A role for cdkn2ab in melanoma of Xiphophorus and Medaka

Manfred Schartl (1, 2) , Janine Regneri (2) , Brigitta Wilde (2) , Barbara Klotz (2) , Michael Hausmann (2) , Susanne Kneitz (2) , Amaury Herpin (3) , Ronald B. Walter (4)

1 Comprehensive Cancer Center
2 Physiological Chemistry
3 LPGP - Laboratoire de Physiologie et Génomique des Poissons
4 Department of Chemistry & Biochemistry, Molecular Biosciences Research Group

Inactivation of the human CDKN2A tumor suppressor gene by mutation or epigenetic changes is a frequent signature of malignant melanoma. The locus encodes two transcripts, Ink4a and Arf, which are important regulators of the cell cycle. In teleost fish a gene has been identified, cdkn2ab, which represents the common ancestor of mammalian cdkn2a and cdkn2b genes. However, the cdkn2ab gene encodes only Ink4a. In platyfish and swordtails the cdkn2ab gene has been mapped to a genomic region that harbors a melanoma suppressor gene. However, an unexpected positive correlation of cdkn2ab and xmrk transcript levels has been reported. As high xmrk expression levels are a reliable molecular readout for melanoma malignancy, simultaneous high levels of a tumor suppressor gene are counterintuitive. To obtain a better understanding of the role of cdkn2ab in melanoma formation in fish we studied the effect of its expression levels in-vitro and in-vivo. First we analyzed cdkn2ab and xmrk expression levels in a large number of melanoma of varying degree of malignancy. Overexpression of cdkn2ab in a melanoma cell line led to decreased proliferation and induction of a senescence-like phenotype. This indicated an analogous melanoma-suppressive function as in mammals. Overexpression of Xiphophorus cdkn2ab under control of the medaka mitfa promoter did not affect pigment cell development in wildtype medaka. Coexpression of this construct in the mitf:xmrk trangenic medaka melanoma model resulted in full suppression of pigment cell tumor development. Interestingly, this occured also in the majority of fish with a p53 negative background. Using CRISPR/cas9 technology a cdkn2ab knock-out was produced, which is fully viable with no detectable major defects. However, unlike in humans, the cdkn2ab deficient medaka did not display spontaneous melanoma development so far. The mitf:xmrk melanoma inducing gene was crossed into the cdkn2a negative background to monitor the effect on melanoma development.

Domaines: Sciences du Vivant [q-bio]

Manfred Schartl, Janine Regneri, Brigitta Wilde, Barbara Klotz, Michael Hausmann, et al.. A role for cdkn2ab in melanoma of Xiphophorus and Medaka. 9. Zebrafish Disease Models Conference, National University of Singapore (NUS). SGP., Oct 2016, NA, Singapore. ⟨hal-01914407⟩