The Role of Polo-like Kinase 1 as a Therapeutic Target in Hepatocellular Carcinoma

Seng-Gee Lim*. National University Health System, Singapore

Polo-like kinase 1 (PLK1) plays important roles in the progression of cell cycle, especially for cells transiting from anaphase to telophase during mitosis. PLK1 has been found to be overexpressed in various cancers, and has important prognostic implications.

Methods: PLK1 gene expression was evaluated in Hepatocellular carcinoma (HCC) and found to be overexpressed frequently in HCC tissues. Gene silencing technology utilizing short interfering RNA (siRNA) was subsequently employed to study the potential of PLK1 to be the therapeutic target in treating HCC. Knockdown with si-RNA was performed to examine for reduction in cell proliferation using MTS and BrdU assays, after validating reduced expression by real-time PCR (RTPCR) and Western Blotting. Confirmation of apoptosis was performed using TUNEL assay, and subsequent pathway of apoptosis was examined using a caspaseinhibition assay. Experiments with transplanted tumour cell lines into nude mice co-cultured with si-RNA against the target gene, and examining for reduced tumour progression compared to controls was performed.

Results: Microarray analysis showed the most common over expressed gene was PLK1 by 12 times, and was also found to be over-expressed in Huh-7 cells. SiRNA against PLK-1 validated by real-time PCR (RTPCR) and Western Blotting showed a reduction of PLKR1 expression up to 96% in huh-7 cells, and reduced cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays respectively. There was 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this may be caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue apoptotic cells. Immnofluorescence studies co-localised endonuclease G to fragmented chromosomes, thus was postulated to be the main apoptotic effector. Knockdown of the FOXM1 transcription factor, thought to be a positive regulating factor did not affect PLK1 gene expression, suggesting other transcription factors may be important. Finally, huh-7 cells transplanted subcutaneously into nude mice using matrigel were treated with si-PLK1 and control si-RNA. Tumour regression of 33% occurred in the mice treated with si-PLK1 but not in controls.

Conclusion: PLK1 overexpression in HCC was shown to be functionally important by gene silencing experiments, and may be a potential therapeutic target.