Epigenetic changes are strongly associated with cancer development. DNA hypermethylation is associated with gene silencing and is often observed in CpG islands. Recently it was suggested that aberrant CpG island methylation in tumors is directed by Polycomb proteins. However, specific mechanisms responsible for methylation of Polycomb target genes in cancer are not known. Chronic infection and inflammation contribute to up to 25% of all cancers worldwide. Using glutathione peroxidase, Gpx1 and Gpx2, double knockout (Gpx1/2-KO) mice as a model of inflammatory bowel disease predisposing to intestinal cancer, we analyzed genome-wide DNA methylation in the mouse ileum during chronic inflammation, aging and cancer. We found that inflammation leads to aberrant DNA methylation in Polycomb (PcG) target genes, with 70% of the ~250 genes methylated in the inflamed tissue being PcG targets in embryonic stem cells and 59% of the methylated genes being marked by H3K27 trimethylation in the ileum of adult wildtype mice. Acquisition of DNA methylation at CpG islands in the ileum of Gpx-1/2-KO mice frequently correlates with loss of H3K27 trimethylation at the same loci. Inflammation-associated DNA methylation occurs preferentially in tissue-specific silent genes and, importantly, is much more frequently represented in tumors than is age-dependent DNA methylation. 60% of aberrant methylation found in tumors is also present in the inflamed tissue. In summary, inflammation creates a signature of aberrant DNA methylation, which is observed later in the malignant tissue and is directed by the PcG complex.

Cyclin-dependent kinase 2 (CDK2) complex is significantly over-activated in many cancers. While it makes CDK2 an attractive target for cancer therapy, most inhibitors against CDK2 are ATP competitors that are either nonspecific or highly toxic, and typically fail clinical trials. One alternative approach is to develop non-ATP competitive inhibitors; they disrupt interactions between CDK2 and either its partners or substrates, resulting in specific inhibition of CDK2 activities. In this report, we identify two potential druggable pockets located in the protein-protein interaction interface (PPI) between CDK2 and Cyclin A. To target the potential druggable pockets, we perform a LIVS in silico screening of a library containing 1925 FDA approved drugs. Using this approach, homoharringtonine (HHT) shows high affinity to the PPI and strongly disrupts the interaction between CDK2 and cyclins. Further, we demonstrate that HHT induces autophagic degradation of the CDK2 protein via tripartite motif 21 (Trim21) in cancer cells, which is confirmed in a leukemia mouse model and in human primary leukemia cells. These results thus identify an autophagic degradation mechanism of CDK2 protein and provide a potential avenue towards treating CDK2-dependent cancers.

Lactoperoxidase (LPO) is known to be present in secreted fluids, such as milk and saliva. Functionally, LPO teams up with dual oxidases (DUOXs) to generate bactericidal hypothiocyanite in the presence of thiocyanate. DUOX2 is expressed in intestinal epithelium, but there is little information on LPO expression in this tissue. To fill the gap of knowledge, we have analyzed Lpo gene expression and its regulation in mouse intestine. In wild-type (WT) C57BL/6 (B6) mouse intestine, an appreciable level of mouse Lpo gene expression was detected in the colon, but not the ileum. However, in the B6 mice deficient in glutathione peroxidase (GPx)-1 and -2, GPx1/2-double knockout (DKO), which had intestinal pathology, the colon Lpo mRNA levels increased 5-to 12-fold depending on mouse age. The Lpo mRNA levels in WT and DKO 129S1/SvlmJ (129) colon were even higher, 9-and 5-fold, than B6 DKO colon. Higher levels of Lpo protein and enzymatic activity were also detected in the 129 mouse colon than B6 colon. Lpo protein was expressed in the differentiated colon epithelial cells, away from crypt base, as shown by immunohistochemistry. Similar to human LPO mRNA, mouse Lpo mRNA had multiple spliced forms, although only the full-length variant 1 (V1) was translated. Higher methylation was found in 129 than B6 strain, DKO than control colon, and older than juvenile mice. However, methylation of Lpo intragenic CpG island was not directly induced by inflammation, since dextran sulfate sodium (DSS)-induced colitis did not increase DNA methylation in B6 DKO colon. Also, Lpo DNA methylation is not correlated with gene expression.

Supplementary Information from Methylation of Polycomb Target Genes in Intestinal Cancer Is Mediated by Inflammation

Supplementary Information from Methylation of Polycomb Target Genes in Intestinal Cancer Is Mediated by Inflammation