Opposing roles of hepatic stellate cell subpopulations in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis1,2. Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts3, during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion.

Androgens drive sexual dimorphism in liver metastasis by promoting hepatic accumulation of neutrophils.

The liver is one of the most-favored distant metastatic sites for solid tumors, and interactions between cancer cells and components of the hepatic microenvironment are essential for liver metastasis (LM). Although sex is one of the determinants for primary liver cancer, sexual dimorphism in LM (SDLM) and the underlying mechanisms remain unclear. We herein demonstrate a significant male-biased SDLM, which is attributed to host androgen/androgen receptor (Ar) signaling that promotes hepatic seeding of tumor cells and subsequent outgrowth in a neutrophil-dependent manner. Mechanistically, androgen/Ar signaling promotes hepatic accumulation of neutrophils by promoting proliferation and development of neutrophil precursors in the bone marrow, as well as modulating hepatic recruitment of neutrophils and their functions. Antagonizing the androgen/Ar/neutrophil axis significantly mitigates LM in males. Our data thus reveal an important role of androgen in LM and suggest that androgen/Ar modulation represents a promising target for LM therapy in men.

4-phenylbutyric acid promotes hepatocellular carcinoma via initiating cancer stem cells through activation of PPAR-α.

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 ß-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 ß-catenin signaling. Peroxisome proliferator-activated receptors (PPAR)-α induced by 4-PBA was responsible for the activation of ß-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 ß-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.

Identification of important genes and drug repurposing based on clinical-centered analysis across human cancers.

Identification of the functional impact of mutated and altered genes in cancer is critical for implementing precision oncology and drug repurposing. In recent years, the emergence of multiomics data from large, well-characterized patient cohorts has provided us with an unprecedented opportunity to address this problem. In this study, we investigated survival-associated genes across 26 cancer types and found that these genes tended to be hub genes and had higher K-core values in biological networks. Moreover, the genes associated with adverse outcomes were mainly enriched in pathways related to genetic information processing and cellular processes, while the genes with favorable outcomes were enriched in metabolism and immune regulation pathways. We proposed using the number of survival-related neighbors to assess the impact of mutations. In addition, by integrating other databases including the Human Protein Atlas and the DrugBank database, we predicted novel targets and anticancer drugs using the drug repurposing strategy. Our results illustrated the significance of multidimensional analysis of clinical data in important gene identification and drug development.

Role of nonresolving inflammation in hepatocellular carcinoma development and progression.

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 gut microbiome and liver cancer: mechanisms and clinical translation.

Hepatocellular carcinoma (HCC) is the third leading cause of worldwide cancer mortality. HCC almost exclusively develops in patients with chronic liver disease, driven by a vicious cycle of liver injury, inflammation and regeneration that typically spans decades. Increasing evidence points towards a key role of the bacterial microbiome in promoting the progression of liver disease and the development of HCC. Here, we will review mechanisms by which the gut microbiota promotes hepatocarcinogenesis, focusing on the leaky gut, bacterial dysbiosis, microbe-associated molecular patterns and bacterial metabolites as key pathways that drive cancer-promoting liver inflammation, fibrosis and genotoxicity. On the basis of accumulating evidence from preclinical studies, we propose the intestinal-microbiota-liver axis as a promising target for the simultaneous prevention of chronic liver disease progression and HCC development in patients with advanced liver disease. We will review in detail therapeutic modalities and discuss clinical settings in which targeting the gut-microbiota-liver axis for the prevention of disease progression and HCC development seems promising.

The Role of Cancer-Associated Fibroblasts and Fibrosis in Liver Cancer.

Liver cancer is the second leading cause of cancer mortality worldwide, causing more than 700,000 deaths annually. Because of the wide landscape of genomic alterations and limited therapeutic success of targeting tumor cells, a recent focus has been on better understanding and possibly targeting the microenvironment in which liver tumors develop. A unique feature of liver cancer is its close association with liver fibrosis. More than 80% of hepatocellular carcinomas (HCCs) develop in fibrotic or cirrhotic livers, suggesting an important role of liver fibrosis in the premalignant environment (PME) of the liver. Cholangiocarcinoma (CCA), in contrast, is characterized by a strong desmoplasia that typically occurs in response to the tumor, suggesting a key role of cancer-associated fibroblasts (CAFs) and fibrosis in its tumor microenvironment (TME). Here, we discuss the functional contributions of myofibroblasts, CAFs, and fibrosis to the development of HCC and CCA in the hepatic PME and TME, focusing on myofibroblast- and extracellular matrix-associated growth factors, fibrosis-associated immunosuppressive pathways, as well as mechanosensitive signaling cascades that are activated by increased tissue stiffness. Better understanding of the role of myofibroblasts in HCC and CCA development and progression may provide the basis to target these cells for tumor prevention or therapy.

Inhibition of SIRPα in dendritic cells potentiates potent antitumor immunity.

