Frequent epigenetic inactivation of chromosome 3p candidate tumor suppressor genes in gallbladder carcinoma

Gallbladder carcinoma (GBC) is a highly malignant neoplasm that represents the leading cause of death for cancer in Chilean females. There is limited information about the molecular abnormalities involved in its pathogenesis. We have identified a number of molecular changes in GBC, including frequent allelic losses at chromosome 3p regions. Four distinct 3p sites (3p12, 3p14.2, 3p21.3 and 3p22-24) with frequent and early allelic losses in the sequential pathogenesis of this neoplasm have been detected. We investigated epigenetic and genetic abnormalities in GBC affecting 6 candidate tumor suppressor genes (TSG) located in chromosome 3p, including DUTT1 (3p12), FHIT (3p14.2), BLU, RASSF1A, SEMA3B and hMLH1 (3p21.3). DNA extracted from frozen tissue obtained from 50 surgical resected GBCs was examined for gene promoter methylation using MSP (methylation-specific PCR) technique after bisulfite treatment in all 6 genes. In addition, we performed PCR-based mutation examination using SSCP in FHIT and RASSF1A genes and loss of heterozygosity (LOH) analysis using microdissected tissue in a subset of tumors for the 3p21.3 region with 8 microsatellite markers. A very high frequency of GBC methylation was detected in SEMA3B (46/50, 92%) and FHIT (33/50, 66%), intermediate incidences in BLU (13/50, 26%) and DUTT1 (11/50, 22%) and very low frequencies in RASSF1A (4/50, 8%) and hMLH1 (2/50, 4%). Allelic loss at 3p21.3 was found in nearly half of the GBCs examined. We conclude that epigenetic inactivation by abnormal promoter methylation is a frequent event in chromosome 3p candidate TSGs in GBC pathogenesis, especially affecting genes SEMA3B (3p21.3) and FHIT (3p14.2).     

Chronic heavy metal exposure and gallbladder cancer risk in India, a comparative study with Japan

Background: High incidence of gallbladder cancer (GBC) is reported from North India, with elevatedconcentrations of heavy metals in water and soil. This Indo-Japan collaborative study compared presence ofheavy metals in gallbladder tissues. Methods: Heavy metal concentrations were estimated in Indian GBC andcholecystitis tissues and compared with Japanese GBC and cholecystitis tissues. Spectrophotometry was donefor 13 Indian gallbladder tissues (8 GBC, 5 cholecystitis) and 9 Japanese (5 GBC, 4 cholecystitis). Transmissionelectron microscopy (TEM) thin foil element analysis was done in 10 Indian samples (6 GBC, 4 cholecystitis).Results: Chromium, lead, arsenic and zinc were significantly high in Indian GBC compared with Japanese GBC.Chromium, lead and arsenic were significantly high in the Indian cholecystitis tissues compared to the Japanese.TEM of Indian tissues demonstrated electron dense deposits in GBC. Conclusion: Heavy metals- chromium,lead, arsenic and zinc are potential carcinogens in Indian GBC from endemic areas. This preliminary studylinks presence of heavy metals in gallbladder cancer tissues in endemic areas.     

SPOCK1 as a potential cancer prognostic marker promotes the proliferation and metastasis of gallbladder cancer cells by activating the PI3K/AKT pathway

Background

Gallbladder cancer (GBC) is a leading cause of cancer-related death worldwide, and its prognosis remains poor, with 5-year survival of approximately 5%. In this study, we analyzed the involvement of a novel proteoglycan, Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1), in the tumor progression and prognosis of human GBC.

Methods

SPOCK1 expression levels were measured in fresh samples and stored specimens of GBC and adjacent nontumor tissues. The effect of SPOCK1 on cell growth, DNA replication, migration and invasion were explored by Cell Counting Kit-8, colony formation, EdU retention assay, wound healing, and transwell migration assays, flow cytometric analysis, western blotting, and in vivo tumorigenesis and metastasis in nude mice.

Results

SPOCK1 mRNA and protein levels were increased in human GBC tissues compared with those in nontumor tissues. Immunohistochemical analysis indicated that SPOCK1 levels were increased in tumors that became metastatic, compared with those that did not, which was significantly associated with histological differentiation and patients with shorter overall survival periods. Knockdown of SPOCK1 expression by lentivirus-mediated shRNA transduction resulted in significant inhibition of GBC cell growth, colony formation, DNA replication, and invasion in vitro. The knockdown cells also formed smaller xenografted tumors than control GBC cells in nude mice. Overexpression of SPOCK1 had the opposite effects. In addition, SPOCK1 promoted cancer cell migration and epithelial-mesenchymal transition by regulating the expression of relevant genes. We found that activation of the PI3K/Akt pathway was involved in the oncogenic functions of SPOCK1 in GBC.

