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Background & Aims: Mature transfer RNAs (tRNA) charged with amino acids decode mRNA to synthesize proteins. Dysregulation of translational machineries has a fundamental impact on cancer biology. This study aims to map the tRNAome landscape in liver cancer patients and to explore potential therapeutic targets at the interface of charging amino acid with tRNA. Methods: Resected tumour and paired tumour‐free (TFL) tissues from hepatocellular carcinoma (HCC) patients (n = 69), and healthy liver tissues from organ transplant donors (n = 21), HCC cell lines, and cholangiocarcinoma (CC) patient‐derived tumour organoids were used. Results: The expression levels of different mature tRNAs were highly correlated and closely clustered within individual tissues, suggesting that different members of the tRNAome function cooperatively in protein translation. Interestingly, high expression of tRNA‐Lys‐CUU in HCC tumours was associated with more tumour recurrence (HR 1.1; P =.022) and worse patient survival (HR 1.1; P =.0037). The expression of Lysyl‐tRNA Synthetase (KARS), the enzyme catalysing the charge of lysine to tRNA‐Lys‐CUU, was significantly upregulated in HCC tumour tissues compared to tumour‐free liver tissues. In HCC cell lines, lysine deprivation, KARS knockdown or treatment with the KARS inhibitor cladosporin effectively inhibited overall cell growth, single cell‐based colony formation and cell migration. This was mechanistically mediated by cell cycling arrest and induction of apoptosis. Finally, these inhibitory effects were confirmed in 3D cultured patient‐derived CC organoids. Conclusions: The biological process of charging tRNA‐Lys‐CUU with lysine sustains liver cancer cell growth and migration, and is clinically relevant in HCC patients. This process can be therapeutically targeted and represents an unexplored territory for developing novel treatment strategies against liver cancer. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are thought to be the main drivers for disease progression and treatment resistance across various cancer types. Identifying and targeting these rare cancer cells, however, remains challenging with respect to therapeutic benefit. Here, we report the enrichment of LGR5 expressing cells, a well-recognized stem cell marker, in mouse liver tumors, and the upregulation of LGR5 expression in human hepatocellular carcinoma. Isolated LGR5 expressing cells from mouse liver tumors are superior in initiating organoids and forming tumors upon engraftment, featuring candidate TICs. These cells are resistant to conventional treatment including sorafenib and 5-FU. Importantly, LGR5 lineage ablation significantly inhibits organoid initiation and tumor growth. The combination of LGR5 ablation with 5-FU, but not sorafenib, further augments the therapeutic efficacy in vivo. Thus, we have identified the LGR5+ compartment as an important TIC population, representing a viable therapeutic target for combating liver cancer. Cancer stem cells (CSCs) are thought to be the main drivers for disease progression and treatment resistance in liver cancer. This study identifies the LGR5+ compartment as an important CSC population, representing a viable therapeutic target for combating liver cancer. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming universally occurs in cancer. Mitochondria act as the hubs of bioenergetics and metabolism. The morphodynamics of mitochondria, comprised of fusion and fission processes, are closely associated with mitochondrial functions and are often dysregulated in cancer. In this study, we aim to investigate the mitochondrial morphodynamics and its functional consequences in human liver cancer. We observed excessive activation of mitochondrial fusion in tumor tissues from hepatocellular carcinoma (HCC) patients and in vitro cultured tumor organoids from cholangiocarcinoma (CCA). The knockdown of the fusion regulator genes, OPA1 (Optic atrophy 1) or MFN1 (Mitofusin 1), inhibited the fusion process in HCC cell lines and CCA tumor organoids. This resulted in inhibition of cell growth in vitro and tumor formation in vivo, after tumor cell engraftment in mice. This inhibitory effect is associated with the induction of cell apoptosis, but not related to cell cycle arrest. Genome-wide transcriptomic profiling revealed that the inhibition of fusion predominately affected cellular metabolic pathways. This was further confirmed by the blocking of mitochondrial fusion which attenuated oxygen consumption and cellular ATP production of tumor cells. In conclusion, increased mitochondrial fusion in liver cancer alters metabolism and fuels tumor cell growth. [ABSTRACT FROM AUTHOR]

