Laminin-5 with transforming growth factor-beta1 induces epithelial to mesenchymal transition in hepatocellular carcinoma

BACKGROUND & AIMS: How hepatocellular carcinoma (HCC) cells acquire the ability to invade surrounding tissue is unknown, but epithelial mesenchymal transition (EMT) likely plays a role. We investigate how transforming growth factor (TGF)-beta1 and extracellular matrix protein Laminin-5 (Ln-5) induce EMT and cancer invasion. METHODS: Snail, Slug, E-cadherin, beta-catenin and Ln-5 were investigated on HCC tissues and on HCC cell lines. RESULTS: We show that in HCC but not in peritumoral tissue of the same HCC patients, Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated and beta-catenin is translocated into the nuclei. In vitro, HCC "invasive" cells, partially EMT-transformed, show low levels of E-cadherin. In presence of Ln-5, Snail, and Slug are up-regulated, E-cadherin is down-regulated, beta-catenin is translocated into the nuclei, and cells undergo a dramatic morphological change, becoming scattered and undergoing a complete EMT. This effect is reversed by anti-alpha3 but not by anti-alpha6 integrin blocking antibody. HCC "noninvasive" cells are not EMT-transformed, and have constitutively high levels of E-cadherin. In presence of Ln-5, cells undergo partial EMT, Snail, and Slug are up-regulated, E-cadherin is down-regulated but cells do not scatter. However, the presence of both Ln-5 and TGF-beta1 completes the EMT process, beta-catenin is translocated into the nuclei, cells scatter and become invasive, recalling the "invasive" cells. In this case, too, the effect is reversed by anti-alpha3 integrin blocking antibody. CONCLUSIONS: Our study shows that Ln-5 and TGF-beta1 cooperatively induce EMT in HCC, suggesting the microenvironment as a potential target for new biological therapies.     

Analysis of tumor-infiltrating gamma delta T cells in rectal cancer

AIM: To investigate the regulatory effect of Vδ1 T cells and the antitumor activity of Vδ2 T cells in rectal cancer.METHODS: Peripheral blood, tumor tissues and paracarcinoma tissues from 20 rectal cancer patients were collected. Na?ve CD4 T cells from the peripheral blood of rectal cancer patients were purified by negative selection using a Naive CD4+ T Cell Isolation Kit Ⅱ(Miltenyi Biotec). Tumor tissues and para-carcinoma tissues were minced into small pieces and digested in a triple enzyme mixture containing collagenase type Ⅳ, hyaluronidase, and deoxyribonuclease for 2 h at room temperature. After digestion, the cells were washed twice in RPMI1640 and cultured in RPMI1640 containing 10% human serum supplemented with L-glutamine and 2-mercaptoethanol and 1000 U/m L of IL-2 for the generation of T cells. Vδ1 T cells and Vδ2 T cells from tumor tissues and para-carcinoma tissues were expanded by anti-TCR gδ antibodies. The inhibitory effects of Vδ1 T cells on na?ve CD4 T cells were analyzed using the CFSE method. The cytotoxicity of Vδ2 T cells on rectal cancer lines was determined by the LDH method.RESULTS: The percentage of Vδ1 T cells in rectal tumortissues from rectal cancer patients was significantly increased, and positively correlated with the T stage. The percentage of Vδ2 T cells in rectal tumor tissues from rectal cancer patients was significantly decreased, and negatively correlated with the T stage. After culture for 14 d with 1 mg/m L anti-TCR gδ antibodies, the percentage of Vδ1 T cells from para-carcinoma tissues was 21.45% ± 4.64%, and the percentage of Vδ2 T cells was 38.64% ± 8.05%. After culture for 14 d, the percentage of Vδ1 T cells from rectal cancer tissues was 67.45% ± 11.75% and the percentage of Vδ2 T cells was 8.94% ± 2.85%. Tumor-infiltrating Vδ1 T cells had strong inhibitory effects, and tumor-infiltrating Vδ2 T cells showed strong cytolytic activity. The inhibitory effects of Vδ1 T cells from para-carcinoma tissues and from rectal cancer tissue were not significantly different. In addition, the cytolytic activities of Vδ2 T cells from para-carcinoma tissues and from rectal cancer tissues were not significantly different.CONCLUSION: A percentage imbalance in Vδ1 and Vδ2 T cells in rectal cancer patients may contribute to the development of rectal cancer.     

