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Role of Jun Activation Domain-Binding Protein (JAB1) in Breast Tumorigenesis
Background: In normal breast cell development, oncogenes, growth suppressor genes, and cell-cycle regulatory genes affect cellular differentiation and growth rate; however, in a tumorigenic state, these dictate metastatic patterns and response to treatment. The normal mammalian cell proliferation is under the strict control of cell-cycle regulatory genes. The cyclin-dependent kinase inhibitory protein p27Kip1, which specifically inhibits the cyclin E-Cdk2 and cyclin A-Cdk2 complexes, regulates the G1/S transition of the cell-cycle. Although mutations in p27Kip1 are rarely found in human tumors, reduced protein expression of p27Kip1 correlates with poor survival among breast cancer patients. Lack of p27Kip1 expression, which is associated with poor prognosis, in about 50% of human tumors, is mainly due to accelerated proteolysis, suggesting that disruption of p27Kip1 regulatory mechanisms contributes to neoplasia. The Jun activation domain-binding protein (JAB1), which we previously identified as a protein that potentiates transactivation by c-Jun and promotes cellular proliferation (Nature, 1996), has been shown to mediate the translocation of p27Kip1 from the nucleus to the cytoplasm, thus decreasing the amount of p27Kip1 in the cell and accelerating its degradation through the ubiquitin/proteasome pathway (Nature, 1999). Moreover, our recently published results showed that JAB1 protein levels inversely correlated with those of p27Kip1 in breast cancer thus indicating that JAB1 may be a marker for breast cancer (Cancer Res, 2003). To understand the importance of these biological parameters as prognostic indicators and predictors as targets in response to therapy, it is necessary to examine the role of JAB1 in normal and tumor cell growth.
Hypothesis: It is our hypothesis that JAB1 plays a pivotal role in the proliferation of breast cancer by mediating p27Kip1 degradation. To gain better insight into the clinical relevance of JAB1 and to address the role of JAB1 in breast cancer, we propose to examine whether deregulated expression of the jab1 gene leads to the genesis of breast tumors. We expect that JAB1 overexpression in breast tissue would lead to formation of tumors. We also hypothesize JAB1 as a good therapeutic target in breast cancer, as there is evidence that JAB1 is inappropriately activated in breast cancers. We expect that targeting inactivation of JAB1 would reinstate growth arrest and apoptosis in breast cancer cells.
Specific Aims: (1) To study the effects of oncogenic activation of the jab1 gene on breast tumor formation; (2) To evaluate the role of JAB1 as a therapeutic target in breast cancer cells using small interference RNA (siRNA); (3) To further evaluate the effect of JAB1 siRNA, in Ex Vivo tumor inhibition assay and on tumor regression in human breast cancer xenograft model in mice.
Study Design: We will generate transgenic mouse line specifically expressing full-length jab1 gene in a mammary-tissue specific vector, to evaluate its role in breast tumor formation. The transgenic mice along with wild-type mice will be evaluated by histological analysis to determine any potential pathological changes associated with JAB1. We will target inhibition of JAB1 activity in breast cancer cells to reinstate growth arrest by using (i) JAB1 siRNA oligonucleotides and (ii) the pSuppressor adenoviral siRNA vector system. In the ex vivo assay for tumor suppression effect of JAB1 siRNA, breast cancer cells will be transduced with adeno-JAB1 siRNA or control siRNA, prior to subcutaneous injection into nude mice and the volume of the resulting tumor will be evaluated. As a xenograft model, human breast cancer cells will be injected into athymic nude mice to establish subcutaneous tumors. We will then study the effects of the intratumoral pSuppressor adeno-JAB1 siRNA on tumor regression in these mice.
Benefits of Research: Lack of expression of p27Kip1 in human breast cancer is associated with poor prognosis. Expression of the JAB1 protein is inversely correlated with that of p27Kip1 in breast cancer, which suggests that JAB1-mediated p27Kip1 degradation may play an important role in oncogenesis. Since there is a crucial need for better prognostic markers in breast cancer, understanding the role of JAB1 in breast tumor development will dictate if JAB1 is a good potential prognostic marker. Targeting JAB1 to prevent p27Kip1 degradation to reinstate its cell-cycle inhibitor activity, may be a new powerful therapeutic approach.
Breast cancer is the most commonly diagnosed cancer in women, and it accounts for 30% of all cancers in women. An estimated 211,300 new invasive cases of breast cancer are expected to occur among women in the United States, this year, while 55,700 new cases of the noninvasive and earliest form of breast cancer account for each year. Although, breast cancer in men is rare, 1,300 cases of males are expected to be diagnosed, this year. Breast cancer is the second leading cause of cancer deaths in women, exceeded only by lung cancer and the leading overall cause of cancer death in women between the ages of 20 and 59. This year, about 39,000 women and 400 men are expected to die of breast cancer, which calls for better understanding of the cause, development and for the advancement of therapy.
Breast cancer development is a multistage process. Alterations in the expression of genes, including oncogenes, growth suppressor genes, and cell-cycle regulatory genes, are observed at each stage of development and these are associated with abnormal cell proliferation. Normal mammalian cell proliferation is under the strict control of cell-cycle regulatory proteins. One such regulatory protein is p27Kip1 which is a tumor-suppressor protein that is able to block cell proliferation. Mutations in p27Kip1 are rarely found in human tumors but its expression is reduced in human carcinomas and correlates with the poor survival among breast cancer patients. Lack of p27Kip1 expression in about 50% of human tumors is associated with poor prognosis. Alteration in the expression of p27Kip1 is mainly due to accelerated degradation of this protein. A new protein JAB1, discovered by our research group, mediates this degradation of p27Kip1. JAB1 overexpression in cells resulted in degradation of p27Kip1. Moreover, our recent published results showed that high expression of JAB1 protein inversely correlated with either reduced or lack of expression of p27Kip1 in breast cancer, suggesting that JAB1 mediated disruption of p27Kip1 contributes to cancer. A thorough understanding of the nature of genes and proteins highly expressed and activated in breast cancer is important for rational drug design, as these proteins represent excellent targets for therapeutic drugs.
In this application, we propose to examine the role of tumorigenic activation of jab1 gene on breast tumor development. We propose to generate transgenic mice that overexpress this gene in breast tissue. If this gene were to play an important role in the origin of breast tumors, these mice would likely be predisposed to the development of cancer. Establishment of a role for this gene in breast tumors will possibly aid in a direction towards the development of drugs and therapies that inhibit JAB1 activity. We also propose to examine the role of JAB1 in breast cancer cells by developing cell lines that lack the expression of JAB1 and also by inhibiting JAB1 expression in a mouse breast cancer model. If JAB1 were to play an important role in breast cancer, inhibition of its expression would likely impair cell proliferation, promote growth arrest and cell death of cancer cells. Thus, establishing JAB1 as a novel therapeutic target to cure breast cancer.