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Regulation of JAB1 and its Role in Cellular Proliferation
Regulation of JAB1 and its Role in Cellular Proliferation
Background: The protein p27Kip1 is an inhibitor of cell division. 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. An abnormally low amount of p27 or its degradation is observed in up to 50% of tumors and is associated with a poor prognosis, suggesting that a disruption of p27 regulatory mechanisms contributes to neoplasia. In normal and tumor cells, p27 is regulated primarily by the rate of translation, changes in protein turnover and its localization in the cell. Cytoplasmic mislocation of p27 occurs in 40% of breast tumors, indicating the importance of this cell-cycle regulator. Many genes known to control the cell cycle are mutated in cancer. However, despite its potential role as a tumor suppressor, mutations in p27 are rarely found in human tumors. Lack of p27 protein in tumors is a powerful independent indicator of a poor clinical prognosis among patients with breast carcinoma or primary ovarian, colorectal, prostate, or lung carcinoma. This suggests that a disruption of p27 regulatory mechanisms contributes to neoplasia. Claret et al. identified the Jun-activation binding-protein 1 (JAB1) (Nature, 383:453 1996), which was recently found to control the activity of p27 by facilitating its degradation (Nature, 398:160 1999). JAB1 promotes p27 relocalization from the nucleus to the cytoplasm, thus accelerating its degradation through the ubiquitin/proteasome pathway. Our recent studies have shown that JAB1 protein levels are higher in breast transformed cells and tumor tissues than in normal samples. JAB1 overexpression also correlates with a decrease in p27 protein levels in breast tumor samples related to a certain degree of tumor invasiveness in vivo. These results suggest that changes in expression levels of JAB1 and p27 may contribute to deregulation of the cell cycle and precede the progression of breast tumors. It has been noted that activation of the Her-2/neu receptor, which is overexpressed in 30% of breast cancers, leads to a decrease in p27 protein levels, allowing cells to proliferate. Treatment with trastuzumab (Herceptin) to block Her-2/neu signaling leads to an increase in p27 protein levels and therefore induces G0 arrest. However, only 26% of HER2 overexpressing tumors respond to Herceptin treatment. Thus, JAB1-facilitated degradation of p27 may be one mechanism of breast tumor survival through a resistance mechanism against Herceptin treatment.
Objective/Hypothesis: Although JAB1 is known to facilitate p27 degradation; the regulation of JAB1 and its mechanism of action are still unknown. The objective of this proposal is to characterize the regulation of JAB1 activity and its role in cellular proliferation, offering further insight to the mechanism of JAB1 involvement in breast cancer. We hypothesize that the overexpression of JAB1 in breast cancer protects tumors against Herceptin treatment by facilitating p27 degradation, therefore offering a novel strategy by which the down-regulation of JAB1 might sensitize tumors to Herceptin-induced tumor growth arrest and apoptosis.
Specific Aims: (1) To characterize the jab1 promoter and transcriptional regulation of the jab1 gene, (2) to determine the role of JAB1-mediated p27 degradation and the significance on cell proliferation rate, and (3) to investigate the role of JAB1 in resistance to Herceptin.
Study Design: We will identify the promoter region of jab1 by primer extension analysis followed by cloning of luciferase reporter constructs. The regulatory region will be identified by 5’ and 3’ deletions and confirmed by electrophoretic mobility shift assays. Next, we will determine whether JAB1-mediated p27 degradation prevents p27-induced cell cycle arrest using cells stably expressing inducible p27 and JAB1. Further, we will investigate the role of JAB1 in resistance to Herceptin treatment. We will use siRNA technology to downregulate the endogenous level of JAB1 to increase Herceptin induced growth arrest in a panel of breast cancer cells.
Relevance: Expression of p27 is a powerful independent indicator of poor clinical prognosis for breast cancer. Thus, an improved comprehension of the mechanisms regulating p27 expression and function will contribute to a better understanding of cell growth and tumorigenesis. The link between high JAB1 and low p27 expression in breast cancer suggests that JAB1 plays an important role as a potential prognostic marker for tumor aggressiveness. Further, JAB1 overexpression in breast cancer may provide a mechansism of drug resistance. Development of new therapeutic approaches to downregulate JAB1 to enhance the efficiency of Herceptin treatment will be an exciting venue to explore.
