Objective Sorafenib is the only effective therapy for advanced hepatocellular carcinoma (HCC). Combinatory approaches targeting mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)- and phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)/protein-kinase B(AKT) signalling yield major therapeutic improvements. RAS proteins regulate both RAF/MAPK and PI3K/AKT signalling. However, the most important RAS isoform in carcinogenesis, Kirsten rat sarcoma (KRAS), remains unexplored in HCC.
Design Human HCC tissues and cell lines were used for expression and functional analysis. Sorafenib-resistant HCC cells were newly generated. RNA interference and the novel small molecule deltarasin were used for KRAS inhibition both in vitro and in a murine syngeneic orthotopic HCC model.
Results Expression of wild type KRAS messenger RNA and protein was increased in HCC and correlated with extracellular-signal regulated kinase (ERK) activation, proliferation rate, advanced tumour size and poor patient survival. Bioinformatic analysis and reporter assays revealed that KRAS is a direct target of microRNA-622. This microRNA was downregulated in HCC, and functional analysis demonstrated that KRAS-suppression is the major mediator of its inhibitory effect on HCC proliferation. KRAS inhibition markedly suppressed RAF/ERK and PI3K/AKT signalling and proliferation and enhanced apoptosis of HCC cells in vitro and in vivo. Combinatory KRAS inhibition and sorafenib treatment revealed synergistic antitumorigenic effects in HCC. Sorafenib-resistant HCC cells showed elevated KRAS expression, and KRAS inhibition resensitised sorafenib-resistant cells to suppression of proliferation and induction of apoptosis.
Conclusions KRAS is dysregulated in HCC by loss of tumour-suppressive microRNA-622, contributing to tumour progression, sorafenib sensitivity and resistance. KRAS inhibition alone or in combination with sorafenib appears as novel promising therapeutic strategy for HCC.
- hepatocellular carcinoma
- molecular mechanisms
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Contributors PD, AKB, and CH conceived the project, analyzed the data, and wrote the manuscript. PD designed and performed most of the experiments. AK helped panning, performing, and analyzing mouse experiments. VF was involved in functional cell culture experiments. AH provided material and contributed to data anlysis. All authors approved the final version of the manuscript.
Funding This work was supported by grants from the German Research Association (DFG) (Research Training Group RTG 1962/1, HE2458 and BO1573 in FOR 2127 and HE2458 in KFO262), the German Cancer Aid (Deutsche Krebshilfe), the Bavarian Research Network for Molecular Biosystems (BioSysNet) and the Interdisciplinary Center for Clinical Research (IZKF) Erlangen (J55 (PD), D24 (AB)).
Disclaimer None of the pictures/images has been published before. All data/pictures shown have been newly generated by the authors.
Competing interests None declared.
Patient consent Obtained.
Ethics approval HTCR (Human Tissue and Cell Research) foundation and the local ethics commitee of the University of Regensburg.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement There are no additional data.