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Background and aims 4‐phenylbutyric acid (4‐PBA) is a low molecular weight fatty acid that is used in clinical practice to treat inherited urea cycle disorders. In previous reports, it acted as a chemical chaperone inhibiting endoplasmic reticulum (ER) stress and unfolded protein response signaling. A few studies have suggested its function against hepatic fibrosis in mice models. However, its role in hepatocarcinogenesis remained unknown. Methods 4‐PBA was administered alone or in combination with diethylnitrosamine to investigate its long‐term effect on liver tumorigenesis. The role of 4‐PBA in oncogene‐induced hepatocellular carcinoma (HCC) mice model using sleeping beauty system co‐expressed with hMet and β‐catenin point mutation (S45Y) was also observed. RNA‐seq and PCR array were used to screen the pathways and genes involved. In vitro and in vivo studies were conducted to explore the effect of 4‐PBA on liver and validate the underlying mechanism. Results 4‐PBA alone didn't cause liver tumor in long term. However, it promoted liver tumorigenesis in HCC mice models via initiation of liver cancer stem cells (LCSCs) through Wnt5b‐Fzd5 mediating β‐catenin signaling. Peroxisome proliferator‐activated receptors (PPAR)‐α induced by 4‐PBA was responsible for the activation of β‐catenin signaling. Thus, intervention of PPAR‐α reversed 4‐PBA‐induced initiation of LCSCs and HCC development in vivo. Further study revealed that 4‐PBA could not only upregulate the expression of PPAR‐α transcriptionally but also enhance its stabilization via protecting it from proteolysis. Moreover, high PPAR‐α expression predicted poor prognosis in HCC patients. Conclusions 4‐PBA could upregulate PPAR‐α to initiate LCSCs by activating β‐catenin signaling pathway, promoting HCC at early stage. Therefore, more discretion should be taken to monitor the potential tumor‐promoting effect of 4‐PBA under HCC‐inducing environment., 4‐PBA could promote HCC at early stage of tumor development in vivo.4‐PBA initiated CSCs via activating Wnt5b‐Fzd5‐β‐catenin signaling pathway dependent of PPAR‐α activation.4‐PBA could not only regulate the expression of PPAR‐α transcriptionally, but also stabilize it via direct binding.

Hepatocellular carcinoma (HCC) has become a leading cause of cancer-related death, making the elucidation of its underlying mechanisms an urgent priority. Inflammation is an adaptive response to infection and tissue injury under strict regulations. When the host regulatory machine runs out of control, nonresolving inflammation occurs. Nonresolving inflammation is a recognized hallmark of cancer that substantially contributes to the development and progression of HCC. The HCC-associated inflammation can be initiated and propagated by extrinsic pathways through activation of pattern-recognition receptors (PRRs) by pathogen-associated molecule patterns (PAMPs) derived from gut microflora or damage-associated molecule patterns (DAMPs) released from dying liver cells. The inflammation can also be orchestrated by the tumor itself through secreting factors that recruit inflammatory cells to the tumor favoring the buildup of a microenvironment. Accumulating datas from human and mouse models showed that inflammation promotes HCC development by promoting proliferative and survival signaling, inducing angiogenesis, evading immune surveillance, supporting cancer stem cells, activating invasion and metastasis as well as inducing genomic instability. Targeting inflammation may represent a promising avenue for the HCC treatment. Some inhibitors targeting inflammatory pathways have been developed and under different stages of clinical trials, and one (sorafenib) have been approved by FDA. However, as most of the data were obtained from animal models, and there is a big difference between human HCC and mouse HCC models, it is challenging on successful translation from bench to bedside.

