Expression patterns of programmed death ligand 1 correlate with different microenvironments and patient prognosis in hepatocellular carcinoma.

BACKGROUND: Recent clinical studies have suggested that programmed death ligand 1 (PD-L1) expression in a tumour could be a potential biomarker for PD-L1/PD-1 blockade therapies. METHODS: To better characterise PD-L1 expression in hepatocellular carcinoma (HCC), we analysed its expression patterns in 453 HCC patients by double staining for CD68 and PD-L1 using the Tyramide Signal Amplification Systems combined with immunohistochemistry. We also investigated its correlation with clinical features, prognosis and immune status. RESULTS: The results showed that PD-L1 expression on tumour cells (TCs) was negatively associated with patients' overall survival (OS; P = 0.001) and relapse-free survival (RFS; P = 0.006); however, PD-L1 expression on macrophages (Mφs) was positively correlated with OS (P = 0.017). Multivariate analysis revealed that PD-L1 expression on TCs and Mφs were both independent prognostic factors for OS (hazard ratio (HR) = 1.168, P = 0.004 for TC-PD-L1; HR = 0.708, P = 0.003 for Mφ-PD-L1). Further studies showed that Mφ-PD-L1+ tumours exhibited an activated immune microenvironment, with high levels of CD8+ T-cell infiltration and immune-related gene expression. CONCLUSION: Our study provided a novel methodology to evaluate PD-L1 expression in the tumour microenvironment, which might help to select patients who would benefit from anti-PD-1/PD-L1 immunotherapies.

Distinct patterns and prognostic values of tumor-infiltrating macrophages in hepatocellular carcinoma and gastric cancer.

BACKGROUND: Macrophages (Mφs) constitute a major component of the leukocyte infiltrate and perform distinct roles in different tumor microenvironments. This study aimed to characterize the distribution, composition and prognostic value of Mφs in hepatocellular carcinoma (HCC) and gastric cancer (GC). METHODS: Immunohistochemistry and immunofluorescence were used to identify Mφ subsets in HCC and GC tissues. Kaplan-Meier analysis and Cox regression models were applied to estimate the overall survival (OS) for HCC and GC patients. RESULTS: The results showed that the density of Mφs decreased in the intra-tumor region (IT) of HCC, but remarkably increased in the IT of GC, as compared with their non-tumor regions (NT). In HCC, most CD68+ Mφs were CD204+ and CD169+ cells in the NT region; however, there was a significant decrease in the percentage of CD169+ Mφ in the IT region. In contrast, CD68+ Mφs comprised a smaller percentage of CD204+ than the CD169+ subpopulation in the NT region, while more CD204+ but fewer CD169+ cells were present in the IT region of GC. The density of CD204+ Mφs correlated with poor prognosis in HCC, and CD169+ Mφs were associated with good survival in both HCC and GC. Moreover, the combination of low numbers of CD204+ and high numbers of CD169+ Mφs was associated with improved OS in both GC and HCC. CONCLUSIONS: Mφs display tissue-specific distributions and distinct composition patterns in HCC and GC tissues. Our results suggested that different types of tumors might use diverse strategies to reconstitute Mφ patterns to promote tumor progression.

Circulating hematopoietic stem and progenitor cells are myeloid-biased in cancer patients.

Cancer is associated with a profound perturbation in myelopoiesis that results in the accumulation of myeloid-derived suppressor cells (MDSCs) to promote disease progression. Recent studies in mice suggest that tumor-derived factors could regulate the differentiation of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and subsequently contribute to dysregulation of hematopoiesis. However, the nature and role of HPSCs in patients with cancer remain unknown. Here we show, in detailed studies of the peripheral blood from 133 untreated patients with seven different types of tumors, that the composition of circulating HSPCs was significantly altered in patients with solid tumors. The frequencies of circulating granulocyte-monocyte progenitors (GMPs) were increased four to seven fold in all types of tumors examined, and the circulating hematopoietic precursors exhibited myeloid bias with a skew toward granulocytic differentiation in patients with solid tumors. These myeloid precursors are selectively enriched in tumor tissues, and the high levels of circulating GMPs were positively correlated with disease progression. By using cord blood-derived CD34(+) cells, we developed an in vitro short-term culture model to effectively induce the rapid generation of MDSCs. We found that, among the factors produced by various tumors, GM-CSF, granulocyte colony-stimulating factor, and IL-6 could not only promote the myeloid-biased differentiation, but also induce the differentiation of myeloid precursors into functional MDSCs. These findings suggest that the altered circulating HSPCs may serve as an important link between dysregulated bone marrow hematopoiesis and accumulated MDSCs in patients with cancer.

