Integrin alpha(v)beta6-associated ERK2 mediates MMP-9 secretion in colon cancer cells

There is general consensus that matrix metalloproteinases are involved in tumour progression. We show herein that inhibition of integrin alpha(v)beta6 expression in colon cancer cells suppresses MMP-9 secretion. This integrin-mediated event is dependent upon direct binding between the beta6 integrin subunit and extracellular signal-regulated kinase 2. Targetting either beta6 or its interaction with extracellular signal-regulated kinase in order to inhibit matrix metalloproteinase activity may offer a useful therapeutic approach in preventing growth and spread of colon cancer.     

Suppression of integrin alphaupsilonbeta6 by RNA interference in colon cancer cells inhibits extracellular matrix degradation through the MAPK pathway

Integrin alphaupsilonbeta6 plays a very important role in the progression of colon cancer cells and is now defined as a novel, independent prognostic indicator for aggressive colon cancer in humans. Herein, we use the RNA interfering technology to downregulate the expression of alphaupsilonbeta6 in colon cancer cells. Our data demonstrate that plasmid vector based shRNA can effectively down-regulate alphaupsilonbeta6 expression in protein and mRNA levels. Supression of integrin alphaupsilonbeta6 inhibits the phosphorylation and nonphosphorylation level of ERK1/2, the secretion of uPA, pro-MMP-9 and pro-MMP-2 in tumor conditioned medium, and more important, inhibits MAPK-dependent [(3)H] labeled collagen IV degradation via the plasminogen activation cascade. Our study demonstrates in vitro that supression of integrin alphaupsilonbeta6 inhibits extracellular matrix degradation through the MAPK pathway.     

YES oncogenic activity is specified by its SH4 domain and regulates RAS/MAPK signaling in colon carcinoma cells

Members of the SRC family of tyrosine kinases (SFK) display important functions in human cancer, but their specific role in tumorigenesis remains unclear. We previously demonstrated that YES regulates a unique oncogenic signaling important for colorectal cancer (CRC) progression that is not shared with SRC. Here, we addressed the underlying mechanism involved in this process. We show that YES oncogenic signaling relies on palmitoylation of its SH4 domain that controls YES localization in cholesterol-enriched membrane micro-domains. Specifically, deletion of the palmitoylation site compromised YES transforming activity, while addition of a palmitoylation site in the SH4 domain of SRC was sufficient for SRC to restore the transforming properties of cells in which YES had been silenced. Subsequently, SILAC phosphoproteomic analysis revealed that micro-domain-associated cell adhesive components and receptor tyrosine kinases are major YES substrates. YES also phosphorylates upstream regulators of RAS/MAPK signaling, including EGFR, SHC and SHP2, which were not targeted by SRC due to the absence of palmitoylation. Accordingly, EGFR-induced MAPK activity was attenuated by YES down-regulation, while increased RAS activity significantly restored cell transformation that was lost upon YES silencing. Collectively, these results uncover a critical role for the SH4 domain in the specification of SFK oncogenic activity and a selective role for YES in the induction of RAS/MAPK signaling in CRC cells.     

Expression of alpha(v)beta6 integrin in oral leukoplakia

The distribution of alpha(v)beta6 integrin was examined in oral leukoplakia, lichen planus and squamous cell carcinomas using immunohistochemistry. Controls included oral mucosal wounds, chronically inflamed and normal oral mucosa. Integrins beta1, beta3, beta4, beta5, fibronectin and tenascin were also studied. The integrin alpha(v)beta6 was highly expressed throughout the whole lesion of 90% of the squamous cell carcinomas but was not present in any of the normal specimens. alpha(v)beta6 integrin was also expressed in 41% of the leukoplakia specimens, and 85% of the lichen planus samples, but in none of the tissues with inflammatory hyperplasia or chronic inflammation. The expression of beta1 integrins was localized in the basal layer, and that of the beta4 at the cell surface facing the basement membrane of all specimens. The integrins beta3 and beta5 were absent from all normal and leukoplakia specimens. Fibronectin and tenascin were present in the connective tissue underneath the epithelium of all the sections, and their expression was similar in both alpha(v)beta6-positive and alpha(v)beta6-negative tissues. A group of 28 leukoplakia patients were followed 1-4 years after first diagnosis. In this group, initially alpha(v)beta6 integrin-positive leukoplakia specimens had high tendency for disease progression while alpha(v)beta6-negative specimens did not progress. These results suggest that the expression of alpha(v)beta6 integrin could be associated in the malignant transformation of oral leukoplakias.     

