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    JAB1-Mediated p27Kip1 Degradation as a Prognostic Factor and a Therapeutics Target for Breast Cancer

    Scientific Abstract:
    JAB1-Mediated p27 Degradation as a Prognostic Factor and a Therapeutics Target for Breast Cancer Background: The uncontrolled proliferation of cancer cells results from disruption of cell-cycle control. In normal cells, the cell cycle is controlled by the interaction of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs). p27, a CKI regulates the G1/S phase transition of the cell cycle. An increase in p27 causes proliferating cells to exit the cell cycle, and a decrease in p27 is necessary for quiescent cells to resume division. Despite the putative role of p27 as a tumor suppressor, mutations in p27 are rarely seen in human tumors. However, abnormally low amounts or absence of p27 protein are associated with pathological states of excessive cell proliferation such as tumor aggressiveness and poor patient survival in about 50% of human breast cancers. In tumors, p27 activity appears to be lost not only by increased degradation but also by cytoplasmic mislocalization or sequestration. These findings suggest that disruption of p27 regulation may contribute to neoplasia. We previously identified Jun activation domain-binding protein 1 (JAB1), which interacts with and potentiates transactivation by c-Jun to promote cellular proliferation. JAB1 was recently found to control the activity of p27 by facilitating relocation of p27 from the nucleus to the cytoplasm, thereby accelerating its degradation by the ubiquitin/proteasome pathway. Furthermore, we found that stably overexpressing JAB1 can override G1 phase arrest mediated by p27, promote colonies formation in soft agar, and is tumorigenic in nude mice. The Jab1 locus has been mapped, and Jab1 gene amplification has been found in breast carcinoma cells. We recently conducted a preliminary clinical study of 53 women with invasive breast carcinoma and found that JAB1 protein levels were inversely correlated with p27 protein levels, and clinical outcome indicating that JAB1 may be a marker for breast cancer. Further studies are needed to validate these potential uses of JAB1. JAB1 may also be an important target for breast cancer therapy. Objective: Our goal is to determine whether JAB1 is a good prognostic marker in 250 patients with early stage to late stage metastatic breast cancer who underwent surgery at M. D. Anderson Cancer Center. Jab1 gene amplification will also be evaluated by using the same tissue arrays. Finally, will use an innovative therapeutic approach to block p27 degradation by targeting JAB1 and determine whether this approach promotes growth arrest and apoptosis in nude mice bearing human carcinoma xenografts. This innovative approach, using JAB1 to reestablish the G1 checkpoint in tumor cells, could be a powerful new therapeutic tool. Specific Aims: (1) To determine whether JAB1 and p27 protein expression is a good marker in breast carcinoma; (2) To examine whether the Jab1 gene is amplified, and (3) To evaluate whether JAB1 can be a therapeutic target in cancer. We will use an innovative gene therapy approach to downregulate the endogenous level of JAB1 or to re-express a degradation-resistant form of p27 in mouse xenograft models, to reestablish a G1 checkpoint. We have already successfully used both strategies in cell lines and obtained growth arrest and apoptosis. Study Design: We will use tissue microarrays and immunohistochemical and statistical methods to analyze the expression of p27 and JAB1 in archival sections from approximately 250 tissue samples from patients who underwent surgery prior any chemotherapy at M. D. Anderson. Samples from normal breast tissues and tumor tissues (early, late, and mestastatic stages, from grades I-III) will be selected. We will also include in situ ductal carcinoma as early stage. Jab1 amplification will be evaluated on tissue arrays by fluorescence in situ hybridization. We will evaluate the efficacy of an adenovirus vector driven antisense-JAB1 and a nondegradable form of p27 in a xenograft model and test whether this approach promotes growth arrest and apoptosis. Potential Outcomes: More knowledge about the mechanisms regulating p27 expression and function could improve our understanding of cell growth and tumorigenesis. The identification of JAB1 in metastatic breast cancer has a potential biomarker is novel. Development of a new prognosis marker for early or late stage breast cancer would be invaluable. Our preliminary clinical study demonstrated that the JAB1:p27 ratio may be a novel indicator of aggressive, high-grade tumor behavior and that JAB1 could be a novel target for therapy.

    Lay Abstract:
    "JAB1-Mediated p27 Degradation as a Prognostic Factor and a Therapeutics Target for Breast Cancer" Breast cancer is the most frequently diagnosed cancer in Western women and the leading cause of death in U.S. women ages 40 to 55. Currently, more than 200,000 women in the United States and almost 1 million women worldwide are diagnosed with breast cancer every year. Advances in treatment for breast cancer are progressing, but research is still critical for the improvement of therapies, the development of new biological markers, and the identification of susceptibility genes for breast cancer. Cancer progression is often attributed to the misregulation of the cell cycle. Mammalian cell-cycle progression is regulated by the combined action of cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors. Any alteration of this balance can result in abnormal cell proliferation, which may lead to cancer. The cyclin-dependent kinase inhibitor p27 is a potent inhibitor of cell-cycle progression. The p27 level is regulated by its rate of protein synthesis and protein breakdown and by its subcellular localization; as p27 is active in the nucleus and inactive in the cytoplasm. Despite its potential role as a tumor suppressor, mutations in p27 are rarely found in human breast tumors. However, a lack of p27 protein in human tumors is a powerful independent indicator of poor survival among patients with breast cancer and patient with ovarian, colorectal, prostate or lung carcinoma, suggesting that a disruption of p27 regulation contributes to neoplasia. For example, accelerated p27 degradation is observed in as many as half of all tumors and is associated with a poor prognosis, and cytoplasmic mislocalization of p27 and subsequent inactivation occurs in about 40% of all tumors. The loss of p27 function in human breast cancer and the prognostic significance associated with it suggest that it would be a valuable target for both stratifying patient risk and selecting treatment. Thus, better comprehension of the mechanisms regulating p27 expression and function could contribute to a better understanding of cell growth and tumorigenesis. We identified Jun activation binding-protein 1 (JAB1), which was recently found to control the activity of p27 by facilitating its relocalization from the nucleus to the cytoplasm and therefore promoting its degradation. We found that the overexpression of JAB1 could override cell-cycle arrest mediated by p27. Moreover, we found that JAB1 protein levels were higher in transformed breast cells and that the increase correlated with tumor aggressiveness in vivo. In addition, when endogenous JAB1 suppression was elicited, it resulted in an increase in endogenous p27 protein. Thus, JAB1 may participate directly in regulating the endogenous levels and stability of this key cell-cycle regulator. We recently conducted a preliminary clinical study of 53 women with invasive breast carcinoma and found that JAB1 expression inversely correlated with the level of p27. JAB1 was detected mainly in tumor samples or carcinoma cells and not in normal tissue. It is of critical importance to identify and validate a biological marker that would assist in the decisions that must me make about breast cancer patients receiving therapeutic treatment. Thus, there is much interest in the identification and development of such prognostic tools that would allow us to better estimate the risk of disease recurrence. We propose to evaluate the expression of JAB1 and p27 as prognostic markers in a larger group of patients with breast cancer, from early-stage to late-stage metastatic disease. We will use a tissue array composed of 250 tumor samples. Immunohistochemical and statistical analysis of p27 and JAB1 in these archival sections will determine whether the JAB1-to-p27 ratio correlates with outcome and whether it could be used as a novel prognostic marker in breast cancer. We will also evaluate whether the JAB1 gene is amplified in these tumors. We will also use a gene therapy approach to restabilize p27 activity levels to reinstate a cell-cycle checkpoint in breast tumors. This innovative approach of using JAB1 to reestablish cell-cycle arrest in tumor cells could be a powerful new therapeutic tool.