Hepatic Stellate Cell Modulates the Immune Microenvironment in the Progression of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a major cause of increases in the mortality rate due to cancer that usually develops in patients with liver fibrosis and impaired hepatic immunity. Hepatic stellate cells (HSCs) may directly or indirectly crosstalk with various hepatic cells and subsequently modulate extracellular remodeling, cell invasion, macrophage conversion, and cancer deterioration. In this regard, the tumor microenvironment created by activated HSC plays a critical role in mediating pathogenesis and immune escape during HCC progression. Herein, intermediately differentiated human liver cancer cell line (J5) cells were co-cultured with HSC-conditioned medium (HSC-CM); changes in cell phenotype and cytokine profiles were analyzed to assess the impact of HSCs on the development of hepatoma. The stage of liver fibrosis correlated significantly with tumor grade, and the administration of conditioned medium secreted by activated HSC (aHSC-CM) could induce the expression of N-cadherin, cell migration, and invasive potential, as well as the activity of matrix metalloproteinases in J5 cells, implying that aHSC-CM could trigger the epithelial-mesenchymal transition (EMT). Next, the HSC-CM was further investigated and network analysis indicated that specific cytokines and soluble proteins, such as activin A, released from activated HSCs could remarkably affect the tumor-associated immune microenvironment involved in macrophage polarization, which would, in turn, diminish a host’s immune surveillance and drive hepatoma cells into a more malignant phenotype. Together, our findings provide a novel insight into the integral roles of HSCs to enhance hepatocarcinogenesis through their immune-modulatory properties and suggest that HSC may serve as a potent target for the treatment of advanced HCC.     

Metformin Enhances the Anti-Cancer Efficacy of Sorafenib via Suppressing MAPK/ERK/Stat3 Axis in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) incidence, as well as related mortality, has been steadily increasing in the USA and across the globe, partly due to the lack of effective therapeutic options for advanced HCC. Though sorafenib is considered standard-of-care for advanced HCC, it only improves median survival by a few months when compared to placebo. Sorafenib is also associated with several unpleasant side effects that often lead to early abatement of therapy. Here, we investigate whether a combination regimen including low-dose sorafenib and a non-toxic dose of anti-diabetic drug metformin can achieve effective inhibition of HCC. Indeed, combining metformin with low-dose sorafenib inhibited growth, proliferation, migration, and invasion potential of HCC cells. We observed a 5.3- and 1.9-fold increase in sub-G1 population in the combination treatment compared to sorafenib alone. We found that the combination of metformin enhanced the efficacy of sorafenib and inhibited the MAPK/ERK/Stat3 axis. Our in vivo studies corroborated the in vitro findings, and mice harboring HepG2-derived tumors showed effective tumor reduction upon treatment with low-dose sorafenib and metformin combination. This work sheds light on a therapeutic strategy aiming to augment sorafenib efficacy or dose-de-escalation that may prove beneficial in circumventing sorafenib resistance as well as minimizing related side effects.     

Activation of ERK and p38 Reduces AZD8055-Mediated Inhibition of Protein Synthesis in Hepatocellular Carcinoma HepG2 Cell Line

Protein synthesis is important for maintaining cellular homeostasis under various stress responses. In this study, we screened an anticancer drug library to select compounds with translational repression functions. AZD8055, an ATP-competitive mechanistic target of rapamycin complex 1/2 (mTORC1/2) inhibitor, was selected as a translational suppressor. AZD8055 inhibited protein synthesis in mouse embryonic fibroblasts and hepatocellular carcinoma HepG2 cells. Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) were activated during the early phase of mTORC1/2 inhibition by AZD8055 treatment. Combined treatment of AZD8055 with the MAPK kinase1/2 (MEK1/2) inhibitor refametinib or the p38 inhibitor SB203580 markedly decreased translation in HepG2 cells. Thus, the inhibition of ERK1/2 or p38 may enhance the efficacy of AZD8055-mediated inhibition of protein synthesis. In addition, AZD8055 down-regulated the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and AZD8055-induced phosphorylation of ERK1/2 and p38 had no effect on phosphorylation status of 4E-BP1. Interestingly, AZD8055 modulated the 4E-BP1 mRNA pool by up-regulating ERK1/2 and p38 pathways. Together, these results suggest that AZD8055-induced activation of MAPKs interferes with inhibition of protein synthesis at an early stage of mTORC1/2 inhibition, and that it may contribute to the development of resistance to mTORC1/2 inhibitors.     