Conclusions

SPOCK1 activates PI3K/Akt signaling to block apoptosis and promote proliferation and metastasis by GBC cells in vitro and in vivo. Levels of SPOCK1 increase with the progression of human GBC. SPOCK1 acts as an oncogene and may be a prognostic factor or therapeutic target for patients with GBC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12943-014-0276-y) contains supplementary material, which is available to authorized users.     

Do TNFA -308 G/A and IL6 -174 G/C gene polymorphisms modulate risk of gallbladder cancer in the north Indian population?

OBJECTIVES: Gallbladder carcinoma (GBC) is highly aggressive neoplasm which arises in the background of gall stones and inflammation. GBC affects women 2-3 times more frequently than men. Pro-inflammatory TNFA and IL6 gene polymorphism has been associated with various inflammatory diseases. The aim of this study was to investigate whether TNFA -308 (G/A) and IL6 -174 G/C polymorphisms within flanking region of the genes are associated with GBC susceptibility. METHODS: The promoter polymorphisms were genotyped using PCR-RFLP in 200 healthy subjects and 124 GBC patients. RESULTS: Frequency distribution of TNFA -308 (G/A) and IL6 -174 G/C were not significantly different in GBC patients in comparison to healthy controls. However, frequency of TNFA -308 (G/A) polymorphism in female GBC patients without gallstone were significantly different (p-value= 0.006) when compared to healthy female subjects (OR=3.054; 95% CI=1.39-6.72). CONCLUSION: These results suggest that TNFA -308 (G/A) polymorphism may influence the susceptibility of female gender gallbladder cancer in absence of gallstones while IL6 -174 G/C polymorphism does not seem to be playing significant role in the susceptibility to gallbladder cancer.     

Celecoxib inhibits interleukin-6/interleukin-6 receptor-induced JAK2/STAT3 phosphorylation in human hepatocellular carcinoma cells

Growing evidence shows an association between chronic liver inflammation and hepatocellular carcinoma (HCC) development. STAT3, which is associated with inflammation and cellular transformation, is constitutively activated in human HCC tissues but not in normal human liver tissues. Although interleukin-6 (IL-6) is elevated in the serum of patients with HCC, it is not fully understood whether STAT3 constitutive activation is positively correlated with autocrine IL-6 secreted by HCC cells. Here, we reported that in HCC cells, the elevated levels of both IL-6 and IL-6 receptor (IL-6R, gp80), not IL-6 alone, correlated with STAT3 activation. We also explored whether the anticancer effects of celecoxib, an anti-inflammatory drug, may be due to the inhibition of the IL-6/STAT3 pathway in HCC cells. Our results showed that celecoxib decreased STAT3 phosphorylation by reducing Janus-activated kinase (JAK2) phosphorylation and caused apoptosis in HCC cells. Celecoxib could also block exogenous IL-6-induced STAT3 phosphorylation and nuclear translocation. Moreover, we observed more significant inhibition of cell viability when celecoxib was combined with doxorubicin or sorafenib. We conclude that the elevated levels of IL-6/IL-6R may be correlated with STAT3 activation in HCC cells. Celecoxib may be a candidate for HCC therapy through blocking IL-6/STAT3 pathway and can be combined with other anticancer drugs to reduce drug resistance caused by IL-6/STAT3 signals.     

NDRG2 down-regulation and CD24 up-regulation promote tumor aggravation and poor survival in patients with gallbladder carcinoma

