Smad4在大肠癌细胞中对Wnt/β-catenin信号通路调控机制的研究
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摘要
背景与目的
大肠癌(colorectal cancer,CRC)是临床最常见的恶性肿瘤之一,在全世界范围内,大肠癌的发病率男女均处于恶性肿瘤的第3位,其中在西方发达国家,大肠癌的发病率处于第2位。在我国,随着生活水平的不断提高以及高蛋白、高脂肪、低纤维素等饮食结构的改变,大肠癌的发病率呈上升态势,排在恶性肿瘤和致死因素的第4位,对人民健康和生命构成严重威胁。目前大肠癌发生的分子机制尚不十分清楚,其病因和危险因素复杂,一般认为其发生是机体的内因与环境的外因相互作用的结果。流行病学研究认为大肠癌的发生主要与饮食、遗传、环境等因素关系密切。
目前研究证实大肠癌的发生、发展是一个多阶段、多基因参与的过程,涉及到多条信号转导途径的异常激活、失活及多种癌基因、抑癌基因的异常表达。转化生长因子β(transforming growth factor-β,TGF-β)及其受体与下游信号蛋白Smads组成了一个非常复杂的信号传导网络,其信号转导需要TGF-β受体和Smads的参与,此信号通路的异常是肿瘤的发生发展以及浸润和转移的重要因素。Smad4是Smads家族成员之一,是转导TGF-β信号的重要胞浆内信号级联分子,是TGF-β信号通路上的重要调节靶点,在传导信号由细胞膜到细胞核内调节靶基因的转录过程中起重要作用,其失功能,不管是表达缺失还是基因突变均与大肠肿瘤增强的侵袭和转移能力有关,它能够与活化的受体激活型Smads分子形成复合物,移位到细胞核与其他转录因子协同作用,调节TGF-β应答基因的转录。
Wnt/β-catenin信号传导通路是一条在生物进化中极为保守的通路,这条包括Wnt族基因及其产物与β-连环素(β-catenin)基因等许多其他相关基因的蛋白质产物构成的极为复杂的信号通路控制了从果蝇到人类胚胎发育、细胞命运及组织器官形态形成以及肿瘤发生的诸多重大事件,它与细胞的发育分化密切相关,对正常和肿瘤细胞生长都至关重要。Wnt/β-catenin信号转导通路的失控在大肠肿瘤发生发展过程中具有重要作用,在约80%的结直肠肿瘤细胞中发现存在β-catenin的高转录活性,β-catenin蛋白在细胞核内异常聚集,与T细胞因子/淋巴增强因子(T cell factor/lymphoid enhancer factor,Tcf/Lef)结合形成复合物激活Wnt下游靶基因的转录,如细胞周期素D1(cyclinD1)、紧密连接蛋白-1(Claudin-1)、c-myc等,导致细胞增殖失控而致癌。
TGF-β/Smad4、Wnt/β-catenin信号通路异常与大肠肿瘤的发生、发展及转移密切相关,两条通路在结肠上皮细胞分化的调节过程中都起着重要作用。目前关于两条通路之间交互通话并且共同作用于大肠癌发生机制的研究很多,如Smad4基因可以与β-catenin/Tcf复合物在某些共同靶基因的调节过程中相互作用、聚合,又如有研究证实大鼠胃泌素启动子可被β-catenin/Tcf、TGF-β/Smads信号通路共同调节,这些都证明TGF-β信号介导者Smads家族成员可能参与了Wnt/β-catenin信号发放的调节,TGF-β途径可能与Wnt途径协同的调节某些基因的表达,但具体相互作用机制尚不明确。Smad4是否存在不依赖于TGF-β信号通路而通过作用于Wnt/β-catenin通路发挥其肿瘤抑制作用呢?已有研究初步支持这一论点。例如,有研究报道把Smad4导入Smad4缺失的SW480细胞可以导致这些细胞在裸鼠体内致瘤性的缺失,并伴随有E-钙黏蛋白(E-cadherin)和P-钙黏蛋白(P-cadherin)的诱导转录,E-cadherin可以抑制β-catenin介导的信号转导,提示Smad4可能通过E-cadherin对β-catenin的调节间接地影响Wnt通路的进程,亦有研究显示在大肠癌细胞中Smad4能够以一种不依赖于TGF-β信号通路的方式通过对肿瘤转移促进基因Claudin-1的调节来发挥其侵袭抑制功能,这些研究结果都提示Smad4可能参与调控了Wnt/β-catenin信号通路的传导过程。
因此,本课题我们以Smad4是否参与调控Wnt/β-catenin信号通路的转导过程及探究其机制为线索进行研究。研究的目的在于阐明除了经典的TGF-β信号通路,Smad4还能否通过其它的信号通路对细胞增殖发挥负调控作用。在研究的第一部分我们构建Smad4真核表达载体并稳定转染Smad4基因缺失的大肠癌SW480细胞使之成功表达,第二部分中首先应用Topflash/Fopflash荧光素酶报告基因方法检测Smad4转染大肠癌细胞前后Wnt/β-catenin信号通路的活性,再分别以RT-PCR、Western Blot方法检测转染前后Wnt通路关键基因的mRNA及蛋白表达变化以探索Smad4对Wnt/β-catenin调节的机制,并重点观察Smad4是否影响Wnt通路关键分子β-catenin的细胞内转位情况。本研究初步阐明Smad4能以一种不依赖于TGF-β信号途径的方式发挥其肿瘤生长抑制作用,研究结果将为未来大肠癌的分子靶向治疗提供理论基础和新的思路。
材料与方法
1.构建Smad4真核表达载体
从人宫颈癌HeLa细胞中克隆Smad4基因,以pcDNA3.1(+)为载体构建真核表达质粒。按照Genbank上公布的Smad4 mRNA序列(NM__005359),在其编码区(CDS)两端设计分别带有BamHI、EcoRI酶切位点的引物,利用聚合酶链式反应扩增Smad4 CDS区,BamHI、EcoRI双酶切回收PCR产物和真核表达载体pcDNA3.1(+),T4连接酶连接pcDNA3.1(+)与目的DNA片段,转化到感受态Top10菌液中,挑取单个阳性克隆菌落扩大培养,SDS碱裂解法制备重组质粒DNA后行BamHI、EcoRI双酶切鉴定,选择插入片段正确的克隆测序鉴定。
2.Smad4转染大肠癌SW480细胞系及建立稳定细胞克隆
用Lipofectamine 2000脂质体介导重组质粒转染大肠癌SW480细胞,以转染pcDNA3.1(+)空载体及未转染细胞作为对照。转染后24h用筛选剂量1000μg/mlG418进行抗性筛选,分离收集稳定克隆株细胞扩大培养,维持选择压力。转染Smad4重组质粒的命名为Smad4-SW480细胞、转染pcDNA3.1(+)空载体的命名为pc-SW480细胞,RT-PCR、免疫荧光检测转染前后Smad4基因表达及分布。
3.Topflash/Fopflash瞬时转染检测Smad4对Wnt/β-catenin信号活性的影响
Lipofectamine 2000脂质体介导双荧光素酶报告质粒瞬时转染SW480、Smad4-SW480及pc-SW480细胞,每组细胞分别转染Topflash或Fopflash,并以pRL-SV40作为内对照。48h后用Dual-Luciferase Reporter Assay System Kit在GloMax Luminometry System下检测各组细胞荧光值,每组细胞Topflash/Fopflash的比值即代表该细胞Wnt/β-catenin信号活性,实验重复3次,每次每组细胞设3个复孔。
4.RT-PCR检测Smad4对Wnt通路关键基因mRNA表达的影响
Smad4转染对大肠癌SW480细胞中Wnt/β-catenin信号通路上关键基因β-catenin、Claudin-1、MMP-7 mRNA表达的影响用RT-PCR方法检测。
5.Western Blot检测Smad4对Wnt通路相关蛋白表达的影响
Smad4对Wnt/β-catenin信号通路上重要因子β-catenin(胞浆、胞核)、Claudin-1蛋白表达的影响用Western Blot检测,另外检测上皮细胞钙黏蛋白E-cadherin的表达变化。
6.免疫荧光观察Smad4对Wnt通路相关蛋白细胞内分布的影响
荧光显微镜下分别观察β-catenin、E-cadherin、Claudin-1在转染前后不同组别细胞内的表达分布。
7.统计学分析
数据采用SPSS13.0版统计学软件进行处理。每一实验结果至少重复3次,计量资料以均数±标准差表示,多样本均数的比较采用单因素方差分析(one-wayanalysis of variance),组间多重比较采用Bonferroni法,以P＜0.05视为差异有显著性意义。
结果
1.重组质粒pcDNA3.1(+)-Smad4的酶切鉴定及序列测定结果