C/EBPα mediates the maturation and antitumor functions of macrophages in human hepatocellular carcinoma.

Recent studies have suggested that therapeutic upregulation of CCAAT/enhancer binding protein α (C/EBPα) prevents hepatocellular carcinoma (HCC) progression. However, the mechanisms underlying this outcome are not fully understood. In this study, we investigated the expression and functional roles of C/EBPα in human HCC, with a focus on monocytes/macrophages (Mφs). Paraffin-embedded tissues were used to visualize C/EBPα expression and analyze the prognostic value of C/EBPα+ monocytes/Mφs in HCC patients. The underlying regulatory mechanisms were examined using human monocyte-derived Mφs. The results showed that the expression of C/EBPα on monocytes/Mφs was significantly decreased in intra-tumor tissues compared to the corresponding peri-tumor tissues. C/EBPα+ monocytes/Mφs displayed well-differentiation and antitumor capacities, and the accumulation of these cells in tissue was associated with antitumor immune responses and predicted longer overall survival (OS) of HCC patients. Mechanistic studies demonstrated that C/EBPα was required for Mφ maturation and HLA-DR, CD169 and CD86 expression, which initiates antitumor cytotoxic T-cell responses; however, these effects were inhibited by monocyte autocrine IL-6- and IL-1ß-induced suppression of mTOR1 signaling. Reprogramming Mφs via the upregulation of C/EBPα may provide a novel strategy for cancer immunotherapy in patients with HCC.

Lipid droplet accumulation mediates macrophage survival and Treg recruitment via the CCL20/CCR6 axis in human hepatocellular carcinoma.

Metabolic changes play a crucial role in determining the status and function of macrophages, but how lipid reprogramming in macrophages contributes to tumor progression is not yet fully understood. Here, we investigated the phenotype, contribution, and regulatory mechanisms of lipid droplet (LD)-laden macrophages (LLMs) in hepatocellular carcinoma (HCC). Enriched LLMs were found in tumor tissues and were associated with disease progression in HCC patients. The LLMs displayed immunosuppressive phenotypes (with extensive expression of TREM2, PD-L1, CD206, and CD163) and attenuated the antitumor activities of CD8+ T cells. Mechanistically, tumor-induced reshuffling of cellular lipids and TNFα-mediated uptake of tumoral fatty acids contribute to the generation of triglycerides and LDs in macrophages. LDs prolong LLM survival and promote CCL20 secretion, which further recruits CCR6+ Tregs to HCC tissue. Inhibiting LLM formation by targeting DGAT1 and DGAT2, which catalyze the synthesis of triglycerides, significantly reduced Treg recruitment, and delayed tumor growth in a mouse hepatic tumor model. Our results reveal the suppressive phenotypes and mechanisms of LLM enrichment in HCC and suggest the therapeutic potential of targeting LLMs for HCC patients.

Genome-wide scan for familial nasopharyngeal carcinoma reveals evidence of linkage to chromosome 4.