Recent studies have demonstrated that N-Myc downstream-regulated gene 2 (NDRG2) may reduce the metastatic potential of breast cancer and hepatocellular carcinoma cells by regulating the expression of CD24, which is expressed in a large variety of solid tumors. The aim of this study was to clarify the clinical value of NDRG2 and CD24 expression in primary gallbladder carcinoma (GBC). One hundred and thirty GBC tissues were evaluated by immunohistochemistry for NDRG2 and CD24 expression. The associations of NDRG2 and CD24 expression with the clinicopathological characteristics and the overall survival of patients with GBC were analyzed. NDRG2 and CD24 were positively expressed in 49/130 (37.69%) and 107/130 (82.31%) of GBC tissues, respectively. In addition, the tumors with the down-regulation of NDRG2 and the up-regulation of CD24 more frequently had lymph node metastasis and lymphovascular invasion. Moreover, the tumors with the down-regulation of NDRG2 and the up-regulation of CD24 tended to show deeper invasion depth and higher TNM stage. There was a negative correlation between NDRG2 expression and CD24 expression in GBC tissues (r = -0.86, P < 0.001). The patients with NDRG2 negative expression correlated with poor prognosis of GBC (P = 0.01), as opposed to CD24 (P = 0.01). The survival rate of the patients with NDRG2-/CD24+ expression was the lowest (P < 0.001), and conjoined expression of NDRG2-/CD24+ was an independent prognostic indicator of GBC (P = 0.003). Our data suggest that NDRG2 down-regulation or CD24 up-regulation is an important feature of GBC. A combined detection of NDRG2/CD24 co-expression may benefit us in prediction of the prognosis in GBC.     

AJCC第八版Ⅲ期不同亚组分型的胆囊癌临床特征及预后分析—基于SEER数据库的研究

目的 研究美国癌症联合委员会(AJCC)第8版Ⅲ期胆囊癌(GBC)亚组分型的临床特征、治疗方式及预后.方法 收集3?485名AJCC第8版Ⅲ期胆囊癌患者的临床资料、随访结果并进行对比.绘制并比较ⅢA和ⅢB,T3N0M0(ⅢA)、T1～2N1M0(ⅢB)和T3N1M0(ⅢB)的Kaplan-Meier生存曲线.单因素和C...     

Relationship between HER2 and JAK/STAT-SOCS3 signaling pathway and clinicopathological features and prognosis of ovarian cancer

Objective: The study aims to explore the relationship between expressions of HER2 and JAK/STAT3-SOCS3 signaling pathway and clinicopathological features and prognosis of ovarian cancer (OC).Methods: A total of 136 OC patients were collected. Immunohistochemistry was applied to measure the expressions of STAT3, p-STAT3, SOCS3, HER2 and p-HER2 in the tumor tissues and adjacent normal tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expressions of HER2, SOCS3 and STAT3 and western blotting was applied for protein expressions of HER2, p-HER2, SOCS3, STAT3 and p-STAT3 in the tumor tissues and adjacent normal tissues. Flow cytometry was used for the cell apoptosis in the blank, afatinib (A), ruxolitinib (R) and afatinib + ruxolitinib (A + R) groups. Follow-up was performed to explore relationship of HER2, SOCS3, and STAT3 expressions with survival time of OC patients.Results: HER2, p-HER2, STAT3, and p-STAT3 expressions were higher while SOCS3 expression was lower in the tumor tissues. The positive expressions of STAT3, HER2, p-HER2 and p-STAT3 were lower while the positive expression of SOCS3 was higher in the adjacent normal tissues. The expressions of HER2, SOCS3, and p-STAT3 were associated with clinical stage and lymph node metastasis (LNM), and STAT3 expression has correlation with histological grade and LNM. The mRNA and protein expressions of HER2, STAT3 and p-STAT3 in the tumor tissues were higher than those in the adjacent normal tissues, but SOCS3 expression was significantly decreased. The positive expressions of HER2, p-HER2 and STAT3, the negative expression of SOCS3 and pathological stages were important risk factors for the prognosis of patients with OC.Conclusion: Our study showed that the expressions of HER2, STAT3, and SOCS3 are associated with the progression of OC, and higher expressions of HER2 and STAT3 and lower expression of SOCS3 predict poor prognosis of OC.     

E1A,E1B double-restricted adenovirus for oncolytic gene therapy of gallbladder cancer