The communication between tumor-derived elements and stroma in the metastatic niche has a critical role in facilitating cancer metastasis. Yet, the mechanisms tumor cells use to control metastatic niche formation are not fully understood. Here we report that in the lung metastatic niche, high-metastatic hepatocellular carcinoma (HCC) cells exhibit a greater capacity to convert normal fibroblasts to cancer-associated fibroblasts (CAFs) than low-metastatic HCC cells. We show high-metastatic HCC cells secrete exosomal miR-1247-3p that directly targets B4GALT3, leading to activation of β1-integrin–NF-κB signaling in fibroblasts. Activated CAFs further promote cancer progression by secreting pro-inflammatory cytokines, including IL-6 and IL-8. Clinical data show high serum exosomal miR-1247-3p levels correlate with lung metastasis in HCC patients. These results demonstrate intercellular crosstalk between tumor cells and fibroblasts is mediated by tumor-derived exosomes that control lung metastasis of HCC, providing potential targets for prevention and treatment of cancer metastasis., How tumor cells control metastatic niche formation is not fully understood. Here, the authors show in a lung metastatic niche, high-metastatic hepatocellular carcinoma cells secrete exosomal miR-1247-3p that leads to activation of β1-integrin-NF-κBsignalling, converting fibroblasts to cancer-associated fibroblasts.

Increasing evidence suggests that the presence of endotoxemia is of substantial clinical relevance to patients with cirrhosis, but it is unclear whether and how gut-derived LPS amplifies the tumorigenic response of the liver. We found that the circulating levels of LPS were elevated in animal models of carcinogen-induced hepatocarcinogenesis. Reduction of LPS using antibiotics regimen in rats or genetic ablation of its receptor Toll-like receptor 4 (TLR4) in mice prevented excessive tumor growth and multiplicity. Additional investigation revealed that TLR4 ablation sensitizes the liver to carcinogen-induced toxicity via blocking NF-κB activation and sensitizing the liver to reactive oxygen species (ROS)-induced toxicity, but lessens inflammation-mediated compensatory proliferation. Reconstitution of TLR4-expressing myeloid cells in TLR4-deficient mice restored diethylnitrosamine (DEN)-induced hepatic inflammation and proliferation, indicating a paracrine mechanism of LPS in tumor promotion. Meanwhile, deletion of gut-derived endotoxin suppressed DEN-induced cytokine production and compensatory proliferation, whereas in vivo LPS pre-challenge promotes hepatocyte proliferation. Conclusion: Our data indicate that sustained LPS accumulation represents a pathological mediator of inflammation-associated hepatocellular carcinoma (HCC) and manipulation of the gut flora to prevent pathogenic bacterial translocation and endotoxin absorption may favorably influence liver function in patients with cirrhosis who are at risk of developing HCC. (Hepatology 2010.)

Adult hepatic progenitor (oval) cells are facultative stem cells in liver, which participate in a range of human liver diseases, including hepatocellular carcinoma (HCC). However, the molecular pathways regulating the expansion and differentiation of these cells are poorly understood. We show that active Wnt/β-catenin signaling occurs preferentially within the oval cell population, and forced expression of constitutively active β-catenin mutant promotes expansion of the oval cell population in the regenerated liver. More importantly, we identify a subpopulation of less differentiated progenitor-like cells in HCC cell lines and primary HCC tissues, which are defined by expression of the hepatic progenitor marker OV6 and endowed with endogenously active Wnt/β-catenin signaling. These OV6+ HCC cells possess a greater ability to form tumor in vivo and show a substantial resistance to standard chemotherapy compared with OV6− tumor cells. The fraction of tumor cells expressing OV6 is enriched after Wnt pathway activation, whereas inhibition of β-catenin signaling leads to a decrease in the proportion of OV6+ cells. In addition, the chemoresistance of OV6+ HCC progenitor-like cells can be reversed by lentivirus-delivered stable expression of microRNA targeting β-catenin. These results highlight the importance of the Wnt/β-catenin pathway in activation and expansion of oval cells in normal rodent models and human HCCs. OV6+ tumor cells may represent the cellular population that confers HCC chemoresistance, and therapies targeted to the Wnt/β-catenin signaling may provide a specific method to disrupt this resistance mechanism to improve overall tumor control with chemotherapy. [Cancer Res 2008;68(11):4287–95]

