Le-Xing Yu

Wnt/β-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.

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

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.)

Prognostic Significance of AMPK Activation and Therapeutic Effects of Metformin in Hepatocellular Carcinoma

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 metformins potential in treatment of HCC. Clin Cancer Res; 19(19); 5372–80. ©2013 AACR.

LPS-induced down-regulation of signal regulatory protein α contributes to innate immune activation in macrophages

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

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

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)

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.

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.

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.

Signal regulatory protein α 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.

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.