New treatments, such as gene therapy, are necessary for advanced gallbladder cancer (GBC), but little has been studied. Recent studies have introduced mutant adenoviruses (Ads) with either defective E1B-55kD or mutated E1A, focusing on tumor-specific replication, and the results have been promising. To enhance the safety of this approach, we constructed AxdAdB-3, a double-restricted Ad with a mutant E1A and E1B-55kD deletion. We studied the effects of this Ad in vitro and in vivo on GBC, as well as its safety for normal human cells. We compared the replication and cytopathic effects of AxdAdB-3 in several lines of GBC and primary normal cells with those of wild-type Ad or of AxE1AdB, an E1B-55kD-deleted Ad. The efficacy in vivo was examined in nude mice with s.c. implanted or i.p. disseminated GBC. AxdAdB-3 replicated in and caused oncolysis of GBC cell lines (TGBC-44TKB and Mz-ChA2) as efficiently as wild-type Ad or AxE1AdB in vitro. By contrast, AxdAdB-3 replicated much less effectively in primary normal cells (e.g., epithelial cells, endothelial cells, and hepatocytes) than in GBC cells and had only mild cytopathic effects, unlike wild-type Ad. Furthermore, cytotoxicity of AxdAdB-3 in normal cells was milder than that of AxE1AdB. AxdAdB-3 significantly (P < 0.01) suppressed the growth of GBC (TGBC-44TKB) xenografts. AxdAdB-3 was also effective in the treatment of mice with peritoneally disseminated GBC (TGBC-44TKB), demonstrating tumor-selective replication and oncolysis that resulted in significantly (P < 0.05) prolonged survival. The present study shows that the E1 double-restricted Ad effectively and selectively replicates in and causes oncolysis of GBC in vitro and in vivo with reduced negative effects on normal cells, suggesting that this approach could be a promising tool for gene therapy of GBC.     

Adenovirus-mediated gene transfer of tissue factor pathway inhibitor-2 inhibits gallbladder carcinoma growth in vitro and in vivo

Tissue factor pathway inhibitor-2 (TFPI-2) has been identified as a tumor suppressor gene in several types of cancers, but its role in gallbladder carcinoma (GBC) is yet to be determined. In the present study, TFPI-2 expression in GBC tissues was examined, and its inhibitory activities against GBC growth were evaluated in vitro and in vivo after adenovirus-mediated gene transfer of TFPI-2 (Ad5-TFPI-2) was constructed to restore the expression of TFPI-2 in GBC cell lines (GBC-SD, SGC-996, NOZ) and xenograft tumors. Immunohistochemical staining showed that TFPI-2 was significantly downregulated in GBC tissue specimens. Ad5-TFPI-2 could significantly inhibit GBC growth both in vitro and in vivo. Apoptosis analysis and western blotting assay demonstrated that Ad5-TFPI-2 could induce the apoptosis of both GBC cell lines and tissues by promoting the activities of cytochrome c, Bax, caspase-3 and -9 and suppressing Bcl-2 activity. These data indicated that TFPI-2 acts as a tumor suppressor in GBC, and may have a potential role in gene therapy for GBC.     

CYP1A1, GSTM1, GSTT1 and TP53 Polymorphisms and Risk of Gallbladder Cancer in Bolivians

The Plurinational State of Bolivia (Bolivia) has a high incidence rate of gallbladder cancer (GBC). However, the genetic and environmental risk factors for GBC development are not well understood. We aimed to assess whether or not cytochrome P450 (CYP1A1), glutathione S-transferase mu 1 (GSTM1), theta 1 (GSTT1) and tumor suppressor protein p53 (TP53) genetic polymorphisms modulate GBC susceptibility in Bolivians. This case-control study covered 32 patients with GBC and 86 healthy subjects. GBC was diagnosed on the basis of histological analysis of tissues at the Instituto de Gastroenterologia Boliviano Japones (IGBJ); the healthy subjects were members of the staff at the IGBJ. Distributions of the CYP1A1 rs1048943 and TP53 rs1042522 polymorphisms were assayed using PCR-restriction fragment length polymorphism assay. GSTM1 and GSTT1 deletion polymorphisms were detected by a multiplex PCR assay. The frequency of the GSTM1 null genotype was significantly higher in GBC patients than in the healthy subjects (odds ratio [OR], 2.35; 95% confidence interval [CI], 1.03-5.37; age-adjusted OR, 3.53; 95% CI, 1.29-9.66; age- and sex-adjusted OR, 3.40; 95% CI, 1.24-9.34). No significant differences were observed in the frequencies of CYP1A1, GSTT1, or TP53 polymorphisms between the two groups. The GSTM1 null genotype was associated with increased GBC risk in Bolivians. Additional studies with larger control and case populations are warranted to confirm the association between the GSTM1 deletion polymorphism and GBC risk suggested in the present study.     

