Human Human Hepatocellular Carcinoma Research Articles

Neutrophil extracellular traps induce tumor metastasis through dual effects on cancer and endothelial cells

ABSTRACT Neutrophils constitute a major component in human hepatocellular carcinoma (HCC) and can facilitate disease progression via poorly understood mechanisms. Here, we show that neutrophil extracellular traps (NETs) formation was increased in human HCC tumor tissues than in paired non-tumor liver tissues. Mechanism study revealed that tumor-induced metabolic switch toward glycolysis and pentose phosphate pathway in tumor infiltrating neutrophils promoted NETs formation in a reactive oxygen species dependent-manner. NETs subsequently induced the migration of cancer cells and down-regulation of tight junction molecules on adjacent endothelial cells, thus facilitating tumor intravasation and metastasis. Accordingly, NETs depletion could inhibit tumor metastasis in mice in vivo, and the infiltration levels of NETs-releasing neutrophils were negatively associated with patient survival and positively correlated with tumor metastasis potential of HCC patients. Our results unveiled a pro-metastatic role of NETs in the milieu of human HCC, and pointed to the importance of metabolic reprogramming in shaping their characteristics, thus providing an applicable efficient target for anti-cancer therapies.

Niuhuang (Bovis Calculus)-Shexiang (Moschus) combination induces apoptosis and inhibits proliferation in hepatocellular carcinoma via PI3K/AKT/mTOR pathway

ObjectiveTo investigate the effects of Niuhuang (Bovis Calculus, BC) and Shexiang (Moschus) (BC-Moschus) on human hepatocellular carcinoma (HCC) cells SMMC-7721 and a nude mouse model of subcutaneous xenografts, and to explore its anti-HCC mechanism. MethodsThe BC-Moschus combination was applied to two liver cancer models in vivo and in vitro. SMMC-7721 was divided into the BC-Moschus group and the control group, and different doses (rude drug dosage 0.625, 1.25, 2.5, and 5 mg/mL) of BC-Moschus extract were used for the intervention. The proliferation ability of HCC cells was detected using the Cell Counting Kit-8 (CCK-8) assay, and the migration ability was detected by a wound healing assay. A subcutaneous xenograft model was prepared using nude mice with human HCC. Specific pathogen-free-grade BALB/c nude mice (5-week-old) were randomly divided into the following groups (n = 6 per group): control (0.9% physiological saline 0.2 mL/d), BC-Moschus [BC 45.5 mg/(kg·d)+ Moschus 13 mg/(kg·d)], and cisplatin (DDP, intraperitoneal injection 5 mg/kg per week) groups. All groups were administered for 14 d. The volume and mass of the subcutaneous xenografts in nude mice were observed. The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, apoptosis-associated factor p70 S6 Kinase (S6K), Bax, Bcl-2, caspase-3, and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR (RT-qPCR) and Western blot. Terminal Deoxynucleotidy Transferase-Mediated dUTP Nick-End Labeling (TUNEL) was used for quantitative analysis of apoptotic cells. ResultsThe CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone, and the inhibition effect was dose- and time-dependent (P < 0.01). The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration (P < 0.01). In the subcutaneous xenograft model of nude mice with human HCC, we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group (P < 0.01). The levels of the PI3K/AKT/mTOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased (P < 0.01). The expression level of the anti-apoptotic gene Bcl-2 was downregulated (P < 0.05), and the expression of the pro-apoptotic gene Bax and apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated (P < 0.01). The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC (P < 0.01). ConclusionThe BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice. The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway, regulation of apoptosis-related protein caspase-3, caspase-9, Bcl-2, and Bax expression, and promotion of apoptosis.

β-Catenin signaling in hepatocellular carcinoma.