Insulin-induced phosphorylation of the beta-4 integrin subunit expressed on murine metastatic carcinoma cells

A tumor surface protein (TSP-180) that is highly expressed on highly malignant metastatic cells has been identified on murine lung carcinomas. On SDS-PAGE under reducing conditions, TSP-180 shows a complex banding pattern corresponding to 204, 183, 150, 135, and 116 kDa. All bands of the TSP-180 complex are glycosylated and are labeled by lactoperoxidase-catalyzed radioiodination of viable cells. The mouse TSP-180 complex described here is homologous to the human integrin alpha 6 beta 4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the beta 4 subunit of the integrin complex. It has been shown recently that monoclonal antibody to TSP-180 (MoAb 135-13C) stimulates cell growth in vitro and induces phosphorylation of the 204-kDa protein. We now report that insulin increases the phosphorylation of the 204-kDa protein 30-fold in intact carcinoma cells and epidermal growth factor (EGF) causes a threefold increase. Insulin-like growth factor (IGF-I) and platelet-derived growth factor have no effect. The effect of insulin and of IGF-I on phosphorylation of their own receptors was studied using solubilized cell membranes. Insulin and IGF-I each induced a fivefold increase in the phosphorylation of their respective receptor beta subunits. In order to test if phosphorylation of the 204-kDa protein was induced by direct binding of growth factors to TSP-180 and to identify growth factor receptors on line 1 cells, affinity cross-linking studies were performed. Affinity labeling of receptors demonstrated that insulin and IGF-I both bind to a 135-kDa protein that corresponds to the insulin and IGF-I receptor alpha subunits. Affinity labeling of EGF receptors failed to demonstrate EGF receptor molecules (175-kDa protein) on line 1 cells. Further investigations by using a different approach confirmed the very low amount of EGF receptors on line 1 cells. Direct phosphoamino acid analysis of the 204-kDa protein purified from insulin-stimulated cells demonstrated that this beta 4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that beta 4 integrin molecule is a target for phosphorylation through an indirect receptor-mediated mechanism.     

Axin通过激活p53的表达抑制结肠癌细胞的生长

目的： 观察过表达Axin对结肠癌SW480细胞生长的影响,并探讨其作用机制。方法：将pCMV5-HA-Axin质粒瞬时转染至结肠癌SW480细胞中,并设置转染空载体pCMV5-HA及未转染空白对照组,采用噻唑蓝(MTT)比色法及克隆形成实验检测细胞增殖状态的变化,流式细胞仪检测细胞周期变化;RT-PCR检测Axin及p53 mRNA的表达;Western blot检测Axin及P53蛋白的表达。结果：与转染空载体及空白对照组比较,过表达Axin显著抑制结肠癌SW480细胞的增殖。MTT实验显示转染Axin后细胞生长明显受到抑制,存活的瘤细胞明显减少(P<0.05);克隆形成实验结果显示瞬时转染Axin质粒组细胞集落形成能力明显下降(P<0.05);流式细胞仪分析检测结果显示转染Axin质粒后结肠癌SW480细胞周期G1前期比例升高,G1期明显被阻滞,S期比例下降(P均<0.05)。RT-PCR结果显示转染Axin的SW480细胞中p53 mRNA的表达较转染空载体组升高约1倍(P＜0.05);同时,Western blot结果显示转染质粒Axin后结肠癌SW480细胞中P53的蛋白表达量明显增加,较转染空载体组几近升高1倍(P＜0.05)。结论：过表达Axin抑制结肠癌SW480细胞增殖,其作用机制是通过激活癌基因p53的表达而发挥效应的。     

Butyrate elicits a metabolic switch in human colon cancer cells by targeting the pyruvate dehydrogenase complex

Butyrate, a short‐chain fatty acid produced by the colonic bacterial fermentation is able to induce cell growth inhibition and differentiation in colon cancer cells at least partially through its capacity to inhibit histone deacetylases. Since butyrate is expected to impact cellular metabolic pathways in colon cancer cells, we hypothesize that it could exert its antiproliferative properties by altering cellular metabolism. We show that although Caco2 colon cancer cells oxidized both butyrate and glucose into CO₂, they displayed a higher oxidation rate with butyrate as substrate than with glucose. Furthermore, butyrate pretreatment led to an increase cell capacity to oxidize butyrate and a decreased capacity to oxidize glucose, suggesting that colon cancer cells, which are initially highly glycolytic, can switch to a butyrate utilizing phenotype, and preferentially oxidize butyrate instead of glucose as energy source to produce acetyl coA. Butyrate pretreated cells displayed a modulation of glutamine metabolism characterized by an increased incorporation of carbons derived from glutamine into lipids and a reduced lactate production. The butyrate‐stimulated glutamine utilization is linked to pyruvate dehydrogenase complex since dichloroacetate reverses this effect. Furthermore, butyrate positively regulates gene expression of pyruvate dehydrogenase kinases and this effect involves a hyperacetylation of histones at PDK4 gene promoter level. Our data suggest that butyrate exerts two distinct effects to ensure the regulation of glutamine metabolism: it provides acetyl coA needed for fatty acid synthesis, and it also plays a role in the control of the expression of genes involved in glucose utilization leading to the inactivation of PDC.     

