In human malignances, DNA amplification leading to oncogene activation represents one of the major forms of genomic alterations that plays a causative role in tumorigenesis. The amplified genes may be viewed as primary oncogenic targets in the development of human cancer. The gain-of-function effect of gene amplification makes them ideal therapeutic targets for cancer. Amplification of ch13q34, the region that Cullin 4A (Cul4A) resides in, is amplified in several human cancers, including breast and hepatocellular cancer. The amplification of this region has also been observed in a variety of human cancers including esophageal, gastric, head and neck, bladder, small cell and non-small cell cancers. Since such the amplification involves multiple genes, the identification of the functional target has been difficult. To date, the study on the potential oncogenic role of Cul4A has been limited. Our hypothesis is that Cul4A is the key cancer-causing oncogene in this amplified region. In our preliminary study, we have identified frequent Cul4A amplification and overexpression in malignant pleural mesothelioma cell lines and tumors. Knockdown of Cul4A by shRNA leads to increased p21 protein, and subsequently induces cell cycle arrest and inhibits mesothelioma cell growth. Forced expression of Cul4A decreases p21 protein and promotes cell growth. The molecular genetics of mesothelioma are relatively homogenous, e.g., homozygous deletion of 9p21 (contains the INK4a/ARF locus) was found in more than 70% of mesothelioma tumors[12], thus make mesothelioma an unique model to study the mechanisms of human carcinogenesis. Currently, there is no Cul4A transgenic mouse tumor model available. The following specific aims outline our detailed plan to prove our hypothesis. To determine the role of Cul4A amplification in mesothelioma, we plan to perform additional Cul4A knockdown and overexpression study using more mesothelioma cell lines (Specific Aim 1). To elucidate the potential mechanisms through which Cul4A is a proto-oncogene, we have the detailed plan on the important Cul4A-related cell cycle and genome stability analysis (Specific Aim 2). To evaluate the potential oncogenic role of Cul4A in vivo and mimic the human mesothelioma, we have generated a conditional Cul4A transgenic mouse model and we plan to cross this model with three conditional tumor suppressor knockout models (Specific Aim 3).
The specific aims to test our hypothesis:
In aim 1, Investigate the role of Cul4A in human mesothelioma cell lines and tissues. We plane to investigate and validate the role of Cul4A in mesothelioma cell lines with amplification in the 13q34 region by knocking-down Cul4A and investigate the effects of enhanced Cul4A expression in normal and mesothelioma cell lines. In addition, we plan to determine the role of Cul4A amplification in large number of mesothelioma tissues;
In aim 2, the goal is to investigate the potential mechanisms through which Cul4A plays an oncogenic role. We plan to investigate the mechanisms through which Cul4A regulates cell proliferation and genome stability;
In aim 3, we plan to elucidate the role of Cul4A in mesothelioma development using knockout and transgenic mouse models. We have generated a Cul4A transgenic mouse model. We are now ready to use the Cul4A transgenic mice to test our hypothesis that Cul4A is an oncogene.

Public Health Relevance

Malignant pleural mesothelioma is a highly aggressive and challenging cancer arising primarily from the pleural lining of the lung. Approximately 3,000 patients are diagnosed with mesothelioma in the United States annually and the incidence of this tumor is predicted to increase over the next decade in both Europe and developing nations. Since mesothelioma usually presents at an advanced stage, a curative resection is rarely possible. Radiotherapy has failed to show clinical benefit as a single treatment modality, and the administration of chemotherapy is mostly restricted to the advanced stage with limited efficiency. Alternative strategies based on pleural injections of recombinant cytokines have similarly proven unsatisfactory. Most patients die within 2 years after diagnosis. Since current interventions offer only limited benefit and overall survival is low, there is an urgent need to develop new therapeutic agents based on a greater understanding of mesothelioma's underlying molecular mechanisms. Loss of cell cycle control is a hallmark of human cancer including mesothelioma. For instance, the most predominant genetic change in human malignant pleural mesothelioma (>70%) is the homozygous deletion of INK4a/ARF locus within the 9p21 chromosome region. Despite that p21 is one of the most important effectors of p53, p21 can also be regulated by many p53-independent mechanisms including posttranslational modifications. For the majority of mesotheliomas that lacks both p14ARF and p16INK4A, up-regulating or stabilizing p21 protein may serve as a novel therapeutic strategy. In addition, functional loss of p21 can mediate drug-resistance phenotype, and that may be one of the key reasons why mesothelioma is resistant to classical chemotherapy and radiation therapy. Mesothelioma provides a very unique and homogeneous genetic model for human cancer. The current mesothelioma mouse models focused only on the conditional knockout of tumor suppressor genes, e.g., INK4a/ARF. Cul4A amplification represents the activation of a distinct proto-oncogene class in mesothelioma. To date, there is no Cul4A transgenic mouse model. Therefore, we have just established a conditional Cul4A transgenic mouse model to mimic human mesothelioma by incorporating this oncogenic event. This model may be used to study the role that Cul4A plays in mesothelioma carcinogenesis, and may also be potentially useful for further drug discovery, e.g., to screen for molecules that inhibiting Cul4A-mediated p21 degradation.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Cancer Genetics Study Section (CG)
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Mietz, Judy
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University of California San Francisco
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