Limin Zheng

Identification of miRNomes in Human Liver and Hepatocellular Carcinoma Reveals miR-199a/b-3p as Therapeutic Target for Hepatocellular Carcinoma

The full scale of human miRNome in specific cell or tissue, especially in cancers, remains to be determined. An in-depth analysis of miRNomes in human normal liver, hepatitis liver, and hepatocellular carcinoma (HCC) was carried out in this study. We found nine miRNAs accounted for ∼88.2% of the miRNome in human liver. The third most highly expressed miR-199a/b-3p is consistently decreased in HCC, and its decrement significantly correlates with poor survival of HCC patients. Moreover, miR-199a/b-3p can target tumor-promoting PAK4 to suppress HCC growth through inhibiting PAK4/Raf/MEK/ERK pathway both in vitro and in vivo. Our study provides miRNomes of human liver and HCC and contributes to better understanding of the important deregulated miRNAs in HCC and liver diseases.

Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1

Macrophages (Mφ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mφ to trigger transient activation of monocytes in peritumoral stroma. We showed that a fraction of monocytes/Mφ in peritumoral stroma, but not in cancer nests, expresses surface PD-L1 (also termed B7-H1) molecules in tumors from patients with hepatocellular carcinoma (HCC). Monocytes activated by tumors strongly express PD-L1 proteins with kinetics similar to their activation status, and significant correlations were found between the levels of PD-L1+ and HLA-DRhigh on tumor-infiltrating monocytes. Autocrine tumor necrosis factor α and interleukin 10 released from activated monocytes stimulated monocyte expression of PD-L1. The PD-L1+ monocytes effectively suppressed tumor-specific T cell immunity and contributed to the growth of human tumors in vivo; the effect could be reversed by blocking PD-L1 on those monocytes. Moreover, we found that PD-L1 expression on tumor-infiltrating monocytes increased with disease progression, and the intensity of the protein was associated with high mortality and reduced survival in the HCC patients. Thus, expression of PD-L1 on activated monocytes/Mφ may represent a novel mechanism that links the proinflammatory response to immune tolerance in the tumor milieu.

Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients

BACKGROUND/AIMS To characterize IL-17-producing cells, a newly defined T helper cell subset with potent pro-inflammatory properties, in hepatocellular carcinoma (HCC) and to determine their prognostic values. METHODS One hundred and seventy-eight HCC patients were enrolled randomly. Distribution and phenotypic features of IL-17-producing cells were determined by flow cytometry and/or immunohistochemistry. RESULTS Compared with corresponding non-tumor regions, the levels of Th17 cells were significantly increased in tumors of HCC patients (P<0.001). Most intratumoral Th17 cells exhibited an effector memory phenotype with increased expression of CCR4 and CCR6. Intratumoral IL-17-producing cell density was associated with overall survival (OS, P=0.001) and disease-free survival (DFS, P=0.001) in HCC patients. Multivariate Cox analysis revealed that intratumoral IL-17-producing cell density was an independent prognostic factor for OS (HR=2.351, P=0.009) and DFS (HR=2.256, P=0.002). Moreover, the levels of intratumoral Th17 cells were positively correlated with microvessel density in tumors (r=0.616, P=0.001). CONCLUSION Accumulation of intratumoral IL-17-producing cells may promote tumor progression through fostering angiogenesis, and intratumoral IL-17-producing cell could serve as a potential prognostic marker and a novel therapeutic target for HCC.

Peritumoral neutrophils link inflammatory response to disease progression by fostering angiogenesis in hepatocellular carcinoma

BACKGROUND & AIMS Substantial evidence indicates that inflammation is a critical component of tumor progression. Hepatocellular carcinoma (HCC) is usually derived from inflamed cirrhotic liver with extensive leukocyte infiltration. Neutrophils are the common inflammatory infiltrate in tumors, but their nature and regulation in human cancers remain elusive. METHODS A total of 238 HCC patients were enrolled randomly. Immunohistochemistry and SuperArray Real-Time PCR were used to analyze the distribution and clinical relevance of neutrophils in different microanatomical areas. The regulation and function of neutrophils were assessed by both in vitro and in vivo studies. RESULTS Neutrophils were enriched predominantly in peritumoral stroma of HCC tissues and their levels could serve as a powerful predictor for poor survival in HCC patients. Proinflammatory IL-17 is a critical mediator of the recruitment of neutrophils into peritumoral stroma of HCC tissues by epithelial cell-derived CXC chemokines. The accumulated peritumoral neutrophils were the major source of matrix metalloproteinase-9 in HCC tissues; this secreted protein stimulated proangiogenic activity in hepatoma cells. Accordingly, high infiltration of peritumoral neutrophils was positively correlated with angiogenesis progression at tumor-invading edge of HCC patients. Furthermore, we found that selective depletion of neutrophils effectively inhibited tumor angiogenesis and growth, in vivo. CONCLUSIONS These data provide direct evidence supporting the critical role of neutrophils in human tumor progression and reveal a fine-tuned collaborative action between cancer cells and immune cells in distinct tumor milieu, which reroutes the inflammatory response into a tumor-promoting direction.