Nasopharyngeal carcinoma (NPC) occurs with high frequency in Asian populations, especially among people of Cantonese ancestry. In areas with high incidence, NPC clusters in families, which suggests that both geography and genetics may influence disease risk. Although the HLA-Bw46 locus is associated with increased risk of NPC, no predisposing genes have been identified so far. Here we report the results of a genome-wide search carried out in families at high risk of NPC from Guangdong Province, China. Parametric analyses provide evidence of linkage to the D4S405 marker on chromosome 4 with a logarithm of odds for linkage (lod) score of 3.06 and a heterogeneity-adjusted lod (hlod) score of 3.21. Fine mapping with additional markers flanking D4S405 resulted in a lod score of 3.54 and hlod score of 3.67 for the region 4p15.1-q12. Multipoint nonparametric linkage analysis gives lod scores of 3.54 at D4S405 (P = 5.4 x 10(-5)) and 4.2 at D4S3002 (P = 1.1 x 10(-5)), which is positioned 4.5 cM away from D4S405. When Epstein Barr virus antibody titer was included as a covariate, the lod scores reached 4.70 (P = 2.0 x 10(-5)) and 5.36 (P = 4.36 x 10(-6)) for D4S405 and D4S3002, respectively. Our findings provide evidence of a major susceptibility locus for NPC on chromosome 4 in a subset of families.

Cholesterol Efflux Drives the Generation of Immunosuppressive Macrophages to Promote the Progression of Human Hepatocellular Carcinoma.

Cholesterol is often enriched in tumor microenvironment (TME); however, its impact on disease progression varies in different tissues and cells. Monocytes/macrophages (Mφ) are major components and regulators of the TME and play pivotal roles in tumor progression and therapeutic responses. We aimed to investigate the profile, effects, and regulatory mechanisms of Mφ cholesterol metabolism in the context of human hepatocellular carcinoma (HCC). Here, we found that patients with high serum levels of cholesterol had shorter survival times and lower response rates to anti-PD-1 treatment. However, the cholesterol content in tumor-infiltrating monocytes/Mφ was significantly lower than that in their counterparts in paired nontumor tissues. The expression of the cholesterol efflux transporter, ABCA1, was upregulated in tumor monocytes/Mφ, and ABCA1 upregulation positively associated with decreased cellular cholesterol content and increased serum cholesterol levels. Mechanistically, autocrine cytokines from tumor-treated monocytes increased LXRα and ABCA1 expression, which led to the generation of immature and immunosuppressive Mφ. Although exogenous cholesterol alone had little direct effect on Mφ, it did act synergistically with tumor-derived factors to promote ABCA1 expression in Mφ with more immunosuppressive features. Moreover, high numbers of ABCA1+ Mφ in HCC tumors associated with reduced CD8+ T-cell infiltration and predicted poor clinical outcome for patients. Our results revealed that dysregulated cholesterol homeostasis, due to the collaborative effects of tumors and exogenous cholesterol, drives the generation of immunosuppressive Mφ. The selective modulation of cholesterol metabolism in Mφ may represent a novel strategy for cancer treatment.

An in situ molecular signature to predict early recurrence in hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND & AIMS: To develop an in situ molecular signature to predict postsurgical recurrence in hepatocellular carcinoma (HCC) patients. METHODS: Immunohistochemistry was performed using tissue microarrays containing both tumoral and peri-tumoral regions of the advancing tumor edge from 336 HCC patients (289 were positive for hepatitis B virus) who underwent curative resection. Forty-nine variables were analyzed in the training set (n=151) using support vector machine and stepwise algorithms to develop a classifier to predict recurrence within 1 year, which was mainly caused by invasion or metastasis from the primary tumors. The classifier was further validated in an independent cohort of 185 patients (71 internal and 114 external). RESULTS: The final signature was composed of eight IHC features: CD80(T), B7-DC(T), HLA-DR(P), FasL(P), Bcl-2(T), Ki-67(T), cyclin D1(T), and CK19(T). In the independent test set, this classifier reliably predicted recurrence within 1 year (sensitivity, 69.1%; specificity, 65.0%) with an odds ratio of 4.149 (95% CI, 2.189-7.864). Based on a multivariate logistic model, the in situ molecular signature provided significant predictive power independent of tumor number, tumor size, vascular invasion and BCLC classification (p=0.001). The highest potential clinical impact of the classifier was observed in early-stage (BCLC classification 0-A) patients (p<0.0001), and the classifier was also predictive of the time-to-recurrence and overall survival (both p<0.0001). CONCLUSIONS: This in situ molecular classifier could provide a novel approach to identify patients who are at greatest risk for postsurgical recurrence of HCC and may benefit from intensive clinical follow-up or chemopreventive strategies.

