Combined effects of polo-like kinase 1 and harvey rat sarcoma in hepatocellular carcinoma reveal novel therapeutic strategies

Introduction:

Inhibition of RAS-RAF-signaling is the major mechanism mediated by sorafenib, the only effective therapeutic approach for advanced hepatocellular carcinoma (HCC). This underlines the importance of RAS-RAF-signaling in HCC. Several studies indicate that the RAS isoform Harvey rat sarcoma (HRAS) can function as an oncogene in HCC. Moreover, the cell cycle promoting polo-like kinase 1 (PLK1) is an increasingly recognized therapeutic target in HCC that can be activated by RAS-RAF-signaling. A recently developed small molecule inhibitor, ON-01910 (“rigosertib”), was shown to interfere with both RAS- and PLK1-signaling. The aim of this study was to analyze the unknown effects of rigosertib in HCC and to assess PLK1 and HRAS expression in HCC.

Methods:

For functional analysis, the HCC cell lines PLC (ATCC CRL-8024) and Hep3B (ATCC HB-8064) were used. Moreover, primary human hepatocytes (PHH) were isolated and cultured for expression analysis. For inhibition of RAS/PLK1 signaling, we used a recently developed non-ATP competitive small molecule inhibitor (ON-01910.Na, also “rigosertib”) (Selleck Chemicals, Houston, USA). Expression analysis was performed using qRT-PCR and Western blot analysis. Cell proliferation was analyzed using florescence-activated cell sorting (FACS) and real-time cell proliferation assays. Toxicity assays were performed using microscopic analysis of cell morphology and quantification of lactate dehydrogenase (LDH) amounts in cell supernatants using a commercial ELISA kit. Several databases were analyzed in silico to investigate the effects of PLK1 and HRAS on patient survival.

Results:

Rigosertib reduced cell proliferation and anchorage-dependent and -independent clonogenicity. Moreover, Rigosertib treatment induced a G2/M- cell cycle arrest in several HCC cell lines. Rigosertib also strongly inhibited both ERK- and AKT-activation in HCC cells, indicating disruption of RAS-signaling. In silico analysis of HCC patient datasets showed that PLK1 and HRAS expression are upregulated during HCC development and in advanced HCC. High expression levels of PLK1 correlated with poor patient survival in HCC patients. Furthermore, high levels of both HRAS and PLK1 revealed combined effects on patient outcome.

Conclusions:

In this study, we newly demonstrate the therapeutic potential of rigosertib in HCC by inhibition of both PLK1 activation and major RAS-pathways, thereby revealing a novel therapeutic “dual-hit” approach for HCC.