Deregulated Wnt/β-catenin signaling is one of the main genetic alterations in human hepatocellular carcinoma (HCC). Comprehensive genomic analyses have revealed that gain-of-function mutation of CTNNB1, which encodes β-catenin, and loss-of-function mutation of AXIN1 occur in approximately 35% of human HCC samples. Human HCCs with activation of the Wnt/β-catenin pathway demonstrate unique gene expression patterns and pathological features. Activated Wnt/β-catenin synergizes with multiple signaling cascades to drive HCC formation, and it functions through its downstream effectors. Therefore, strategies targeting Wnt/β-catenin have been pursued as possible therapeutics against HCC. Here, we review the genetic alterations and oncogenic roles of aberrant Wnt/β-catenin signaling during hepatocarcinogenesis. In addition, we discuss the implication of this pathway in HCC diagnosis, classification, and personalized treatment.

VLCAD inhibits the proliferation and invasion of hepatocellular cancer cells through regulating PI3K/AKT axis.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is an essential mediator in fatty acid metabolism. The progression of human hepatocellular carcinoma (HCC) is closely associated with the disorder of energy supply. Here, we aimed to investigate the role and underlying molecule mechanism of VLCAD in pathological process of HCC. In this study, VLCAD was induced silencing and overexpression using small hairpin RNA (shRNA) and lentiviral-mediated vector in HCC cell lines. The proliferation of HCC cells was determined using CCK-8 assay. Transwell assay and lung metastasis were performed to analysis cell metastasis in vitro and in vivo. ECAR and OCR were used to evaluate the activity of glycolysis and mitochondrial oxidative phosphorylation. Our data indicated that VLCAD was downregulated in human HCC tissues and cells. VLCAD overexpression strongly suppressed the proliferation and metastasis of HCC cells associating with the decrease of ATP accumulation and glycolysis activity. Importantly, the PI3K/AKT inhibitor LY294002 strongly abolished the role of shVLCAD in HCC cells. Our results suggested that VLCAD suppressed the growth and metastasis in HCC cells by inhibiting the activities of glycolysis and mitochondrial oxidative phosphorylation metabolism via PI3K/AKT pathway. Together, present findings not only demonstrated the protective role of and molecular network of VLCAD in HCC cells but also indicated its and potential use as a target in the therapy of HCC.

Prognostic comparative genes predict targets for sorafenib combination therapies in hepatocellular carcinoma

With the increasing incidence and mortality of human hepatocellular carcinoma (HCC) worldwide, revealing innovative targets to improve therapeutic strategies is crucial for prolonging the lives of patients. To identify innovative targets, we conducted a comprehensive comparative transcriptome analysis of 5,410 human HCCs and 974 mouse liver cancers to identify concordantly expressed genes associated with patient survival. Among the 664 identified prognostic comparative HCC (pcHCC) genes, upregulated pcHCC genes were associated with prognostic clinical features, including large tumor size, vascular invasion and late HCC stages. Interestingly, after validating HCC patient prognoses in multiple independent datasets, we matched the 664 aberrant pcHCC genes with the sorafenib-altered genes in TCGA_LIHC patients and found these 664 pcHCC genes were enriched in sorafenib-related functions, such as downregulated xenobiotic and lipid metabolism and upregulated cell proliferation. Therapeutic agents targeting aberrant pcHCC genes presented divergent molecular mechanisms, including suppression of sorafenib-unrelated oncogenic pathways, induction of sorafenib-unrelated ferroptosis, and modulation of sorafenib transportation and metabolism, to potentiate sorafenib therapeutic effects in HCC combination therapy. Moreover, the pcHCC genes NCAPG and CENPW, which have not been targeted in combination with sorafenib treatment, were knocked down and combined with sorafenib treatment, which reduced HCC cell viability based on disruption to the p38/STAT3 axis, thereby hypersensitizing HCC cells. Together, our results provide important resources and reveal that 664 pcHCC genes represent innovative targets suitable for developing therapeutic strategies in combination with sorafenib based on the divergent synergistic mechanisms for HCC tumor suppression.