REGULATION OF JAB1 AND ITS ROLE IN CELLULAR PROLIFERATION
Breast cancer is the second only to lung cancer as the leading cause of death in women due to cancer. This year, 211,300 new cases of breast cancer are expected, while killing nearly 39,800 women in the same year. Advances in treatment for breast cancer are progressing, but research is still critical for the advancement of therapy. Cancer progression is often attributed to the misregulation of cell cycle. The cyclin-dependent kinase inhibitor p27 is a potent inhibitor of cell cycle progression. Abnormally low levels of the p27 protein are frequently found in 50% of breast cancer and others. Cytoplasmic mislocation of p27 and subsequent inactivation occurs in 40% of breast tumors, suggesting the importance of this cell-cycle regulator.
Many genes known to control the cell cycle are mutated in cancer. Despite its potential role as a tumor suppressor, mutations in p27 are rarely found in human tumors. However, lack of the p27 protein in tumors is a powerful independent indicator of poor clinical prognosis among patients with breast carcinoma and also primary ovarian, colorectal, prostate or lung carcinoma, suggesting that disruption of p27 regulatory mechanisms contributes to neoplasia. Claret, et.al. identified the Jun-activation binding-protein 1 (JAB1) (Nature, 1996), which was recently found to control the activity of p27 by facilitating its degradation (Nature, 1999). JAB1 is able to transport p27 from the nucleus to the cytoplasm and thus accelerating its degradation. Our studies have shown that JAB1 protein levels are increased in breast transformed cells and tumor tissues compared to normal samples. JAB1 overexpression is also correlated with a decrease in p27 protein levels in breast tumor samples and this correlates with a certain degree of tumor invasiveness in vivo. These results suggest that changes in expression levels of JAB1 and p27 may contribute to deregulation of the cell cycle and precede the progression of breast tumors. Thus, an understanding of the regulation of function and activity of p27 has tremendous potential to improve our knowledge of p27 and tumorigenesis.
The human epidermal growth factor receptor 2 (HER2-neu) is overexpressed in 30% of breast tumors and has recently been shown to upregulate p27 protein levels. A humanized monoclonal antibody, trastuzumab or Herceptin®, has been successfully used in clinics to treat patients with metastatic breast cancers that overexpress HER2. However, the response rate to Herceptin treatment is only 26% percent, suggesting that there is a mechanism of resistance downstream of this signaling pathway. Further studies have proved that HER-2/neu causes a decrease in p27 protein level by inducing the mislocation of p27 in the cytoplasm for degradation through JAB1 regulation. This suggests a link between HER-2/neu signaling and JAB1 regulation affecting the turnover rate of p27. Thus providing yet another mechanism of p27 degradation in breast cancer. It is possible that a JAB1 overexpressing tumor would have a protective barrier against Herceptin mediated upregulation of p27. Therefore, strategies aiming to inhibit JAB1 level may provide a novel target for experimental therapies to increase the efficiency of Herceptin treatment. The proposed research is relevant to the study of breast cancer as well as others. Our recent research has shown an inverse correlation between expression of p27 and JAB1 protein levels in breast tumors and other carcinomas underlying its potential as a biological marker for tumor invasiveness. The research proposed here will elucidate the transcriptional control of JAB1 that has been overactivated in breast transformed cells and tumor tissues. Understanding JAB1 regulation may help to design new strategies to block some of the upstream signaling pathways that inappropriately activated JAB1. In addition, because JAB1 promotes cell cycle progression through inhibition of a cell-cycle regulator, characterization of JAB1 protein will add further to our knowledge of cell-cycle regulation. We will also explore whether JAB1 overexpression is a mechanism of Herceptin resistance in breast cancer. Development of new therapeutic approaches to down regulate JAB1 may therefore enhance the efficiency of Herceptin treatment in breast cancer. In summary, the proposed research addresses many important questions whose answers will contribute to the advancement of breast cancer research.