Reprogramming immunosuppressive myeloid cells by activated T cells promotes the response to anti-PD-1 therapy in colorectal cancer.

Overcoming local immunosuppression is critical for immunotherapy to produce robust anti-tumor responses. Myeloid-derived suppressor cells (MDSCs) are key regulators of immunosuppressive networks and promote tumor progression. However, it remains unclear whether and how tumor-infiltrating MDSCs are shaped in response to anti-PD-1 treatment and what their impact on therapeutic efficacy is in colorectal cancer (CRC). In this study, the levels of infiltrating MDSCs were significantly higher in the non-responding organoids and were selectively reduced in the responding group, with MDSCs showing increased apoptosis and attenuated functional activity after anti-PD-1 treatment. A negative correlation between T-cell activation and MDSC function was also observed in fresh human CRC tissues. Mechanistic studies revealed that autocrine IFN-α/ß upregulated TRAIL expression on activated T cells to elicit MDSC apoptosis via the TRAIL-DR5 interaction and acted synergistically with TNF-α to inhibit MDSC function of suppressing the T-cell response through the JNK-NMDAR-ARG-1 pathway. Moreover, blockade of IFN-α/ß and TNF-α abolished the therapeutic efficacy of anti-PD-1 treatment by preserving the frequency and suppressive activity of infiltrating MDSCs in a CRC mouse model. This result suggested that reprogramming MDSCs by IFN-α/ß and TNF-α from activated T cells was necessary for successful anti-PD-1 treatment and might serve as a novel strategy to improve the response and efficacy of anticancer therapy.

Retinoic Acid Synthesis Deficiency Fosters the Generation of Polymorphonuclear Myeloid-Derived Suppressor Cells in Colorectal Cancer.

Metabolism is reprogrammed in cancer to fulfill the demands of malignant cells for cancer initiation and progression. Apart from its effects within cancer cells, little is known about whether and how reprogramed metabolism regulates the surrounding tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSC) are key regulators of the TME and greatly affect tumor progression and therapeutic responses. In this study, our results revealed that retinol metabolism-related genes and enzymes were significantly downregulated in human colorectal cancer compared with adjacent colonic tissues, and tumors exhibited a defect in retinoic acid (RA) synthesis. Reduced ADH1-mediated retinol metabolism was associated with attenuated RA signaling and accumulated MDSCs in colorectal cancer tumors. Using an in vitro model, generating MDSCs from CD34+ myeloid precursors, we found that exogenous RA could abrogate the generation of polymorphonuclear MDSCs (PMN-MDSC) with negligible impact on myeloid differentiation. Mechanistically, RA could restrain the glycolytic capacity of myeloid cells, which in turn activated the AMP-activated protein kinase (AMPK) pathway, further impairing the suppressive capacity of myeloid cells. Supplementation with RA could significantly delay tumor growth, with reduced arginase-1-expressing myeloid cells and increased CD8+ and granzyme B+ T cells in both colitis-associated and implanted MC38 mouse colorectal cancer models. Our results indicated that the defect in ADH1-mediated RA synthesis could provide a possible mechanism that fosters the generation of PMN-MDSCs in colorectal cancer and that restoring RA signaling in the TME could serve as a promising therapeutic strategy to abrogate the generation of PMN-MDSCs.

Co-expression of nuclear and cytoplasmic HMGB1 is inversely associated with infiltration of CD45RO+ T cells and prognosis in patients with stage IIIB colon cancer.