SDS碱裂解法制备重组质粒DNA后行BamHI、EcoRI双酶切鉴定,证实有大小正确的插入片段;以pcDNA3.1(+)-Smad4重组质粒为模板进行PCR鉴定在预计目的片段处有明显亮带;重组质粒经Invitrogen公司测序及通过美国国家生物技术信息中心(NCBI)在线比对,重组克隆与目的基因序列同源性100%,证明重组成功。
2.稳定表达Smad4基因的SW480细胞模型构建结果
重组质粒转染Smad4基因缺失的大肠癌SW480细胞,通过G418抗性筛选得到细胞克隆Smad4-SW480及其对照pc-SW480细胞。RT-PCR结果证实通过基因转染得到了外源性Smad4高表达的SW480细胞,荧光显微镜观察下可见Smad4蛋白在SW480细胞中主要为细胞核和细胞浆表达,说明重组质粒在SW480中表达成功,转染pcDNA3.1(+)空质粒、未转染的SW480细胞均无可见荧光。以上结果证明Smad4基因高表达的结肠癌细胞模型构建成功。
3.Smad4对Wnt/β-catenin信号转录活性的影响
检测结果经单因素方差分析得出SW480细胞转染前后双荧光素酶报告基因活性有显著差异(P=0.000＜0.05),采用Bonferroni法进行组间多重比较得出,稳定转染Smad4基因的Smad4-SW480(1)(2574.44±449.725)、Smad4-SW480(2)(2768.89±699.383)细胞荧光素酶活性相对值显著低于SW480细胞(8506.67±910.536)(P=0.000＜0.05),提示Smad4表达重建显著降低了SW480细胞中Wnt/β-catenin信号转录活性,而SW480与转染空载体的pc-SW480细胞荧光素酶活性相比较差异无统计学意义(P=1.000＞0.05)。
4.Smad4重建对β-catenin mRNA、蛋白表达及在细胞内分布的影响
β-catenin在Wnt/β-catenin信号通路上起着关键的调控作用,其表达水平及细胞内分布情况能直接反应Wnt通路的状态。RT-PCR结果用比较β-catenin与GADPH的比值得出,Western Blot(胞浆、胞核)结果用β-catenin与β-actin的比值得出。无论在mRNA还是蛋白水平,Smad4-SW480(1)、(2)细胞组β-catenin表达量与SW480细胞组相比差异均有显著统计学意义(P＜0.05),提示Smad4重建降低了SW480细胞β-catenin的转录活性以及胞浆、胞核的蛋白表达水平,而SW480与转染空载体的pc-SW480细胞相比较差异无统计学意义(P＞0.05)。
因为β-catenin在Wnt通路信号传导过程中发挥作用与其在胞浆、胞核的分布密切相关,我们应用间接免疫荧光法进一步分析β-catenin在Smad4基因转染前后不同细胞内的分布情况。荧光显微镜观察下可见在无Smad4基因表达的SW480细胞中,β-catenin蛋白主要分布于胞浆,少量分布在胞核,转染Smad4基因后,β-catenin发生自胞浆、胞核向胞膜的转移。
5.Smad4重建对E-cadherin、Claudin-1蛋白表达的影响
E-cadherin已知能够与β-catenin结合成为复合体起着负性调节Wnt/β-catenin信号活性的作用。稳定表达Smad4的Smad4-SW480(1)、(2)细胞组E-cadherin蛋白表达水平与SW480细胞组比较差异有显著统计学意义(P=0.000＜0.05),Smad4重建提高了SW480细胞E-cadherin蛋白表达水平,而SW480与pc-SW480细胞E-cadherin蛋白水平比较无显著差异(P=0.202＞0.05)。
Claudin-1蛋白是一种已知的β-catenin信号下游重要的靶蛋白,对Claudin-1蛋白相对表达量进行多重比较得出Smad4-SW480(1)、(2)细胞组与SW480细胞组之间均有显著差异(P=0.000＜0.05),提示Smad4表达降低了大肠癌SW480细胞Wnt通路下游Claudin-1蛋白水平,间接证明其对Wnt通路起着负性调节的作用。
6.Smad4对Claudin-1、MMP-7 mRNA表达的作用
Claudin-1、MMP-7是Wnt/β-catenin信号通路下游重要的代表分子。Smad4-SW480细胞Claudin-1 mRNA水平与SW480细胞相比差异有显著性意义(P=0.000＜0.05),提示Smad4重建降低了SW480细胞中Claudin-1的mRNA表达。同法比较各组间MMP-7 mRNA表达,Smad4-SW480与SW480之间有显著差异(P=0.024＜0.05),提示Smad4能够降低SW480细胞中MMP-7 mRNA表达,以上结果提示Smad4对Wnt通路下游基因转录起着负性调节的作用。
结论
1.运用基因重组技术构建得到pcDNA3.1(+)-Smad4真核表达质粒。
2.重组质粒稳定转染Smad4基因缺失的大肠癌SW480细胞得到Smad4-SW480细胞克隆,经RT-PCR及免疫荧光技术证实得到外源性Smad4高表达的大肠癌SW480细胞系,表明成功构建了Smad4基因稳定表达的大肠癌细胞模型。
3.通过瞬时转染双荧光素酶报告基因的方法测定Wnt/β-catenin信号,证实Smad4对大肠癌细胞系中Wnt/β-catenin通路的活性有抑制作用。
4.Smad4重建能降低Wnt/β-catenin通路分子开关β-catenin mRNA及蛋白(胞浆、胞核)表达水平,并导致β-catenin发生自胞浆、胞核向胞膜的转位。
5.Smad4在大肠癌细胞中能诱导上皮细胞钙黏蛋白E-cadherin表达增强,同时能降低紧密连接蛋白Claudin-1的蛋白表达水平。
6.Smad4能抑制Wnt/β-catenin通路下游靶基因Claudin-1、MMP-7的mRNA表达。
7.Smad4在大肠癌细胞中参与调控了Wnt/β-catenin信号通路的转导过程,对Wnt信号有负性调节作用。本研究提示Smad4除了参与经典的TGF-β信号通路外,还可能通过Wnt/β-catenin信号途径发挥肿瘤抑制作用。
Background and objectives
Colorectal cancer(CRC) is one of the major malignancies in the world,the incidence rate of CRC is the third most common type of cancer.With the many changes having taken place in people's diet and lifestyle,CRC has become the fourth most frequent cause of death due to cancer.in china,and the number of new cases arising each year is still increasing.
At present,it is considered that the development of cancer has the relation to the failure to anti-oncogene、activation of oncogene because of deletion and mutation. Smad4,originally characterized as a central intracellular signal transduction component of the transforming growth factor-β(TGF-β) family in epithelial cells,is regarded as a tumor suppressor gene predominantly involved in gastrointestinal carcinogenesis.Loss of Smad4 function either due to loss of its expression or genetic mutation is considered to be a genetically late step and occurs in a significant proportion of colon and pancreatic cancer.
