Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential
therapeutic cancer targets. In particular, HDMs of class 5 (KDM5) ,also known as JARID, are over-expressed in different types of cancers and have a prominent role in cancer cells ploriferation and DNA damage repair. In particular, KDM5b (JARID 1B) is up-regulated in 90% of human breast cancers. In order to understand its role in breast cancer cells physiology, we propose:
a - to use different chemical inhibitors of KDM5 catalytic activity and to test their effects on transcriptome, cell survival and DNA damage repair.
b - to study the post-transcriptional regulation of JARID 1B expression by miRNAs potentially targeting the protein and significantly modulated in breast cancer cells and their effects on its functional activities
Oncogenesis is strictly associated with epigenetic modifications and enzymes controlling crucial histone modifications are very frequently differentially expressed in cancer as compared with normal tissue. JARID HDMs in particular are over-expressed in different types of tumours and have a prominent role in cancer cells proliferation and DNA damage repair. In particular, KDM5B (JARID 1B) is up-regulated in 90% of human breast cancers and has been shown to be a luminal lineage-driving oncogene. The mechanism of its oncogenic potential is not completely understood. It could be mediated by transcriptomic modulation and/or by its demethylation action at the damaged genomic sites. To really understand its mechanism we have to interfere with its activity in cancer cells. Silencing or knocking out the gene could be informative but not resolutive, since besides their catalytic activities JARID HDMs have subsidiary roles in multiprotein complexes hosting other regulatory proteins. It is therefore crucial to use chemical inhibitors of their catalytic activities. The worldwide search of such molecules in the last five years produced many candidate molecules but few of them are able to enter cancer cells and efficiently and specifically inhibit JARID enzymes without affecting other classes' HDMs. We previously found two different specific inhibitors which we will use together with a commercial one, which also looks extremely specific, in order to investigate on their effects on global transcriptomic regulation and DNA repair efficiency. It is very important to use inhibitors with different chemical structures in order to rule out that the observed effects are due to side effects rather than to an effective catalytic inhibition.
It is also very important to understand the mechanism leading to over-expression of JARID HDMs in cancer cells. With a bioinformatic analysis of the transcriptomes of breast cancer samples contained in the TCGA Cancer Atlas database we were able to correlate up-regulation of JARID1B mRNA with down-regulation of two miRNAs potentially able to target it. Preliminary data suggest that both microRNAs if transfected into breast cancer cell lines which over-express JARID1B can down-regulate the level of the protein. We plan to validate these data by using contructs carrying the JARID1B 3'-UTR downstream of a luciferase reporter construct and testing the effect of transfection with wild type and mutant microRNAs. Dissecting the network of post-transcriptional regulation of JARID1B by microRNAs in breast cancer cell lines can be very informative on its overall regulation and useful as an additional tool to understand its contrubution to cancer cells ploriferation and resistance to DNA damage. Futher bioinformatic analyses will be carried out to find other putative regulators of JARID expression and putative targets of its regulative action.