Implications of NOTCH3 expression and signaling for cholangiocarcinogenesis

Question The NOTCH pathway is an evolutionary conserved signaling pathway, which has a pivotal role for physiological liver development and regeneration. Persistent hyperactivation of NOTCH signaling is a potential driver of liver cancer development and progression, particularly of the cholangiocarcinoma (CCA) subtype. However, compared to the well-studied NOTCH1 receptor, the role of NOTCH3 is poorly characterized. Similarly, the expression of NOTCH receptors in benign precursor lesions including biliary intraepithelial neoplasia (BillN) or intraductal papillary neoplasms of the bile duct (IPNB) was not yet investigated. Here, we elucidated the function of the atypical NOTCH3 receptor during cholangiocarcinogenesis.

Methods Gene expression analysis was performed to quantify the mRNA levels of normal bile duct epithelium, precursor lesions and invasive CCA in a matched design using a well-characterized human CCA cohort. Tissue microarray analysis was conducted to determine the expression, localization and activity levels of the NOTCH receptors on protein level in human CCA specimens. The activity and function of NOTCH1 and NOTCH3 were investigated in vitro in CCA cell lines. To study the impact on liver tumorigenesis in vivo, hydrodynamic tail vein injection (HDTV) of mice was utilized to stably overexpress hyperactive NOTCH1 or NOTCH3 in combination with an AKT oncogene in hepatocytes.

Results Analyzing patient data, we detected a gradual upregulation of NOTCH3 expression during cholangiocarcinogenesis, while the mRNA levels of the NOTCH1 and NOTCH2 receptor were not significantly or gradually altered. Analyzing protein abundance, a subgroup of CCA patients with active nuclear NOTCH1 and NOTCH3 staining were identified. Accordingly, in vitro analysis showed that NOTCH1 and NOTCH3 were differentially expressed among multiple human CCA cell lines. In addition, in vivo experiments demonstrated that NOTCH3 has a similarly potent oncogenic function relative to NOTCH1, leading to tumor formation 7 weeks after HDTV when combined with AKT overexpression. By immunohistochemistry, all NOTCH/AKT tumors were classified as CCA subtype.

Conclusions Our data suggest that particularly NOTCH1 and NOTCH3 signaling might be implicated in CCA development. Investigating the differences of both receptors on downstream signaling and tumor features is subject of our current research.

Publication History

Article published online:
04 January 2021