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 proliferation and DNA damage repair. In particular, KDM5B (JARID1B) is upregulated in 90% of human breast cancers and in aggressive forms of melanoma. Moreover, there are three splicing isoforms of KDM5B, but protein structure and function of these isoforms are not clear.
In order to understand their role in cancer cells physiology, we propose:
1) To use different chemical inhibitors of KDM5B catalytic activity and to test their effects on transcriptome, cell survival and DNA damage repair in breast cancer and melanoma derived cell lines.
2) To study the functional effects of two different miRNAs which, as we previously showed, target KDM5B mRNA and significantly modulate its abundance in breast cancer cells, in melanoma cancer cells which over-express a less frequent isoform of KDM5B.
3) Using publicly available transcriptomic datasets, to perform a comprehensive bioinformatic analysis in order to evaluate the expression levels of all canonical and putative transcript isoforms of KDM5B from breast and melanoma cancer patients and cell lines and validate the results by rapid amplification of cDNA ends (RACE).
4) To quantify KDM5B protein isoforms and to localize the subcellular distribution of proteins by immunofluorescence assay in the cell lines over-expressing one or more isoforms of KDM5B
5) To set-up a co-immunoprecipitation protocol for the alternative isoforms of KDM5B in order to identify their interactors in cancer cells through mass spectromic analysis.
We previously showed that JARID1B over-expression in human breast cancer tissues correlates with the down-regulation of miRNAs targeting its mRNA. In human cell lines a causal relationship between the two phenomena has been demonstrated (Mocavini et al., 2018). Moreover, in MCF7 breast cancer cell line miRNAs induced JARID1B down-regulation, although not changing the level of bulk chromatin H3K4 tri-methylation. Our preliminary results suggest that inhibition of JARID HDMs activity by chemical inhibitors and/or miRNAs can cause hypersensitivity to genotoxic damage and BRCA1 derepression. These results, if confirmed, could shed light on JARID HDMs role in protecting cancer cells against genotoxic damage and open the way for possible therapeutic use of miRNAs which specifically target these enzymes and/or their specific chemical inhibitors.
We will also investigate on a plausible mechanism for the role of HDMs in DNA damage repair. Considering the emerging role of lncRNAs at the sites of DSB we will explore the influence of HDMs activity in favoring the pervasive transcription which is required for their synthesis.
Then we want to test the significance of KDM5B and its splicing variants in melanoma and we will try to extend analyses on DNA damage accumulation to melanoma cell lines in which KDM5B has been shown to be crucial for chemoresistance. Integrating a comprehensive bioinformatic analysis on transcriptomic data from breast cancer and melanoma cell lines, a targeted rapid amplification of cDNA ends for mapping KDM5B transcripts and quantify protein isoforms in selected cell lines we will able to better characterize the deregulated expression of KDM5B isoforms in cancer cells and begin to characterize their biological roles identifying their binding partners.