TITLE: PARK2, a Large common Fragile Site Gene, Is Part of a Stress Response Network in Normal Cells that is Disrupted During the Development of Ovarian Cancer PRINCIPAL INVESTIGATOR:

PARK2 (Parkin) is an extremely large gene that spans greater than 1.3 megabases of genomic sequence within chromosomal band 6q26. This gene is derived from within the distal end of the highly unstable FRA6E (6q26) common fragile site (CFS). CFSs are large chromosomal regions that are highly unstable, found in all individuals, and prone to deletions and other alterations, especially in developing cancer cells. The central two questions that we want to address with this work are what role does inactivation of Parkin and other large CFS genes play in the development of ovarian cancer and whether these genes function as part of a stress response network. In order to address these two questions, we have analyzed the effect of re-introducing Parkin (and several other large CFS genes) into ovarian cancer cell lines that do not express it. We have now shown that the re-introduction of Parkin, and several other CFS genes, is associated with greater sensitivity to the induction of apoptosis. This is consistent with our hypothesis that the inactivation of these genes contributes of ovarian cancer development. We have now identified 20 extremely large genes like Parkin that reside within CFS regions. To determine if these genes are randomly inactivated during cancer development, we have utilized real-time RT-PCR analysis to measure the expression of a number of these genes including Parkin in panels of cancer cell lines and primary tumors for cancers of the prostate, ovary, breast, brain and liver. This analysis reveals a decidedly non-random inactivation of the expression of these genes in different cancers. In addition, we’ve found that there is a greater inactivation of expression of the large CFS genes (and greater numbers of these genes inactivated) in cancers that are generally more aggressive and have a poorer overall clinical prognosis. This may offer a prognostic test of individual ovarian cancers based upon the number of large CFS genes that are inactivated in each cancer. The second part of our studies was to examine Parkin and other large CFS genes as being part of a stress response system within cells. We have utilized the newly developed whole-genome tiling arrays which contain tiled oligonucleotides across the non-redundant portion of the genome to characterize transcripts within and around Parkin (and several other large CFS genes) and their response to two stresses, hypoxia and treatment with the carcinogen NNK. These studies reveal that there are stress-responsive non-coding transcripts within the large CFS genes and may begin to explain why these genes are so large in the first place. We’ve also now observed that the expression of many of these non-coding transcripts are altered in ovarian tumors. These studies thus support our overall hypothesis that the large CFS genes function as a stress response system within cells and this system, and the large genes, are uniquely susceptible to genomic instability. Introduction Parkin is a gene that spans an extremely large chromosomal region of 1.36 megabases (Mbs). This large gene spans the distal half within the highly unstable FRA6E CFS (6q26) and our novel hypothesis that received funding from the Department of Defense Ovarian Cancer Research Program funding was that Parkin and other large CFS genes were part of a stress response system that is disrupted during the development of ovarian cancer. There were two major goals to this proposal. The first was to determine if inactivation of expression of Parkin and other large CFS genes could contribute to the development of ovarian cancer. The second was to demonstrate that Parkin and other large CFS genes functioned as part of a stress response system within cells. We will now summarize the work that we’ve completed after our third year of Department of Defense funding. Our key findings are as follows: (1) We’ve demonstrated that Parkin is indeed inactivated in many ovarian and other cancers and that the reintroduction of Parkin results in growth inhibition of ovarian cancer cell lines; (2) We’ve now identified