Ricerc@Sapienza

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.