Canonical Wnt signaling regulates Slug activity and links epithelial–mesenchymal transition with epigenetic Breast Cancer 1, Early Onset (BRCA1) repression

Slug (Snail2) plays critical roles in regulating the epithelial–mesenchymal transition (EMT) programs operative during development and disease. However, the means by which Slug activity is controlled remain unclear. Herein we identify an unrecognized canonical Wnt/GSK3β/β-Trcp1 axis that controls Slug activity. In the absence of Wnt signaling, Slug is phosphorylated by GSK3β and subsequently undergoes β-Trcp1–dependent ubiquitination and proteosomal degradation. Alternatively, in the presence of canonical Wnt ligands, GSK3β kinase activity is inhibited, nuclear Slug levels increase, and EMT programs are initiated. Consistent with recent studies describing correlative associations in basal-like breast cancers between Wnt signaling, increased Slug levels, and reduced expression of the tumor suppressor Breast Cancer 1, Early Onset (BRCA1), further studies demonstrate that Slug—as well as Snail—directly represses BRCA1 expression by recruiting the chromatin-demethylase, LSD1, and binding to a series of E-boxes located within the BRCA1 promoter. Consonant with these findings, nuclear Slug and Snail expression are increased in association with BRCA1 repression in a cohort of triple-negative breast cancer patients. Together, these findings establish unique functional links between canonical Wnt signaling, Slug expression, EMT, and BRCA1 regulation.     

Identification of a panel of ten cell surface protein antigens associated with immunotargeting of leukemias and lymphomas by peripheral blood �æ� T cells

Background

Vγ9Vδ2 T lymphocytes are regarded as promising mediators of cancer immunotherapy due to their capacity to eliminate multiple experimental tumors, particularly within those of hematopoietic origin. However, Vγ9Vδ2 T-cell based lymphoma clinical trials have suffered from the lack of biomarkers that can be used as prognostic of therapeutic success.

Design and Methods

We have conducted a comprehensive study of gene expression in acute lymphoblastic leukemias and non-Hodgkin’s lymphomas, aimed at identifying markers of susceptibility versus resistance to Vγ9Vδ2 T cell-mediated cytotoxicity. We employed cDNA microarrays and quantitative real-time PCR to screen 20 leukemia and lymphoma cell lines, and 23 primary hematopoietic tumor samples. These data were analyzed using state-of-the-art bioinformatics, and gene expression patterns were correlated with susceptibility to Vγ9Vδ2 T cell mediated cytolysis in vitro.

Results

We identified a panel of 10 genes encoding cell surface proteins that were statistically differentially expressed between “γδ-susceptible” and “γδ-resistant” hematopoietic tumors. Within this panel, 3 genes (ULBP1, TFR2 and IFITM1) were associated with increased susceptibility to Vγ9Vδ2 T-cell cytotoxicity, whereas the other 7 (CLEC2D, NRP2, SELL, PKD2, KCNK12, ITGA6 and SLAMF1) were enriched in resistant tumors. Furthermore, some of these candidates displayed a striking variance of expression among primary follicular lymphomas and T-cell acute lymphoblastic leukemias.

Conclusions

Our results suggest that hematopoietic tumors display a highly variable repertoire of surface proteins that can impact on Vγ9Vδ2 cell-mediated immunotargeting. The prognostic value of the proposed markers can now be evaluated in upcoming Vγ9Vδ2 T cell-based lymphoma/leukemia clinical trials.     

Fas信号通路通过诱导EMT促进胃癌细胞的侵袭转移

目的探讨Fas信号通路促进胃癌细胞侵袭转移的可能相关机制。方法以低浓度FasL处理胃癌细胞株AGS,免疫印迹及ELISA检测上皮间质转化(epithelial-mesenehymal transition,EMT)的分子生物学标记改变;稳定沉默Snail及Twist转录因子,transwell小室侵袭实验检测FasL处理后胃癌细胞的侵袭能力;免疫印迹检测信号通路激活状态及相应的抑制效应。结果 FasL可以诱导AGS细胞株出现EMT表型,并可促进胃癌细胞的侵袭转移能力。同时该过程中出现ERK1/2信号通路的激活,而抑制ERK1/2信号通路,可以抑制FasL诱导EMT及提高肿瘤侵袭能力的作用。胃癌组织中相应分子生物学标记的表达变化符合EMT的发生。结论 Fas信号通路能够激活ERK1/2通路诱导EMT的发生,并且能通过该机制增强胃癌细胞AGS的活动能力。     

γδ T-cell killing of primary follicular lymphoma cells is dramatically potentiated by GA101, a type II glycoengineered anti-CD20 monoclonal antibody

Background

Anti-CD20 monoclonal antibodies are major therapeutic agents for patients with follicular lymphoma and work through complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. Optimization of antibody-dependent cellular cytotoxicity, in particular by amplifying its effectors, could further increase the efficacy of anti-CD20 monoclonal antibodies.

