Jie-Yi Shi

CXCR6 Upregulation Contributes to a Proinflammatory Tumor Microenvironment That Drives Metastasis and Poor Patient Outcomes in Hepatocellular Carcinoma

CXC chemokines and their cognate receptors have been implicated widely in cancer pathogenesis. In this study, we report a critical causal relationship between CXCR6 expression and tumorigenesis in the setting of human hepatocellular carcinoma (HCC). Among the CXC chemokine receptors, only CXCR6 was detected in all the hepatoma cell lines studied. Moreover, in HCC tissue, CXCR6 expression was significantly higher than in noncancerous liver tissues. Reduction of CXCR6 or its ligand CXCL16 in cancer cells reduced cell invasion in vitro and tumor growth, angiogenesis, and metastases in vivo. Importantly, loss of CXCR6 led to reduced Gr-1+ neutrophil infiltration and decreased neoangiogenesis in hepatoma xenografts via inhibition of proinflammatory cytokine production. Clinically, high expression of CXCR6 was an independent predictor of increased recurrence and poor survival in HCCs. Human HCC samples expressing high levels of CXCR6 also contained an increased number of CD66b+ neutrophils and microvessels, and the combination of CXCR6 and neutrophils was a superior predictor of recurrence and survival than either marker used alone. Together, our findings suggest that elevated expression of CXCR6 promotes HCC invasiveness and a protumor inflammatory environment and is associated with poor patient outcome. These results support the concept that inhibition of the CXCR6-CXCL16 pathway may improve prognosis after HCC treatment.

Margin-Infiltrating CD20+ B Cells Display an Atypical Memory Phenotype and Correlate with Favorable Prognosis in Hepatocellular Carcinoma

Purpose: The role of infiltrating B cells in hepatocellular carcinoma has been overlooked for many years. This study is aimed to delineate the distribution, prognostic value, and functional status of B cells in human hepatocellular carcinoma. Experimental design: Immunohistochemistry was used to investigate the distribution and clinical significance of infiltrating CD20+ B cells in a series of 120 patients with hepatocellular carcinoma. The results were further tested in an independent series of 200 patients with hepatocellular carcinoma. The functional status of CD20+ B cells was determined by flow cytometry, immunofluorescence, and in vitro coculture assay. Results: Infiltrating CD20+ B cells were predominantly concentrated in the tumor invasive margin, compared with the peri- and intratumor areas. High density of margin-infiltrating B lymphocytes (MIL-B) positively correlated with small tumor size, absence of vascular invasion, and increased density of CD8+ T cells (P < 0.05). Survival analyses revealed that increased number of MIL-Bs and their penetration through the tumor capsule were significantly associated with improved overall and recurrence-free survival, and were identified as independent prognosticators for patients with hepatocellular carcinoma (P < 0.05). Importantly, the results were further validated in another independent hepatocellular carcinoma cohort. Moreover, we found that MIL-Bs featured an atypical memory phenotype (IgD−IgG+CD27−CD38−), expressed surface markers characteristic of antigen-presenting cells, possessed tumor-killing potential by producing IFN-γ, interleukin 12p40 (IL-12p40), granzyme B, and TRAIL, and acted in cooperation with CD8+ T cells. Conclusions: The profile of CD20+ B cells in situ is a new predictor of prognosis for patients with hepatocellular carcinoma and provides a novel target for an optimal immunotherapy against this fatal malignancy. Clin Cancer Res; 19(21); 5994–6005. ©2013 AACR.

