Examination of the epidermal growth factor receptor 3 (HER3) dysregulations in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the fourth leading cause of cancer-related death globally. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are the major risk factors accounting for 75% of all HCC cases. Currently, curative treatments including surgical resection and liver transplantation can only be applied to early diagnosed patients. For advanced HCC patients, the currently available first-line molecularly targeted medications showed limited survival benefits and low response rates, possibly due to high inter-patient variabilities and development of drug resistance. Therefore, there is an urgent unmet medical need for identifying new therapeutic targets in HCC.

Receptor tyrosine kinases (RTKs) are plasma membrane-associated protein kinases which control the activation of intracellular signal transductions in response to the extracellular stimuli. Abnormal activation of specific RTKs has been implicated in various kinds of human cancers, and pharmacological inhibition of cancer-associated RTKs is one of the key strategies of modern targeted therapy for cancer treatment. However, a comprehensive and systematic assessment of the RTKs dysregulations in terms of expression and mutation in HCC is still much awaited.

In this study, RNA-sequencing analysis was performed in paired HCC tissues to examine the expression profiles of all 58 RTKs. Also, the mutational status of these RTKs in HCC tissues was further examined by targeted DNA-sequencing (targeted DNA-seq) analysis. Our systematic analyses have revealed that epidermal growth factor receptor 3 (HER3), the third member of the EGFR family, was significantly upregulated and recurrently mutated in HCC. HER3 dysregulations have been implicated to play a pro-oncogenic role in some cancers, however, the potential functional roles of HER3 in supporting HCC development are still unknown.

By performing clinicopathological correlation analysis, HER3 overexpression in HCC was significantly correlated with the presence of metastatic features of HCC including tumour microsatellite formation and venous invasion. Functionally, HER3 was demonstrated to support aggressive HCC-associated phenotypes. It was found that treatment with Neuregulin-1 (NRG-1), a major ligand of the HER3 receptor, could stimulate the activation of HER3 and the downstream PI3K/Akt and MAPK signalling pathways. More importantly, CRISPR-Cas9-mediated HER3 knockout in HCC cells suppressed NRG-1-induced cell proliferation and cell migration in vitro. The functional changes induced by NRG-1 was inhibited in the presence of an Akt inhibitor, MK2206, suggesting that HER3 can regulate these functional changes via the PI3K/Akt pathway. Moreover, HER3 knockout significantly reduced the in vivo tumorigenicity in a subcutaneous xenograft model.

The potential role of HER3 mutations in HCC was examined by using a lentiviral bicistronic expression system to clone and overexpress HER3 mutants in HCC cells. Specific HER3 mutants were shown to induce a stronger EGFR phosphorylation upon NRG-1 treatment, suggesting that these mutations of HER3 might exhibit a relatively higher biological activity in promoting EGFR signalling upon growth factor stimulation, and potentially supporting HCC-associated functions.

Taken together, these findings have elucidated the clinical relevance and the pro-oncogenic roles of HER3 dysregulations in HCC development. Targeting HER3 could potentially be an alternative therapeutic strategy which can broaden for the treatment landscape of HCC.