Abstract 1581: Tumor suppressor functions of the zebrafish ink4ab: a novel cyclin-dependent kinase inhibitor.

Abstract The human INK4b-ARF-INK4a genetic locus encodes two closely related members of the INK4 family of cyclin dependent kinase inhibitors, p15INK4b and p16INK4a regulating RB phosphorylation and subsequently cell proliferation, and a p53 stabilizer, known as ARF. All of the three products play a pivotal role in tumor suppression and are frequently deleted or inactivated in a wide spectrum of human cancers. Despite the critical role of the mammalian INK4b-ARF-INK4a locus in tumor suppression, its counterpart in zebrafish has not yet been characterized. Zebrafish is an advantageous vertebrate to model human cancers, specifically because of the high fecundity, the ease of performing high-throughput screens, and the genetic conservation with mammals. We identified a syntenic zebrafish ink4ab locus that consists of a single gene orthologous to both mammalian CDKN2a (INK4A) and CDKN2b (INK4B), and functions to activate senescence in response to oxidative stress. Utilizing morpholino targeted knockdown and zebrafish mutants for the ink4ab gene, we have developed a zebrafish model for tumorigenesis. We demonstrate that the zebrafish ink4ab gene functions as a tumor suppressor by controlling cell cycle regulation under stress. Senescence was activated by oxidative stress in wild type embryos but not in embryos with ink4ab deficiency when examined with a whole embryo senescence-associated beta-galactosidase (SA-β-gal) assay. Surprisingly, we find that ink4ab deficient embryos also display significantly higher levels of apoptosis than controls. We determined that the apoptosis associated with ink4ab deficiency is p53-dependent. Furthermore, ink4ab deficiency led to significantly lower survival rates, both overall and upon radiation. Ink4ab deficient zebrafish had splenomegaly, increased lymphocyte proliferation, and developed spontaneous tumors including metastatic melanoma, osteosarcoma, leukemia and myelodysplastic disorders. Thus, the zebrafish ink4ab protein functions as a tumor suppressor similarly to the human p15INK4B and p16INK4A. To study the upstream control of zebrafish ink4ab expression, we investigated the orchestrated regulation by the Bmi-1 oncogene, and identified bmi-1a as a repressor of ink4ab expression. Zebrafish heterozygous for both p53 and ink4ab mutations displayed significantly higher tumor incidence compared to p53 heterozygotes with wild-type ink4ab, indicating that ink4ab haploinsufficiency promotes tumorigenesis. To this end, the combined deficiency of ink4ab and p53 accelerates the latency and broadens the tumor spectrum including the formation of retinoblastoma. Collectively, the zebrafish ink4ab model of tumor suppression provides a platform to perform large-scale screens for small molecules that modulate tumorigenesis in the ink4ab deficient fish, and permit defining the genetic pathways of CDKN2a- and CDKN2b-mediated tumor suppression. Citation Format: Stephani Davis, Hatem E. Sabaawy. Tumor suppressor functions of the zebrafish ink4ab: a novel cyclin-dependent kinase inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1581. doi:10.1158/1538-7445.AM2013-1581