The Wnt/β-catenin signaling is implicated in colon carcinogenesis and suppression of this signal pathway is associated with a reduction of cellular proliferation and the induction of cellular differentiation.β-catenin performs dual functions,which include a crucial role in cell-cell adhesion and the Wnt/β-catenin pathway.Activation of Wnt signaling inhibits GSK dependent phosphorylation ofβ-catenin and results in an increase inβ-catenin protein levels.The accumulation ofβ-catenin promotes its nuclear translocation where it regulates Wnt target genes transcription in collaboration with factors from the Tcf/Lef family.
Both TGF-βand Wnt signaling pathways are thought to play critical roles in regulating the differentiation processes of colonic epithelial cells.Increasing evidence of cross talk between Smad signaling and the Wnt pathway has been reported during the past years.Nevertheless,the relationship between the migration-suppressive responses of Smad4 and the activity of Wnt signaling pathway is not thoroughly understood.Recent evidences have suggested additional mechanisms underlying the effects of Smad4,for example,re-expression of Smad4 in the Smad4-deficient SW480 colon carcinoma cells resulted in loss of tumorigenicity of these cells in nude mice that was accompanied by the restoration of a more epithelioid morphology and induced transcription of E-cadherin and P-cadherin,in addition,it was recently shown that a novel mechanism underlying Smad4 invasion-suppressive function through regulation of a potential metastatic modulator,Claudin-1,in a TGF-βindependent manner.Cell-cell adhesion in epithelial cell sheets is maintained mainly through adherens junctions and tight junctions.E-cadherin is a key player in cell—cell adhesion connecting adjacent cells via the cadherin—catenin adhesion complex. Claudin-1,the most apical cell-cell contacts and the most important tight junction for barrier function in colon cancer,was recently identified as a target of Wnt/β-catenin signaling.Based on these studies,we hypothesized that there was a relationship between Smad4,a tumor suppressor protein,and Wnt/β-catenin signaling in colorectal carcinoma.
The objective of this study was to investigate the impact of enhanced Smad4 expression on Wnt/β-catenin signaling activity in colon cancer cells.In this study,we reported that Smad4 directly suppressed the Wnt/β-catenin signal pathway in Smad4-responsive human colon carcinoma cells by down-regulating the cytoplasmic and nucleus expression ofβ-catenin and increasing localization ofβ-catenin to the plasma membrane.We further showed that Smad4 induced the expression of the intercellular adhesion molecule E-cadherin and reduced transcriptional activity ofβ-catenin/Tcf target genes,such as Claudin-1 and MMP-7.The data suggested that restoration of Smad4 in Smad4-deficient cells may provide a potential therapeutic strategy for intervention of colon cancer progress.
Materials and methods
1.Construction of eukaryotic expression plasmid
A cDNA of human Smad4 was amplified by RT-PCR and constructed into the eukaryotic expression vector pcDNA3.1(+),the recombinant plasmid pcDNA3.1(+)-Stand4 was identified by enzyme digestion、PCR amplification and DNA sequencing.
2.Overexpression of Stand4 by stable transfection
Cells were transfected using Lipofectamine2000 according to the manufacturer's instructions and selected in complete medium containing 1mg/ml G418 sulfate at 48h after transfection,G418-resistant cell clones were isolated for 2 weeks.Two clones that expressed similar levels of Smad4 in protein concentrations were selected for subsequent experiments.SW480 cells that were stably transfected with pcDNA3.1(+) were used as a control.The expression changes of Smad4 were detected using RT-PCR.A fluorescence microscope was used to detected the distribution of Smad4.
