p53, the “guardian of the genome” is a major tumor suppressor protein in humans. p53 functions mainly as a transcription factor activated by different cellular stress signals, such as DNA damage, oncogene activation, hypoxia, nucleotide depletion, or chemotherapeutic drugs. Upon activation, p53 induce synthesis of various downstream targets, which regulate processes like cell cycle arrest, senescence, apoptosis and/or DNA repair depending on the extent and duration of the stress signal. The importance of p53 for regulation of cells decision between life and death is revealed by the frequent mutations of the TP53 gene in cancer cells and the inactivation of p53 by viral proteins, which is also linked to the development of cancer. Consequently, activation of p53 in cancer cells, which pushes them towards senescence or apoptosis, is considered an efficient anti-cancer therapeutic strategy.
Our group has recently identified the HZ compounds, a novel class of molecules that not only activate p53 transcription factor activity but also increase p53 protein synthesis. These molecules inhibit the enzyme dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway. Other known DHODH inhibitors also lead to an increase of p53 protein synthesis and transcription factor activity. Initial mRNA studies, however, showed that p53 mRNA levels do not change significantly after short-term treatment with the HZ compounds suggesting that, in this case, increased p53 mRNA translation is the cause of increased p53 protein levels.
The aim of this project is understanding the mechanisms that underlie increased translation of p53 mRNA after DHODH inhibition. For that purpose, we will study whether inhibition of DHODH by different DHODH inhibitors alters the expression of a set of selected proteins and small non-coding RNAs that regulate p53 translation. The selection will be based on mRNA and small RNA sequencing data. The expression of small RNAs, mRNAs, and proteins of interest will be investigated using RT-qPCR, siRNA and WB analysis in different cell lines. Additionally, interactions of the selected molecules with p53 mRNA will be confirmed with Proximity Ligation Assays (PLA) and RNA scope.