Design and Methods

We investigated the cytotoxic activity of Vγ9Vδ2 T cells against follicular lymphoma cells and whether this killing could be increased by promoting antibody-dependent cellular cytotoxicity with anti-CD20 monoclonal antibodies, in particular a type-II glycoengineered anti-CD20. Vγ9Vδ2 T cells were expanded in vitro in the presence of bromohydrin pyrophosphate (Phosphostim) and interleukin-2 and their ability to kill follicular lymphoma primary cells or cell lines was evaluated by flow cytometry cytotoxic T-lymphocyte assays in the presence or absence of three anti-CD20 monoclonal antibodies: the afucosylated GA101, the chimeric rituximab or the humanized ofatumumab. The ability of these cells to release perforin/granzyme and secrete interferon-γ when co-cultured with follicular lymphoma primary cells or cell lines in the presence or not of the three anti-CD20 monoclonal antibodies was also evaluated by CD107a staining and Elispot assays.

Results

Phosphostim and interleukin-2 expanded Vγ9Vδ2 T cells were cytotoxic to primary follicular lymphoma cells and their cytotoxic potential was dramatically increased by GA101, a type II glycoengineered anti-CD20 monoclonal antibody, and to a lesser extent, by rituximab and ofatumumab. The increased cytotoxicity was associated with increased secretion of perforin/granzyme and interferon-γ.

Conclusions

In-vitro expanded Vγ9Vδ2 T cells efficiently kill primary follicular lymphoma cells and express CD16; anti-CD20 monoclonal antibodies, in particular GA101, dramatically increase the cytotoxic activity of expanded Vγ9Vδ2 T cells. These preclinical results prompt the development of clinical trials using this antibody dependent cellular cytotoxicity property of Vγ9Vδ2 T cells and anti-CD20 monoclonal antibodies.     

Interaction between cyclooxygenase-2,Snail,and E-cadherin in gastric cancer cells

AIM:To investigate the mechanisms of how cyclooxygenase-2(COX-2)regulates E-cadherin in gastric cancer cells.METHODS:COX-2 expression in human gastric cancer cell lines SGC-7901,BGC-823,MGC-803 and AGS were measured at the mRNA and protein level.COX-2 rich cell line SGC-7901 was chosen for subsequent experiments.siRNA mediated gene knockdown was used to investigate the impact of COX-2 on nuclear factor-κB (NF-κB),Snail,and E-cadherin in gastric cancer cells.Gene expression was determined by Western blot and real-time polymerase chain reaction.To analyze whether NF-κB inhibition could interrupt the modulatory effect of COX-2 or prostaglandin E2(PGE2)on E-cadherin,gastric cancer cells were treated with celecoxib or PGE2,in the presence of NF-κB specific siRNA.RESULTS:Highest expression level of COX-2 was found in SGC-7901 cells,both at mRNA and protein levels.siRNA mediated down-regulation of COX-2 led to a reduced expression of NF-κB and Snail,but an increased expression of E-cadherin in SGC-7901 cells.siRNA mediated down-regulation of NF-κB also led to a reduced expression of E-cadherin and Snail in SGC-7901 cells.However,COX-2 expression did not alter after cells were treated with NF-κB specific siRNA in SGC-7901 cells.Treatment of SGC-7901 cells with celecoxib led to a reduced expression of Snail but an increased expression of E-cadherin.In contrast,treatment of SGC-7901 cells with PGE2 led to an increased Snail and a decreased E-cadherin.However,siRNAmediated knockdown of NF-κB partially abolished the effect of celecoxib and PGE2 on the regulation of E-cadherin and Snail in SGC-7901 cells.CONCLUSION:COX-2 likely functions upstream of NF-κB and regulates the expression of E-cadherin via NF-κB/Snail signaling pathway in gastric cancer cells.     