BACKGROUND: The intratumoral infiltration of T cells, especially memory T cells, is associated with a favorable prognosis in early colorectal cancers. However, the mechanism underlying this process remains elusive. This study examined whether high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, is involved in the infiltration of T cells and disease progression in locally advanced colon cancer. METHODS: Seventy-two cases of pathologically-confirmed specimens were obtained from patients with stage IIIB (T3N1M0) colon cancer who underwent radical resection between January 1999 and May 2002 at the Cancer Center of Sun Yat-Sen University. The density of tumor-infiltrating lymphocytes (TILs) within the tumor tissue and the expression of HMGB1 in the cancer cells were examined via immunohistochemical analysis. The phenotype of CD45RO+ cells was confirmed using a flow cytometric assay. The association between HMGB1 expression, the density of TILs, and the 5-year survival rate were analyzed. RESULTS: The density of CD45RO+ T cells within the tumor was independently prognostic, although a higher density of CD3+ T cells was also associated with a favorable prognosis. More importantly, the expression of HMGB1 was observed in both the nucleus and the cytoplasm (co-expression pattern) in a subset of colon cancer tissues, whereas nuclear-only expression of HMGB1 (nuclear expression pattern) existed in most of the cancer tissues and normal mucosa. The co-expression pattern of HMGB1 in colon cancer cells was inversely associated with the infiltration of both CD3+ and CD45RO+ T cells and 5-year survival rates. CONCLUSIONS: This study revealed that the co-expression of HMGB1 is inversely associated with the infiltration of CD45RO+ T cells and prognosis in patients with stage IIIB colon cancer, indicating that the distribution patterns of HMGB1 might contribute to the progression of colon cancer via modulation of the local immune response.

Glutamine Deprivation Promotes the Generation and Mobilization of MDSCs by Enhancing Expression of G-CSF and GM-CSF.

Solid tumors are often challenged by hypoxic and nutrient-deprived tumor microenvironments (TME) as tumors progress, due to limited perfusion and rapid nutrient consumption. While cancer cells can demonstrate the ability to survive in nutrient-deprived conditions through multiple intrinsic alterations, it is poorly understood how nutrient-deprived cancer cells co-opt the TME to promote cancer cell survival and tumor progression. In the present study, we found that glutamine deprivation markedly potentiated the expression of G-CSF and GM-CSF in mouse mammary cancer cells. The IRE1α-JNK pathway, which is activated by glutamine starvation, was found to be important for the upregulation of these cytokines. G-CSF and GM-CSF are well-known facilitators of myelopoiesis and mobilization of hematopoietic progenitor cells (HPC). Consistently, as tumors progressed, we found that several myeloid HPC compartments were gradually decreased in the bone marrow but were significantly increased in the spleen. Mechanistically, the HPC-maintaining capacity of the bone marrow was significantly impaired in tumor-bearing mice, with lower expression of HPC maintaining genes (i.e., CXCL12, SCF, ANGPT1, and VCAM1), and reduced levels of mesenchymal stem cells and CXCL12-producing cells. Furthermore, the mobilized HPCs that displayed the capacity for myelopoiesis were also found to accumulate in tumor tissue. Tumor-infiltrating HPCs were highly proliferative and served as important sources of immunosuppressive myeloid-derived suppressor cells (MDSCs) in the TME. Our work has identified an important role for glutamine starvation in regulating the expression of G-CSF and GM-CSF, and in facilitating the generation of immunosuppressive MDSCs in breast cancer.

The dominance of China 1 in the spectrum of Epstein-Barr virus strains from Cantonese patients with nasopharyngeal carcinoma.

Nasopharyngeal carcinoma is a disease with a remarkable geographic and ethnic distribution, and has a high incidence in southern China. Infection with Epstein-Barr virus (EBV) is an important contributing factor. The profile of EBV strains in Cantonese patients from Guangdong, the nasopharyngeal carcinoma endemic region in southern China, is described on the sequence variations in latent membrane protein 1 carboxyl-terminus. The results show that China 1 was the dominant EBV strain detected in both the tumor biopsies and samples of throat washings, whereas multiple strains, including China 1, China 2, B95-8, and Med, were detected in blood samples. In addition, a new strain named China 4 was found in blood samples. These findings suggest that the host population is susceptible to the predominant China 1 strain in the nasopharyngeal carcinoma endemic region of China, but its relationship with the host remains to be characterized further.

