Master’s Degree Thesis Proposal

(Návrh tématu diplomové práce)

Lab: Molecular Mechanisms of Cancer, Institute of Biology, Faculty of Medicine MU

PI: Zdenek Andrysik, Ph.D.

Project title:

The role of p53 target gene PARADA in carcinogenesis

(Úloha p53-indukovaného genu PARADA v karcinogenezi)

TP53 gene is the most frequently mutated tumor suppressor in human cancers. However, in about 60% of tumors TP53 remains unmutated and is silenced by some other mechanism, such as increased activity of a negative upstream regulator. TP53 encodes transcription factor p53, which controls induction of hundreds of target genes participating in several programs with anti-tumoral functions like cell cycle arrest, DNA repair, senescence, and apoptosis. Our multiomics study of the p53 network revealed an understudied conserved target among genes directly regulated by p53 PARADA (p53-activated Regulator of Apoptotic Death in Adenomas, Fig. 1) linking p53 to a novel function in ion channel biology relevant to both physiological functions of numerous tissues expressing PARADA and cancer treatment.

Rationale

Transcription factor p53 is notorious for its role in proliferation arrest in response to a variety pf stress signals. However, target gene PARADA seems to have an opposite function – it was reported that cells depleted of PARADA displayed cell cycle arrest. Since drugs activating p53 are candidates for novel targeted tumor therapies, thorough understanding of the complex p53 response is critical for designing an efficient treatment. Therefore, we propose to study i) the p53-driven induction of the PARADA gene, ii) the impact of p53 activity on PARADA function, iii) the role of p53-induced PARADA in proliferation arrest, and iv) the effect of PARADA inhibitor on cellular response to p53 activation. We will investigate implications of the p53-PARADA axis in human colorectal cancer (CRC) as PARADA expression is strongly increased in tumor tissue of both colonic and rectal origins.

Hypothesis

PARADA counteracts p53-driven anti-proliferative effects.

Specific Aims

To test the hypothesis, we propose following Specific Aims:

Aim 1. To document PARADA protein levels in three CRC cell lines HCT116, RKO, and HCT8.

Aim 2. To characterize ion flux in CRC lines upon p53 induction, PARADA depletion with shRNA, and PARADA pharmacological inhibition.

Aim 3. To test the effect of PARADA inhibition, knock-down, and ectopic expression on CRC cell lines proliferation.

Methods

Tissue cultures, DNA/RNA/protein isolation, cloning, PCR, qRT-PCR, flow cytometry, lentiviral expression vectors, western blotting, agarose electrophoresis, fluorescence microscopy, proliferation/cell metabolism assays, functional genomics (shRNA and/or CRISPR).

Who can apply

Everyone is welcome to apply, any background, any level of experience, any year of study (including students in their 1st or 2nd year). Feel free to reach out to the PI for details.

Students of Molecular Biology and Genetics, Medical Genetics and Molecular Diagnostics, Human Biology, and Experimental Animal Biology and Immunology are strongly encouraged to submit their applications.

Conclusion

This is an ambitious project in a high impact field, informative for cancer therapy. You will have an opportunity to familiarize yourself with a portfolio of both basic and advanced methods in molecular biology. Upon completion, you will be well-positioned for the next step in your career, regardless if you prefer to stay in academia or head for a position in bioindustry. Finally, this project represents an uncharted territory of the p53 biology with a great relevance to carcinogenesis and cancer therapy.