Effect of RBM10 on regulating the proliferation and metastasis activity of human hepatocellular carcinoma cells by affecting the stability of miR-21

In recent decades, RNA binding motif (RBM) proteins have been widespread concerned by researchers. Among them, RBM5 is considered as a potential tumor suppressor gene in HCC. RBM10, also belonging to the RBM family, have similar structure and high homology with RBM5, indicating its potential as potential tumor suppressor genes. However, the role of RBM10 in tumors is controversial. The purpose of this study was to analyze the expression correlation and functional relationship of miR-21 and RBM10 in human hepatocellular carcinoma (HCC) tissues and corresponding tumor cells. Bioinformatics analysis showed that miR-21 and RBM10 were both highly expressed in HCC; similarly, the expression levels of miR-21 and RBM10 in HCC cells were significantly higher than those in normal hepatocytes. There was a positive correlation between miR-21 and RBM10. Furthermore, knockdown of RBM10 inhibited the proliferation, migration and invasion of SNU-398 cells, and simultaneous overexpression of miR-21 attenuated this inhibitory effect. Meanwhile, overexpressing RBM10 could promote the proliferation, migration and invasion of Hep G2 cells; while the stability of miR-21 could be reduced by knocking down RBM10. On the whole, the findings of this study indicate that RBM10 is involved in regulating the function and activity of human HCC cells by affecting the stability of miR-21. RBM10 cannot be simply summarized as a tumor suppressor or oncogene. Further studies are needed to clarify the role of RBM10 in tumors.

From the Editor’s Desk...

From the Editor’s Desk...

Elevated FBXO45 promotes liver tumorigenesis through enhancing IGF2BP1 ubiquitination and subsequent PLK1 upregulation.

Dysregulation of tumor-relevant proteins may contribute to human hepatocellular carcinoma (HCC) tumorigenesis. FBXO45 is an E3 ubiquitin ligase that is frequently elevated expression in human HCC. However, it remains unknown whether FBXO45 is associated with hepatocarcinogenesis and how to treat HCC patients with high FBXO45 expression. Here, IHC and qPCR analysis revealed that FBXO45 protein and mRNA were highly expressed in 54.3% (57 of 105) and 52.2% (132 of 253) of the HCC tissue samples, respectively. Highly expressed FBXO45 promoted liver tumorigenesis in transgenic mice. Mechanistically, FBXO45 promoted IGF2BP1 ubiquitination at the Lys190 and Lys450 sites and subsequent activation, leading to the upregulation of PLK1 expression and the induction of cell proliferation and liver tumorigenesis in vitro and in vivo. PLK1 inhibition or IGF2BP1 knockdown significantly blocked FBXO45-driven liver tumorigenesis in FBXO45 transgenic mice, primary cells, and HCCs. Furthermore, IHC analysis on HCC tissue samples revealed a positive association between the hyperexpression of FBXO45 and PLK1/IGF2BP1, and both had positive relationship with poor survival in HCC patients. Thus, FBXO45 plays an important role in promoting liver tumorigenesis through IGF2BP1 ubiquitination and activation, and subsequent PLK1 upregulation, suggesting a new strategy for treating HCC by targeting FBXO45/IGF2BP1/PLK1 axis.

Nuclear localization dictates hepatocarcinogenesis suppression by glycine N-methyltransferase

GNMT (glycine N-methyltransferase) is a tumor suppressor gene, but the mechanisms mediating its suppressive activity are not entirely known. We investigated the oncosuppressive mechanisms of GNMT in human hepatocellular carcinoma (HCC). GNMT mRNA and protein levels were evaluated by quantitative RT-PCR and immunoblotting. GNMT effect in HCC cell lines was modulated through GNMT cDNA induced overexpression or anti-GNMT siRNA transfection. GNMT was expressed at low level in human HCCs with a better prognosis (HCCB) while it was almost absent in fast-growing tumors (HCCP). In HCCB, the nuclear localization of the GNMT protein was much more pronounced than in HCCP. In Huh7 and HepG2 cell lines, GNMT forced expression inhibited the proliferation and promoted apoptosis. At the molecular level, GNMT overexpression inhibited the expression of CYP1A (Cytochrome p450, aromatic compound-inducible), PREX2 (Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2), PARP1 [Poly (ADP-ribose) polymerase 1], and NFKB (nuclear factor-kB) genes. By chromatin immunoprecipitation, we found GNMT binding to the promoters of CYP1A1, PREX2, PARP1, and NFKB genes resulting in their strong inhibition. These genes are implicated in hepatocarcinogenesis, and are involved in the GNMT oncosuppressive action. Overall, the present data indicate that GNMT exerts a multifaceted suppressive action by interacting with various cancer-related genes and inhibiting their expression.