Genetic Polymorphism of the Kinesin-Like Protein KIF1B Gene and the Risk of Hepatocellular Carcinoma

Background Frequent deletions of the kinesin-like protein gene 1B (KIF1B) have been reported in neural tumors. Recently, a genome-wide association study revealed an association between polymorphisms in the KIF1B gene and the risk of hepatocellular carcinoma (HCC), and several case-control studies have further investigated this relationship. However, these studies have yielded controversial results. We therefore performed a meta-analysis to derive a more precise estimation of the association between the KIF1B gene polymorphisms and HCC risk. Methodology/Principal Finding PubMed, EMBASE, the ISI Web of Science and the CNKI databases were systematically searched to identify relevant studies. A total of 5 studies containing 13 cohorts with 5,773 cases and 6,404 controls were included. Odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were used to assess the strength of the associations. Subgroup analyses were conducted based on ethnicities, sample sizes and quality scores. Overall, the G allele at rs17401966 of the KIF1B gene was associated with a significantly decreased risk for HCC (OR  = 0.81, 95%CI: 0.70–0.93; P = 0.003). Furthermore, subgroup analyses showed that the G allele at rs17401966 of the KIF1B gene significantly reduced the risk for HCC in Chinese cohorts (OR  = 0.76, 95%CI: 0.64–0.90; P = 0.002), large-sample-size cohorts (OR  = 0.80, 95%CI: 0.73–0.88, P<0.01) and high-quality cohorts (OR  = 0.78, 95%CI: 0.71–0.87, P<0.01). However, no significant associations were found in small-sample-size cohorts, studies with low-quality scores and when excluding the cohorts from the study reporting the original discovery. Conclusion/Significance These findings demonstrate that the presence of the G allele at rs17401966 of the KIF1B gene may decrease the risk for HCC and suggest that KIF1B may play a critical role in the development of HCC. High-quality studies with larger sample sizes and different ethnic populations will be of great value to further confirm these findings.

RANKL Promotes Migration and Invasion of Hepatocellular Carcinoma Cells via NF-κB-Mediated Epithelial-Mesenchymal Transition

Background Metastasis accounts for the most deaths in patients with hepatocellular carcinoma (HCC). Receptor activator of nuclear factor kappa B ligand (RANKL) is associated with cancer metastasis, while its role in HCC remains largely unknown. Methods Immunohistochemistry was performed to determine the expression of RANK in HCC tissue (n = 398). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to examine the expression of RANK, E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist and MMPs in HCC cells. Wound healing and Transwell assays were used to evaluate cell migration and invasion ability. Results We found that expression of RANK, the receptor of RANKL, was significantly higher in HCC tumor tissues than in peritumor liver tissues (p<0.001). Constitutive expression of RANK was detected in HCC cell lines, which can be up-regulated when HCC cells were stimulated with RANKL. Notably, in vitro experiments showed that activation of RANKL-RANK axis significantly promoted migration and invasion ability of HCC cells. In addition, RANKL stimulation increased the expression levels of N-cadherin, Snail, and Twist, while decreased the expression of E-cadherin, with concomitant activation of NF-κB signaling pathway. Moreover, administration of the NF-κB inhibitor attenuated RANKL-induced migration, invasion and epithelial-mesenchymal transition of HCC cells. Conclusions RANKL could potentiate migration and invasion ability of RANK-positive HCC cells through NF-κB pathway-mediated epithelial-mesenchymal transition, which means that RANKL-RANK axis could be a potential target for HCC therapy.

Diverse modes of clonal evolution in HBV-related hepatocellular carcinoma revealed by single-cell genome sequencing