Abstract: Interferons (IFNs) with antiviral and immune-stimulatory functions have been widely used in prevention and treatment of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 1 (STAT1) is a key element of the IFN signaling, and the function of STAT1 is critically determined by its phosphorylation state. This study aims to understand the functions of phosphorylated (p-) and unphosphorylated (u-) STAT1 in HCC. We found that u-STAT1 is significantly elevated in patient HCC tumor tissues and predominantly expressed in cytoplasm; while p-STAT1 is absent. Loss of u-STAT1 potently arrested cell cycle and inhibited cell growth in HCC cells. Induction of p-STAT1 by IFN-α treatment effectively triggers the expression of interferon-stimulated genes (ISGs), but has moderate effect on HCC cell growth. Interestingly, both u-STAT1 and p-STAT1 are induced by IFN-α, through with distinct time-dependent process. Furthermore, the ISG induction patterns mediated by p-STAT1 and u-STAT1 are also distinct. Importantly, artificial blocking of the induction of u-STAT1, but not p-STAT1, sensitizes HCC cells to treatment of IFNs. Therefore, p-STAT1 and u-STAT1 exert dichotomal functions and coordinately regulate the responsiveness to IFN treatment in HCC.Key Messages: STAT1 is upregulated and predominantly presented as u-STAT1 in HCC, while p-STAT1 is absent.U-STAT1 sustains but p-STAT1 inhibits HCC growth.The dynamic change of phosphorylation state of STAT1 control the responsiveness to IFN treatment. [ABSTRACT FROM AUTHOR]

The current understanding of cancer biology and development of effective treatments for cancer remain far from satisfactory. This in turn heavily relies on the availability of easy and robust model systems that resemble the architecture/physiology of the tumors in patients to facilitate research. Cancer research in vitro has mainly been based on the use of immortalized 2D cancer cell lines that deviate in many aspects from the original primary tumors. The recent development of the organoid technology allowing generation of organ-buds in 3D culture from adult stem cells has endowed the possibility of establishing stable culture from primary tumors. Although culturing organoids from liver tumors is thought to be difficult, we now convincingly demonstrate the establishment of organoids from mouse primary liver tumors. We have succeeded in culturing 91 lines from 129 liver tissue/tumors. These organoids can be grown in long-term cultures in vitro. About 20% of these organoids form tumors in immunodeficient mice upon (serial) transplantation, confirming their tumorigenic and self-renewal properties. Interestingly, single cells from the tumor organoids have high efficiency of organoid initiation, and a single organoid derived from a cancer cell is able to initiate a tumor in mice, indicating the enrichment of tumor-initiating cells in the tumor organoids. Furthermore, these organoids recapitulate, to some extent, the heterogeneity of liver cancer in patients, with respect to phenotype, cancer cell composition and treatment response. These model systems shall provide enormous opportunities to advance our research on liver cancer (stem cell) biology, drug development and personalized medicine. [ABSTRACT FROM AUTHOR]

Recently, we reported the accumulation of CD4+FOXP3+ regulatory T cells (Tregs) within the tumor mass of patients bearing liver cancer. Tumor-infiltrating Tregs (TiTreg) are active and potent suppressors of antitumor immunity. Importantly, treatment with GITR L reduced the immunosuppression mediated by TiTreg. [ABSTRACT FROM AUTHOR]

Novel systemic treatments for hepatocellular carcinoma (HCC) are strongly needed. Immunotherapy is a promising strategy that can induce specific antitumor immune responses. Understanding the mechanisms of immune resistance by HCC is crucial for development of suitable immunotherapeutics. We used immunohistochemistry on tissue-microarrays to examine the co-expression of the immune inhibiting molecules PD-L1, Galectin-9, HVEM and IDO, as well as tumor CD8+lymphocyte infiltration in HCC, in two independent cohorts of patients. We found that at least some expression in tumor cells was seen in 97% of cases for HVEM, 83% for PD-L1, 79% for Gal-9 and 66% for IDO. In the discovery cohort (n = 94), we found that lack of, or low, tumor expression of PD-L1 (p< 0.001), Galectin-9 (p< 0.001) and HVEM (p< 0.001), and low CD8+TIL count (p= 0.016), were associated with poor HCC-specific survival. PD-L1, Galectin-9 and CD8+TIL count were predictive of HCC-specific survival independent of baseline clinicopathologic characteristics and the combination of these markers was a powerful predictor of HCC-specific survival (HR 0.29;p<0.001). These results were confirmed in the validation cohort (n = 60). We show that low expression levels of PD-L1 and Gal-9 in combination with low CD8+TIL count predict extremely poor HCC-specific survival and it requires a change in two of these parameters to significantly improve prognosis. In conclusion, intra-tumoral expression of these immune inhibiting molecules was observed in the majority of HCC patients. Low expression of PD-L1 and Galectin-9 and low CD8+TIL count are associated with poor HCC-specific survival. Combining immune biomarkers leads to superior predictors of HCC mortality. [ABSTRACT FROM AUTHOR]