HMBS is the most suitable reference gene for RT-qPCR in human HCC tissues and blood samples.

Reverse transcription-quantitative (RT-q) PCR is the most feasible and useful technique for identifying and evaluating cancer biomarkers; however, the method requires suitable reference genes for gene expression analysis. The aim of the present study was to identify the most suitable reference gene for the normalization of relative gene expression in human hepatocellular carcinoma (HCC) tissue and blood samples. First, 14 candidate reference genes were selected through a systematic literature search. The expression levels of these genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, PGK1, PPIA, RPLP0, RPL13A, SDHA, TBP, TFRC and YWHAZ) were evaluated using human multistage HCC transcriptome data (dataset GSE114564), which included normal liver (n=15), chronic hepatitis (n=20), liver cirrhosis (n=10), and early (n=18) and advanced HCC (n=45). From the 14 selected genes, five genes, whose expression levels were stable in all liver disease statuses (ACTB, GAPDH, HMBS, PPIA and RPLP0), were further assessed using RT-qPCR in 40 tissues (20 paired healthy tissues and 20 tissues from patients with HCC) and 40 blood samples (20 healthy controls and 20 samples from patients with HCC). BestKeeper statistical algorithms were used to identify the most stable reference genes, of which HMBS was found to be the most stable in both HCC tissues and blood samples. Therefore, the results of the present study suggest HMBS as a promising reference gene for the normalization of relative RT-qPCR techniques in HCC-related studies.

Xanthomicrol suppresses human hepatocellular carcinoma cells migration and invasion ability via Μu-opioid receptor.

Xanthomicrol is one of the methoxylated flavones and a promising cancer chemopreventive agent, but its anti-migration and anti-invasion ability on human hepatocellular carcinoma (HCC) remains unknown. This study aims to explore Xanthomicrol's effects on migration and invasion ability of the human HCC Huh7 cell line. Viability of Huh7 cells was measured by cell counting kit-8 (CCK8) assay. Cell apoptosis was assayed with flow cytometry analysis. The ability of migration and invasion of Huh7 cells was then detected through Transwell assays. Epithelial-mesenchymal transition (EMT)-related proteins were also detected through Western blot. Xanthomicrol inhibits the migration and invasion of Huh7 cells. The overexpression of Μu-opioid receptor (MOR) increases Huh7 cells' proliferation and enhances migration and invasion ability, while xanthomicrol treatment decreases the expression of MOR. Moreover, xanthomicrol can reverse migration, invasion and EMT-related protein expression by overexpressed MOR. These results suggest that xanthomicrol is a potential MOR antagonist, and it possesses potent anti-migration and anti-invasion ability on Huh7 cells.

Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo

Background aimsTracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression. MethodsEx vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The authors administered 1,1’-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry. ResultsThe fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45+CD56+CD3− NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D. ConclusionsAdministered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45+CD56+CD3− NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.

HIF-1α-activated TM4SF1-AS1 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by enhancing TM4SF1 expression