Despite their central function in tumor immunity, dendritic cells (DCs) can respond to inhibitory signals and become tolerogenic, curtailing T cell responses in vivo. Here, we provide the evidence for an inhibitory function of signal regulatory protein (SIRP) α in DC survival and activation. In tumors from human liver cancer patients, infiltrative DCs expressed elevated levels of SIRPα, which is correlated with the induction of immune tolerance within the tumors. Silencing of SIRPα resulted in a significant increase in the longevity of antigen-pulsed DCs in the draining lymph nodes. In addition, SIRPα controls the activation and output of DCs. Silencing of DC-expressed SIRPα induced spontaneous and enhanced production of IL12 and costimulatory molecules, resulting in more potent cytotoxic T lymphocyte responses, including the eradication of previously established solid tumors. SIRPα exerted such effects, at least in part, via the association and sequestration of p85 subunit of PI3K. Thus, SIRPα is a critical regulator of DC lifespan and activity, and its inhibition might improve the clinical efficacy of DC-based tumor vaccines.

ADRB2 signaling promotes HCC progression and sorafenib resistance by inhibiting autophagic degradation of HIF1α.

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.

Platelets promote tumour metastasis via interaction between TLR4 and tumour cell-released high-mobility group box1 protein.

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.

Role of ß-Catenin in regulating the balance between TNF-α- and Fas-induced acute liver injury.

ß-Catenin plays many critical roles during various liver physiological and pathological processes. However, the role of ß-Catenin in acute liver failure remains unclear. Using hepatocyte specific ß-Catenin knockout mice, we found that loss of ß-Catenin in hepatocyte significantly reduced GalN/LPS-induced liver damage and hepatocyte apoptosis, but exacerbated Jo2-mediated liver injury. Mechanistically, the dual effects of ß-Catenin attributes on its function of inhibiting NF-κB signaling, which aggravates oxidative stress but decreases Fas expression under injury conditions. In conclusion, ß-Catenin plays an important role in regulating the balance between TNF-α and Fas-induced liver injury via its effect on NF-κB.

Profound impact of gut homeostasis on chemically-induced pro-tumorigenic inflammation and hepatocarcinogenesis in rats.

BACKGROUND & AIMS: Due to its anatomic connection, the liver is constantly exposed to gut-derived bacterial products or metabolites. Disruption of gut homeostasis is associated with many human diseases. The aim of this study was to determine the role of gut homeostasis in initiation and progression of hepatocellular carcinoma (HCC). METHODS: Disruption of intestinal homeostasis by penicillin or dextran sulfate sodium (DSS) and its restoration by probiotics were applied in a diethylnitrosamine (DEN) model of rat hepatocarcinogenesis. RESULTS: Patients with liver cirrhosis and HCC had significantly increased serum endotoxin levels. Chronic DEN treatment of rats was associated with an imbalance of subpopulations of the gut microflora including a significant suppression of Lactobacillus species, Bifidobacterium species and Enterococcus species as well as intestinal inflammation. Induction of enteric dysbacteriosis or intestinal inflammation by penicillin or DSS, respectively, significantly promoted tumor formation. Administration of probiotics dramatically mitigated enteric dysbacteriosis, ameliorated intestinal inflammation, and most importantly, decreased liver tumor growth and multiplicity. Interestingly, probiotics not only inhibited the translocation of endotoxin, which bears pathogen-associated molecular patterns (PAMPs) but also the activation of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1). As a result, the production of pro- and anti-inflammatory cytokines was skewed in favor of a reduced tumorigenic inflammation in the liver. CONCLUSIONS: The data highlights the importance of gut homeostasis in the pathogenesis of HCC. Modulation of the gut microbiota by probiotics may represent a new avenue for therapeutic intervention to treat or prevent HCC development.

Gut-derived lipopolysaccharide promotes T-cell-mediated hepatitis in mice through Toll-like receptor 4.

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.

OV6⁺ tumor-initiating cells contribute to tumor progression and invasion in human hepatocellular carcinoma.

BACKGROUND & AIMS: Accumulating evidence suggests the involvement of tumor-initiating cells (T-ICs) in cancer genesis, but whether liver T-ICs contribute to HCC invasion and metastasis remains unclear. METHODS: OV6(+) T-ICs were isolated from SMMC7721 and HuH7 cell lines by magnetic sorting. Characteristics of T-ICs were assessed by in vitro and mouse xenograft assays. Expression of OV6 was determined by immunostaining in specimens from 218 HCC patients, and Kaplan-Meier survival analysis was used to determine the correlation of OV6 expression with prognosis. RESULTS: OV6(+) T-ICs isolated from HCC cell lines not only possess a higher capacity to form tumor spheroids in vitro, but also had a greater potential to form tumors when implanted in non-obese diabetic/severe combined immunodeficient mice, suggesting their elevated self-renewal capacity and tumorigenicity. Moreover, OV6(+) T-ICs exhibited more invasive and metastatic potentials both in vitro and in vivo. Patients with more OV6(+) tumor cells were associated with aggressive clinicopathologic features and poor prognosis. CXCR4 is expressed at higher levels in OV6(+) cells. Recombinant stromal cell-derived factor-1 (SDF-1) treatment expanded the OV6(+) HCC T-ICs population, by sustaining the stem cell property of OV6(+) cells. The SDF-1 effect was blocked by a specific CXCR4 inhibitor, AMD3100, or transfection of siRNA targeting CXCR4. CONCLUSIONS: OV6(+) HCC cells may represent a subpopulation of T-ICs with augmented invasion and metastasis potential, which contribute to progression and metastasis of HCC. The SDF-1/CXCR4 axis also provides therapeutic targets for elimination of liver T-ICs.

