Investigation of new tumor suppressor genes in pancreatic ductal adenocarcinoma (PDAC)

Aim: The endocytic and the methionine pathway which provides the main methyl donor for methylation reactions play a pivotal role in cancer pathogenesis. The genes involved in these pathways have sporadic mutations in humans and based on in silico analyses some of these genes may play a role as tumor-suppressors. The main purpose of this study is to investigate the genes that involved in endocytosis and methionine cycle which are Methionine Adenosyltransferase 2A (Mat2a), Protein arginine methyltransferase 6 (Prmt6), Protein arginine methyltransferase 5 (Prmt5), Protein arginine methyltransferase 7 (Prmt7), protein 4 (Prmt7) SET and MYND4 domain containing protein (Smyd4), DNA methyltransferase 1 (Dnmt1), Ras-related protein 11b (Rab11b), Ras-related protein 4b (Rab4b), Selenoprotein I (SelenoI), Selenoprotein N (SelenoN) as a candidate tumor suppressor genes for the in vivo KRAS induced PDAC. In addition, a second aim is to demonstrate in vitro and in vivo that Smyd 4, one of the listed genes, acts as a tumor suppressor in the development of PDAC. Material and methods: We took advantage of a somatic CRISPR/Cas9 genome engineering method to screen for tumor-suppressors between a set of 10 genes involved in the above mentioned pathways. Results: In these sets of genes one of the identified tumor-suppressors is the lysine methyltransferase Smyd4 in breast cancer. In our study CRISPR/Cas9 mediated Smyd4 deletion in mouse pancreatic cell lines increased cell proliferation, colony formation in soft agar and tumor formation in an orthotopic mouse model. Discussion: Mechanistically Smyd4 deletion led to a specific downregulation of the histone H3K9m2, suggesting the histone H3K9me3 to be the specific substrate for the Smyd4 demethylase activity. Furthermore, in silico analyses and cell culture experiments link the deletion of Smyd4 with changes in glucose metabolism. Together, we identify the lysine methyltransferase Smyd4 as a novel tumor-suppressor for PDAC and suggest an underlying mechanism by which Smyd4 specifically demethylates the histone H3K9me3 and regulates glucose metabolism.