Long non-coding RNAs (lncRNAs) are essential drivers or suppressors in human hepatocellular carcinoma (HCC) by participating in controlling transcription, translation, mRNA stability, and protein degradation protein-protein interaction. TM4SF1-AS1 is recently identified as a tumor-promoting factor in lung cancer. Nevertheless, its function in HCC and related molecular mechanisms remain unknown. Here, our data indicated that either hypoxia or hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor (DMOG) induced the upregulation of TM4SF1-AS1 in HCC cells. HIF-1α knockdown rather than HIF-2α silencing remarkably abrogated hypoxia-upregulated TM4SF1-AS1 expression. Furthermore, we confirmed the elevated expression of TM4SF1-AS1 in HCC tissue samples and cell lines. The silencing of TM4SF1-AS1 prominently inhibited the proliferative, migratory, and invasive abilities of HCC cells. TM4SF1-AS1 depletion significantly blocked hypoxia-enhanced Hep3B cell proliferation and mobility. Interfering TM4SF1-AS1 remarkably reduced TM4SF1 mRNA and protein levels in HCC cells. But TM4SF1-AS1 knockdown did not impact the stability of TM4SF1 mRNA. Hypoxia enhanced the expression of TM4SF1 mRNA, which was subsequently decreased by TM4SF1-AS1 knockdown in HCC cells. We confirmed the positive correlation between TM4SF1 mRNA and TM4SF1-AS1 expression in HCC specimens. Finally, TM4SF1 prominently reversed the inhibitory role of TM4SF1-AS1 depletion in Hep3B cells. In summary, hypoxia-responsive TM4SF1-AS1 was overexpressed in human HCC and contributed to the malignant behaviors of tumor cells by enhancing TM4SF1-AS1 expression.

Phosphodiesterase 4D Depletion/Inhibition Exerts Anti-Oncogenic Properties in Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Drug resistance is a serious problem in the treatment of HCC. Therefore, it is of high clinical impact to discover targeted therapies that may overcome drug-related resistance and improve the survival of patients affected by HCC. In the present study, we investigated the role of Isoform D of type 4 phosphodiesterase (PDE4D) in HCC development and progression. We found that PDE4D is over-expressed HCCs in vitro and in vivo and the depletion of the gene by silencing or the pharmacological inhibition of protein activity exerted anti-tumorigenic activities.Isoform D of type 4 phosphodiesterase (PDE4D) has recently been associated with several human cancer types with the exception of human hepatocellular carcinoma (HCC). Here we explored the role of PDE4D in HCC. We found that PDE4D gene/protein were over-expressed in different samples of human HCCs compared to normal livers. Accordingly, HCC cells showed higher PDE4D activity than non-tumorigenic cells, accompanied by over-expression of the PDE4D isoform. Silencing of PDE4D gene and pharmacological inhibition of protein activity by the specific inhibitor Gebr-7b reduced cell proliferation and increased apoptosis in HCC cells, with a decreased fraction of cells in S phase and a differential modulation of key regulators of cell cycle and apoptosis. PDE4D silencing/inhibition also affected the gene expression of several cancer-related genes, such as the pro-oncogenic insulin growth factor (IGF2), which is down-regulated. Finally, gene expression data, available in the CancerLivER data base, confirm that PDE4D over-expression in human HCCs correlated with an increased expression of IGF2, suggesting a new possible molecular network that requires further investigations. In conclusion, intracellular depletion/inhibition of PDE4D prevents the growth of HCC cells, displaying anti-oncogenic effects. PDE4D may thus represent a new biomarker for diagnosis and a potential adjuvant target for HCC therapy.

Imaging, Pathology, and Immune Correlates in the Woodchuck Hepatic Tumor Model.

BackgroundWoodchucks chronically infected with woodchuck hepatitis virus (WHV), which resembles human hepatitis B virus, develop spontaneous hepatic tumors and may be an important biological and immunological model for human HCC. Nonetheless, this model requires further validation to fully realize its translational potential.MethodsWoodchucks infected at birth with WHV that had developed HCC (n=12) were studied. Computed tomography, ultrasound, and magnetic resonance imaging were performed under anesthesia. LI-RADS scoring and correlative histologic analysis of sectioned tissues were performed. For immune characterization of tumors, CD3 (T cells), CD4 (T helpers), NCAM (Natural killers), FOXP3 (T-regulatory), PDL-1 (inhibitory checkpoint protein), and the human hepatocellular carcinoma (HCC) biomarker alpha-fetoprotein (AFP) immunohistochemical stains were performed.ResultsForty tumors were identified on imaging of which 29 were confirmed to be HCC with 26 categorized as LR-4 or 5. The remainder of the tumors had benign histology including basophilic foci, adenoma, and lipidosis as well as pre-malignant dysplastic foci. LR-4 and LR-5 lesions showed high sensitivity (90%) and specificity (100%) for malignant and pre-malignant tumors. Natural killers count was found to be 2–5 times lower in tumors relative to normal parenchyma while other immune cells were located in the periphery of tumors. Tumors expressed AFP and did not express PD-L1.ConclusionWoodchucks chronically infected with WHV developed diverse hepatic tumor types with diagnostic imaging, pathology, and immune patterns comparable to that in humans. This unique animal model may provide a valuable tool for translation and validation of novel image-guided and immune-therapeutic investigations.