Hepatocellular carcinoma (HCC) is a cancer of substantial morphologic, genetic and phenotypic diversity. Yet we do not understand the relationship between intratumor heterogeneity and the associated morphologic/histological characteristics of the tumor. Using single-cell whole-genome sequencing to profile 96 tumor cells (30-36 each) and 15 normal liver cells (5 each), collected from three male patients with HBV-associated HCC, we confirmed that copy number variations occur early in hepatocarcinogenesis but thereafter remain relatively stable throughout tumor progression. Importantly, we showed that specific HCCs can be of monoclonal or polyclonal origins. Tumors with confluent multinodular morphology are the typical polyclonal tumors and display the highest intratumor heterogeneity. In addition to mutational and copy number profiles, we dissected the clonal origins of HCC using HBV-derived foreign genomic markers. In monoclonal HCC, all the tumor single cells exhibit the same HBV integrations, indicating that HBV integration is an early driver event and remains extremely stable during tumor progression. In addition, our results indicated that both models of metastasis, late dissemination and early seeding, have a role in HCC progression. Notably, early intrahepatic spreading of the initiating clone leads to the formation of synchronous multifocal tumors. Meanwhile, we identified a potential driver gene ZNF717 in HCC, which exhibits a high frequency of mutation at both single-cell and population levels, as a tumor suppressor acting through regulating the IL-6/STAT3 pathway. These findings highlight multiple distinct tumor evolutionary mechanisms in HCC, which suggests the need for specific treatment strategies.

CCL15 recruits suppressive monocytes to facilitate immune escape and disease progression in hepatocellular carcinoma

Chemokines play a key role in orchestrating the recruitment and positioning of myeloid cells within the tumor microenvironment. However, the tropism regulation and functions of these cells in hepatocellular carcinoma (HCC) are not completely understood. Herein, by scrutinizing the expression of all chemokines in HCC cell lines and tissues, we found that CCL15 was the most abundantly expressed chemokine in human HCC. Further analyses showed that CCL15 expression was regulated by genetic, epigenetic, and microenvironmental factors, and negatively correlated with patient clinical outcome. In addition to promoting tumor invasion in an autocrine manner, CCL15 specifically recruited CCR1+ cells toward HCC invasive margin, approximately 80% of which were CD14+ monocytes. Clinically, a high density of marginal CCR1+CD14+ monocytes positively correlated with CCL15 expression and was an independent index for dismal survival. Functionally, these tumor‐educated monocytes directly accelerated tumor invasion and metastasis through bursting various pro‐tumor factors and activating signal transducer and activator of transcription 1/3, extracellular signal‐regulated kinase 1/2, and v‐akt murine thymoma viral oncogene homolog signaling in HCC cells. Meanwhile, tumor‐derived CCR1+CD14+ monocytes expressed significantly higher levels of programmed cell death‐ligand 1, B7‐H3, and T‐cell immunoglobulin domain and mucin domain‐3 that may lead to immune suppression. Transcriptome sequencing confirmed that tumor‐infiltrating CCR1+CD14+ monocytes were reprogrammed to upregulate immune checkpoints, immune tolerogenic metabolic enzymes (indoleamine and arginase), inflammatory/pro‐angiogenic cytokines, matrix remodeling proteases, and inflammatory chemokines. Orthotopic animal models confirmed that CCL15‐CCR1 axis forested an inflammatory microenvironment enriched with CCR1+ monocytes and led to increased metastatic potential of HCC cells. Conclusion: A complex tumor‐promoting inflammatory microenvironment was shaped by CCL15‐CCR1 axis in human HCC. Blockade of CCL15‐CCR1 axis in HCC could be an effective anticancer therapy.

Telomere length variation in tumor cells and cancer‐associated fibroblasts: potential biomarker for hepatocellular carcinoma

The role of telomere dysfunction and aberrant telomerase activities in hepatocellular carcinoma (HCC) has been overlooked for many years. This study aimed to delineate the variation and prognostic value of telomere length in HCC. Telomere‐specific fluorescence in situ hybridization (FISH) and qPCR were used to evaluate telomere length in HCC cell lines, tumor tissues, and isolated non‐tumor cells within the tumor. Significant telomere attrition was found in tumor cells and cancer‐associated fibroblasts (CAFs) compared to their normal counterparts, but not in intratumor leukocytes or bile duct epithelial cells. Clinical relevance and prognostic value of telomere length were investigated on tissue microarrays of 257 surgically treated HCC patients. Reduced intensity of telomere signals in tumor cells or CAFs correlated with larger tumor size and the presence of vascular invasion (p < 0.05). Shortened telomeres in tumor cells or CAFs associated with reduced survival and increased recurrence, and were identified as independent prognosticators for HCC patients (p < 0.05). These findings were validated in an independent HCC cohort of 371 HCC patients from The Cancer Genome Atlas (TCGA) database, confirming telomere attrition and its prognostic value in HCC. We also showed that telomerase reverse transcriptase promoter (TERTp) mutation correlated with telomere shortening in HCC. Telomere variation in tumor cells and non‐tumor cells within the tumor microenvironment of HCC was a valuable prognostic biomarker for this fatal malignancy.

