Macrophage-secreted TNF-α Triggers The Malignant Transformation Of Liver Progenitor Cells And Initiates Liver Cancer Development

PartⅠMacrophage-secreted TNF-a Triggers the Malignant Transformation of Liver Progenitor Cells and Initiates Liver Cancer DevelopmentPrimary liver cancer (PLC) is the sixth most common cancer worldwide and second leading cause of cancer deaths in man. PLC is traditionally classified into three pathological types:hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined HCC-CC (CHC). HCC and ICC are believed to be derived from hepatocytes and cholangiocytes respectively, while CHC is thought to originate from liver progenitor cells (LPCs). However, the observation that LPC markers express in certain human HCCs, and substantial heterogeneity and hierarchical organizations in these HCCs favor the hypothesis of progenitor cell origin for some HCCs. Nevertheless, the cell of origin of HCC is currently debated and the scientific evidence remains to be scarce at present. Therefore, clarifying the cell of origin and refining the pathological classification is urgently required to improve HCC treatment in particular personalized therapy.Liver progenitor cells, also termed oval cells in rodent, are distinguished by specific cell surface marker such as OV6 and are capable of differentiating into hepatocytes and cholangiocytes. Compensatory LPC proliferation is usually observed in livers with chronic injuries, such as viral hepatitis, alcoholic hepatitis, and nonalcoholic fatty liver disease (NAFLD), and the degree of LPC activation positively correlates with the stage of these diseases. Evidence in favor of the involvement of LPCs in hepatocarcinogenesis included that LPCs isolated from p53-null mice formed HCC in nude mice, and Ras-triggered transformation of mouse primary LPCs gave rise to hepatic T-ICs. Our previous study also demonstrated that long-term TGF-β exposure drove the transformation of liver progenitor cells into hepatic T-ICs, which contributes to cirrhosis-elicited hepatocarcinogenesis. However, whether LPCs are involved in the chronic inflammation-triggered hepatocarcinogenesis remains unkown.Chronic non-resolving inflammation is considered to be the distinct feature and causal factor of hepatocarcinogenesis. Accumulating studies indicates that inflammation exerts multiple effects on carcinogenesis, but the cellular and molecular machinery linking chronic inflammation to tumorigenesis remain not fully elucidated. Kupffer cells, hepatic tissue-resident macrophages, is the largest population of innate immune cells residing in the liver. The genetic depletion of Toll-like receptor 4 (TLR4) of Kupffer cells in mice attenuated hepatic inflammatory responses and HCC development triggered by diethylnitrosamine (DEN), suggesting an essential role for Kupffer cells in hepatocarcinogenesis. Tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) are the most essential proinflammatory cytokines secreted by kupffer cells in liver. Ik-B transgenic or IL-6 knockout mice developed much less HCC upon chronic liver inflammation or DEN exposure, suggesting the important role of TNF-a and IL-6 in HCC development. However, role of TNF-αand IL-6 in LPCs transformation and LPCs-derived PLC development remains largely unknown.Herein we reported that 65% of cytokeratin 19 positive (CK19+) HCC presented the expression of OV6, a LPCs marker, suggesting the progenitor cell origin of these HCCs. Expression of hepatic tumor initiating cell (T-ICs) markers was detected on the LPCs in cirrhotic liver of patients suggesting the transformation of LPCs during hepatocarcinogenesis. LPCs isolated from DEN-induced fibrotic livers possessed tumorigenicity indicating the chronic inflammation-triggered malignant transformation of LPCs. Intriguingly, depletion of kupffer cells attenuated the self-renewal capacity of LPCs and suppressed OV6+ HCC initiation. On the contrary, LPCs co-cultured with macrophages or treated by the supernatant of activated macrophages exhibited increased T-IC marker expression and enhanced self-renewal ability. We further demonstrated that low dosage of TNF-abut not IL-6 triggered the transformation of LPCs. Mechanistically, long-term TNF-a treatment led to the dysregulation of ubiquitin D and Checkpoint kinase 2, and thus triggered the chromosomal instability