Serum Response Factor (SRF) Drives the Transcriptional Upregulation of the MDM4 Oncogene in HCC.

Simple SummaryHepatocellular carcinoma (HCC) represents the most common type of liver cancer and has a poor prognosis. Therefore, there is an urgent need for the identification of new therapeutic options. The mouse double minute homolog 4 (MDM4) gene, a known p53 inhibitor, is upregulated in most HCCs. Here, we aimed to investigate the mechanisms leading to MDM4 transcriptional upregulation and to evaluate whether therapeutic targeting of these mechanisms might represent a suitable approach for future therapy. Using human HCC cell lines, a mouse model, and human HCC cohorts, we have identified serum response factor (SRF), ETS transcription factors ELK1 and ELK4 as transcription factors (TFs) driving MDM4 expression. Treatment of HCC cell lines with XI-011, a pharmaceutical inhibitor of MDM4 transcription, reduced the expression of both the TFs and MDM4 and impaired tumor growth, suggesting that targeting the MDM4 transcription may provide a rationale for future targeted therapy of HCC. Different molecular mechanisms support the overexpression of the mouse double minute homolog 4 (MDM4), a functional p53 inhibitor, in human hepatocellular carcinoma (HCC). However, the transcription factors (TFs) leading to its transcriptional upregulation remain unknown. Following promoter and gene expression analyses, putative TFs were investigated using gene-specific siRNAs, cDNAs, luciferase reporter assays, chromatin immunoprecipitation, and XI-011 drug treatment in vitro. Additionally, MDM4 expression was investigated in SRF-VP16iHep transgenic mice. We observed a copy-number-independent upregulation of MDM4 in human HCCs. Serum response factor (SRF), ELK1 and ELK4 were identified as TFs activating MDM4 transcription. While SRF was constitutively detected in TF complexes at the MDM4 promoter, presence of ELK1 and ELK4 was cell-type dependent. Furthermore, MDM4 was upregulated in SRF-VP16-driven murine liver tumors. The pharmacological inhibitor XI-011 exhibited anti-MDM4 activity by downregulating the TFs driving MDM4 transcription, which decreased HCC cell viability and increased apoptosis. In conclusion, SRF drives transcriptional MDM4 upregulation in HCC, acting in concert with either ELK1 or ELK4. The transcriptional regulation of MDM4 may be a promising target for precision oncology of human HCC, as XI-011 treatment exerts anti-MDM4 activity independent from the MDM4 copy number and the p53 status.