Activated monocytes in peritumoral stroma of hepatocellular carcinoma promote expansion of memory T helper 17 cells

Although cancer patients exhibit a generalized immunosuppressive status, substantial evidence indicates that the inflammatory reaction at a tumor site can promote tumor growth and progression. Hepatocellular carcinoma (HCC) is usually derived from inflamed cirrhotic liver with extensive leukocyte infiltration. We recently found that proinflammatory T helper (Th)17 cells are accumulated in HCC tissue, where they promote disease progression by fostering angiogenesis. Here we show that interleukin (IL)‐17‐producing cells were enriched predominantly in peritumoral stroma of HCC tissues, and their levels were well correlated with monocyte/macrophage density in the same area. Most peritumoral CD68+ cells exhibited an activated phenotype. Accordingly, tumor‐activated monocytes were significantly superior to the suppressive tumor macrophages in inducing expansion of Th17 cells from circulating memory T cells in vitro with phenotypic features similar to those isolated from HCCs. Moreover, we found that tumor‐activated monocytes secreted a set of key proinflammatory cytokines that triggered proliferation of functional Th17 cells. Inhibition of monocytes/macrophages inflammation in liver markedly reduced the level of tumor‐infiltrating Th17 cells and tumor growth in vivo. Conclusion: The proinflammatory Th17 cells are generated and regulated by a fine‐tuned collaborative action between different types of immune cells in distinct HCC microenvironments, and allows the inflammatory response of activated monocytes to be rerouted in a tumor‐promoting direction. Selectively modulating the “context” of inflammatory response in tumors might provide a novel strategy for anticancer therapy. (HEPATOLOGY 2009.)

Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization

Lysosomal membrane permeabilization (LMP) is emerging as an important regulator of cell apoptosis. Human neutrophils are highly granulated phagocytes, which respond to pathogens by exhibiting increased production of reative oxygen species (ROS) and lysosomal degranulation. In a previous study, we observed that intracellular, nonphagosomal generation of ROS triggered by adherent bacteria induced ROS‐dependent neutrophil apoptosis, whereas intraphagosomal production of ROS during phagocytosis had no effect. In the present study, we measured lysosomal membrane stability and leakage in human neutrophils and found that adherent, noningested, Type 1‐fimbriated Escherichia coli bacteria induced LMP rapidly in neutrophils. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium markedly blocked the early LMP and apoptosis in neutrophils stimulated with Type 1‐fimbriated bacteria but had no effect on the late LMP seen in spontaneously apoptotic neutrophils. The induced lysosomal destabilization triggered cleavage of the proapoptotic Bcl‐2 protein Bid, followed by a decrease in the antiapoptotic protein Mcl‐1. Involvement of LMP in initiation of apoptosis is supported by the following observations: Bid cleavage and the concomitant drop in mitochondrial membrane potential required activation of cysteine‐cathepsins but not caspases, and the differential effects of inhibitors of cysteine‐cathepsins and cathepsin D on apoptosis coincided with their ability to inhibit Bid cleavage in activated neutrophils. Together, these results indicate that in microbe‐induced apoptosis in neutrophils, ROS‐dependent LMP represents an early event in initiation of the intrinsic apoptotic pathway, which is followed by Bid cleavage, mitochondrial damage, and caspase activation.

Increased intratumoral regulatory T cells are related to intratumoral macrophages and poor prognosis in hepatocellular carcinoma patients.

Immunosuppression mediated by regulatory T cells (Tregs) is a key facilitator of tumor immune evasion, but the source of these Tregs and their contribution to human cancer progression remains unclear. This study investigated the properties of FoxP3+ Tregs, their prognostic value in patients with hepatocellular carcinoma (HCC) and the underlying mechanisms of FoxP3+ Treg intratumoral accumulation. In addition to an increased number of circulating FoxP3+ Tregs, the results also showed that FoxP3+ Tregs gathered in the tumor site, where they suppressed tissue‐derived CD4+CD25− T‐cell activation (p < 0.001), promoting disease progression and poor prognosis in HCC patients (< 0.01). The intratumoral prevalence of FoxP3+ Tregs was associated with a high density of macrophages (Mφ) (p < 0.001). Depletion of tissue Mφ thus attenuated the increase of liver FoxP3+ Treg frequency attributed to in vivo inoculation with hepatoma (p = 0.01), whereas Mφ exposed to tumor culture supernatants from hepatoma‐derived cell lines increased FoxP3+ Treg frequency in vitro (p < 0.001). This increase was partially blocked by antiinterleukin‐10 antibody (p < 0.01). In conclusion, tumor‐associated Mφ may trigger a rise of the intratumoral FoxP3+ Treg population, which in turn may promote HCC progression.

