Molecular Mechanisms Of Congenital And Acquired Resistance To TRAIL In Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is one of the most common cancers and one of the leading causes of cancer-related death worldwide. Existing treatment modalities such as surgery, radiofrequency ablation and chemotherapy have demonstrated limited therapeutic efficacy. Developing new cancer therapeutic strategies is particularly important for human HCC. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) displays potent anticancer effects in a wide range of cancers resistant to conventional therapy without apparent toxic side effects to normal cells. Binding of homotrimeric TRAIL to DR4 and DR5 induces oligomerization of the receptors and promotes formation of the death inducing signaling complex (DISC), resulting in activation of the initiator caspase-8. In type Ⅰ cells, death receptor-initiated caspase-8 activation generates a signal strong enough to activate downstream effector caspase-3 that triggers execution of apoptosis. In contrast, in type Ⅱ cells, the magnitude of caspase-8 activation is not sufficient to directly activate caspase-3. Active caspase-8 cleaves Bid, which induces activation of Bax, followed by permeabilization of mitochondria. Pro-apoptotic proteins such as cytochrome c, Smac and DIABLO are released from mitochondria, activating caspase-9 and caspase-3. Several TRAIL-based novel drugs targeting its receptors are in the preclinical or clinical development. Nevertheless, resistance to TRAIL-induced apoptosis in cancer cells is one impediment to the use of TRAIL-based agents as antitumor drugs. Though many cancer cells are sensitive to TRAIL-induced apoptosis congenitally, they can acquire TRAIL resistance after long-time treatment by TRAIL. So, investigations of the congenital and acquired resistance mechanisms of TRAIL, finding potential molecules which may play key roles in resistance processes and improve the cytotoxic effects of TRAIL on cancer cells by modulating there expression is of great significance.Interferon exercises its biological functions by regulating downstream ISGs (IFN-stimulated genes) expression. Recent studies have shown that dysregulation of IFN-signaling pathway resulting in overexpression of ISGs and leading to tumorigenesis and possibly drug resistance. Interferon induces extrinsic or mitochondria-dependent intrinsic apoptosis by stimulating produce of death ligand such as TRAIL or Fas. There are many small molecular weight proteins with unknown functions such as ISG12a, G1P3, IFITM3, and IFITM2, which may be responsible for the disruption of mitochondria and the release of cytochrome c during the apoptosis process induced by IFN are still unknown. Though the regulation of ISG12a was investigated in many studies, the biochemical function of ISG12a is still unclear. Recent studies showed that ISG12a localizes on the outermembrane of mitochondria and regulates drug-induced apoptosis, but little is known about its expression status in liver cells and how it regulates TRAIL-induced apoptosis. In the present study, we will detect ISG12a expression levels in liver tissues and in many liver cancer cell lines. We will further investigate its role in TRAIL-induced apoptosis in liver cancer cells.To investigate the mechanisms of congenital resistance to TRAIL in hepatocellular carcinoma, we treated different HCC cell lines with TRAIL and detected apoptosis by western blot, flow cytometry and DNA ladder assays. The results showed that Huh7 and HLCZ01 cells were resistant to TRAIL. In contrast, LH86 and HLCZ02 cells were sensitive to TRAIL treatment. We also demonstrated that TRAIL-induced apoptosis was dependent on caspase-9 activity and cytochrome c release. The flow cytometry results indicated that the pan caspase inhibitor Z-VAD-FMK inhibited TRAIL-induced apoptosis. We found that the expression level of IFN-stimulated gene ISG12a was higher in TRAIL sensitive cells than in TRAIL resistant cells by qPCR assay. Forced expression of ISG12a in resistant cells sensitized the cells to TRAIL treatment. In contrast, knockdown of ISG12a in sensitive cells changed the TRAIL sensitive phenotype to a resistant one. To test the effects of ISG12a on TRAIL-induced apoptosis in vivo, we silenced ISG12a in LH86 or overexpressed ISG12a in Huh7 cells and implanted these cells into the right dorsal sides of immunodeficiency mice respectively. Then TRAIL treatment via tail vein injections was performed and tumor size was measured. The results showed that tumor size was smaller in mice which injected with ISG12a overexpression Huh7 cell groups than control groups. In contrast, tumor size was larger in ISG12a knockdown LH86 cells than in control groups. To identify which miRNA matches the seed sequence of ISG12a mRNA, we performed bioinformatics search (Targetscan) and found that ISG12a is a putative target of miR-942. We confirmed that miR-942 targeted the 3’UTR of ISG12a and regulated its expression by luciferase assay and western blot assay. Meanwhile, we observed inverse relationship of ISG12a and miR-942 in HCC cells and human liver tissues. Further study showed that miR-942 decreased TRAIL-induced apoptosis through downregulation of ISG12a and was regulated by AKT.To investigate the mechanisms of acquired resistance to TRAIL in hepatocellular carcinoma, we isolated a stable TRAIL-resistant sub-population of HCC cell line LH86, designated LH86-TR. We observed decreased Akt activity in TRAIL resistant cells. Inhibition of Akt activity by using Akt specific inhibitor could not overcome TRAIL resistance. These results indicated that differential activation of AKT is not responsible for acquisition of TRAIL resistance. We studied several pluripotency factors by qPCR analysis and found that Msil was elevated in TRAIL resistant cells. This finding was further confirmed by western blot analysis. Forced expression of Msil decreased the sensitivity of HCC cells to TRAIL both in vitro and in vivo. Conversely, shRNA-mediated depletion of Msil enhanced TRAIL efficacy. Elevated expression of Msil activates ERK. SiRNA-mediated depletion of ERK overcame TRAIL resistance. The data suggested that Msil confers resistance to TRAIL by activating ERK in cancer cells.