in LPCs. Moreover, TNF-a treatment increased Nanog and Lin28 expression via TNFR1/Src/STAT3 pathway resulting in enhanced self-renewal of LPCs, which should be responsible at least partially the LPC transformation and hepatic T-IC expansion. Collectively, we proposed that exposure of TNF-a from macrophage triggers chromosomal instability and enhances self-renewal of LPCs contributing to the generation and expansion of hepatic T-ICs. These data not only provide new insight into the pathogenesis of inflammation-associated HCC development, but also propose novel molecular classification of HCC, which is critical for HCC personalized treatment.Part ⅡCyclin G1 Expands Liver Tumor-Initiating Cells by Sox2 Induction via Akt/mTOR SignalingAlthough surgical resection and transplantation have significantly improved the survival of HCC patients, long-term survival remains unsatisfactory due to the high incidence of recurrence after hepatectomy. It has been reported that 5-year recurrence rate of liver cancer exceeds 70% to date. Moreover, most of the HCC patients was diagnosed at advanced stages and not eligible for surgery. Unfortunately, either conventional chemotherapy or transarterial chemoembolization could eradicate cancer cells due to the chemo-resistance nature of HCC. Thus it is urgent to elucidate the molecular mechanism of HCC recurrence and chemo-resistance, so that novel strategy of treatment can be developed.Recent studies strongly support the hypothesis that tumors are generated and maintained by a small subpopulation of undifferentiated cells which are termed "tumor initiating cells" or "cancer stem cells". These cells are capable of self-renewal, differentiating, and maintaining tumor growth and heterogeneity. Being a built-in population of tumor, these tumor initiating cells can survive chemotherapy and repopulate the tumor. Numerous studies have shown that T-ICs exist in various tumors, including leukemia, glioma, colon cancer, breast cancer etc. Liver T-ICs have been identified by several cell surface markers such as CD 133, CD90 and epithelial cell adhesion molecule, CD24 etc. The presence of T-ICs in HCC is likely one of the main reasons why current oncologic therapies are poorly effective in preventing tumor progression, metastasis and recurrence. Eradication of liver T-ICs should be critical to achieve stable, long-lasting remission of HCC. However, no therapy regimen targeting T-ICs was developed in HCC due to lack of specific targets to the small reservoir of tumor cells. It is thereby urgent to clarify the molecular mechanism of T-IC regulation so that novel strategy for HCC treatment can be developed.Cyclin G1 was initially discovered as a novel cyclin family member with homology to c-src. Importantly, cyclin G1 is transactivated by p53, and in turn negatively regulates p53 family proteins. Loss of cyclin G1 led to a lower tumor incidence after carcinogenic challenge in experimental hepatocarcinogenesis. Interaction between cyclin G1 and miR-122a might impair p53-mediated inhibition of HBV replication and therefore contribute to viral persistence and carcinogenesis. Our previous data suggest that cyclin G1 promotes epithelial-mescenchymal transition of hepatoma cells via PI3-K/Akt pathway and facilitates HCC metastasis. However, the role of cyclin G1 in HCC chemo-resistance and recurrence remains largely unknown.In this study, we found that elevated expression of cyclin Gl was associated with drug resistance of hepatoma cells and higher recurrence rate of HCC patients. Correlated expression of cyclin G1 and T-IC markers was observed in human HCC tissues. Forced cyclin Gl expression remarkably enhanced self-renewal and tumorigenicity of hepatoma cells. Overexpression of cyclin G1 dramatically up-regulated Sox2 expression, which was inhibited by chemical inhibitors of Akt/mTOR signaling. Furthermore, suppression of Akt/mTOR signaling or interference of Sox2 expression attenuated cyclin G1-enhanced self-renewal, chemo-resistance and tumurigenicity of hepatoma cells, indicating that cyclin G1 promotes liver T-ICs expansion through Akt/mTOR/Sox2 signaling pathway. Our findings suggest that cyclin G1-induced expansion of liver T-IC contributes to the chemo-resistance and recurrence of HCC, and cyclin G1 may be a promising biomarker for personalized therapy of HCC patients.