Background & Aims Considerable evidence suggests that adrenergic signaling played an essential role in tumor progression. However, its role in hepatocellular carcinoma (HCC) and the underlying mechanisms remain unknown. Methods The effect of adrenaline in hepatocarcinogenesis was observed in a classical diethylnitrosamine-induced HCC mouse model. Effects of ADRB2 signaling inhibition in HCC cell lines were analyzed in proliferation, apoptosis, colony formation assays. Autophagy regulation by ADRB2 was assessed in immunoblotting, immunofluorescence and immunoprecipitation assays. In vivo tumorigenic properties and anticancer effects of sorafenib were examined in nude mice. Expression levels of ADRB2 and hypoxia-inducible factor-1α (HIF1α) in 150 human HCC samples were evaluated by immunohistochemistry. Results We uncovered that adrenaline promoted DEN-induced hepatocarcinogenesis, which was reversed by the ADRB2 antagonist ICI118,551. ADRB2 signaling also played an essential role in sustaining HCC cell proliferation and survival. Notably, ADRB2 signaling negatively regulated autophagy by disrupting Beclin1/VPS34/Atg14 complex in an Akt-dependent manner, leading to HIF1α stabilization, reprogramming of HCC cells glucose metabolism, and the acquisition of resistance to sorafenib. Conversely, inhibition of ADRB2 signaling by ICI118,551, or knockdown ADRB2 expression, led to enhanced autophagy, HIF1α destabilization, tumor growth suppression, and improved anti-tumor activity of sorafenib. Consistently, ADRB2 expression correlated positively with HIF1α in HCC specimens and was associated with HCC outcomes. Conclusions Our results uncover an important role of ADRB2 signaling in regulating HCC progression. Given the efficacy of ADRB2 modulation on HCC inhibition and sorafenib resistance, adrenoceptor antagonist appears to be a putative novel treatment for HCC and chemoresistance. Lay summary ADRB2 signaling played an essential role in sustaining hepatocellular carcinoma cell proliferation and survival. ADRB2 signaling negatively regulated autophagy, leading to hypoxia-inducible factor-1α stabilization, reprogramming of hepatocellular carcinoma cells glucose metabolism, and the acquisition of resistance to sorafenib. Adrenoceptor antagonist appears to be a putative novel treatment for hepatocellular carcinoma and chemoresistance.

Hepatocellular carcinoma (HCC) is a prototype of inflammation-associated cancer. Oncoprotein Gankyrin, which mostly increases in HCC, plays a critical role in HCC development and metastasis. However, the exact mechanism of Gankyrin up-regulation in HCC remains unclear. A Gankyrin luciferase reporter was developed to screen a potential regulator for Gankyrin from a list of proinflammatory cytokines, and interleukin (IL)-1β was found as one of its activators. In clinical premalignant and malignant liver disease samples, enhanced IL-1β/interleukin-1 receptor-associated kinase 1 (IRAK-1) signaling accompanied by increased Gankyrin was observed. Lower expression of Gankyrin and phospho-IRAK-1 are favorable prognostic markers for HCC. A similar correlation was observed in the diethylnitrosamine (DEN) model of rat hepatocarcinogenesis. The results from Gankyrin reporter activity, real-time polymerase chain reaction, or immunoblotting further confirmed the up-regulation of Gankyrin by IL-1β/IRAK-1 inflammatory signaling. Moreover, a series of Gankyrin's truncated reporters were constructed, and electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were performed to analyze the properties of Gankyrin promoter. Mechanistically, the core promoter of Gankyrin contains the binding site of nuclear factor Y (NF-Y) family members, which can recruit histone acetyltransferase coactivator E1A-binding protein p300 (p300) or CREB-binding protein (CBP) to promote Gankyrin transcription. Conversely, knockdown of NF-Y, p300, or CBP inhibits Gankyrin expression. IL-1β stimulation causes sequential phosphorylation of IRAK-1, c-Jun N-terminal kinase (JNK), and p300 and enhances recruitment of the p300/CBP/NF-Y complex to Gankyrin promoter. Inhibition of phospho-JNK impairs IL-1β/IRAK-1 signaling-mediated up-regulation of Gankyrin. Conclusion: The finding of IL-1β/IRAK-1 signaling promoting Gankyrin expression through JNK and NF-Y/p300/CBP complex provides a fresh view on inflammation-enhanced hepatocarcinogenesis. (Hepatology 2015;61:585-597)