Epithelial-mesenchymal transition in colorectal cancer tissue of patients with Lynch syndrome

AIM:To explore the epithelial-mesenchymal transition(EMT)in tissue from patients with Lynch syndrome,and to interpret biological behaviour of Lynch syndrome.METHODS:Sixty-eight formalin-fixed and paraffin embedded tissue blocks were analyzed in this study,including tissues from Lynch syndrome(n=30),sporadic colorectal carcinoma(CRC)(n=30),and tumoradjacent tissues(n=8).Tissue sections were stained for human mutS homolog 2(hMSH2),human mutL homolog 1(hMLH1),transforming growth factor-βtypeⅡreceptor(TGFβRⅡ),E-cadherin,β-catenin,matrix metalloproteinase-7(MMP-7)and tissue inhibitor of metalloproteinase-2(TIMP-2)by immunohistochemical staining.Furthermore,clinical data such as age,gender and tumor-node-metastasis stage were also collected retrospectively.RESULTS:The positive expression rates of hMSH2,hMLH1,TGFβRⅡ,E-cadherin,β-catenin,MMP-7 and TIMP-2 were significantly related to the depth of invasion and lymph node metastasis,but not to sex or tumour size or location.The differences in the positive expression rates of hMSH2,hMLH1,TGFβRⅡ,E-cadherin,cytomembraneβ-catenin,cytoplasmicβ-catenin,MMP-7 and TIMP-2 were significant between sporadic CRC and Lynch syndrome.The expression of hMSH2 had a positive correlation with that of hMLH1 in Lynch syndrome and sporadic CRC.The expression of TGFβRⅡhad a positive correlation with that of hMSH2,hMLH1 and MMP-7,and a negative correlation with that of TIMP-2.The expression of MMP-7 had a negative correlation with that of TIMP-2 in Lynch syndrome and sporadic CRC.The expression of E-cadherin was positively correlated with that of cytomembraneβ-catenin.However,the expression of cytomembraneβ-catenin was negatively correlated with that of cytoplasmicβ-catenin,and the expression of cytoplasmicβ-catenin was positively correlated with that of MMP-7.CONCLUSION:EMT may play an important role in the development and progression of Lynch syndrome.Lynch syndrome was caused by the mutations of mismatch repair genes,mainly hMSH2 and hMLH1,which also beget the mutational inactivation of TGFβRⅡ.Therefore,the colorectal cancer of Lynch syndrome can escape the inhibitory effect of TGFβ1.However,TGFβ1 can up-regulate the expression of MMP-7 and down-regulate the expression of TIMP-2 in tumors by disassembling the E-cadherin/β-catenin complex in the cytomembrane.     

Curcumin协同ABT-737对肝癌细胞上皮间质转化的抑制作用及相关机制初步研究

目的探讨Curcumin协同ABT-737对肝癌7404细胞上皮间质转化(epithelial-mesenchymal transition,EMT)的抑制作用及机制研究。方法2μmol/L Curcumin、5μmol/L ABT-737或2μmol/L Curcumin+5μmol/L ABT-737作用肝癌7404细胞后,观察细胞形态的变化,细胞划痕实验检测7404细胞的迁移能力,蛋白质印迹法检测7404细胞中E-cadherin、Vimentin、N-cadherin、ZEB1蛋白的表达和p-beta-catenin、p-JNK、Snail、Twist蛋白的表达。构建肝癌动物模型(Alb-Cre;P53f/f;Ras),在苏木精伊红染色下观察肝转移瘤情况,并对在肝脏形成的转移灶进行数量统计。结果相比2μmol/L Curcumin、5μmol/L ABT-737,2μmol/L Curcumin+5μmol/L ABT-737作用后,7404细胞梭形化明显减少(P<0.05),细胞迁移能力显著降低(P<0.05),Vimentin、N-cadherin、ZEB1蛋白的表达水平明显抑制(P<0.05),E-cadherin蛋白的表达水平明显上调(P<0.05),beta-catenin和JNK的磷酸化水平明显上调(P<0.05),beta-catenin下游靶基因Snail、Twist的表达水平明显抑制(P<0.05)。相比对照组,Curcumin+ABT-737作用后,肝癌动物模型(Alb-Cre;P53f/f;Ras)中肝转移瘤数量明显减少(P<0.05)。结论Curcumin协同ABT-737能抑制肝癌细胞上皮间质转化,JNK-beta-catenin可能参与EMT转化的抑制。