HBx Promotes Expansion And Tfumorigenicity Of Hepatic Progenitor Cells

Part I Hepatitis B Virus X (HBx) Promotes Expansion andTumorigenicity of Hepatic Progenitor CellsHepatic progenitor cells (HPCs) are considered to have bipotential ability to differentiateinto hepatocyte or cholangiocyte. Clinical evidence has also shown that the degree ofprogenitor/stem cell activation is associated with the severity of inflammation and fibrosisin chronic hepatitis. In addition, HPCs are closely related to hepatocellular carcinoma(HCC). Recent studies reported that aberrant activation of Wnt/β-catenin, transforminggrowth factor beta (TGF-β), interleukin (IL)-6signaling, Bmi1, and Hippo-Salvadorpathway contribute to the expansion and activation of HPCs, as well as transformation ofHPCs. However, the specific mechanisms of expansion, activation and transformation ofHPCs are need further investigation.Hepatitis B virus X (HBx), a small17-kDa soluble protein, is known to be essential forHBV-induced carcinogenesis. It is one of four defined overlapping open reading frames(ORFs) in HBV genomic DNA and has been found in both nucleus and cytoplasm.Hepatitis B virus X (HBx) protein is implicated in hepatitis B virus (HBV)-associated livercarcinogenesis. However, it remains unclear whether HBx-expressing hepatic progenitorcells (HPCs) are attributed to liver tumor formation.In this study, by using HBx transgenic mice and a3,5-diethoxycarbonyl-1,4-dihydrocol-collidine (DDC)-induced liver injury model, the relationship between HBx expression andtumorigenicity of HPCs was analyzed.Compared with control mice, an elevated number ofEpCAM+cells with characteristics of HPCs was observed in HBx mice after1month and4months of DDC diet feeding. All HBx transgenic mice developed liver tumorscharacterized by histological features of both hepatocellular carcinoma (HCC) andcholangiocarcinoma after7months of DDC feeding. Notably, EpCAM+HPCs isolatedfrom premalignant HBx mice exposed to a DDC diet for4months formed subcutaneousmixed-lineage tumors (four out of six) in nonobese diabetic/severe-combinedimmunodeficient (NOD/SCID) mice, and none of the cells from wildtype (WT) inducedtumors, indicating that HBx may induce malignant transformation of HPCs that contributesto tumorigenesis. We also found higher titers of circulating interleukin (IL)-6, activities ofIL-6/STAT3, and Wnt/β-catenin signaling pathways in HBx transgenic mice, suggesting HBx may induce intrinsic changes in HPCs by way of the above signaling that enablesHPCs with tumorigenicity potential. Finally, clinical evidence showed that high HBxexpression in human HBV-related HCC was statistically associated with expansion ofEpCAM+or OV6+tumor cells and aggressive clinicopathologic features.Conclusion: HBx induces intrinsic cellular transformation promoting the expansion andtumorigenicity of HPCs in DDC-treated mice, which may be a possible origin for livercancer induced by chronic hepatitis infection. Part II Autophagy Promotes Stemness Characteristics of LiverTumor-Initiating CellsThe cancer stem cell hypothesis suggests that cancers are maintained in a hierarchicalorganization of few ''cancer stem cells''(or "tumor-initiating cells", T-ICs), which divideinto many transit-amplifying cells and very many differentiated tumor cells. It is reportedlyknown that T-ICs which possess self renewal, chemoresistent and tumorigenic capabilities,play an important role in hepatocarcinogenesis. In recent years, liver T-ICs have beenidentified by several markers such as CD133, EpCAM, CD90, OV6, CD13and CD24.However, the mechanisms that regulate stemness characteristics of liver T-ICs are not wellunderstood.Macroautophagy, hereafter autophagy, which is an evolutionally conserved degradationsystem among all eukaryotes, produces vesicles called autophagosomes that capture anddeliver cytoplasmic material to lysosomes. Autophagy is activated in by a broad range ofstressors such as starvation, high temperature, low oxygen, and accumulation of mutantproteins. Autophagy-related genes (Atg), which were first identified through geneticscreens in yeast, are required for the execution of the autophagy pathway. It is reported thatAtg8/LC3contributes to the completion of autophagosome formation. LC3is cleaved byAtg4to form cytosolic LC3-I, which is covalently conjugated to thephosphatidylethanolamine to form membrane-associated LC3-II. And LC3-II is the onlyknown protein that specifically associates with autophagosomes. It is widely accepted thatautophagy is involved in many liver diseases including acute liver injury, alcoholic liverdisease and hepatocellular carcinoma.Recent discoveries have shown that autophagy plays a critical role in stem cellsmaintenance such as embryonic stem cells and hematopoietic stem cells. And autophagycan positively regulate the CD44+CD24-/low breast cancer stem-like phenotype andmediate survival of pancreatic tumour-initiating cells in a hypoxic microenvironment. Inaddition, autophagy is required for self-renewal and differentiation of adult human stemcells. Thus, we hypothesized that autophagy can regulate stemness characteristics of liverT-ICs.In this study, we investigated whether autophagy regulate stemness characteristics ofliver T-ICs. First of all, we found that autophagy activity is enhanced in T-ICs of liver cancer cell lines. Then we showed that pharmacological inhibition of autophagy orknockdown of autophagy related genes virtually suppressed stem-like properties of liverT-ICs. Finally, we found that inhibition of autophagy can reverse percentage ofcisplatin-enriched T-ICs.Conclusin: Autophagy promotes stemness characteristics of liver tumor-initiating cells,and inhibition of autophagy can reverse enriched T-ICs by cisplatin. These results mayprovide a possible target for eliminating liver T-ICs.