3.Transient transfection and luciferase assays
Cells were cotransfected with a firefly luciferase reporter construct Topflash or Fopflash,and a reference construct that contains Renilla reniformis luciferase, pRL-SV40,using Lipofectamine 2000.Forty-eight hours later,luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit in a GloMax Luminometry System.Tcf-mediated gene transcription was determined by the ratio of pTopflash to pFopflash luciferase activity.Firefly luciferase activity was normalized to Renilla reniformis luciferase activity and plotted as mean±SD from three independent experiments.
4.Semiquantitative RT-PCR
Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit.PCR analysis was performed using gene-specific primers,such asβ-catenin、Claudin-1、MMP-7,etc.
5.Immunoblot analysis
Equal amounts of protein samples were electrophoretically separated by SDS-PAGE in separation gels and transferred to nitrocellulose sheets.They were incubated overnight at 4℃with the primary antibodies diluted in the same buffer (Smad4 1:400;E-cadherin 1:600;β-catenin 1:1000;Claudin-1 1:1000;β-actin 1:3000 dilutions).Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies(1:5000 dilutions) for enhanced chemiluminescence detection using an ECL detection kit.
6.Immunofluorescence
Immunofluorescence were performed with the following primary antibodies: anti-β-catenin、-claudin-1 and -E-cadherin.FITC anti- mouse and anti-rabbit、Rhodamine Red-X anti-rabbit were used as secondary antibodies at 1:200 dilutions, respectively.Stained glass coverslips were viewed and photographed using an OLYMPUS fluorescence microscope.
7.Statistical analysis
Each experiment was repeated at least three times.Differences in mean values were evaluated using One-way ANOVA tests,Bonferrori analysis were performed in groups comparison.All statistical analyses were performed using SPSS 13.0 software. P＜0.05 was considered to be statistically significant.
Results
1.The DNA oligonucleotides encoding Smad4 mRNA were synthesized,and cloned into the eukaryotic expression plasmid,which were identified by restriction enzyme digestion analysis and DNA sequencing.Recombinant Smad4 eukaryotic expression vector pcDNA3.1(+)-Smad4 was constructed successfully.
2.SW480 cells which were transfected with pcDNA3.1(+)-Smad4 were screened by G418,two clones of SW480 with stable expression of Smad4 were established using Smad4 eukaryotic expression plasmid.The results of RT-PCR demonstrated that the mRNA level of Smad4 was increased notably in the Smad4-SW480 cells, and the result of immunofluorescence showed that Smad4 was predominantly localized in cytoplasm and nucleus.
3.We analyzed Wnt/β-catenin signaling activity in SW480、pc-SW480 and Smad4-SW480 cells by transiently transfecting Tcf and mutant Tcf reporter plasmids(Topflash and Fopflash,respectively).Relative luciferase activities were calculated as described in Methods.The data show that the relative transcriptional activity of theβ-catenin/Lef complex was decreased(3- to 4-fold) in Smad4-SW480 cells compared with SW480 or pc-SW480 cells.This analysis confirmed that Wnt signaling activity was significantly suppressed by Smad4 expression.
4.There was lessβ-catenin mRNA expression in Smad4-transfected cells than Smad4-negative colon carcinoma cells(P＜0.05).Relatively lessβ-catenin was detected in the cytoplasmic and nuclear fractions of Smad4-transfected cells compared with SW480 and pc-SW480 groups(P＜0.05).As the function ofβ-catenin in signal transduction depends on its cytoplasmic/nuclear localization, we further analyzed the distribution of theβ-catenin protein through indirect immunofluorescence.SW480 cells showed strong staining forβ-catenin in the cytoplasm and nucleus.Smad4 re-expression cells,in contrast,showed staining in the plasma membrane.The data demonstrated that up-regulation of Smad4 caused the relocalization ofβ-catenin from cytoplasm and nucleus to the plasma membrane in SW480 cells.
5.Low levels of E-cadherin protein were observed in SW480 cells and its control cell lines(pc-SW480) with relatively more E-cadherin detected in cells that had increased Smad4(P＜0.05).Inverse data were found in cellular Claudin-1 concentration,we observed reduced expression of claudin-1 in clones transfected with Smad4 when compared with the Smad4-negative cells(P＜0.05).We performed immunofluorescence analysis to further investigate whether up-regulation of Smad4 expression changed the localization of E-cadherin and Claudin-1.The localization of E-cadherin and Claudin-1 was not altered in Smad4-SW480 cells.The results of these two studies indicated a decrease in the expression of Claudin-1 but a robust increase in E-cadherin expression in Smad4-SW480 clones compared with SW480 cells.
6.There was less MMP-7 and Claudin-1 mRNA in Smad4-SW480 clones than SW480 or pc-SW480 cells(P＜0.05).Transfection of Smad4 resulted in reduced MMP-7 and Claudin-1 mRNA concentration as compared to cells transfected with the control or nonsense.
Conclusions
1.Recombinant Smad4 eukaryotic expression vector pcDNA3.1(+)-Smad4 was constructed successfully.
2.Stable colon cancer cells that have increased Smad4 expression were created using a eukaryotic vector containing Smad4-pcDNA3.1(+).
3.Re-expression of Smad4 in SW480 cells leads to a reduction in Wnt/β-catenin signaling activity.
4.Smad4 restoration decreasesβ-catenin expression(cytoplasm and nuclus) and alters its localization from cytoplasm to the plasma membrane in SW480 cells.
5.Up-regulation of Smad4 also increased the expression of E-cadherin and decreased the expression of Claudin-1.
6.Smad4 restoration in SW480 cells decreases mRNA expression of Wnt/β-catenin signaling target genes,such as Claudin-1 and MMP-7.
7.The data suggested that restoration of Smad4 in Smad4-deficient cells may provide a potential therapeutic strategy for intervention of colon cancer progress.
