Functional characterisation of cell cycle-related kinase (CCRK) in colorectal cancer carcinogenesis.

ABSTRACT

Cell cycle-related kinase (CCRK) is a newly identified protein kinase homologous to Cdk7. We have previously shown that CCRK is a candidate oncogene in human glioblastoma. However, whether CCRK is a bona fide oncogene remains to be tested. The aim of this study was to investigate the role of CCRK in human colorectal cancer carcinogenesis. By Western blotting, we analysed the expression profile of CCRK protein in 10 colorectal cancer tissue samples and their adjacent normal colon tissues and in seven colorectal cancer cell lines. CCRK protein expression was also investigated by immunohistochemistry in a colorectal tissue microarray, which contained 120 cases of primary colorectal cancer and adjacent normal colorectal mucosa. The effects of CCRK knock-down on cell cycle profile and proliferation of colorectal cancer cells were examined by transfecting LoVo and DLD1 human colorectal cancer cell lines by either short-hairpin RNA (shCCRK) or small interfering RNA targeting CCRK (siCCRK). We found that CCRK protein levels were elevated by more than 1.5-fold in 70% of colorectal cancer patient samples examined and CCRK was detectable in all seven colorectal cancer cell lines tested. Colorectal tissue microarray indicated that overexpression of CCRK was detected in 62/109 (56.9%) of informative colorectal cancer cases and was significantly associated with the tumour pT and pN status (p<0.05). Suppression of CCRK by siCCRK led to G1 phase cell cycle arrest and reduced cell growth. Consistently, stable clones of LoVo and DLD1 cells expressing shCCRK exhibited decreased cell proliferation rates. Furthermore, we showed that CCRK is required for the phosphorylation of Cdk2 (on Thr-160) and Rb (on Ser-795) and the expression of cyclin E. These results suggest for the first time that CCRK is involved in colorectal cancer carcinogenesis and G1/S cell cycle transition by regulating Cdk2, cyclin E and Rb.