Clinical Impact of Herpesvirus Entry Mediator (HVEM) in Malignant Liver Tumors

Background: Herpes virus entry mediator (HVEM), also known as tumor necrosis factor receptor (TNFR) superfamily 14, regulates a variety of physiological and pathological responses in both innate and acquired immunity. Recently, HVEM is also suggested to be a critical regulator in tumor immunity. This study aimed to clarify clinical importance of HVEM in human hepatocellular carcinoma (HCC) and colorectal liver metastasis (CRLM). Methods: This study examined 150 patients with HCC and 104 patients with CRLM who underwent curative liver resection at Nara Medical University between 2000 and 2014. Immunohistochemical staining was performed using antibodies against HVEM, CD4, CD8, and CD45RO. Results: High HVEM expression was observed in 66 of 150 patients (44.0%) with HCC, and 49 of 104 patients (47.1%) with CRLM. Expression of HVEM was not associated tumor size, number of tumors, or histologic differentiation. The high-HVEM group exhibited significantly worse overall survival (OS) than the low-HVEM group (HCC: P=0.002, CRLM: P = 0.002). Multivariate analysis showed that independent poor prognostic factors of OS for HCC were high HVEM expression in HCC and tumor size >5 cm, while in CRLM, high HVEM expression in CRLM, age of 70 years or older, and having five or more tumors were prognostic factors of OS. HVEM status was inversely correlated with tumor-infiltrating CD8+ and CD45RO+ lymphocytes in both HCC and CRLM. Conclusions: Tumour-expressing HVEM might play a critical role in human HCC and CRLM, possibly through regulating immune evasion. Therefore, targeting HVEM may be a novel promising therapeutic strategy for HCC and CRLM.

Praeruptorin A reduces metastasis of human hepatocellular carcinoma cells by targeting ERK/MMP1 signaling pathway.

Praeruptorin A (PA) is one of the active ingredients found in the dried root of Peucedanum praeruptorum Dunn, has been reported to possess anticancer effects against various types of cancer. However, the effect of PA on human hepatocellular carcinoma (HCC) remains uncleared. In this study, our results indicated that PA did not induce cytotoxicity or alter cell cycle distribution in human HCC cells (Huh-7, SK-Hep-1, and PLC/PRF/5 cells). Instead, PA inhibited the migration and invasion of human HCC cells while downregulating the expression of matrix metalloproteinase-1 (MMP1) and activating the extracellular signal-regulated kinase (ERK) signaling pathways. Furthermore, blocking the ERK signaling pathway through siERK restored the expression of MMP1 and the invasive ability of PA-treated HCC cells. In conclusion, our results demonstrate the antimetastatic activity of PA against human HCC cells, supporting its potential as a therapeutic agent of HCC treatments.

Yes-associated protein (YAP) induces a secretome phenotype and transcriptionally regulates plasminogen activator Inhibitor-1 (PAI-1) expression in hepatocarcinogenesis

BackgroundOverexpression and nuclear enrichment of the oncogene yes-associated protein (YAP) cause tumor initiation and support tumor progression in human hepatocellular carcinoma (HCC) via cell autonomous mechanisms. However, how YAP expression in tumor cells affects intercellular communication within the tumor microenvironment is not well understood.MethodsTo investigate how tumor cell-derived YAP is changing the paracrine communication network between tumor cells and non-neoplastic cells in hepatocarcinogenesis, the expression and secretion of cytokines, growth factors and chemokines were analyzed in transgenic mice with liver-specific and inducible expression of constitutively active YAP (YAPS127A). Transcriptomic and proteomic analyses were performed using primary isolated hepatocytes and blood plasma. In vitro, RNAinterference (RNAi), expression profiling, functional analyses and chromatin immunoprecipitation (ChIP) analyses of YAP and the transcription factor TEA domain transcription factor 4 (TEAD4) were performed using immortalized cell lines. Findings were confirmed in cohorts of HCC patients at the transcript and protein levels.ResultsYAP overexpression induced the expression and secretion of many paracrine-acting factors with potential impact on tumorous or non-neoplastic cells (e.g. plasminogen activator inhibitor-1 (PAI-1), C-X-C motif chemokine ligand 13 (CXCL13), CXCL16). Expression analyses of human HCC patients showed an overexpression of PAI-1 in human HCC tissues and a correlation with poor overall survival as well as early cancer recurrence. PAI-1 statistically correlated with genes typically induced by YAP, such as connective tissue growth factor (CTGF) and cysteine rich angiogenic inducer 61 (CYR61) or YAP-dependent gene signatures (CIN4/25). In vitro, YAP inhibition diminished the expression and secretion of PAI-1 in murine and human liver cancer cell lines. PAI-1 affected the expression of genes involved in cellular senescence and oncogene-induced senescence was confirmed in YAPS127A transgenic mice. Silencing of TEAD4 as well as treatment with the YAP/TEAD interfering substance Verteporfin reduced PAI-1 expression. ChIP analyses confirmed the binding of YAP and TEAD4 to the gene promoter of PAI-1 (SERPINE1).ConclusionsThese results demonstrate that the oncogene YAP changes the secretome response of hepatocytes and hepatocyte-derived tumor cells. In this context, the secreted protein PAI-1 is transcriptionally regulated by YAP in hepatocarcinogenesis. Perturbation of these YAP-dependent communication hubs including PAI-1 may represent a promising pharmacological approach in tumors with YAP overexpression.99z8_srgZpQ-KvP6pFLwuFVideo abstract