大肠癌(colorectal cancer,CRC)是临床最常见的恶性肿瘤之一,在全世界范围内,大肠癌的发病率男女均处于恶性肿瘤的第3位,其中在西方发达国家,大肠癌的发病率处于第2位。在我国,随着生活水平的不断提高以及高蛋白、高脂肪、低纤维素等饮食结构的改变,大肠癌的发病率呈上升态势,排在恶性肿瘤和致死因素的第4位,对人民健康和生命构成严重威胁。目前大肠癌发生的分子机制尚不十分清楚,其病因和危险因素复杂,一般认为其发生是机体的内因与环境的外因相互作用的结果。流行病学研究认为大肠癌的发生主要与饮食、遗传、环境等因素关系密切。
目前研究证实大肠癌的发生、发展是一个多阶段、多基因参与的过程,涉及到多条信号转导途径的异常激活、失活及多种癌基因、抑癌基因的异常表达。转化生长因子β(transforming growth factor-β,TGF-β)及其受体与下游信号蛋白Smads组成了一个非常复杂的信号传导网络,其信号转导需要TGF-β受体和Smads的参与,此信号通路的异常是肿瘤的发生发展以及浸润和转移的重要因素。Smad4是Smads家族成员之一,是转导TGF-β信号的重要胞浆内信号级联分子,是TGF-β信号通路上的重要调节靶点,在传导信号由细胞膜到细胞核内调节靶基因的转录过程中起重要作用,其失功能,不管是表达缺失还是基因突变均与大肠肿瘤增强的侵袭和转移能力有关,它能够与活化的受体激活型Smads分子形成复合物,移位到细胞核与其他转录因子协同作用,调节TGF-β应答基因的转录。
Wnt/β-catenin信号传导通路是一条在生物进化中极为保守的通路,这条包括Wnt族基因及其产物与β-连环素(β-catenin)基因等许多其他相关基因的蛋白质产物构成的极为复杂的信号通路控制了从果蝇到人类胚胎发育、细胞命运及组织器官形态形成以及肿瘤发生的诸多重大事件,它与细胞的发育分化密切相关,对正常和肿瘤细胞生长都至关重要。Wnt/β-catenin信号转导通路的失控在大肠肿瘤发生发展过程中具有重要作用,在约80%的结直肠肿瘤细胞中发现存在β-catenin的高转录活性,β-catenin蛋白在细胞核内异常聚集,与T细胞因子/淋巴增强因子(T cell factor/lymphoid enhancer factor,Tcf/Lef)结合形成复合物激活Wnt下游靶基因的转录,如细胞周期素D1(cyclinD1)、紧密连接蛋白-1(Claudin-1)、c-myc等,导致细胞增殖失控而致癌。
TGF-β/Smad4、Wnt/β-catenin信号通路异常与大肠肿瘤的发生、发展及转移密切相关,两条通路在结肠上皮细胞分化的调节过程中都起着重要作用。目前关于两条通路之间交互通话并且共同作用于大肠癌发生机制的研究很多,如Smad4基因可以与β-catenin/Tcf复合物在某些共同靶基因的调节过程中相互作用、聚合,又如有研究证实大鼠胃泌素启动子可被β-catenin/Tcf、TGF-β/Smads信号通路共同调节,这些都证明TGF-β信号介导者Smads家族成员可能参与了Wnt/β-catenin信号发放的调节,TGF-β途径可能与Wnt途径协同的调节某些基因的表达,但具体相互作用机制尚不明确。Smad4是否存在不依赖于TGF-β信号通路而通过作用于Wnt/β-catenin通路发挥其肿瘤抑制作用呢?已有研究初步支持这一论点。例如,有研究报道把Smad4导入Smad4缺失的SW480细胞可以导致这些细胞在裸鼠体内致瘤性的缺失,并伴随有E-钙黏蛋白(E-cadherin)和P-钙黏蛋白(P-cadherin)的诱导转录,E-cadherin可以抑制β-catenin介导的信号转导,提示Smad4可能通过E-cadherin对β-catenin的调节间接地影响Wnt通路的进程,亦有研究显示在大肠癌细胞中Smad4能够以一种不依赖于TGF-β信号通路的方式通过对肿瘤转移促进基因Claudin-1的调节来发挥其侵袭抑制功能,这些研究结果都提示Smad4可能参与调控了Wnt/β-catenin信号通路的传导过程。
因此,本课题我们以Smad4是否参与调控Wnt/β-catenin信号通路的转导过程及探究其机制为线索进行研究。研究的目的在于阐明除了经典的TGF-β信号通路,Smad4还能否通过其它的信号通路对细胞增殖发挥负调控作用。在研究的第一部分我们构建Smad4真核表达载体并稳定转染Smad4基因缺失的大肠癌SW480细胞使之成功表达,第二部分中首先应用Topflash/Fopflash荧光素酶报告基因方法检测Smad4转染大肠癌细胞前后Wnt/β-catenin信号通路的活性,再分别以RT-PCR、Western Blot方法检测转染前后Wnt通路关键基因的mRNA及蛋白表达变化以探索Smad4对Wnt/β-catenin调节的机制,并重点观察Smad4是否影响Wnt通路关键分子β-catenin的细胞内转位情况。本研究初步阐明Smad4能以一种不依赖于TGF-β信号途径的方式发挥其肿瘤生长抑制作用,研究结果将为未来大肠癌的分子靶向治疗提供理论基础和新的思路。
材料与方法
1.构建Smad4真核表达载体
从人宫颈癌HeLa细胞中克隆Smad4基因,以pcDNA3.1(+)为载体构建真核表达质粒。按照Genbank上公布的Smad4 mRNA序列(NM__005359),在其编码区(CDS)两端设计分别带有BamHI、EcoRI酶切位点的引物,利用聚合酶链式反应扩增Smad4 CDS区,BamHI、EcoRI双酶切回收PCR产物和真核表达载体pcDNA3.1(+),T4连接酶连接pcDNA3.1(+)与目的DNA片段,转化到感受态Top10菌液中,挑取单个阳性克隆菌落扩大培养,SDS碱裂解法制备重组质粒DNA后行BamHI、EcoRI双酶切鉴定,选择插入片段正确的克隆测序鉴定。
2.Smad4转染大肠癌SW480细胞系及建立稳定细胞克隆
用Lipofectamine 2000脂质体介导重组质粒转染大肠癌SW480细胞,以转染pcDNA3.1(+)空载体及未转染细胞作为对照。转染后24h用筛选剂量1000μg/mlG418进行抗性筛选,分离收集稳定克隆株细胞扩大培养,维持选择压力。转染Smad4重组质粒的命名为Smad4-SW480细胞、转染pcDNA3.1(+)空载体的命名为pc-SW480细胞,RT-PCR、免疫荧光检测转染前后Smad4基因表达及分布。
3.Topflash/Fopflash瞬时转染检测Smad4对Wnt/β-catenin信号活性的影响
Lipofectamine 2000脂质体介导双荧光素酶报告质粒瞬时转染SW480、Smad4-SW480及pc-SW480细胞,每组细胞分别转染Topflash或Fopflash,并以pRL-SV40作为内对照。48h后用Dual-Luciferase Reporter Assay System Kit在GloMax Luminometry System下检测各组细胞荧光值,每组细胞Topflash/Fopflash的比值即代表该细胞Wnt/β-catenin信号活性,实验重复3次,每次每组细胞设3个复孔。
4.RT-PCR检测Smad4对Wnt通路关键基因mRNA表达的影响
Smad4转染对大肠癌SW480细胞中Wnt/β-catenin信号通路上关键基因β-catenin、Claudin-1、MMP-7 mRNA表达的影响用RT-PCR方法检测。
5.Western Blot检测Smad4对Wnt通路相关蛋白表达的影响
Smad4对Wnt/β-catenin信号通路上重要因子β-catenin(胞浆、胞核)、Claudin-1蛋白表达的影响用Western Blot检测,另外检测上皮细胞钙黏蛋白E-cadherin的表达变化。
