◥Natural killer (NK) cells are enriched within the liver. Apart from conventional NK (cNK) cells, recent studies identified a liverresident NK (LrNK) subset, which constitutes about half of hepatic NK cells and exhibits distinct developmental, phenotypic, and functional features. However, it remains unclear whether and how LrNK cells, as well as cNK cells, participate in the development of hepatocellular carcinoma (HCC) individually. Here, we report that both LrNK and cNK cells are significantly decreased in HCC. The T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) was significantly upregulated in both tumor-infiltrating LrNK and cNK cells and suppressed their cytokine secretion and cytotoxic activity. Mechanistically, phosphatidylserine (PtdSer) engagement promoted phosphorylation of Tim-3, which then competed with PI3K p110 to bind p85, inhibiting downstream Akt/mTORC1 signaling and resulting in malfunctioning of both NK-cell subsets. Tim-3 blockade retarded HCC growth in a NKcell-dependent manner. These studies for the first time report the presence and dysfunction of LrNK cells in HCC and show that Tim-3-mediated PI3K/mTORC1 interference is responsible for the dysfunction of both tumor-infiltrating cNK and LrNK cells, providing a new strategy for immune checkpoint-based targeting.Significance: Tim-3 enhances hepatocellular carcinoma growth by blocking natural killer cell function.

Non-alcoholic fatty liver disease (NAFLD) leads to hepatocellular carcinoma (HCC). However, the underlying mechanism remains largely unclear. Here, we investigated the role of the tumor suppressor Zinc fingers and homeoboxes 2 (ZHX2) in the progression of NAFLD to HCC. ZHX2 expression was significantly decreased in fatty liver tissues, especially in the liver with NAFLD-HCC. ZHX2 overexpression disturbed lipid homeostasis of cultured HCC cells, and inhibited lipid deposition in hepatocytes both in vitro and in vivo. Moreover, ZHX2 inhibited uptake of exogenous lipids through transcriptional suppression of lipid lipase (LPL), leading to retarded proliferation of HCC cells. Importantly, LPL overexpression significantly reversed ZHX2-mediated inhibition of HCC cell proliferation, xenograft tumor growth, lipid deposition, and spontaneous liver tumor formation. Consistently, IHC staining demonstrated a negative correlation of ZHX2 with LPL in an HCC cohort. Collectively, ZHX2 protects hepatocytes from abnormal lipid deposition in NAFLD through transcriptional repression of LPL, which subsequently retards cell growth and NAFLD-HCC progression. These findings illustrate a novel mechanism of NAFLD progression into HCC.

Initiation of hepatocellular carcinoma (HCC) by chronic hepatitis B virus (HBV) infection is a complex process that includes both oncogene activation and tumor suppressor inhibition. The HBV X (HBx) protein has an important and complex role in processes leading to HCC. We previously identified the mammalian Zinc fingers and homeoboxes 2 (ZHX2) gene as an HCC-associated tumor suppressor gene. In the present study, we investigated whether the oncogenic properties of HBV and, more specifically, HBx, involved ZHX2 silencing. Our data indicates that ZHX2 expression is significantly decreased in tumor tissues from HBV-positive HCC patients and livers from HBV transgenic mice. In vitro and in vivo studies confirmed that HBV-encoded proteins, particularly HBx, inhibits both the expression and tumor suppression properties of ZHX2. Further analyses identified miR-155, a well-known oncomiR in various cancers, as an important link between HBx and ZHX2 inhibition. Increased miR-155 levels were found in HBV-positive tumors, livers of HBV transgenic mice and HBx-overexpressing hepatoma cell lines. MiR-155 overexpression reduced ZHX2 levels via miR-155 seed sites in the ZHX2 3'UTR, whereas blocking miR-155 levels led to increased ZHX2 levels. Taken together, our data indicate that HCC-promoting properties of HBV may include ZHX2 silencing via a miR-155 dependent pathway and suggests a novel therapy for HBV-related HCC.

As an immune checkpoint, Tim-3 plays roles in the regulation of both adaptive and innate immune cells including macrophages and is greatly involved in chronic liver diseases. However, the precise roles of Tim-3 in nonalcoholic steatohepatitis (NASH) remain unstated. In the current study, we analyzed Tim-3 expression on different subpopulations of liver macrophages and further investigated the potential roles of Tim-3 on hepatic macrophages in methionine and choline-deficient diet (MCD)-induced NASH mice. The results of flow cytometry demonstrated the significantly increased expression of Tim-3 on all detected liver macrophage subsets in MCD mice, including F4/80CD11b, F4/80CD68, and F4/80CD169 macrophages. Remarkably, Tim-3 knockout (KO) significantly accelerated MCD-induced liver steatosis, displaying higher serum ALT, larger hepatic vacuolation, more liver lipid deposition, and more severe liver fibrosis. Moreover, compared with wild-type C57BL/6 mice, Tim-3 KO MCD mice demonstrated an enhanced expression of NOX2, NLRP3, and caspase-1 p20 together with increased generation of IL-1β and IL-18 in livers. In vitro studies demonstrated that Tim-3 negatively regulated the production of reactive oxygen species (ROS) and related downstream pro-inflammatory cytokine secretion of IL-1β and IL-18 in macrophages. Exogenous administration of N-Acetyl-L-cysteine (NAC), a small molecular inhibitor of ROS, remarkably suppressed caspase-1 p20 expression and IL-1β and IL-18 production in livers of Tim-3 KO mice, thus significantly reducing the severity of steatohepatitis induced by MCD. In conclusion, Tim-3 is a promising protector in MCD-induced steatohepatitis by controlling ROS and the associated pro-inflammatory cytokine production in macrophages.

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