整合素相互作用蛋白kindlin-1在大肠癌演化过程中的作用
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摘要
背景／目的
Kindlin家族蛋白包括Kindlin-1、Kindlin-2、Kindlin-3，得名来源于一种常染色体隐性遗传性皮肤病—Kindler综合症。Kindler综合症临床表现为：皮肤的光敏感、皱缩、异常色素沉着、脆性增加、肢端大庖，少数病例伴有出血性肠炎以及直乙状结肠腐蚀、溃烂并覆盖有假膜。随后研究表明导致Kindler综合症的原因是由于编码Kindlin-1的新基因KIND1的功能丧失性突变从而导致Kindlin-1失去连接细胞外基质(ECM)与细胞骨架actin的功能而致。Kindlin家族蛋白的氨基酸序列高度同源，结构上都具有两个FERM((?)our pointone-(?)zrin-(?)adxin-(?)oesin)结构域，中间包含一个PH(pleckstrin homology)结构域。FERM结构域是把胞浆内蛋白质带到细胞膜的一个独特的结构域，含有FERM结构域的蛋白质是连接细胞膜糖蛋白和细胞骨架actin的桥梁，参与调节细胞的信号传导和细胞膜结构的完整性。PH结构域则广泛存在于和细胞膜相连的信号蛋白。由于这种独特的结构以及Kindlin-1被发现定位于角化细胞的粘附结构(focal adhesion)，预示着Kindlin-1和整合素的相互作用并调节整合素介导的信号传递、细胞的粘附以及运动。由于Kindlin-1的mRNA在70％的大肠癌病人和60％的肺癌病人中高度表达，特别是Kindlin-1的表达受转化生长因子TGF-β的上调，同时伴随细胞—细胞粘附连接(adhere junction)蛋白E-cadherin的下调以及E-cadherin的转录抑制因子Snail的表达上调，从而促进细胞发生上皮—间质转换(epithelial-mesenchymal transition，EMT)。因此本课题拟研究Kindlin-1在大肠癌演化过程中的表达以及在整合素介导的大肠癌细胞粘附、运动以及侵袭的影响，并探讨Kindlin-1参与大肠癌演化的可能机制。
方法
1、收集代表大肠癌演化过程中不同阶段的组织标本：正常粘膜、大肠增生性息肉、大肠腺瘤，不同Dukes分期大肠癌以及部分同一大肠癌病人相应的淋巴结转移癌，远处转移癌，用免疫组化法检测Kindlin-1的表达水平；2、在连续切片上用免疫组化检测Kindlin-1及核增殖标记物Ki-67的表达水平，分析二者的相关性；3、采用GST下拉实验以及免疫共沉淀实验验证Kindlin-1和整合素β1的相互作用，并采用免疫荧光双染色法检测二者是否在细胞粘附结构、片状伪足／丝状伪足(lamellipodia／fillopodia)以及大肠癌组织共定位；4、运用基因转染的手段，在大肠癌细胞中过度表达Kindlin-1，或者采用siRNA(smallinterference RNA)敲低Kindlin-1在大肠癌细胞中的表达水平，从获得性功能实验(gain of function)以及缺失性功能实验(loss of function)正反两个方面检测Kindlin-1对整合素介导的细胞粘附、运动以及Kindlin-1对大肠癌细胞侵袭以及增殖能力的影响；5、在用获得性功能实验以及缺失性功能实验检测Kindlin-1对大肠癌细胞生物学行为影响的同时，运用Western blot检测Kindlin-1对Wnt／β-catenin信号通路中关键蛋白表达的调节作用，并用Topflash／Fopflashluciferase报告基因检测Kindlin-1对Wnt／β-catenin信号通路下游转录因子TCF激活的影响，探讨Kindlin-1参与大肠癌演化的可能机制。
结果
1、Kindlin-1的表达水平随着大肠癌的演化过程逐渐升高：Kindlin-1在正常大肠粘膜表达阴性或者只在正常大肠干细胞存在的大肠腺管隐窝底部表达。在良性增生性息肉中Kindlin-1低表达或者阴性。在大肠腺瘤中，Kindlin-1表达开始增加，但表达仍低于原发灶大肠癌，在转移性大肠癌中表达进一步升高(p＜0.0001)。在不同Dukes分期的大肠癌中，Kindlin-1在早期的Dukes A期大肠癌中的表达低于处于进展期的Dukes B以及发生转移的Dukes C和Dukes D期大肠癌(p＜0.05)。另外，Kindlin-1在大肠粘液腺癌中的表达低于大肠腺癌(p＜0.0001)；2、Kindlin-1和核增殖标记物Ki-67在大肠癌中的表达呈正相关(p＜0.0001)，MTT增殖实验结果显示：与转染Flag空载体的SW480细胞相比，转染Flag-Kindlin-1的SW480细胞增殖能力增强(p＜0.05)；3、用GST下拉实验，发现GST-Integrinβ1 tail可以下拉外源性的Flag-Kindlin-1。在大肠癌细胞株中，用免疫共沉淀实验发现内源性的整合素β1可以和内源性的Kindlin-1相互作用并被结合有整合素β1抗体的珠子沉淀。采用免疫荧光双染色，发现Kindlin-1和整合素β1共定位于SW480细胞的粘附结构、片状伪足以及大肠癌组织侵袭前缘(invasive front)的细胞；4、和转染Flag空载体的SW480细胞相比，转染Flag-Kindlin-1增强了细胞在Collagen I上的粘附能力(p＜0.05)以及运动能力(p＜0.001)，并且细胞侵袭穿透Matrigel的能力显著增强(p＜0.01)。而用Kindlin-1 siRNA敲低Kindlin-1在SW480细胞中的表达水平后，细胞在Collagen I上的粘附能力(p＜0.05)及运动能力(p＜0.01)均下降。5、用siRNA敲低HCT116细胞中Kindlin-1的表达水平后(p＜0.01)，同时也下调磷酸化GSK-3β的水平(p＜0.05)，增强GSK-3β的活性，导致β-catenin(p＜0.01)及其下游蛋白cyclin D1(p＜0.01)、Snail(p＜0.001)的下调。在SW480细胞中，用另外2个siRNA也获得相同的结果。并且在SW480细胞中，表达丢失的E-cadherin又重新表达。另一方面，在SW480细胞中过度表达Kindlin-1，可以上调Wnt／β-catenin的下游靶蛋白cyclin D1(p＜0.01)、c-myc(p＜0.05)以及Snail(p＜0.05)。在HCT116细胞中转染Flag-Kindlin-1后，Topflash／Fopflash比值明显高于转染Flag空载体的细胞(p＜0.05)，表明过度表达Kindlin-1激活Wnt／β-catenin信号通路，增强了Tcf的活性。