6.免疫荧光观察Smad4对Wnt通路相关蛋白细胞内分布的影响
荧光显微镜下分别观察β-catenin、E-cadherin、Claudin-1在转染前后不同组别细胞内的表达分布。
7.统计学分析
数据采用SPSS13.0版统计学软件进行处理。每一实验结果至少重复3次,计量资料以均数±标准差表示,多样本均数的比较采用单因素方差分析(one-wayanalysis of variance),组间多重比较采用Bonferroni法,以P＜0.05视为差异有显著性意义。
结果
1.重组质粒pcDNA3.1(+)-Smad4的酶切鉴定及序列测定结果
SDS碱裂解法制备重组质粒DNA后行BamHI、EcoRI双酶切鉴定,证实有大小正确的插入片段;以pcDNA3.1(+)-Smad4重组质粒为模板进行PCR鉴定在预计目的片段处有明显亮带;重组质粒经Invitrogen公司测序及通过美国国家生物技术信息中心(NCBI)在线比对,重组克隆与目的基因序列同源性100%,证明重组成功。
2.稳定表达Smad4基因的SW480细胞模型构建结果
重组质粒转染Smad4基因缺失的大肠癌SW480细胞,通过G418抗性筛选得到细胞克隆Smad4-SW480及其对照pc-SW480细胞。RT-PCR结果证实通过基因转染得到了外源性Smad4高表达的SW480细胞,荧光显微镜观察下可见Smad4蛋白在SW480细胞中主要为细胞核和细胞浆表达,说明重组质粒在SW480中表达成功,转染pcDNA3.1(+)空质粒、未转染的SW480细胞均无可见荧光。以上结果证明Smad4基因高表达的结肠癌细胞模型构建成功。
3.Smad4对Wnt/β-catenin信号转录活性的影响
检测结果经单因素方差分析得出SW480细胞转染前后双荧光素酶报告基因活性有显著差异(P=0.000＜0.05),采用Bonferroni法进行组间多重比较得出,稳定转染Smad4基因的Smad4-SW480(1)(2574.44±449.725)、Smad4-SW480(2)(2768.89±699.383)细胞荧光素酶活性相对值显著低于SW480细胞(8506.67±910.536)(P=0.000＜0.05),提示Smad4表达重建显著降低了SW480细胞中Wnt/β-catenin信号转录活性,而SW480与转染空载体的pc-SW480细胞荧光素酶活性相比较差异无统计学意义(P=1.000＞0.05)。
4.Smad4重建对β-catenin mRNA、蛋白表达及在细胞内分布的影响
β-catenin在Wnt/β-catenin信号通路上起着关键的调控作用,其表达水平及细胞内分布情况能直接反应Wnt通路的状态。RT-PCR结果用比较β-catenin与GADPH的比值得出,Western Blot(胞浆、胞核)结果用β-catenin与β-actin的比值得出。无论在mRNA还是蛋白水平,Smad4-SW480(1)、(2)细胞组β-catenin表达量与SW480细胞组相比差异均有显著统计学意义(P＜0.05),提示Smad4重建降低了SW480细胞β-catenin的转录活性以及胞浆、胞核的蛋白表达水平,而SW480与转染空载体的pc-SW480细胞相比较差异无统计学意义(P＞0.05)。
因为β-catenin在Wnt通路信号传导过程中发挥作用与其在胞浆、胞核的分布密切相关,我们应用间接免疫荧光法进一步分析β-catenin在Smad4基因转染前后不同细胞内的分布情况。荧光显微镜观察下可见在无Smad4基因表达的SW480细胞中,β-catenin蛋白主要分布于胞浆,少量分布在胞核,转染Smad4基因后,β-catenin发生自胞浆、胞核向胞膜的转移。
5.Smad4重建对E-cadherin、Claudin-1蛋白表达的影响
E-cadherin已知能够与β-catenin结合成为复合体起着负性调节Wnt/β-catenin信号活性的作用。稳定表达Smad4的Smad4-SW480(1)、(2)细胞组E-cadherin蛋白表达水平与SW480细胞组比较差异有显著统计学意义(P=0.000＜0.05),Smad4重建提高了SW480细胞E-cadherin蛋白表达水平,而SW480与pc-SW480细胞E-cadherin蛋白水平比较无显著差异(P=0.202＞0.05)。
Claudin-1蛋白是一种已知的β-catenin信号下游重要的靶蛋白,对Claudin-1蛋白相对表达量进行多重比较得出Smad4-SW480(1)、(2)细胞组与SW480细胞组之间均有显著差异(P=0.000＜0.05),提示Smad4表达降低了大肠癌SW480细胞Wnt通路下游Claudin-1蛋白水平,间接证明其对Wnt通路起着负性调节的作用。
6.Smad4对Claudin-1、MMP-7 mRNA表达的作用
Claudin-1、MMP-7是Wnt/β-catenin信号通路下游重要的代表分子。Smad4-SW480细胞Claudin-1 mRNA水平与SW480细胞相比差异有显著性意义(P=0.000＜0.05),提示Smad4重建降低了SW480细胞中Claudin-1的mRNA表达。同法比较各组间MMP-7 mRNA表达,Smad4-SW480与SW480之间有显著差异(P=0.024＜0.05),提示Smad4能够降低SW480细胞中MMP-7 mRNA表达,以上结果提示Smad4对Wnt通路下游基因转录起着负性调节的作用。
结论
1.运用基因重组技术构建得到pcDNA3.1(+)-Smad4真核表达质粒。
2.重组质粒稳定转染Smad4基因缺失的大肠癌SW480细胞得到Smad4-SW480细胞克隆,经RT-PCR及免疫荧光技术证实得到外源性Smad4高表达的大肠癌SW480细胞系,表明成功构建了Smad4基因稳定表达的大肠癌细胞模型。
3.通过瞬时转染双荧光素酶报告基因的方法测定Wnt/β-catenin信号,证实Smad4对大肠癌细胞系中Wnt/β-catenin通路的活性有抑制作用。
4.Smad4重建能降低Wnt/β-catenin通路分子开关β-catenin mRNA及蛋白(胞浆、胞核)表达水平,并导致β-catenin发生自胞浆、胞核向胞膜的转位。
5.Smad4在大肠癌细胞中能诱导上皮细胞钙黏蛋白E-cadherin表达增强,同时能降低紧密连接蛋白Claudin-1的蛋白表达水平。
6.Smad4能抑制Wnt/β-catenin通路下游靶基因Claudin-1、MMP-7的mRNA表达。
7.Smad4在大肠癌细胞中参与调控了Wnt/β-catenin信号通路的转导过程,对Wnt信号有负性调节作用。本研究提示Smad4除了参与经典的TGF-β信号通路外,还可能通过Wnt/β-catenin信号途径发挥肿瘤抑制作用。
Background and objectives
Colorectal cancer(CRC) is one of the major malignancies in the world,the incidence rate of CRC is the third most common type of cancer.With the many changes having taken place in people's diet and lifestyle,CRC has become the fourth most frequent cause of death due to cancer.in china,and the number of new cases arising each year is still increasing.