In liver cancer tumor-infiltrating regulatory T cells (Ti-Treg) are potent suppressors of tumor-specific T-cell responses and express high levels of the Treg-associated molecules cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR). In this study, we have evaluated the capacity of GITR-ligation, CTLA-4-blockade and a combination of both treatments to alleviate immunosuppression mediated by Ti-Treg. Usingex vivoisolated cells from individuals with hepatocellular carcinoma (HCC) or liver metastases from colorectal cancer (LM-CRC) we show that treatment with a soluble form of the natural ligand of GITR (GITRL), or with blocking antibodies to CTLA-4, reduces the suppression mediated by human liver tumor-infiltrating CD4+Foxp3+ Treg, thereby restoring proliferation and cytokine production by effector T cells. Importantly, combined treatment with low doses of both molecules exhibited stronger recovery of T cell function compared with either treatment alone. Our data suggest that in patients with primary and secondary liver cancer both GITR-ligation and anti-CTLA-4 mAb can improve the antitumor immunity by abrogating Ti-Treg mediated suppression. [ABSTRACT FROM AUTHOR]

CD4+type 1 T regulatory (Tr1) cells have a crucial role in inducing tolerance. Immune regulation by these cells is mainly mediated through the secretion of high amounts of IL-10. Several studies have suggested that this regulatory population may be involved in tumor-mediated immune-suppression. However, direct evidence of a role for Tr1 cells in human solid tumors is lacking. Usingex vivoisolated cells from individuals with hepatocellular carcinoma (HCC;n= 39) or liver metastases from colorectal cancer (LM-CRC;n= 60) we identify a CD4+FoxP3−IL-13−IL-10+T cell population in tumors of individuals with primary or secondary liver cancer that is characterized as Tr1 cells by the expression of CD49b and the lymphocyte activation gene 3 (LAG-3) and strong suppression activity of T cell responses in an IL-10 dependent manner. Importantly, the presence of tumor-infiltrating Tr1 cells is correlated with tumor infiltration of plasmacytoid dendritic cells (pDCs). pDCs exposed to tumor-derived factors enhance IL-10 production by Tr1 cells through up-regulation of the inducible co-stimulatory ligand (ICOS-L). These findings suggest a role for pDCs and ICOS-L in promoting intra-tumoral immunosuppression by Tr1 cells in human liver cancer, which may foster tumor progression and which might interfere with attempts of immunotherapeutic intervention. [ABSTRACT FROM AUTHOR]

• NK cells and cytotoxic T cells infiltrate poorly into cholangiocarcinoma. • Regulatory T cells accumulate in cholangiocarcinoma. • PD1, CTLA4 and GITR are over-expressed on tumor-infiltrating T cells in cholangiocarcinoma. • Blocking PD1 or CTLA4 or stimulating GITR enhances effector functions of tumor-infiltrating T cells in cholangiocarcinoma. Cholangiocarcinoma is an aggressive hepatobiliary malignancy originating from biliary tract epithelium. Whether cholangiocarcinoma is responsive to immune checkpoint antibody therapy is unknown, and knowledge of its tumor immune microenvironment is limited. We aimed to characterize tumor-infiltrating lymphocytes (TILs) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TILs. We isolated TILs from resected tumors of patients with cholangiocarcinoma and investigated their compositions compared with their counterparts in tumor-free liver (TFL) tissues and blood, by flow cytometry and immunohistochemistry. We measured expression of immune co-stimulatory and co-inhibitory molecules on TILs, and determined whether targeting these molecules improved ex vivo functions of TILs. Proportions of cytotoxic T cells and natural killer cells were decreased, whereas regulatory T cells were increased in tumors compared with TFL. While regulatory T cells accumulated in tumors, the majority of cytotoxic and helper T cells were sequestered at tumor margins, and natural killer cells were excluded from the tumors. The co-stimulatory receptor GITR and co-inhibitory receptors PD1 and CTLA4 were over-expressed on tumor-infiltrating T cells compared with T cells in TFL and blood. Antagonistic targeting of PD1 or CTLA4 or agonistic targeting of GITR enhanced effector molecule production and T cell proliferation in ex vivo stimulation of TILs derived from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated with differences in TIL immune phenotype. Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells that over-express co-inhibitory receptors suggest that the tumor microenvironment in cholangiocarcinoma is immunosuppressive. Targeting GITR, PD1 or CTLA4 enhances effector functions of tumor-infiltrating T cells, indicating that these molecules are potential immunotherapeutic targets for patients with cholangiocarcinoma. The defense functions of immune cells are suppressed in cholangiocarcinoma tumors. Stimulating or blocking "immune checkpoint" molecules expressed on tumor-infiltrating T cells can enhance the defense functions of these cells. Therefore, these molecules may be promising targets for therapeutic stimulation of immune cells to eradicate the tumors and prevent cancer recurrence in patients with cholangiocarcinoma. [ABSTRACT FROM AUTHOR]