Abnormal expression of activating/inhibitory receptors leads to natural killer (NK) cells dysfunction in tumor. Here we show that programmed cell death protein 1 (PD-1), a well-known immune checkpoint of T cells, is highly expressed on peripheral and tumor-infiltrating NK cells from patients with digestive cancers including esophageal, liver, colorectal, gastric and biliary cancer. The increased PD-1 expression on NK cells indicates poorer survival in esophageal and liver cancers. Blocking PD-1/PD-L1 signaling markedly enhances cytokines production and degranulation and suppresses apoptosis of NK cells in vitro. PD-1/PD-L1 exerts inhibitory effect through repressing the activation of PI3K/AKT signaling in NK cells. More importantly, a PD-1 blocking antibody was found to significantly suppress the growth of xenografts in nude mice, and this inhibition of tumor growth was completely abrogated by NK depletion. These findings strongly suggested that PD-1 is an inhibitory regulator of NK cells in digestive cancers. PD-1 blockade might be an efficient strategy in NK cell-based tumor immunotherapy.

Tim-3 displays critical roles in microenvironment-induced activation and protumoral effects of TAMs in HCC. Interference of Tim-3 might be great potential in HCC therapy.

No abstract

BACKGROUND & AIMS Zinc-fingers and homeoboxes 2 (ZHX2) represses transcription of several genes associated with liver cancer. However, little is known about the role of ZHX2 in development of hepatocellular carcinoma (HCC). We investigated the mechanisms by which ZHX2 might affect proliferation of HCC cells. METHODS We overexpressed and knocked down ZHX2 in HCC cells and analyzed the effects on proliferation, colony formation, and the cell cycle. We also analyzed the effects of ZHX2 overexpression in growth of HepG2.2.15 tumor xenografts in nude mice. Chromatin immunoprecipitation and luciferase reporter assays were used to measure binding of ZHX2 target promoters. Levels of ZHX2 in HCC samples were evaluated by immunohistochemistry. RESULTS ZHX2 overexpression significantly reduced proliferation of HCC cells and growth of tumor xenografts in mice; it led to G1 arrest and reduced levels of cyclins A and E in HCC cell lines. ZHX2 bound to promoter regions of CCNA2 (which encodes Cyclin A) and CCNE1 (which encodes cyclin E) and inhibited their transcription. Knockdown of cyclin A or cyclin E reduced the increased proliferation mediated by ZHX2 knockdown. Nuclear localization of ZHX2 was required for it to inhibit proliferation of HCC cells in culture and in mice. Nuclear localization of ZHX2 was reduced in human HCC samples, even in small tumors (diameter<5 cm), compared to adjacent non-tumor tissues. Moreover, reduced nuclear levels of ZHX2 correlated with reduced survival times of patients, high levels of tumor microvascularization, and hepatocyte proliferation. CONCLUSIONS ZHX2 inhibits HCC cell proliferation, by preventing expression of cyclins A and E, and reduces growth of xenograft tumors in mice. Loss of nuclear ZHX2 might be an early step in the development of HCC.

Hepatitis B virus (HBV) infection afflicts &gt;300 million people worldwide and is a leading cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. While the morphological characteristics of dying hepatocytes are well documented, the molecular mechanisms leading to the death of hepatocytes during HBV infection are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in the death of hepatocytes under certain inflammatory but not normal conditions. To determine the potential roles of TRAIL in HBV-induced hepatitis, we examined the effects of HBV and its X protein (HBx) on TRAIL-induced hepatocyte apoptosis both in vivo and in vitro. We found that hepatitis and hepatic cell death in HBV transgenic mice were significantly inhibited by a soluble TRAIL receptor that blocks TRAIL function. We also found that HBV or HBx transfection of a hepatoma cell line significantly increased its sensitivity to TRAIL-induced apoptosis. The increase in TRAIL sensitivity were associated with a dramatic up-regulation of Bax protein expression. Knocking down Bax expression using Bax-specific small interference RNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases 3 and 9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific small interference RNA can be used to inhibit HBV-induced hepatic cell death.

TIPE1 (tumor necrosis factor-α-induced protein 8-like 1 or TNFAIP8L1) is a newly identified member of the TIPE (TNFAIP8) family, which play roles in regulating cell death. However, the biologic functions of TIPE1 in physiologic and pathologic conditions are largely unknown. Here, we report the roles of TIPE1 in hepatocellular carcinoma (HCC). Evaluated by immunohistochemical staining, HCC tissues showed significantly downregulated TIPE1 expression compared with adjacent non-tumor tissues, which positively correlated with tumor pathologic grades and patient survival. Using a homograft tumor model in Balb/c mice, we discovered that TIPE1 significantly diminished the growth and tumor weight of murine liver cancer homografts. Consistently, TIPE1 inhibited both cell growth and colony formation ability of cultured HCC cell lines, which was further identified to be due to TIPE1-inducing apoptosis in a caspase-independent, necrostatin-1 (Nec-1)-insensitive manner. Furthermore, mechanistic investigations revealed that TIPE1 interacted with Rac1, and inhibited the activation of Rac1 and its downstream p65 and c-Jun N-terminal kinase pathway. Moreover, overexpression of constitutively active Rac1 partially rescued the apoptosis induced by TIPE1, and Rac1 knockdown significantly restored the deregulated cell growth induced by TIPE1 small interfering RNA. Our findings revealed that TIPE1 induced apoptosis in HCC cells by negatively regulating Rac1 pathway, and loss of TIPE1 might be a new prognostic indicator for HCC patients.

No abstract

Activation of Wnt/β-catenin signalling is frequently observed in many types of cancer including hepatocellular carcinoma (HCC). We recently reported that cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, is overexpressed in many types of solid tumours and contributes to epigenetic silencing of tumour suppressors. In this study, we characterized the function of CUL4B in HCC and investigated whether CUL4B is involved in the regulation of Wnt/β-catenin signalling. CUL4B and β-catenin were frequently up-regulated and positively correlated in HCC tissues. CUL4B activated Wnt/β-catenin signalling by protecting β-catenin from GSK3-mediated degradation, achieved through CUL4B-mediated epigenetic silencing of Wnt pathway antagonists. Knockdown of CUL4B resulted in the up-regulation of Wnt signal antagonists such as DKK1 and PPP2R2B. Simultaneous knockdown of PPP2R2B partially reversed the down-regulation of β-catenin signalling caused by CUL4B depletion. Furthermore, CRL4B promoted the recruitment and/or retention of PRC2 at the promoters of Wnt antagonists and CUL4B knockdown decreased the retention of PRC2 components as well as H3K27me3. Knockdown of CUL4B reduced the proliferation, colony formation, and invasiveness of HCC cells in vitro and inhibited tumour growth in vivo, and these effects were attenuated by introduction of exogenous β-catenin or simultaneous knockdown of PPP2R2B. Conversely, ectopic expression of CUL4B enhanced the proliferation and invasiveness of HCC cells. We conclude that CUL4B can up-regulate Wnt/β-catenin signalling in human HCC through transcriptionally repressing Wnt antagonists and thus contributes to the malignancy of HCC.