Pathogen-Induced Apoptotic Neutrophils Express Heat Shock Proteins and Elicit Activation of Human Macrophages

Ingestion of aged or irradiated apoptotic neutrophils actively suppresses stimulation of macrophages (Mφ). Many bacterial pathogens can also provoke apoptosis in neutrophils, but little is known about how such apoptotic cells influence Mφ activation. We found that neutrophils undergoing apoptosis induced by UV irradiation, Escherichia coli, or Staphylococcus aureus could either stimulate or inhibit Mφ activation. In contrast to Mφ that had ingested irradiated apoptotic neutrophils, Mφ that had phagocytosed bacteria-induced apoptotic neutrophils exhibited markedly increased production of the proinflammatory cytokine TNF-α, but not the anti-inflammatory cytokine TGF-β. Moreover, ingestion of bacteria, but not UV-induced apoptotic neutrophils, caused increased expression of FcγRI on Mφ, and this effect was not provoked directly by bacteria associated with the apoptotic neutrophils. Instead, we found that a link between pathogen-induced apoptotic neutrophils and up-regulation of the heat shock proteins HSP60 and HSP70, and we also observed that recombinant HSP60 and HSP70 potentiated LPS-stimulated production of TNF-α in Mφ. The opposing macrophage responses to neutrophils undergoing apoptosis induced in different ways may represent a novel mechanism that regulates the extent of the immune response to invading microbes in two steps: first by aiding the functions of Mφ at an early stage of infection, and subsequently by deactivating those cells through removal of uninfected apoptotic neutrophils. HSP induction in neutrophils may provide the danger signals required to generate a more effective macrophage response.

High tumor-infiltrating macrophage density predicts poor prognosis in patients with primary hepatocellular carcinoma after resection ☆,☆☆

Macrophages constitute a major component of the leukocyte infiltrate of tumors and perform distinct roles in different tumor microenvironments. This study attempted to investigate the prognostic values of tumor-infiltrating macrophages in patients with hepatocellular carcinoma after resection, paying particular attention to their tissue microlocalization. The CD68(+) macrophages were assessed by immunohistochemistry in tissues from 137 patients with hepatocellular carcinoma. Prognostic value of intratumoral, marginal, and peritumoral macrophage densities was evaluated by Kaplan-Meier analysis and Cox regression. Both intratumoral and marginal macrophage densities were associated inversely with overall survival (P = .034 and .004, respectively) and disease-free survival (P = .006 and .008, respectively). In contrast, peritumoral macrophage density was associated with neither overall survival nor disease-free survival. Intratumoral macrophage density emerged as an independent prognosticator of overall survival (hazard ratio = 1.721, P = .049) and disease-free survival (hazard ratio = 2.165, P = .007). Marginal macrophage density, but not intratumoral macrophage density, was associated with vascular invasion, tumor multiplicity, and fibrous capsule formation. Our results demonstrate that high macrophage infiltration predicts poor prognosis in patients with hepatocellular carcinoma. These results, together with our previous report showing the distinct activation patterns of macrophages in different areas of tumor tissue, implies that macrophages in those areas may use different strategies to promote the tumor progression.

Tumor-Activated Monocytes Promote Expansion of IL-17–Producing CD8+ T Cells in Hepatocellular Carcinoma Patients

The proinflammatory IL-17–producing CD8+ T cells (Tc17 cells) have recently been detected in tumors, but the nature and regulation of these cells in human tumors are presently unknown. We have recently found that IL-17+ cells are accumulated in human hepatocellular carcinomas (HCC), where they promote disease progression by fostering angiogenesis. In this study, we showed that Tc17 cells constitute a remarkable portion of IL-17–producing cells in human HCC. Although most circulating Tc17 cells were negative for IFN-γ, >80% of Tc17 cells in HCC tissues were positive for IFN-γ, and they were enriched predominantly in invading tumor edge. Most CD68+ cells located in invading tumor edge exhibited an activated phenotype and, accordingly, the activated monocytes isolated from HCC tissues were significantly superior to those isolated from nontumor tissues in inducing expansion of Tc17 cells in vitro with phenotypic features similar to those isolated from tumors. Compared with IL-17−IFN-γ+CD8+ cells, these IFN-γ+Tc17 cells have significantly higher expression of proinflammatory cytokines (IL-2, IL-22, and TNF-α), but reduced expression of granzyme B and perforin. Moreover, we found that tumor-activated monocytes secreted a set of key cytokines (IL-1β, IL-6, and IL-23) to trigger the proliferation of Tc17 cells. These data reveal an intriguing mechanism in which human Tc17 cells are generated by a fine-tuned collaborative action between different types of immune cells in distinct tumor microenvironments.