Macrophages induce CD47 upregulation via IL-6 and correlate with poor survival in hepatocellular carcinoma patients.

CD47 is known to be involved in phagocyte-mediated tumor clearance; however, its expression, clinical significance, and regulatory mechanism in hepatocellular carcinoma (HCC) remain poorly understood. In the present study, we found that upregulation of CD47 expression on tumor cells was correlated with poor overall survival and recurrence-free survival in patients with HCC. Abundance of macrophages (Mϕs) infiltration was found in CD47+ tumor tissues. Mechanistic studies revealed that IL-6 derived from tumor-infiltrating Mϕs could upregulate CD47 expression on hepatoma cells through activation of the STAT3 pathway. Neutralization of CD47 or disruption of the IL-6-STAT3 axis reduced the ability of tumor cells to escape phagocytosis. Moreover, CD47 blockade could enhance Mϕ-mediated phagocytosis in the presence of chemotherapeutic drugs, and HCC patients with lower CD47 expression were more likely to benefit from adjuvant transcatheter arterial chemoembolization (TACE) treatment. These findings revealed that Mϕ-derived IL-6 was responsible for CD47 expression on hepatoma cells, which might be served as a potential prognostic marker and a predictor for patients who might benefit from adjuvant TACE treatment.

EZH2 negatively regulates PD-L1 expression in hepatocellular carcinoma.

BACKGROUND: Accumulating studies suggest that targeting epigenetic modifications could improve the efficacy of tumor immunotherapy; however, the mechanisms underlying this phenomenon remain largely unknown. Here, we investigated the ability of the epigenetic modifier, enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), to regulate the expression of immune checkpoint inhibitor, programmed death-1 ligand 1 (PD-L1) in hepatocellular carcinoma (HCC). METHODS: Immunohistochemistry and multiplex immunofluorescence staining were performed to analyze the expression and correlation of EZH2 and PD-L1 in HCC tissues. Immunoblotting, quantitative real-time PCR, flow cytometry, chromatin immunoprecipitation, and dual-luciferase reporter gene assays were performed to evaluate the regulatory roles of EZH2 on PD-L1 expression. RESULTS: In vitro cell experiments revealed that EZH2 negatively regulated the PD-L1 expression of hepatoma cell lines in IFNγ-dependent manner. Mechanistic studies demonstrated that EZH2 could suppress PD-L1 expression by upregulating the H3K27me3 levels on the promoters of CD274 (encoding PD-L1) and interferon regulatory factor 1 (IRF1), an essential transcription factor for PD-L1 expression, without affecting the activation of the IFNγ-signal transducer and activator of transcription 1 (STAT1) pathway. Clinical samples from HCC patients with immune-activated microenvironments showed negative correlations between EZH2 and PD-L1 expression in hepatoma cells. Multivariate Cox analysis demonstrated that the combination of EZH2 and PD-L1 was an independent prognostic factor for both OS and RFS for patients with HCC. CONCLUSIONS: The epigenetic modificator EZH2 can suppress the expression of immune checkpoint inhibitor PD-L1 by directly upregulating the promoter H3K27me3 levels of CD274 and IRF1 in hepatoma cells, and might serve as a potential therapeutic target for combination of immunotherapy for immune-activated HCC.

Immunosuppressive Immature Myeloid Cell Generation Is Controlled by Glutamine Metabolism in Human Cancer.