At present,it is considered that the development of cancer has the relation to the failure to anti-oncogene、activation of oncogene because of deletion and mutation. Smad4,originally characterized as a central intracellular signal transduction component of the transforming growth factor-β(TGF-β) family in epithelial cells,is regarded as a tumor suppressor gene predominantly involved in gastrointestinal carcinogenesis.Loss of Smad4 function either due to loss of its expression or genetic mutation is considered to be a genetically late step and occurs in a significant proportion of colon and pancreatic cancer.
The Wnt/β-catenin signaling is implicated in colon carcinogenesis and suppression of this signal pathway is associated with a reduction of cellular proliferation and the induction of cellular differentiation.β-catenin performs dual functions,which include a crucial role in cell-cell adhesion and the Wnt/β-catenin pathway.Activation of Wnt signaling inhibits GSK dependent phosphorylation ofβ-catenin and results in an increase inβ-catenin protein levels.The accumulation ofβ-catenin promotes its nuclear translocation where it regulates Wnt target genes transcription in collaboration with factors from the Tcf/Lef family.
Both TGF-βand Wnt signaling pathways are thought to play critical roles in regulating the differentiation processes of colonic epithelial cells.Increasing evidence of cross talk between Smad signaling and the Wnt pathway has been reported during the past years.Nevertheless,the relationship between the migration-suppressive responses of Smad4 and the activity of Wnt signaling pathway is not thoroughly understood.Recent evidences have suggested additional mechanisms underlying the effects of Smad4,for example,re-expression of Smad4 in the Smad4-deficient SW480 colon carcinoma cells resulted in loss of tumorigenicity of these cells in nude mice that was accompanied by the restoration of a more epithelioid morphology and induced transcription of E-cadherin and P-cadherin,in addition,it was recently shown that a novel mechanism underlying Smad4 invasion-suppressive function through regulation of a potential metastatic modulator,Claudin-1,in a TGF-βindependent manner.Cell-cell adhesion in epithelial cell sheets is maintained mainly through adherens junctions and tight junctions.E-cadherin is a key player in cell—cell adhesion connecting adjacent cells via the cadherin—catenin adhesion complex. Claudin-1,the most apical cell-cell contacts and the most important tight junction for barrier function in colon cancer,was recently identified as a target of Wnt/β-catenin signaling.Based on these studies,we hypothesized that there was a relationship between Smad4,a tumor suppressor protein,and Wnt/β-catenin signaling in colorectal carcinoma.
The objective of this study was to investigate the impact of enhanced Smad4 expression on Wnt/β-catenin signaling activity in colon cancer cells.In this study,we reported that Smad4 directly suppressed the Wnt/β-catenin signal pathway in Smad4-responsive human colon carcinoma cells by down-regulating the cytoplasmic and nucleus expression ofβ-catenin and increasing localization ofβ-catenin to the plasma membrane.We further showed that Smad4 induced the expression of the intercellular adhesion molecule E-cadherin and reduced transcriptional activity ofβ-catenin/Tcf target genes,such as Claudin-1 and MMP-7.The data suggested that restoration of Smad4 in Smad4-deficient cells may provide a potential therapeutic strategy for intervention of colon cancer progress.
Materials and methods
1.Construction of eukaryotic expression plasmid
A cDNA of human Smad4 was amplified by RT-PCR and constructed into the eukaryotic expression vector pcDNA3.1(+),the recombinant plasmid pcDNA3.1(+)-Stand4 was identified by enzyme digestion、PCR amplification and DNA sequencing.
2.Overexpression of Stand4 by stable transfection
Cells were transfected using Lipofectamine2000 according to the manufacturer's instructions and selected in complete medium containing 1mg/ml G418 sulfate at 48h after transfection,G418-resistant cell clones were isolated for 2 weeks.Two clones that expressed similar levels of Smad4 in protein concentrations were selected for subsequent experiments.SW480 cells that were stably transfected with pcDNA3.1(+) were used as a control.The expression changes of Smad4 were detected using RT-PCR.A fluorescence microscope was used to detected the distribution of Smad4.
3.Transient transfection and luciferase assays
Cells were cotransfected with a firefly luciferase reporter construct Topflash or Fopflash,and a reference construct that contains Renilla reniformis luciferase, pRL-SV40,using Lipofectamine 2000.Forty-eight hours later,luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit in a GloMax Luminometry System.Tcf-mediated gene transcription was determined by the ratio of pTopflash to pFopflash luciferase activity.Firefly luciferase activity was normalized to Renilla reniformis luciferase activity and plotted as mean±SD from three independent experiments.
4.Semiquantitative RT-PCR
Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit.PCR analysis was performed using gene-specific primers,such asβ-catenin、Claudin-1、MMP-7,etc.
5.Immunoblot analysis
Equal amounts of protein samples were electrophoretically separated by SDS-PAGE in separation gels and transferred to nitrocellulose sheets.They were incubated overnight at 4℃with the primary antibodies diluted in the same buffer (Smad4 1:400;E-cadherin 1:600;β-catenin 1:1000;Claudin-1 1:1000;β-actin 1:3000 dilutions).Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies(1:5000 dilutions) for enhanced chemiluminescence detection using an ECL detection kit.
