Abstract
Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Additionally, the efficacy of targeted molecular therapies with multiple tyrosine kinase inhibitors is limited. In this study, we focused on the cellular signaling pathways common to diverse HCC cells and used quantitative reverse phase protein array (RPPA) and statistical analyses to elucidate the molecular mechanisms determining its malignancy. We examined the heterogeneity of 17 liver cancer cell lines by performing cluster analysis of their expression of CD90 and EpCAM cancer stem cell markers. Gaussian mixture model clustering identified three dominant clusters: CD90-positive and EpCAM-negative (CD90+), EpCAM-positive and CD90-negative (EpCAM+) and EpCAM-negative and CD90-negative (Neutral). A multivariate analysis by partial least squares revealed that the former two cell populations showed distinct patterns of protein expression and phosphorylation in the EGFR and EphA2 signaling pathways. The CD90+ cells exhibited higher abundance of AKT, EphA2 and its phosphorylated form at Ser897, whereas the EpCAM+ cells exhibited higher abundance of ERK, RSK and its phosphorylated form. This demonstrates that pro-oncogenic, ligand-independent EphA2 signaling plays a dominant role in CD90+ cells with higher motility and metastatic activity than EpCAM+ cells. We also showed that an AKT inhibitor reduced the proliferation and survival of CD90+ cells but did not affect those of EpCAM+ cells. Taken together, our results suggest that AKT activation may be a key pro-oncogenic regulator in HCC.
Highlights
IntroductionAdditional tyrosine kinase inhibitor (TKI), lenvatinib [8,9,10] and regorafenib [11,12] have been approved as first- and second-line treatments for advanced Hepatocellular carcinoma (HCC), respectively
To address the heterogeneity of Hepatocellular carcinoma (HCC) cells in terms of cancer stem cells (CSCs), we first applied Gauss3 of 13 ian mixture model (GMM) clustering to examine the potential clusters in 17 liver cancer cell lines based on EpCAM and CD90 expression, as detected on the reverse phase protein array (RPPA) analysis (Figure S1)
For JHH-7, two7 ofthat way analysis of variance (ANOVA) revealed only a significant main effect of day (p < 0.0001). This suggests the inhibitor did not affect the proliferative ability of JHH-7 cells. These results suggest that the effect of AKT inhibition on proliferation is specific to the CD90+ cell cluster
Summary
Additional TKIs, lenvatinib [8,9,10] and regorafenib [11,12] have been approved as first- and second-line treatments for advanced HCC, respectively Their therapeutic response and survival benefits are still low and limited. For patients with advanced HCC, EGFR inhibitors show only modest clinical activity [20] This raises the issue of HCC tumor heterogeneity likely to be due to CSCs; the need to elucidate the regulatory mechanisms responsible for modulating the function of the EGFR signaling cascade should be considered. When unliganded, EphA2 activation stimulates the ERK/MAPK and PI3K/AKT/mTOR signaling pathways. We compared the patterns of protein expression and phosphorylation of the EGFR and EphA2 signaling pathways between EpCAM-positive and CD90-positive cells.
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