Epigenetic silencing of BCL6B inactivates p53 signaling and causes human hepatocellular carcinoma cell resist to 5-FU

BCL6B is a potential tumor suppressor in human gastric cancer, but the regulation and mechanism of BCL6B in human hepatocellular carcinogenesis remain unclear. This study is to explore the epigenetic change and mechanism of BCL6B in human hepatocellular carcinoma (HCC). Nineteen hepatic cancer cell lines, 50 cases of adjacent tissue and 149 cases of HCC samples were employed. BCL6B is methylated in 100% (19/19) of human HCC cell lines, 40.0% (20/50) of adjacent tissue samples and 86.6% (129/149) of primary cancer samples. Methylation of BCL6B is associated with HBV positive (p < 0.05). But no association was found with age, sex, tumor size, differentiation, TNM stage, recurrence and survival. Loss of BCL6B expression was found in 19 of completely methylated HCC cell lines. BCL6B was re-expressed after 5-aza-2′-deoxycytidine treatment. Restoration of BCL6B expression suppressed cell proliferation, induced apoptosis and G1/S arrest in HCC cells. The expression of EGR1, a key component of p53 signaling, was increased after re-expression BCL6B in HCC cells. Re-expression of BCL6B activated p53 signaling and sensitized HCC cells to 5-fluorouracil. BCL6B is frequently methylated in human HCC and the expression of BCL6B is regulated by promoter region hypermethylation. BCL6B activates p53 signaling by increasing EGR1 expression in HCC.     

Upregulation of PP2Ac predicts poor prognosis and contributes to aggressiveness in hepatocellular carcinoma

Protein phosphatase 2A (PP2A) is a heterotrimeric protein phosphatase consisting of a 36-k_D catalytic C subunit (PP2Ac). This study aimed to explore the prognostic and biological significance of PP2Ac in human hepatocellular carcinoma (HCC). High PP2Ac expression was significantly (P < 0.01) associated with serum hepatitis B surface antigen positivity, serum hepatitis B e antigen positivity, liver cirrhosis, moderate to poor differentiation grade, advanced disease stage, intrahepatic metastasis, and early recurrence in HCC. Multivariate analysis revealed PP2Ac as an independent prognostic factor for overall survival. Enforced expression of hepatitis B virus X protein (HBx) and its carboxyl-terminal truncated isoform induced PP2Ac expression in HCC cells. Co-immunoprecipitation assay revealed a direct interaction between PP2Ac and HBx. Small interfering RNA-mediated knockdown of PP2Ac significantly inhibited in vitro cell proliferation, colony formation, migration, and invasion and reduced tumor growth in an xenograft mouse model. In contrast, overexpression of PP2Ac promoted HCC cell proliferation, colony formation, and tumorigenesis. Additionally, silencing of PP2Ac impaired the growth-promoting effects on HepG2 HCC cells elicited by overexpression of carboxyl-terminal truncated HBx. Gene expression profiling analysis showed that PP2Ac downregulation modulated the expression of numerous genes involved in cell cycle and apoptosis regulation. Collectively, PP2Ac upregulation has a poor prognostic impact on the overall survival of HCC patients and contributes to the aggressiveness of HCC. PP2Ac may represent a potential therapeutic target for HCC.     

A study on angiogenesis-related matrix metalloproteinase networks in primary hepatocellular carcinoma

