Validation of citron kinase as a drug target for anti-cancer therapy

One of the hallmarks of cancer is uncontrolled cell proliferation and many cell division regulators are validated targets for the isolation of novel chemotherapeutic drugs for the treatment of cancer pathologies (1). The family of serine/threonine kinases has become one of the most studied classes of anti-cancer drug targets and small molecule inhibitors of mitotic kinases such as Aurora and Polo-like kinases are currently undergoing clinical trials (2). There is preliminary evidence in the literature that another mitotic kinase, the cytokinesis regulator Citron kinase (CIT-K), could be a valid target for the treatment of liver cancer pathologies (3). Moreover, data from the Human Protein Atlas and Oncomine databases suggests that CIT-K is overexpressed in several cancers, including breast, cervical, malignant glioma, ovarian, pancreatic, skin, thyroid and urothelial cancers. Thus there is a need to establish if this kinase could be a target for other cancer types. The aim of this proposal is to gain further understanding of the CIT-K inhibition phenotype, disease linkage, cancer selectivity, and potential clinical applications of CIT-K inactivation.

The objectives of our proposal are:
1. To establish whether CIT-K is misexpressed in diverse cancer cell lines and cancer tissue microarrays (TMAs).
We will generate a CIT-K monoclonal antibody and in collaboration with Dr Paul Edwards (Hutchinson-MRC Centre) will examine CIT-K expression by qPCR and Western blot in cell lines derived from breast, colon, lung and pancreatic carcinomas and compare it to normal immortalised counterparts. In collaboration with Prof Carlos Caldas (CR-UK Cambridge Research Institute) we will also analyse CIT-K expression by immunohistochemistry on TMAs containing about 1,000 invasive breast cancers along with normal breast tissue samples. In parallel, we will also analyse CIT-K expression on commercially available TMAs containing 110 cancer and 55 normal samples covering 11 cancer types: breast, colon, lung, kidney, ovarian, endometrial, stomach, prostate, melanoma, liver, and lymphoma.

2. To investigate the effect of CIT-K knockdown on the proliferation and behaviour of different cancer cell lines.
On the basis of the results obtained from objective 1, we will select a few representative cell lines for each cancer type and test the effects of CIT-K RNAi knockdown on five different processes: cell cycle, cell proliferation, cell division, cell death and ability to form colonies. Moreover, we will analyse the velocity and extent of recovery of some of these cell lines after removal of RNAi inhibition. We will also compare the toxicity of CIT-K RNAi versus knock down of another mitotic kinase, Aurora B, whose inhibitors are currently in clinical trails. Finally, we will test if CIT-K knockdown could reduce the tumourigenicity of xenografts of CIT-K-sensitive cancer cells in nude mice.

Should CIT-K be validated through these experiments, the project could then develop into a drug discovery effort in collaboration with CRT.