Tumor-associated myeloid cells are one of the prominent components of solid tumors, serving as major immune regulators for the tumor microenvironment (TME) and an obstacle for immune-checkpoint blocking (ICB) therapy. However, it remains unclear how metabolic processes regulate the generation of suppressive myeloid cells in the TME. Here, we found that hematopoietic precursor cells are enriched in the tissues of several types of human cancer and can differentiate into immature myeloid cells (IMC). Tumor-infiltrating IMCs are highly immunosuppressive, glycolytic, and proliferative, as indicated by high levels of M-CSFR, Glut1, and Ki67. To elucidate the role of metabolism in regulating the generation of IMCs, we induced suppressive IMCs from hematopoietic precursor cells with GM-CSF and G-CSF in vitro We found that the generation of suppressive IMCs was accompanied by increased glycolysis, but not affected by glucose deprivation due to alternative catabolism. Generation of IMCs relied on glutaminolysis, regardless of glucose availability. Glutamine metabolism not only supported the expansion of IMCs with glutamine-derived α-ketoglutarate but also regulated the suppressive capacity through the glutamate-NMDA receptor axis. Moreover, inhibition of glutaminase GLS1 enhanced the therapeutic efficacy of anti-PD-L1 treatment, with reduced arginase 1+ myeloid cells, increased CD8+, IFNγ+ and granzyme B+ T cells, and delayed tumor growth in an ICB-resistant mouse model. Our work identified a novel regulatory mechanism of glutamine metabolism in controlling the generation of suppressive IMCs in the TME.

Combined prognostic value of the cancer stem cell markers CD47 and CD133 in esophageal squamous cell carcinoma.

BACKGROUND: Treatments based on the inhibition of pivotal signals of cancer stem cells (CSCs) are on a promising track. Recent studies have shown that targeting CSCs with broader immune-based therapeutic methods, for example, the anti-CD47 treatment, may serve as a more potent strategy for eliminating these intractable cells. We aimed to explore the prognostic effects of CD47/CD133 and the potential therapeutic significance of CD47 in esophageal squamous cell carcinoma (ESCC). METHODS: Immunohistochemistry was employed to identify the characteristics of CD47 and CD133 in 26 pairs of tumor tissues and adjacent non-tumor tissues and 136 ESCC tissues. Kaplan-Meier analysis and Cox proportional hazards models were built for estimating the prognostic values of CD47 and CD133 expression and their combined stemness index. Sphere formation assays were undertaken to explore the effects of CD47 inhibition on primary human ESCC CSCs. RESULTS: Results conclude that CD47 and CD133 expression is increased in tumor tissues as compared to adjacent non-tumor tissues. A positive correlation between CD47/CD133 expression and differentiation was found in 136 ESCC patients. Survival analysis indicated that patients with high CD47 or CD133 expression exhibited poor overall survival and progression-free survival (PFS). The combination of high CD47 and CD133 expression was a reliable independent prognostic factor for both OS (HR = 1.940, 95% CI = 1.399-2.690, P < 0.0001) and progression-free survival (HR = 1.883, 95% CI = 1.384-2.562, P < 0.0001). Notably, CD47+ CD133+ ESCC cells were observed to possess the characteristics of CSCs, and anti-CD47 treatment veritably eliminated the CSCs pool. CONCLUSIONS: The stemness index determined by the expression of CD47 and CD133 is a promising prognostic predictor, and CD47 is a potential therapeutic target for CSCs in ESCC patients.

A functional variant in the transcriptional regulatory region of gene LOC344967 cosegregates with disease phenotype in familial nasopharyngeal carcinoma.