Matrix metalloproteinases (MMPs) specially degrade extracellular matrix (ECM) proteins and are involved in tissue remodeling and angiogenesis. Therefore, studies on the role of MMPs in the carcinogenesis, proliferation and infiltration of hepatocellular carcinoma (HCC) may greatly contribute to the development of a new clinically applicable therapeutic approach. In the present study, we immunologically examined the expression rates of various MMPs including MMP-2, 3, 7, 9, membrane type 1-MMP (MT1-MMP), and MT2-MMP in the cancerous and noncancerous areas of resected tumor specimens from 30 patients with primary HCC. The rate of MMP-2 expression was high for both cancerous and noncancerous areas. However, the expression rates of MMP-3, MT1-MMP, and MT2-MMP were significantly higher in cancerous areas than in noncancerous areas. Next, we examined the clinicopathologic features such as the number of tumor nodules, maximal tumor size, presence or absence of capsular infiltration and portal vein invasion, histological grades of HCCs, state of noncancerous areas (chronic hepatitis: CH or liver cirrhosis: LC), and short-term recurrence after resection (within six months). In conclusion, it was found that three main networks of MMPs are predominantly involved in the case of HCC, that is, MMP-2 and MT1-MMP in the carcinogenesis and progression, MMP-7 and MMP-9 in the capsular infiltration and portal vein invasion, as well as MMP-3 and MMP-7 in the progression of HCC. Furthermore, MT1-MMP appeared to be the most important factor in HCC because of its widespread pattern of expression.     

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.     

Quantitative Assessment of CMTM6 in the Tumor Microenvironment and Association with Response to PD-1 Pathway Blockade in Advanced-Stage Non–Small Cell Lung Cancer

IntroductionCKLF like MARVEL transmembrane domain containing 6 (CMTM6) has been described as a programmed death ligand 1 (PD-L1) regulator at the protein level by modulating stability through ubiquitination. In this study, we describe the patterns of CMTM6 expression and assess its association with response to programmed cell death 1 pathway blockade in NSCLC.MethodsWe used multiplexed quantitative immunofluorescence to determine the expression of CMTM6 and PD-L1 in 438 NSCLCs represented in tissue microarrays, including in two independent retrospective cohorts of immunotherapy-treated (n = 69) and non–immunotherapy-treated (n = 258) patients and a third collection of EGFR- and KRAS-genotyped tumors (n = 111).ResultsTumor and stromal CMTM6 expression was detected in approximately 70% of NSCLCs. CMTM6 expression was not associated with clinical features or EGFR/KRAS mutational status and showed a modest correlation with T-cell infiltration (R² < 0.40). We found a significant correlation between CMTM6 and PD-L1, which was higher in the stroma (R² = 0.51) than in tumor cells (R² = 0.35). In our retrospective NSCLC cohort, neither CMTM6 nor PD-L1 expression alone significantly predicted immunotherapy outcomes. However, high CMTM6 and PD-L1 coexpression in the stromal and CD68 compartments (adjusted hazard ratio = 0.38, p = 0.03), but not in tumor cells (p = 0.15), was significantly associated with longer overall survival in treated patients but was not observed in the absence of immunotherapy.ConclusionThis study supports the mechanistic role for CMTM6 in stabilization of PD-L1 in patient tumors and suggests that high coexpression of CMTM6 and PD-L1, particularly in stromal immune cells (macrophages), might identify the greatest benefit from programmed cell death 1 axis blockade in NSCLC.     

B7家族新成员在肝癌中的研究进展

T细胞介导的免疫反应在机体抗肿瘤过程中发挥着重要作用。其中B7-CD28家族在T细胞的活化过程中起到关键作用。随着对B7共刺激分子的深入研究，发现了5个B7家族新成员，包括B7-H3、B7-H4、B7-H5、B7-H6、B7-H7。这5个B7家族新成员对T细胞有共刺激或共抑制作用，介导机体的抗肿瘤免疫，在肿瘤的发生、发展以及基因治疗等方面具有重要的意义。近年来研究发现这些B7家族新成员在肝癌中异常表达并与预后、病情发生进展、治疗等相关。现就B7-CD28家族新成员的基本特性和免疫调节功能及其在肝癌中的最新研究进展作一综述，以期B7家族新成员有可能成为肝癌免疫治疗的新靶点。     

Peroxisome proliferator activated receptor alpha inhibits hepatocarcinogenesis through mediating NF-κB signaling pathway

