Contribution of notch signaling on HCC stem cell status and utilizing TLR agonists and notch inhibition to improve HCC theraphy

Abstract

Hepatocellular carcinoma (HCC) is the seventh most common cancer type worldwide, and ranked third place among cancer-related deaths within both sexes. As in many solid tumors, HCC shelters a cancer stem cell subpopulation, and is held responsible for the resistance developed during chemo-and-radio-therapy of HCC. The only option to cure HCC is liver transplantation, which is the bottleneck to provide a remedy to patients due to limited availability. Understanding the stem cell behavior of HCC would critically contribute to develop effective eradication strategies. In this study, a panel of 17 HCC cell lines was evaluated for their CSC status. Of these cell lines, six of them were determined to be positive for CD133 expression, a cardinal CSC marker. Next, HepG2, Huh7 and Hep3B-TR, (a desensitized TGF-beta-1 receptor clone) were selected and Notch activity vs. CSC fraction was investigated by analyzing CD133+/EpCAM+ levels. Our results revealed that DAPT (a notch inhibitor) led to a drop in CD133+/EpCAM+ levels in HepG2 and Huh7 by half, but not in Hep3B-TR cells, implicating a possible TGFβ1R involvement on CSC generation/maintenance. Treatment of cells with a notch ligand, Jagged-1, however, had little or no positive effect on CD133+/EpCAM+ expressions in all tested cells. Additionally, HCC cells' response to different TLR ligands and the resulting transcript expressions of TLRs were investigated by PCR. Of note, TLRs are widely used in immunotherapy of cancers. Here we aimed to combine Notch inhibitor along with selected TLR ligand, thereby improving tumor clearance in athymic mice xenografted with HCC. We found that in three selected cell lines upon TLR2 ligand stimulation, TLR5 and TLR7 were highly upregulated. Afterwards, treating these HCC cell lines with these ligands we observed that TLR3, TLR7/8 and TLR9 levels were activated. In the final part of this study, tumor-bearing mice with Huh7, were subjected to a combination therapy with TLR ligands +/- DAPT. We demonstrated that combination therapy comprising TLR3, 7/8 and 9 ligands and DAPT (only two injections, a week apart) induced significant tumor regression.