Increasing evidence suggests that TLR4 expression by tumour cells promotes tumour progression, but it is unclear whether TLR4 is involved in metastasis. Here we show that TLR4 deficiency significantly diminishes experimental lung metastasis without affecting primary tumour growth. Bone marrow transplantation experiment and application of antiplatelet agents in mice demonstrate that TLR4 on platelets plays an important role in metastasis. TLR4 is critical for platelet-tumour cell interaction in vitro. Furthermore, high-mobility group box1 (HMGB1) neutralization attenuates platelet-tumour cell interaction in vitro and metastasis in vivo in a TLR4-dependent manner, indicating that tumour cell-released HMGB1 is the key factor that interacts with TLR4 on platelets and mediates platelet-tumour cell interaction, which promotes metastasis. These findings demonstrate a mechanism by which platelets promote tumour cell metastasis and suggest TLR4, and its endogenous ligand HMGB1 as targets for antimetastatic therapies.

Purpose: The AMP-activated protein kinase (AMPK) serves as an energy sensor in eukaryotic cells and occupies a central role in linking metabolism and cancer development. However, the phosphorylation status of AMPK and its therapeutic value in human hepatocellular carcinoma (HCC) remain unclear. Experimental Design: The phosphorylation status of AMPK (Thr172) was determined by immunoblotting and immunostaining in specimens from 273 patients with HCC (including 253 patients with hepatitis B virus -related HCC). Kaplan–Meier survival analysis was used to determine the correlation with prognosis. The effects of therapeutic metformin/AMPK activation were assessed in cultured human HCC cell lines and primary HCC cells in vitro and in xenograft tumors model in vivo. To define the mechanisms of anticancer effects of metformin, we examined its influence on AMPK activation and NF-κB pathway. Results: AMPK is dysfunctional in patients with HCC, and low p-AMPK staining is correlated with aggressive clinicopathologic features and poor prognosis. Activation of AMPK by metformin not only inhibited HCC cells growth in vitro and in vivo, but also augmented cisplatin-induced growth inhibition in HCC cells. Knockdown of AMPKα expression can greatly decrease the inhibitory effect of metformin, indicating that AMPK activation is required for the anticancer action of metformin. Mechanistically, metformin/AMPK activation inhibited NF-κB signaling through upregulation of IκBα. Activation of NF-κB signaling by ectopic expression of P65 or overexpression of an undegradable mutant form of IκBα attenuated the anticancer effects of metformin. Conclusions: These results present novel insight into a critical role of AMPK in HCC progression. Anticancer effects of therapeutic metformin/AMPK activation unravel metformin's potential in treatment of HCC. Clin Cancer Res; 19(19); 5372–80. ©2013 AACR.