Inhibition of integrin alpha5beta1 function with a small peptide (ATN-161) plus continuous 5-FU infusion reduces colorectal liver metastases and improves survival in mice

Integrin alpha(5)beta(1) is expressed on activated endothelial cells and plays a critical role in tumor angiogenesis. We hypothesized that a novel integrin alpha(5)beta(1) antagonist, ATN-161, would inhibit angiogenesis and growth of liver metastases in a murine model. We further hypothesized that combining ATN-161 with 5-fluorouracil (5-FU) chemotherapy would enhance the antineoplastic effect. Murine colon cancer cells (CT26) were injected into spleens of BALB/c mice to produce liver metastases. Four days thereafter, mice were given either ATN-161 (100 mg/kg, every 3rd day) or saline by intraperitoneal injection, with or without combination of continuous-infusion 5-FU (100 mg/kg/2 weeks), which was started on day 7. On day 20 after tumor cell inoculation, mice were killed and liver weights and number of liver metastases were determined. A follow-up study on survival was also conducted in which mice were randomized to receive ATN-161, 5-FU or ATN-161+5-FU. Combination therapy with ATN-161+5-FU significantly reduced tumor burden (liver weight) and number of liver metastases (p<0.02). Liver tumors in the ATN-161 and ATN-161+5-FU groups had significantly fewer microvessels (p<0.05) than tumors in the control or 5-FU-treated groups. Unlike treatment with either agent alone, ATN-161+5-FU significantly increased tumor cell apoptosis and decreased tumor cell proliferation (p<0.03) and improved overall survival (p<0.03, log-rank test). Targeting integrin alpha(5)beta(1) in combination with 5-FU infusion reduced liver metastases formation and improved survival in this colon cancer model. The enhancement of antineoplastic activity from the combination of anti-angiogenic therapy and chemotherapy may be a promising approach for treating metastatic colorectal cancer.     

miR－647在结肠癌细胞中的表达及临床意义

目的：分析miR－647在结肠癌组织及细胞系中的表达情况,探讨其对结肠癌细胞增殖、迁移能力的影响及其可能的作用机理。方法：利用Real－time quantitative Polymerase Chain Reaction（qPCR）技术检测17例结肠癌患者癌及癌旁组织中miR－647的表达;利用qPCR技术检测正常人肠上皮细胞HIEC及结肠癌细胞HT－29中miR－647的表达;将miR－647 antagomir及对照分别转染至结肠癌细胞中,应用MTT实验检测细胞增殖,体外划痕实验检测细胞迁移能力,评价转染miR－647抑制剂对结肠癌细胞增殖能力和迁移能力的影响。结果：qPCR结果显示,与癌旁组织相比,miR－647在结肠癌肿瘤组织中表达明显升高;与正常人肠上皮细胞相比,人结肠癌细胞系HT－29中miR－647表达明显升高;MTT结果显示miR－647抑制剂可以显著抑制SW480和SW620细胞的增殖能力和细胞迁移能力。结论：miR－647通过促进结肠癌细胞的增殖和迁移能力,参与结肠癌的发生发展进程。     

新城疫病毒7793株对人结肠癌细胞的杀伤作用

目的:研究新城疫病毒NDV7793对人结肠癌细胞的体外杀伤作用,为结肠癌的生物疗法奠定基础。方法:通过蚀斑实验纯化病毒并测定纯化的NDV7793株的感染力;用乳酸脱氢酶微量释放法测定纯化病毒对人LoVo和Ls174t结肠癌细胞株的杀伤作用并且通过血凝实验测定病毒在不同细胞中的增殖力。结果:NDV7793在感染细胞96h后出现直径约为0.5mm左右的空斑,PFU为1.25×107个/ml,为弱毒株;NDV7793对LoVo和Ls174t人结肠癌细胞株有明显的杀伤作用,而且杀伤作用的强度与病毒作用的时间和病毒的浓度呈正相关的关系;NDV7793可以在肠癌细胞中生长复制,该病毒株在人结肠癌细胞株LoVo的复制能力强于Ls174t。结论:NDV7793具有较强的选择性杀伤人结肠癌细胞的作用,且为弱毒株,这株病毒具备肿瘤生物治疗的潜能。     