Abstract 2976: Targeting fibroblast growth factor (FGF) signals induced type I IFN regulated response and Immunogenic cell death (ICD) markers in tumor cells with activated FGF signals

Abstract Introduction: The FGF family plays key roles in many developmental and physiological processes. Aberrant activation of FGF signals (receptor mutations, fusions, and amplifications of ligands or receptors) have been reported, and considered to cause malignant cancers. Targeting FGFR suppresses proliferation and survival of tumor cells with activated FGF signal. However, the role of tumor FGFR inhibition in tumor microenvironments (TMEs) is largely still unknown. Lenvatinib (LEN) is a multi-targeted tyrosine kinase inhibitor that mainly inhibits VEGFR1-3 and FGFR1-4. Previously we showed that LEN inhibited tumor FGF signal, and induced cancer cell death, which had a role in antitumor activity. Here, we investigated roles of targeting tumor FGFR in TMEs using LEN or FGFR1-3 specific inhibitor E7090 in tumor cells with activated FGF signals. Methods: To examine effects of FGF signal on gene expression profile (GEP) of tumor cells, mouse breast tumor 4T1 cell lines expressing Luc2 and AcGFP (4T1-Luc), human hepatocellular carcinoma (HCC) Hep3B2.1-7 and HuH-7 cells with FGF19 overexpression, and an endometrial cancer (EC) AN3CA cell line harboring FGFR2 mutation were treated with LEN or E7090. GEP was analyzed by RNA-Seq or qPCR in the 4T1-Luc model. Pathway enrichment analysis using RNA-Seq data was conducted by Ingenuity pathway analysis (IPA). Expression levels of type I IFN-related genes were analyzed in HCC and EC cell lines. Expressions of Cxcl10 and HMGB1 in culture supernatant were measured by ELISA. Cell surface expressions of PD-L1, MHC class I molecules and Calreticulin (CRT) were determined by flow cytometry. Results: Unbiased pathway analysis using RNA-Seq data (fold change &amp;gt;2 or &amp;lt;0.5, TPM in NT-average &amp;gt;1) from 4T1-Luc cells treated with LEN or E7090 identified Interferon (IFN) signal as one of the top 3 most significantly regulated pathways with several genes known to regulate tumor immunity. qPCR analysis showed that LEN and E7090 increased expression of type I IFN-related genes in human HCC and EC cell lines. LEN significantly increased CXCL10 in culture supernatant and expression of MHC class I and PD-L1 on cell surfaces of 4T1-Luc and Hep3B2.1-7 cells. E7090 also increased Cxcl10 in culture sup and expressions of MHC class I and PD-L1 on cell surface of 4T1-Luc cells. In addition to type I IFN-response, we found that LEN and E7090 might regulate ICD evidenced by increased expression levels of cell surface CRT, and Len increased secreted HMGB1 in culture sup of Hep3B2.1-7 cells. Conclusion: Our results suggested that tumor FGF signals modulated immune response in TMEs through regulating interferon signal. Tumor FGF signal inhibition by LEN and E7090 may enhance antitumor immune response in the TMEs of that were immune suppressive with activated FGF signals. Further analyses will be warranted. Citation Format: Taisuke Hoshi, Yu Kato, Yasuhiro Funahashi. Targeting fibroblast growth factor (FGF) signals induced type I IFN regulated response and Immunogenic cell death (ICD) markers in tumor cells with activated FGF signals [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2976.