A New Preoperative Prognostic System Combining CRP and CA199 For Patients with Intrahepatic Cholangiocarcinoma

Objectives:In this study, we aimed at investigating the preoperatively available prognostic factors for intrahepatic cholangiocarcinoma (ICC) patients and proposing a new preoperative prognostic scoring system for ICC.Methods:A total of 246 consecutive ICC patients who underwent curative hepatectomy were enrolled retrospectively and were randomly divided into training (n=164) and validation cohorts (n=82) at a ratio of 2:1. The prognostic factors were investigated in both cohorts using multivariate Cox’s proportional hazards regression model.Results:Multivariate analyses identified that two preoperative factors (serum C-reactive protein (CRP) levels >4.1 mg/l (hazard ratio (HR): 2.75, 95% CI: 1.65–4.73, P<0.001) and carbohydrate antigen 19-9 (CA19-9) levels >300 mg/ml (HR: 3.76, 95% CI: 2.18–6.49)) were independent prognostic factors for postoperative survival in the training cohort. The results were further confirmed in the validation cohort. On the basis of these data, a preoperative prognostic score (PPS) was established by allocating 0 or 1 point to the two factors, respectively. Then, both in the training and validation cohorts, the PPS showed the power to stratify patients into three distinct groups (groups with scores 2, 1, and 0) with significant difference in the risk of postoperative death.Conclusions:A new preoperative scoring system consisting of preoperative CRP and CA19-9 levels could effectively predict postoperative survival of ICC patients.

FOXP3 Is a HCC suppressor gene and Acts through regulating the TGF-β/Smad2/3 signaling pathway

BackgroundFOXP3 has been discovered to be expressed in tumor cells and participate in the regulation of tumor behavior. Herein, we investigated the clinical relevance and biological significance of FOXP3 expression in human hepatocellular carcinoma (HCC).MethodsExpression profile of FOXP3 was analyzed using real-time RT-PCR, western blotting and immunofluorescence on HCC cell lines, and immunostaing of a tissue microarray containing of 240 primary HCC samples. The potential regulatory roles of FOXP3 were dissected by an integrated approach, combining biochemical assays, analysis of patient survival, genetic manipulation of HCC cell lines, mouse xenograft tumor models and chromatin immunoprecipitation (ChIP) sequencing.ResultsFOXP3 was constitutively expressed in HCC cells with the existence of splice variants (especially exon 3 and 4 deleted, Δ3,4-FOXP3). High expression of FOXP3 significantly correlated with low serum α-fetoprotein (AFP) level, absence of vascular invasion and early TNM stage. Survival analyses revealed that increased FOXP3 expression was significantly associated with better survival and reduced recurrence, and served as an independent prognosticator for HCC patients. Furthermore, FOXP3 could potently suppress the proliferation and invasion of HCC cells in vitro and reduce tumor growth in vivo. However, Δ3,4-FOXP3 showed a significant reduction in the tumor-inhibiting effect. The inhibition of FOXP3 on HCC aggressiveness was acted probably by enhancing the TGF-β/Smad2/3 signaling pathway.ConclusionOur findings suggest that FOXP3 suppresses tumor progression in HCC via TGF-β/Smad2/3 signaling pathway, highlighting the role of FOXP3 as a prognostic factor and novel target for an optimal therapy against this fatal malignancy.