6.Immunofluorescence
Immunofluorescence were performed with the following primary antibodies: anti-β-catenin、-claudin-1 and -E-cadherin.FITC anti- mouse and anti-rabbit、Rhodamine Red-X anti-rabbit were used as secondary antibodies at 1:200 dilutions, respectively.Stained glass coverslips were viewed and photographed using an OLYMPUS fluorescence microscope.
7.Statistical analysis
Each experiment was repeated at least three times.Differences in mean values were evaluated using One-way ANOVA tests,Bonferrori analysis were performed in groups comparison.All statistical analyses were performed using SPSS 13.0 software. P＜0.05 was considered to be statistically significant.
Results
1.The DNA oligonucleotides encoding Smad4 mRNA were synthesized,and cloned into the eukaryotic expression plasmid,which were identified by restriction enzyme digestion analysis and DNA sequencing.Recombinant Smad4 eukaryotic expression vector pcDNA3.1(+)-Smad4 was constructed successfully.
2.SW480 cells which were transfected with pcDNA3.1(+)-Smad4 were screened by G418,two clones of SW480 with stable expression of Smad4 were established using Smad4 eukaryotic expression plasmid.The results of RT-PCR demonstrated that the mRNA level of Smad4 was increased notably in the Smad4-SW480 cells, and the result of immunofluorescence showed that Smad4 was predominantly localized in cytoplasm and nucleus.
3.We analyzed Wnt/β-catenin signaling activity in SW480、pc-SW480 and Smad4-SW480 cells by transiently transfecting Tcf and mutant Tcf reporter plasmids(Topflash and Fopflash,respectively).Relative luciferase activities were calculated as described in Methods.The data show that the relative transcriptional activity of theβ-catenin/Lef complex was decreased(3- to 4-fold) in Smad4-SW480 cells compared with SW480 or pc-SW480 cells.This analysis confirmed that Wnt signaling activity was significantly suppressed by Smad4 expression.
4.There was lessβ-catenin mRNA expression in Smad4-transfected cells than Smad4-negative colon carcinoma cells(P＜0.05).Relatively lessβ-catenin was detected in the cytoplasmic and nuclear fractions of Smad4-transfected cells compared with SW480 and pc-SW480 groups(P＜0.05).As the function ofβ-catenin in signal transduction depends on its cytoplasmic/nuclear localization, we further analyzed the distribution of theβ-catenin protein through indirect immunofluorescence.SW480 cells showed strong staining forβ-catenin in the cytoplasm and nucleus.Smad4 re-expression cells,in contrast,showed staining in the plasma membrane.The data demonstrated that up-regulation of Smad4 caused the relocalization ofβ-catenin from cytoplasm and nucleus to the plasma membrane in SW480 cells.
5.Low levels of E-cadherin protein were observed in SW480 cells and its control cell lines(pc-SW480) with relatively more E-cadherin detected in cells that had increased Smad4(P＜0.05).Inverse data were found in cellular Claudin-1 concentration,we observed reduced expression of claudin-1 in clones transfected with Smad4 when compared with the Smad4-negative cells(P＜0.05).We performed immunofluorescence analysis to further investigate whether up-regulation of Smad4 expression changed the localization of E-cadherin and Claudin-1.The localization of E-cadherin and Claudin-1 was not altered in Smad4-SW480 cells.The results of these two studies indicated a decrease in the expression of Claudin-1 but a robust increase in E-cadherin expression in Smad4-SW480 clones compared with SW480 cells.
6.There was less MMP-7 and Claudin-1 mRNA in Smad4-SW480 clones than SW480 or pc-SW480 cells(P＜0.05).Transfection of Smad4 resulted in reduced MMP-7 and Claudin-1 mRNA concentration as compared to cells transfected with the control or nonsense.
Conclusions
1.Recombinant Smad4 eukaryotic expression vector pcDNA3.1(+)-Smad4 was constructed successfully.
2.Stable colon cancer cells that have increased Smad4 expression were created using a eukaryotic vector containing Smad4-pcDNA3.1(+).
3.Re-expression of Smad4 in SW480 cells leads to a reduction in Wnt/β-catenin signaling activity.
4.Smad4 restoration decreasesβ-catenin expression(cytoplasm and nuclus) and alters its localization from cytoplasm to the plasma membrane in SW480 cells.
5.Up-regulation of Smad4 also increased the expression of E-cadherin and decreased the expression of Claudin-1.
6.Smad4 restoration in SW480 cells decreases mRNA expression of Wnt/β-catenin signaling target genes,such as Claudin-1 and MMP-7.
7.The data suggested that restoration of Smad4 in Smad4-deficient cells may provide a potential therapeutic strategy for intervention of colon cancer progress.
引文
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[9] Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in TGF-beta signal transduction. J Cell Sci 2001;114: 4359-4369.
[10] Miyaki M, Kuroki T. Role of Smad4 (DPC4) inactivation in human cancer. Biochem Biophys Res Commun 2003;306: 799-804.
[11] Nusse R. An ancient cluster of Wnt paralogues. Trends Genet 2001 ;17: 443.
[12] Polakis P. Wnt signaling and cancer. Genes Dev 2000;14: 1837-1851.
[13] Liu JH, Wei S, Burnette PK, et al. Functional association of TGF-beta receptor II with cyclin B. Oncogene 1999; 18: 269-275.
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[16] Shiou SR, Singh AB, Moorthy K, et al. Smad4 regulates claudin-1 expression in a transforming growth factor-beta-independent manner in colon cancer cells.Cancer Res 2007;67: 1571-1579.
[17] Hahn SA, Schutte M, Hoque AT, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996;271: 350-353.
[18] Woodford-Richens KL, Rowan AJ, Gorman P, et al. SMAD4 mutations in colorectal cancer probably occur before chromosomal instability, but after divergence of the microsatellite instability pathway. Proc Natl Acad Sci U S A 2001;98:9719-9723.
[19] Martin GS. Cell signaling and cancer. Cancer Cell 2003;4: 167-174.
[20] Moskaluk CA, Hruban RH, Schutte M, et al. Genomic sequencing of DPC4 in the analysis of familial pancreatic carcinoma. Diagn Mol Pathol 1997;6: 85-90.
[21] Caestecker MP, Hemmati P, Larisch-Bloch S, et al. Characterization of functional domains within Smad4/DPC4. J Biol Chem 1997;272: 13690-13696.
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