MicroRNA-182 promotes epithelial-mesenchymal transition by targeting FOXN3 in gallbladder cancer

Increasing evidence has suggested an association between the expression profiles of microRNAs (miRs) and gallbladder cancer (GBC). Recently, miR-182 has been demonstrated to exert tumor-promoting effects. However, the biological activity and molecular mechanisms of miR-182 in GBC remain unclear. The results of the present study demonstrated that miR-182 expression was significantly upregulated in GBC tissues and cell lines (GBC-SD and SGC-996). In addition, miR-182-knockdown attenuated epithelial-mesenchymal transition (EMT) in GBC cells, as indicated by decreased cell migratory and invasive abilities, decreased vimentin expression, and increased E-cadherin expression. The activities of β-catenin and its downstream factors, Cyclin D1 and c-Myc, were also demonstrated to decrease following miR-182-knockdown. Forkhead box N3 (FOXN3) was identified as the direct target of miR-182. Overexpression of FOXN3 ameliorated EMT and the β-catenin pathway. Taken together, the results of the present study suggested that miR-182 promotes EMT in GBC cells by targeting FOXN3, which suppresses the Wnt/β-catenin pathway.     

过表达STAT1与CD74CD44促进结肠癌细胞粘附和迁移的相关性

目的 探讨信号转导和转录激活子1(STAT1)与CD74/CD44在结肠癌中异常表达与肿瘤转移的相关性.方法 培养结肠癌细胞株Ls-174-T,待其生长至对数生长期,传至第3代收集肿瘤细胞,各细胞分为3份.1份行RT-PCR、Western blotting方法检测STAT1与CD74/CD44表达情况;1份检测定量粘附细胞数;1份以划痕实验检测细胞迁移能力.统计分析比较STAT1与CD74/CD44在结肠癌细胞中的异常表达情况及与肿瘤迁移和粘附能力的相关性.结果 RT-PCR、Western blotting结肠癌组织中STAT1与CD74/CD44的蛋白及mRNA表达显著高于正常结肠组织,两组相比具有统计学差异(P＜0.05).MTT法检测结果显示,不同作用时间(0、6、12、24、48 h)不同剂量STAT1与CD74/CD44过表达质粒(0.000、0.001、0.010、0.060、0.100 mg/ml)干预后,结肠癌细胞体外粘附能力明显加强,与对照组(0.000 mg/ml各组)相比,差异有统计学意义;Transwell结果显示,不同作用时间(0、6、12、24、48 h)不同剂量STAT1与CD74/CD44过表达质粒(0.000、0.001、0.010、0.060、0.100 mg/ml)干预后,结肠癌细胞体外迁移能力随着时间及剂量的增加而明显加强,与对照组(0.000mg/ml各组)相比,差异有统计学意义.结论 STAT1与CD74/CD44在结肠癌细胞中高表达,从而调节结肠癌细胞粘附和迁移能力的作用,表明STAT1与CD74/CD44在结肠癌发生进展中可能与肿瘤的侵袭和转移密切相关.     

IL-6通过活化STAT3/Notch信号促进癌相关成纤维细胞衰老及宫颈癌上皮细胞侵袭与放疗抵抗的机理研究

背景与目的：肿瘤微环境中衰老的癌相关成纤维细胞(cancer-associated fibroblasts,CAFs)介导上皮肿瘤的转移和放化疗抵抗,而CAFs分泌的炎性细胞因子IL-6可能促进宫颈癌的侵袭和放疗抵抗,但其作用机制并不清楚。该研究旨在探讨IL-6对CAFs衰老及宫颈癌上皮细胞侵袭与放疗抵抗的作用。方法：以宫颈癌CAFs、宫颈组织正常成纤维细胞(normal ifbroblasts,NFs)、宫颈癌细胞系HeLa、Siha和ME180为材料,采用IL-6、STAT3和Notch抑制剂处理不同细胞,用细胞染色、免疫荧光、蛋白[质]印迹法(Western blot)和流式细胞术等方法测定细胞衰老、STAT3、Notch信号、细胞侵袭能力和对放射线诱导的细胞凋亡变化。结果：CAFs条件培养基(conditioned medium,CM)或IL-6可以激活STAT3和Notch信号诱导细胞衰老或促进宫颈癌细胞的侵袭能力;CAFs与宫颈癌细胞混合培养能促进宫颈癌细胞的侵袭,但IL-6抗体、STAT3抑制剂S31-201或Notch抑制剂DAPT处理细胞则会抑制宫颈癌细胞的侵袭。IL-6/STAT3作为Notch信号的上游分子,可能主要通过自分泌或旁分泌的方式上调成纤维细胞或癌细胞中Notch信号关键配体Jagged-1而活化Notch信号,最后赋予宫颈癌细胞对放射线的抵抗作用。结论：CAFs在肿瘤微环境中可能通过IL-6/STAT3活化Notch信号诱导宫颈癌上皮细胞的侵袭和放疗抵抗,靶向STAT3/Notch信号相关分子有可能提高宫颈癌的放疗效果。