PEBP4 Directs the Malignant Behavior of Hepatocellular Carcinoma Cells via Regulating mTORC1 and mTORC2

Phosphatidylethanolamine binding protein 4 (PEBP4) is an understudied multifunctional small protein. Previous studies have shown that the expression of PEBP4 is increased in many cancer specimens, which correlates to cancer progression. The present study explored the mechanism by which PEBP4 regulates the growth and progression of hepatocellular carcinoma cells. Thus, we showed that knockdown of PEBP4 in MHCC97H cells, where its expression was relatively high, diminished activities of serine/threonine protein kinase B (PKB, also known as Akt), mammalian target of rapamycin complex 1(mTORC1), and mTORC2, events that were not restored by insulin-like growth factor 1 (IGF-1). Conversely, overexpression of PEBP4 in MHCC97L cells with the low endogenous level yielded opposite effects. Furthermore, physical association of PEBP4 with Akt, mTORC1, and mTORC2 was observed. Interestingly, introduction of AktS473D mutant, bypassing phosphorylation by mTORC2, rescued mTORC1 activity, but without effects on mTORC2 signaling. In contrast, the effect of PEBP4 overexpression on the activity of mTORC1 but not that of mTORC2 was suppressed by MK2206, a specific inhibitor of Akt. In conjunction, PEBP4 knockdown-engendered reduction of cell proliferation, migration and invasion was partially rescued by Akt S473D while increases in these parameters induced by overexpression of PEBP4 were completely abolished by MK2206, although the expression of epithelial mesenchymal transition (EMT) markers appeared to be fully regulated by the active mutant of Akt. Finally, knockdown of PEBP4 diminished the growth of tumor and metastasis, whereas they were enhanced by overexpression of PEBP4. Altogether, our study suggests that increased expression of PEBP4 exacerbates malignant behaviors of hepatocellular cancer cells through cooperative participation of mTORC1 and mTORC2.     

Cytotoxic T Cell Expression of Leukocyte-Associated Immunoglobulin-Like Receptor-1 (LAIR-1) in Viral Hepatitis C-Mediated Hepatocellular Carcinoma

Virus-related hepatocellular carcinoma (HCC) pathogenesis involves liver inflammation, therefore, despite successful treatment, hepatitis C virus (HCV) may progress to HCC from initiated liver cirrhosis. Cytotoxic T cells (Tcs) are known to be involved in HCV-related cirrhotic complications and HCC pathogenesis. The inhibitory checkpoint leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is expressed on Tcs. Therefore, we aimed to determine whether the Tc expression level of LAIR-1 is associated with HCC progression and to evaluate LAIR-1 expression as a noninvasive biomarker for HCC progression in the context of liver cirrhosis related to HCV genotype 4 (G4) in Egyptian patients’ peripheral venous blood liquid biopsy. A total of 64 patients with HCC and 37 patients with liver cirrhosis were enrolled in this case-controlled study, and their LAIR-1 expression on Tc related to the progression of liver cirrhosis was examined and compared to that of the apparently healthy control group (n = 20). LAIR-1 expression was analyzed using flow cytometry. Results: The HCC group had significantly higher LAIR-1 expression on Tc and percentage of Tc positive for LAIR-1 (LAIR-1+Tc%) than the HCV G4-related liver cirrhosis group. LAIR-1+Tc% was correlated with the HCC surrogate tumor marker AFP (r = 0.367, p = 0.001) and insulin resistance and inflammation prognostic ratios/indices. A receiver operating characteristic (ROC) curve revealed that adding LAIR-1+Tc% to AFP can distinguish HCC transformation in the Egyptian patients’ cohort. Upregulated LAIR-1 expression on Tc could be a potential screening noninvasive molecular marker for chronic inflammatory HCV G4 related liver cirrhosis. Moreover, LAIR-1 expression on Tc may be one of the players involved in the progression of liver cirrhosis to HCC.     

