Posttranslational regulation of augurin, a new secretory tumor suppressor

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DESCRIPTION (provided by applicant): Glioblastoma is the most common type of primary malignant brain tumor in adults and one of the most aggressive cancers. Even though there have been recent advances in surgical management of this type of cancer, the median survival time for these patients is approximately 14 months, most likely due to the loss of tumor suppressor expression within the tumor genome. Monitoring the level of certain molecules in either plasma or brain biopsies might provide us with information regarding the development/progression of a possible glioblastoma in early stage. Augurin, recently identified as a potential tumor suppressor, is a secretory molecule produced in the pituitary, brain, thyroid, and esophagus. It is implicated in a wide array of physiological processes, from ACTH release to tumor suppression, and is well conserved among all vertebrates. Most interestingly, the expression of augurin is down-regulated by promoter hypermethylation in certain types of cancers, including glioblastoma. Secretory proteins are involved in diverse physiological functions, such as the regulation of metabolism, cell proliferation, differentiation, and cell deat; they require a number of posttranslational modifications during their trafficking through the secretory pathway in order to attain bioactive status. Without these posttranslational modifications, secretory proteins cannot exert their biological activities, resulting in deficiencis in physiological functions. However, information regarding the endogenous forms of proaugurin-derived peptides in various cell types has yet to be reported. The goal of this proposal is to understand the posttranslational modifications of proaugurin as a tumor suppressor. Our recent report has shown that two specific proaugurin-derived peptides are generated by the action of the specific proprotein convertase furin in vitro. Furthermore, we have found that proaugurin is sulfated during trafficking through the secretory pathway. Proliferation assays with a glioblastoma cell line demonstrated that only furin-cleaved proaugurin could suppress cell proliferation, suggesting that proteolytic cleavage is a posttranslational requirement for proaugurin to exhibit bioactivity. In addition, the predicted sulfation sites in augurin are highly conserved among other species, supporting the idea that sulfation in augurin might contribute to the suppressive activity against tumor cell proliferation. In this proposal, we will determine the bioactive species of augurin that function to suppress tumor cell proliferation using its synthetic peptides of proaugurin-derived peptides; perform mass spectrometry to determine the sulfation site(s) in augurin and clarify the importance of sulfation in augurin; determine the augurin levels in normal and glial tumor-bearing mice using radioimmunoassay and immunohistochemical staining, to test the idea that lowered augurin expression may be a potential indicator of tumor-developing cells. These experiments will help to reveal molecular mechanisms involved in proaugurin maturation, and will provide clues to its biological importance as a tumor suppressor.