Peroxisome proliferator-activated receptor alpha (PPARα) ligands have been reported to suppress cancer growth. However, the role of PPARα in hepatocarcinogenesis remains unclear. We investigated the functional significance of PPARα in hepatocellular carcinoma (HCC). PPARα-knockout (PPARα^-/-) mice were more susceptible to diethylnitrosamine (DEN)-induced HCC at 6 months compared with wild-type (WT) littermates (80% versus 43%, P < 0.05). In resected HCCs, TUNEL-positive apoptotic cells were significantly less in PPARα^-/- mice than in WT mice (P < 0.01), commensurate with a reduction in cleaved caspase-3 and caspase-7 protein expression. Ki-67 staining showed increased cell proliferation in PPARα^-/- mice (P < 0.01), with concomitant up-regulation of cyclin-D1 and down-regulation of p15. Moreover, ectopic expression of PPARα in HCC cells significantly suppressed cell proliferation and induced apoptosis. The anti-tumorigenic function of PPARα was mediated via NF-κB as evidenced by inhibition of NF-κB promoter activity, diminution of phosphor-p65, phosphor-p50 and BCL2 levels, and enhancing IkBα protein. Chromatin immunoprecipitation analysis confirmed PPARα directly binds to the IkBα promoter. In conclusion, PPARα deficiency enhances susceptibility to DEN-initiated HCC. PPARα suppresses tumor cell growth by inhibiting cell proliferation and inducing cell apoptosis via direct targeting IκBα and NF-κB signaling pathway.     

Prognostic significance and therapeutic potential of eukaryotic translation initiation factor 5A (eIF5A) in hepatocellular carcinoma

Using comparative proteomic and genomic approaches, the authors identified eukaryotic translation initiation factor 5A (eIF5A) as an oncofetal molecule highly abundant in mouse embryonic livers and human hepatocellular carcinoma (HCC) cell lines. To evaluate the oncogenic role and prognostic significance of eIF5A in HCC, we investigate the expression patterns of the two isoforms (eIF5A1 and eIF5A2) in a cohort of 258 HCC cases by cDNA microarray. Both eIF5A isoforms were expressed in the tumors, and clinically correlated eIF5A1 with more numbers of tumor nodules and eIF5A2 with tumor venous infiltration in HCC. In a separate cohort of 50 HCCs, high level of eIF5A2, but not eIF5A1, was associated with elevated levels of deoxyhypusine synthase and deoxyhypusine hydroxylase that catalyze post‐translational hypusination of eIF5A protein. Interestingly, N1‐guanyl‐1,7‐diaminoheptane (GC7), which is an inhibitor for the first step of eIF5A hypusination, was shown to significantly impair the cell proliferation and invasion of primary HCC cells (HepG2 and Hep3B). To further demonstrate the tumorigenic role associated with eIF5A, a drastic reduction of cell proliferation was associated with suppression of eIF5A2 by transfecting Hep3B, H2‐P and H2‐M HCC cells expressing high level of this isoform using small interfering RNA (siRNA) against eIF5A2. For these assays, a milder response was usually observed in normal hepatocyte cell line. Therefore, these findings suggest that eIF5A plays an important role in HCC tumorigenesis and metastasis, and targeting eIF5A hypusination by GC7 inhibitor or eIF5A2 by RNA interference (RNAi) may offer new therapeutic alternatives to HCC patients.     

Mitotic checkpoint gene MAD1 in hepatocellular carcinoma is associated with tumor recurrence after surgical resection