Nasopharyngeal carcinoma is a common malignancy in Southeast Asian countries, and genetic background is a well-known component of the complexity underlying its tumorigenic process. We have mapped a nasopharyngeal carcinoma susceptibility locus to chromosome 4p15.1-q12 in a previous linkage study on nasopharyngeal carcinoma pedigrees. In this study provided in this communication, we screened all the genes in this region, with a focus on exons, promoters, and the exon-intron boundary to identify nasopharyngeal carcinoma-associated mutations or functional variants. Importantly, we found a novel gene (LOC344967) with a single nucleotide polymorphism -32G/A in the promoter region. This gene is a member of the acyl CoA thioesterase family that plays an important role in fatty acid metabolism and is involved in the progression of various types of tumors. The -32A variant was found cosegregated with the disease phenotype in the nasopharyngeal carcinoma pedigrees that we previously used for the linkage study. Moreover, this -32A variant creates an activator protein (AP-1)-binding site in the transcriptional regulatory region of LOC344967, which significantly enhanced the binding of AP-1 to the promoter region and the transcription activity of the promoter in vivo. Furthermore, the expression of LOC344967 was significantly up-regulated at both mRNA and protein levels in nasopharyngeal carcinoma cells sharing the -32G/A genotype compared with nasopharyngeal carcinoma cells with the -32G/G genotype. Collectively, these results provide evidence that the -32A variant is a functional sequence change and may be related to nasopharyngeal carcinoma susceptibility in the families studied.

Monocytes/Macrophages promote vascular CXCR4 expression via the ERK pathway in hepatocellular carcinoma.

We recently identified CXCR4 as a novel vascular marker for vessel sprouting in hepatocellular carcinoma (HCC) tissues. Thus, CXCR4+ endothelial cells (ECs) could serve as a potential predictor for patients who may benefit from sorafenib treatment; however, the mechanism that regulates vascular CXCR4 expression in HCC remains largely unknown. Here, we revealed a large number of monocytes/macrophages (Mo/Mϕ) to be selectively enriched in the perivascular areas of CXCR4+ vessels in HCC samples. The depletion of Mo/Mϕ with gadolinium chloride (GdCl3) or zoledronic acid (ZA) treatment significantly reduced vascular CXCR4 expression in HCC tumors. This phenomenon was also confirmed in CCR2-KO mice, which exhibited reduced infiltration of inflammatory Mo/Mϕ in tumor tissues. Mechanistic studies revealed that inflammatory cytokines derived from tumor conditioned Mo/Mϕ, especially TNF-α, could up-regulate CXCR4 expression on ECs. TNF-α-induced activation of the Raf-ERK pathway, but not Notch signaling, was responsible for the expression of CXCR4. Moreover, the combination treatment of sorafenib with ZA was associated with improved anti-tumor efficacy by significantly reducing vascular CXCR4 expression. These findings revealed that Mo/Mϕ could regulate CXCR4 expression in the tumor vasculature. Thus, the inhibition of Mo/Mϕ inflammation might enhance the treatment efficacy of sorafenib in HCC.

Haplotype of gene Nedd4 binding protein 2 associated with sporadic nasopharyngeal carcinoma in the Southern Chinese population.

BACKGROUND: Bcl-3 as an oncoprotein is overexpressed in nasopharyngeal carcinoma (NPC). Nedd4 binding protein 2 (N4BP2), which is located in the NPC susceptibility locus, is a Bcl-3 binding protein. This study is aimed to explore the association between N4BP2 genetic polymorphism and the risk of NPC. METHODS: We performed a hospital-based case-control study, including 531 sporadic NPC and 480 cancer-free control subjects from southern China. PCR-sequencing was carried out on Exons, promoter region and nearby introns of the N4BP2 gene. The expression pattern of N4BP2 and Bcl-3 was also analyzed. RESULTS: We observed a statistically significant difference in haplotype blocks ATTA and GTTG between cases and controls. In addition, three novel SNPs were identified, two of which were in exons (loc123-e3l-snp2, position 39868005, A/G, Met171Val; RS17511668-SNP2, position 39926432, G/A, Glu118Lys), and one was in the intron6 (RS794001-SNP1, position 39944127, T/G). Moreover, N4BP2 was at higher levels in a majority of tumor tissues examined, relative to paired normal tissues. CONCLUSION: These data suggest that haplotype blocks ATTA and GTTG of N4BP2 is correlation with the risk of sporadic nasopharyngeal carcinoma in the Southern Chinese population and N4BP2 has a potential role in the development of NPC.