Role of Notch Signaling in Hepatocellular Carcinoma (HCC)

Abstract

Liver cancer is the 6th most common cancer worldwide and Australia’s fastest growing malignancy both in incidence and mortality. Although great improvement in early diagnosis and treatment approach have been achieved during last two decades, patients with HCC still have a dismal prognosis with a mortality-to-incidence ratio close to 1, meaning that the disease is almost uniformly fatal. One of explanations for such a dismal prognosis for HCC is the existence of heterogeneity in HCC, in which HCC is composed of liver cancer stem cells and mature HCC cells. Previous data suggested that liver cancer stem cells are responsible for HCC initiation, early metastasis and treatment resistance. However, accurate characterization of liver cancer stem cells is still required as the reliability of those reported approaches in identify liver cancer stem cells is variable. To better characterization of liver cancer stem cells, we found by qPCR and western blotting that Oct4, a stem cell gene associated with a range of functions in HCC cells with stem cell feature, is ubiquitously expressed in all HCC tumors in our cohort, whereas other liver cancer stem cell markers had high variability in their expression. By utilizing a human Oct4 promoter driving an enhanced green fluorescent protein reporter, we successfully established OCT4+EGFP reporter HCC cells. More importantly, Oct4+ cells had all the classic features of liver cancer stem cells including increased sphere formation in vitro, tumor forming potential in vivo, along with up-regulated expression of stemness genes and increased resistance towards Sorafenib, the only drug approved for advanced HCC. Upon success in characterization for liver cancer stem cell, we, for the first, demonstrated by western blotting that JAG2, one of the five ligands in Notch signaling, is significantly increased in liver cancer stem cells. Moreover, JAG2 is the only Notch ligands aberrantly expressed in liver cancer stem cells characterized by qPCR and western blotting. In addition, Sorafenib resistant HCC cells, which are enriched for liver cancer stem cells, also have profound increase in the expression of JAG2 at both RNA and protein level. Since the reported functions of Notch signaling in HCCs are conflicting and an effective approach targeting Notch signaling with tolerable side effect is missing, we then studied the expression profile of Notch signaling components in HCC by qPCR and western blotting. We found that JAG2 is significantly increased in 65% of HCC tissues in our cohort, which is in consistence with microarray data extracted from NCBI and RNA sequence results provided by TCGA. More importantly, we showed that patients with hyper-Notch activity had worse prognosis than that with hypo-Notch activity demonstrated by immunohistochemical staining and Kaplan-Meier survival assay. These results indicate an important biological role of JAG2 mediated Notch signaling in regulation of liver cancer stem cells and HCC. Using Compound E, a selective γ-secretase inhibitor, we demonstrated that inhibition of Notch signaling leads to slowed cell proliferation and decreased liver cancer stem frequency, which were testified by Brdu assay and flow cytometry respectively. Moreover, selective inhibition of JAG2 alone by shRNA in HCC cells results in a similar phenotype along with impaired function in liver cancer stem cells. We also demonstrated a great translational potential of selective targeting JAG2 in HCC as verified by colony-forming assay that JAG2 knockdown HCC cells became significantly sensitive to the killing effect of Sorafenib. In term of mechanism, we identified that Notch2, one of the four Notch signaling receptors, is solo responsive to JAG2 mediated signaling testified by western blotting and immunofluorescence analysis. Inhibition of Notch2-dependent JAG2 signaling leads to a mature HCC phenotype including less liver cancer stem cells with impaired biological function. Targeting JAG2-initiated Notch2 signaling in HCC would greatly improve prognosis of patients with HCC.