MicroRNA-302b Enhances the Sensitivity of Hepatocellular Carcinoma Cell Lines to 5-FU via Targeting Mcl-1 and DPYD

MiR-302b is a member of miR-302-367 cluster. The miR-302-367 cluster played important roles in maintaining pluripotency in human embryonic stem cells (hESCs) and has been proved to be capable of suppressing cell growth in several types of cancer cell lines including Hepatocellular Carcinoma (HCC) Cell lines. However, the role that miR-302b plays in the 5-Fluorouracil (5-FU) sensitivity of HCC has not been known. This study showed that miR-302b could enhance the sensitivity to 5-FU in HCC cell lines and verified its two putative targeted genes responsible for its 5-FU sensitivity.     

Validation of Bmi1 as a Therapeutic Target of Hepatocellular Carcinoma in Mice

Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. Our previous studies have revealed that Bmi1 acts as an oncogene in hepatic carcinogenesis in an INK4a/ARF locus independent manner. However, whether Bmi1 can be used as a potential target for hepatocellular carcinoma treatment has not been fully confirmed yet. Here, we show that perturbation of Bmi1 expression by using short hairpin RNA can inhibit the tumorigenicity and tumor growth of hepatocellular carcinoma cells both in vitro and in vivo. Importantly, Bmi1 knockdown can block the tumor growth, both in the initiating stages and the fast growing stages. Cellular biology analysis revealed that Bmi1 knockdown induces cell cycle arrest and apoptosis. Our findings verify Bmi1 as a qualified treatment target for hepatocellular carcinoma (HCC) and support Bmi1 targeting treatment with chemotherapeutic agents.     

The Role of Nrf2 Transcription Factor and Sp1-Nrf2 Protein Complex in Glutamine Transporter SN1 Regulation in Mouse Cortical Astrocytes Exposed to Ammonia

Ammonia toxicity in the brain primarily affects astrocytes via a mechanism in which oxidative stress (OS), is coupled to the imbalance between glutamatergic and GABAergic transmission. Ammonia also downregulates the astrocytic N system transporter SN1 that controls glutamine supply from astrocytes to neurons for the replenishment of both neurotransmitters. Here, we tested the hypothesis that activation of Nrf2 is the process that links ammonia-induced OS formation in astrocytes to downregulation and inactivation of SN1 and that it may involve the formation of a complex between Nrf2 and Sp1. Treatment of cultured cortical mouse astrocytes with ammonia (5 mM NH₄Cl for 24 h) evoked Nrf2 nuclear translocation, increased its activity in a p38 MAPK pathway-dependent manner, and enhanced Nrf2 binding to Slc38a3 promoter. Nrf2 silencing increased SN1 mRNA and protein level without influencing astrocytic [³H]glutamine transport. Ammonia decreased SN1 expression in Nrf2 siRNA treated astrocytes and reduced [³H]glutamine uptake. In addition, while Nrf2 formed a complex with Sp1 in ammonia-treated astrocytes less efficiently than in control cells, treatment of astrocytes with hybrid-mode inactivated Sp1-Nrf2 complex (Nrf2 silencing + pharmacological inhibition of Sp1) did not affect SN1 protein level in ammonia-treated astrocytes. In summary, the results document that SN1 transporter dysregulation by ammonia in astrocytes involves activation of Nrf2 but does not require the formation of the Sp1-Nrf2 complex.