新城疫病毒7793株对人结肠癌细胞的杀伤作用

目的：研究新城疫病毒NDV7793对人结肠癌细胞的体外杀伤作用,为结肠癌的生物疗法奠定基础。方法：通过蚀斑实验纯化病毒并测定纯化的NDV7793株的感染力;用乳酸脱氢酶微量释放法测定纯化病毒对人LoVo和Ls174t结肠癌细胞株的杀伤作用并且通过血凝实验测定病毒在不同细胞中的增殖力。结果：NDV7793在感染细胞96h后出现直径约为0.5mm左右的空斑,PFU为1.25×107个/ml,为弱毒株;NDV7793对LoVo和Ls174t人结肠癌细胞株有明显的杀伤作用,而且杀伤作用的强度与病毒作用的时间和病毒的浓度呈正相关的关系;NDV7793可以在肠癌细胞中生长复制,该病毒株在人结肠癌细胞株LoVo的复制能力强于Ls174t。结论：NDV7793具有较强的选择性杀伤人结肠癌细胞的作用,且为弱毒株,这株病毒具备肿瘤生物治疗的潜能。     

Axin在结肠癌中的表达及其临床病理学意义

目的:探讨Axin在结肠癌中的表达及其临床病理学意义。方法:采用免疫组化SP法检测30例结肠正常黏膜组织及150例结肠癌组织中Axin蛋白的表达。结果:Axin在结肠癌中的阳性表达（46%）低于结肠正常黏膜组织的表达（83.3%）,二者差异有统计学意义(P<0.05)。Axin的表达与肿瘤的组织学分级、TNM分期、淋巴结转移正相关（P<0.05）。结论:Axin可作为结肠癌诊断的实验室指标,对结肠癌的诊断和预后评价有重要的参考价值。     

The alpha v beta 6 integrin induces gelatinase B secretion in colon cancer cells

In human cancers, the co-operative role between cell-adhesion receptors and proteases capable of degrading matrix barriers remains poorly understood. We have previously reported that the epithelium-restricted integrin alpha(v)beta6 becomes highly expressed in colon cancer compared with normal mucosa and that heterologous expression of alpha(v)beta6 in colon cancer cells is associated with enhanced cell growth. Herein, we report that alpha(v)beta6 expression in colon cancer cells leads to a relative increase in secretion of the matrix metalloproteinase gelatinase B over its respective inhibitor and that this secretion parallels the level of cell-surface beta6 expression. The alpha(v)beta6-mediated gelatinase B secretion is associated with increased proteolysis of denatured collagen at the cell surface, and inactivation of gelatinase B in beta6-expressing tumour cells inhibits cell spreading and proliferation within 3-dimensional collagen matrices. Our findings suggest that alpha(v)beta6-mediated gelatinase B secretion is important in the progression of human colon cancer.     

Reduced expression of a gene proliferation signature is associated with enhanced malignancy in colon cancer

The association between cell proliferation and the malignant potential of colon cancer is not well understood. Here, we evaluated this association using a colon-specific gene proliferation signature (GPS). The GPS was derived by combining gene expression data obtained from the analysis of a cancer cell line model and a published colon crypt profile. The GPS was overexpressed in both actively cycling cells in vitro and the proliferate compartment of colon crypts. K-means clustering was used to independantly stratify two cohorts of colon tumours into two groups with high and low GPS expression. Notably, we observed a significant association between reduced GPS expression and an increased likelihood of recurrence (P<0.05), leading to shorter disease-free survival in both cohorts. This finding was not a result of methodological bias as we verified the well-established association between breast cancer malignancy and increased proliferation, by applying our GPS to public breast cancer data. In this study, we show that reduced proliferation is a biological feature characterizing the majority of aggressive colon cancers. This contrasts with many other carcinomas such as breast cancer. Investigating the reasons underlying this unusual observation may provide important insight into the biology of colon cancer progression and putative novel therapy options.     

IL-6在不同Dukes分期结肠癌中的表达及临床意义

目的：探讨不同 Dukes 分期结肠癌组织及癌旁组织中的 IL -6以及结肠癌患者外周血 IL -6的表达情况。方法：收集56例结肠癌、癌旁组织的标本及15例正常结肠组织标本。收集结肠癌患者术前外周血标本和正常对照组外周血标本。采用免疫组织化学 SP 法检测 IL -6蛋白的表达及用酶联免疫吸附（ELISA）法检测血清中 IL -6的水平。结果：结肠癌患者组织 IL -6阳性率显著多于癌旁对照组（P <0.05）及正常对照组（P <0.05）。阳性表达率与淋巴结转移、临床 Dukes 分期有关（P <0.05）,与组织学分级、肿瘤大小无关（P>0.05）;结肠癌外周血 IL -6水平显著高于正常对照组（P <0.05）。结论：IL -6在结肠癌中表达明显增高,且与临床分期有关,可作为诊断结肠癌的预警信号和判断预后的有用指标。