Hepatitis B virus X (HBx) induces tumorigenicity of hepatic progenitor cells in 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated HBx transgenic mice.

UNLABELLED: 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/ß-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.

Endotoxin accumulation prevents carcinogen-induced apoptosis and promotes liver tumorigenesis in rodents.

UNLABELLED: 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.

Abrogation of local cancer recurrence after radiofrequency ablation by dendritic cell-based hyperthermic tumor vaccine.

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, with >or=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.

Wnt/beta-catenin signaling contributes to activation of normal and tumorigenic liver progenitor cells.

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/beta-catenin signaling occurs preferentially within the oval cell population, and forced expression of constitutively active beta-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/beta-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 beta-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 beta-catenin. These results highlight the importance of the Wnt/beta-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/beta-catenin signaling may provide a specific method to disrupt this resistance mechanism to improve overall tumor control with chemotherapy.

Signal regulatory protein alpha negatively regulates both TLR3 and cytoplasmic pathways in type I interferon induction.

Recognition of double-stranded RNA (dsRNA) activates interferon-regulatory factor 3 (IRF3)-dependent expression of anti-viral factors. The innate immune system recognizes viral dsRNA through two distinct pathways. First, the Toll-like receptor 3 (TLR3) detects dsRNA phagocytosed in endosomes. In addition, the helicases retinoic acid induced protein I (RIG-I)/melanoma differentiation associated gene 5 (MDA5) binds cytoplasmic dsRNA generated during viral replication. Both RIG-I/MDA5 and TLR3 can bind polyriboinosinic:polyribocytidylic acid (poly(I:C)), the synthetic analog of viral dsRNA, and mediate type I IFN production. Here we show that signal regulatory protein (SIRP) alpha negatively regulates both TLR3- and RIG-1/MDA5-dependent anti-viral pathways. Suppression of SIRPalpha expression by RNA interference results in enhanced activation of IRF3 and MAPK pathways after poly(I:C) treatment, coupled with the up-regulation of IFN-beta and IFN-beta-inducible gene transcriptional activation. The requirement of phosphoinositide 3-kinase (PI3K) activity for the induction of IFN-beta and IFN-beta-inducible genes by dsRNA is supported by the observation that a PI3K inhibitor failed to activate IFN-beta and IFN-beta-inducible gene expression. PI3K, whose activity is essential for activation of IRF3, is recruited to the phosphorylated tyrosine residues of SIRPalpha upon poly(I:C) stimulation, which lead to a reduction in the activity of the downstream kinase AKT. Thus SIRPalpha may accomplish its inhibitory function in type I IFN induction, in part, through its association and sequestration of the signal transducer PI3K.

LPS-induced down-regulation of signal regulatory protein {alpha} contributes to innate immune activation in macrophages.

Activation of the mitogen-activated protein kinases (MAPKs) and nuclear factor kappaB (NF-kappaB) cascades after Toll-like receptor (TLR) stimulation contributes to innate immune responses. Signal regulatory protein (SIRP) alpha, a member of the SIRP family that is abundantly expressed in macrophages, has been implicated in regulating MAPK and NF-kappaB signaling pathways. In addition, SIRPalpha can negatively regulate the phagocytosis of host cells by macrophages, indicating an inhibitory role of SIRPalpha in innate immunity. We provide evidences that SIRPalpha is an essential endogenous regulator of the innate immune activation upon lipopolysaccharide (LPS) exposure. SIRPalpha expression was promptly reduced in macrophages after LPS stimulation. The decrease in SIRPalpha expression levels was required for initiation of LPS-induced innate immune responses because overexpression of SIRPalpha reduced macrophage responses to LPS. Knockdown of SIRPalpha caused prolonged activation of MAPKs and NF-kappaB pathways and augmented production of proinflammatory cytokines and type I interferon (IFN). Mice transferred with SIRPalpha-depleted macrophages were highly susceptible to endotoxic shock, developing multiple organ failure and exhibiting a remarkable increase in mortality. SIRPalpha may accomplish this mainly through its association and sequestration of the LPS signal transducer SHP-2. Thus, SIRPalpha functions as a biologically important modulator of TLR signaling and innate immunity.