OBJECTIVE: Underlying mechanism of mitotic checkpoint gene mitosis arrest deficiency 1 (MAD1) in human hepatocellular carcinoma (HCC) is rarely known. MATERIALS AND METHODS: We studied genetic change of the MAD1 gene as well as protein expression in 44 HCC and their associated non-cancerous surrounding liver tissues. RESULTS: Genotype AG of MAD1 G-1849 A promoter was highly significant in microscopic vascular invasion than other genotypes (P = 0.006). Moreover, the mean tumor size of HCC with genotype AG (7.71 cm) was significantly larger than those of other genotypes (AA, 4.41 cm; GG, 4.59 cm; P = 0.033). After a median follow-up of 22 months, 18 (41%) of the 44 patients relapsed. Eleven (32.4%) of 34 with MAD1 protein expression and 7 (70%) of 10 with no expression of MAD1 protein showed tumor recurrence. The incidence of tumor recurrence in patients with the lost MAD1 expression was significantly higher than in those with the expressed MAD1 protein (P = 0.011). CONCLUSION: These results suggest that MAD1 promoter genotype may be involved in tumor progression. Moreover, the loss of MAD1 protein expression may be related to the tumor recurrence after surgical resection of HCC.     

Tripartite motif-containing 3 (TRIM3) inhibits tumor growth and metastasis of liver cancer

Background:Reduced expression of tripartite motif-containing 3 (TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly reduced in human primary hepatocellular carcinoma (HCC) tissues and that low TRIM3 expression was associated with short survival of HCC patients.However,the role of TRIM3 in liver cancer remains unknown.This study aimed to investigate the function of TRIM3 in liver cancer cells.Methods:The protein levels of TRIM3 in five liver cancer cell lines (SK-Hep1,Hep3B,Huh7,HepG2,Bel-7402) and one normal liver cell line (L02) were detected with Western blotting.HepG2 and Bel-7402 cells with IowTRIM3 expression were infected with recombinant lentiviruses overexpressing TRIM3 (LV-TRIM3),whereas Huh7 and Hep3B cells with high TRIM3 expression were transfected with TRIM3-targeted small interfering RNA (siTRIM3).The functions of TRIM3 in the proliferation,colony formation,cell cycle,migration,invasion,and apoptosis of the above cell lines were examined.The effect of TRIM3 on tumor growth and metastases in nude mice was also investigated.Results:TRIM3 was overexpressed in HepG2 and Bel-7402 cells with LV-TRIM3 infection,which further reduced proliferation,colony formation,migration,and invasion of both cell lines.Cell cycle analysis showed thatTRIM3 overexpression induced G0/G1 phase arrest in HepG2 and Bel-7402 cells.Moreover,apoptosis was not increased in HepG2 or Bel-7402 cells overexpressing TRIM3.Contrarily,silencing TRIM3 expression in Huh7 and Hep3B cells by siTRIM3 led to significantly decreased percentages of both cells in the G0/G1 phase and promoted cell proliferation,colony formation,migration,and invasion.In vivo experiment results confirmed thatTRIM3 overexpression suppressed tumor growth and metastasis.Conclusions:TRIM3 plays a tumor-suppressing role in the regulation of liver cancer development by reducing cell proliferation through cell cycle arrest at the G0/G1 phase.     

B-myb在人肝细胞癌中表达的生物学意义及与cyclin D1相关性的研究

目的：研究人肝细胞癌（hepatocellular carcinoma，HCC）组织中B—myb及细胞周期素D1（cyclin D1）的表达，探讨其与肿瘤发生发展的关系。方法：采用免疫组化法检测60例肝癌组织及癌旁肝组织中B-myb和cyclin D1的表达情况。结果：B—myb、cyclin D1在癌组织中的阳性率分别为56．67％和50％，而在癌旁组织中的阳性率分别为36．67％和31．67％，癌组织与癌旁组织比较差异有统计学意义（P〈0．05）。B-myb在人肝癌组织中的表达与临床分期、肝外转移、术后复发及肿瘤个数相关，而与门静脉癌栓、肿瘤直径、血清AFP水平及肿瘤分化程度无明显相关性。cyclin D1在人肝癌组织中的表达与临床分期、门静脉癌栓、肝外转移、术后复发、肿瘤个数及肿瘤分化程度相关，而与肿瘤直径及血清AFP水平无明显相关。在癌组织中B-myb与cyclin D1的表达呈正相关。结论：肝癌组织中B—myb及cyclin D1高表达，可促使肝癌细胞增殖，与肝癌的发生发展密切相关。