Robust clinical and epidemiologic data support the role of inflammation as a key player in hepatocellular carcinoma (HCC) development. Our previous data showed that gut-derived lipopolysaccharide (LPS) promote HCC development by activating Toll-like receptor 4 (TLR4) expressed on myeloid-derived cells. However, the effects of gut-derived LPS on other types of liver injury models are yet to be studied. The purpose of this study was to determine the importance of gut-derived LPS and TLR4 signaling in a T-cell–mediated hepatitis—Con A–induced hepatitis model, which mimic the viral hepatitis. Reduction of endotoxin using antibiotics regimen or genetic ablation of TLR4 in mice significantly alleviate Con A–induced liver injury by inhibiting the infiltration of T lymphocytes into the liver and the activation of CD4+ T lymphocytes as well as the production of T helper 1 cytokines; in contrast, exogenous LPS can promote Con A–induced hepatitis and CD4+ T cells activation in vivo and in vitro. Reconstitution of TLR4–expressing myeloid cells in TLR4-deficient mice restored Con A–induced liver injury and inflammation, indicating the major cell target of LPS. In addition, TLR4 may positively regulate the target hepatocellular apoptosis via the perforin/granzyme B pathway. These data suggest that gut-derived LPS and TLR4 play important positive roles in Con A–induced hepatitis and modulation of the gut microbiotia may represent a new avenue for therapeutic intervention to treat acute hepatitis induced by hepatitis virus infection, thus to prevent hepatocellular carcinoma. Cancer Prev Res; 5(9); 1090–102. ©2012 AACR.

Hepatitis B virus X (HBx) protein is implicated in hepatitis B virus (HBV)-associated liver carcinogenesis. However, it remains unclear whether HBx-expressing hepatic progenitor cells (HPCs) are attributed to liver tumor formation. In this study, by using HBx transgenic mice and a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury model, the relationship between HBx expression and tumorigenicity of HPCs was analyzed. Compared with control mice, an elevated number of EpCAM+ cells with characteristics of HPCs was observed in HBx mice after 1 month and 4 months of DDC diet feeding. All HBx transgenic mice developed liver tumors characterized by histological features of both hepatocellular carcinoma (HCC) and cholangiocarcinoma after 7 months of DDC feeding. Notably, EpCAM+ HPCs isolated from premalignant HBx mice exposed to a DDC diet for 4 months formed subcutaneous mixed-lineage tumors (four out of six) in nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice, and none of the cells from wildtype (WT) induced tumor, indicating that HBx may induce malignant transformation of HPCs that contributes to tumorigenesis. We also found higher titers of circulating interleukin (IL)-6, activities of IL-6/STAT3, and Wnt/β-catenin signaling pathways in HBx transgenic mice, suggesting HBx may induce intrinsic changes in HPCs by way of the above signaling that enables HPCs with tumorigenicity potential. Finally, clinical evidence showed that high HBx expression in human HBV-related HCC was statistically associated with expansion of EpCAM+ or OV6+ tumor cells and aggressive clinicopathologic features. Conclusion: HBx induces intrinsic cellular transformation promoting the expansion and tumorigenicity of HPCs in DDC-treated mice, which may be a possible origin for liver cancer induced by chronic hepatitis infection. (HEPATOLOGY 2012)

Local recurrence is a therapeutic challenge for radiofrequency ablation (RFA) in treatment of small solid focal malignancies. Here we show that RFA induced heat shock proteins (HSPs) expression and high mobility group box-1 (HMGB1) translocation in xenografted melanoma, which might create a proinflammatory microenvironment that favors tumor antigen presentation and activation of the effector T cells. On this basis, we investigate whether a prime-boost strategy combining a prime with heat-shocked tumor cell lysate-pulsed dendritic cell (HT-DC) followed by an in situ boost with radiofrequency thermal ablation can prevent local tumor recurrence. The combination treatment with HT-DC and RFA showed potent antitumor effects, withor=90% of tumor recurrence abrogated following RFA treatment. By contrast, prevaccination with unheated tumor lysate-pulsed DC had little effect on tumor relapse. Analysis of the underlying mechanism revealed that splenocytes from mice treated with HT-DC plus RFA contained significantly more tumor-specific, IFN-gamma-secreting T cells compared with control groups. Moreover, adoptive transfer of splenocytes from successfully treated tumor-free mice protected naive animals from tumor recurrence following RFA, and this was mediated mainly by CD8(+) T cells. Therefore, the optimal priming for the DC vaccination before RFA is important for boosting antigen-specific T cell responses and prevention of cancer recurrence.