. Also, accumulating dosage of certain drugs can result in life-threatening side effects. Further understanding the mechanisms and identifying approaches to enhance chemosensitivity has been the major challenge in systemic cancer treatment. To this regard, a great promise has been on the concurrent treatment of tumors by chemotherapeutic agents combined with inhibitors of the epidermal growth factor receptor (EGFR), a critical cancer target and prognostic tumor marker. It is known that EGFR activates multiple signaling pathways to dictate the malignant behavior of cancer cells. Like ErbB-2/HER-2, EGFR conveys these biological functions through its activity as a ?protein kinase? to phosphorylate other proteins. The common belief is that EGFR promotes cancer cell growth, prevents cell killing by the chemotherapeutic drugs, thus inhibiting EGFR can make cancer cells more sensitive to chemotherapy. However, recent clinical studies implementing such approaches have not been successful as tumors receiving chemotherapy combined with anti-EGFR agents do not respond better than tumors treated by the chemotherapeutic agents alone. These unexpected and disappointing results cast a serious concern for the development of anti-EGFR-based combination cancer therapy in breast cancer. Thus, there is an urgent need for further understanding of the biological mechanisms of EGFR in regulating cell growth and response to chemotherapeutic stress in cancer cells, and for the development of potential biomarkers to identify patients who might be sensitive to the particular therapeutic regimens. PCNA (proliferative cell nuclear antigen) is the indispensable molecular coordinator during DNA synthesis, the obligated process for cancer cells to proliferate and the most vulnerable stage of cancer cells to DNA-damaging agents, such as doxorubicin, etoposide and cisplatin. Because of its critical role in tumor growth, PCNA has been widely used as a prognostic marker in multiple tumor types, including breast cancer. Intriguingly, however, the correlation between PCNA expression and patient survival has been controversial as studies either challenging or supporting the correlation have been reported. We found that EGFR phosphorylates PCNA at the tyrosine residue 211 (Y211). This phosphorylation is important because it stabilizes PCNA on DNA double helix so that DNA synthesis can be conducted. Y211 phosphorylation earmarks PCNA function and is highly correlated with poor survival in a cohort of breast cancer patients. More importantly, EGFR-positive breast cancer cells expressing the wild-type PCNA are more sensitive to chemotherapeutic agents than the cancer cells expressing the mutant PCNA not subject to the phosphorylation. Pretreatment of EGFR inhibitor resulted in de-sensitization to subsequent chemotherapeutic drugs. We propose that EGFR activates PCNA to promote cancer cell proliferation and subsequently sensitizes these actively growing cells to chemotherapy. To test our hypotheses, in Specific Aim 1, we will determine whether EGFR directly stimulates DNA replication through PCNA phosphorylation in cancer cells. The results of this study will be important for future designing of chemotherapy against EGFR-involved breast cancer and will provide further evidence for the role of phospho-Y211 PCNA as a novel tumor marker of this disease. In Specific Aim 2, we will study exactly how the EGFR-PCNA signaling responds to chemotherapeutic agents in breast cancer cells. The resulted knowledge can be used to develop potential tumor markers to evaluate or predict chemoresponse of cancer cells. In Specific Aim 3, we will study the translational impact of manipulating the EGFR-PCNA signaling for chemosensitization in cancer cells and in nude mice breast tumor model inoculated with human breast cancer cells. We will determine whether the sequential combination therapy starting with the chemotherapeutic drugs followed by inhibiting EGFR can achieve improved therapeutic efficacy compared with the conventional regimens (pre- or concurrent inhibition of EGFR). The proposed studies are likely to unveil a critical insight of how cell proliferation, DNA damage repair, and the response to chemotherapy can be regulated through PCNA functions in breast cancer dictated by receptor tyrosine kinases, and provide an opportunity of identifying novel therapeutic targets and prognostic markers of breast cancer. As mentioned above, deregulation of EGFR is frequently associated with breast cancer development and therefore has been an important target for cancer prevention and therapy. Furthermore, the similar concept of sequentially combined treatments may also be applied to therapies targeting other oncoproteins or employing other common chemotherapeutic agents in breast cancer treatment. Thus, success of the proposed research is expected to benefit a large cohort of breast cancer patients. In addition, because all these reagents have been approved for clinical use, the knowledge resulted from the proposed study can become instrumental in developing therapeutic strategies targeting breast cancer in the near future.