结论
1、Kindlin-1的表达随大肠癌的演化进程逐渐升高；
2、Kindlin-1和整合素β1相互作用，增强整合素介导的大肠癌细胞的粘附力以及运动、侵袭能力。
3、Kindlin-1和整合素相互作用，通过抑制GSK-3β的活性，激活Wnt／β-catenin信号通路，上调其下游蛋白c-myc、cyclin D1的表达，促进大肠癌细胞增殖。
4、Kindlin-1通过激活Wnt／β-catenin信号通路上调Snail表达或者抑制GSK-3β的活性直接抑制Snail的降解从而抑制E-cadherin的表达，促进大肠癌的EMT过程。
Background/Objective
Kindlins are a newly found protein family including Kindlin-1, Kindlin-2 and Kindlin-3. Loss of function mutation of KIND1 gene leads to a defect in actin-ECM linkage, which causes Kindler syndrome, an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Some special cases showed hemorrhagic colitis, erosions and ulcers with pseudomembranous on mucosa of the descending and sigmoid colon and the rectum. The Kindlin family proteins show high homology of their amino acid sequence and share the same protein structure that all of them have two FERM (Four point one-ezrin-radxin-moesin) domains interrupted with a PH (pleckstrin homology) domain. FERM domain is a conservative structure existing in proteins that connect the cytoplasmic domains of transmembrane proteins to the actin cytoskeleton and modulate cell signaling and cell membrane structures while PH domain is a feature of a number of cytoskeletal-associated and/or cell-signaling molecules. The special structure of Kindlin-1 and its localization at focal adhesion imply Kindlin-1 may interact with Integrins and modulate Integrin mediated cell adhesion and migraton. Interestingly, Kindlin-1 mRNA was found overexpressed in 70% of human cancers and 60% of lung cancer patients. What's more, Kindlin-1 can be upregulated by TGF-131 and suppress expression of E-cadherin, upregulating Snail expression simultaneously, which contributes to EMT (epithelial-mesenchymal transition). In our study, we will focus on Kindlin-1 expression during colon cancer progression and its role in Integrin mediated colon cancer cell adhesion, migration and invasion and elucidate the signaling pathway in which Kindlin-1 involves and contributes to colon cancer progression.
Methods
1. Immunohistochemistry was performed to examine Kindlin-1 expression in benign polyps, adenomas, primary colon cancers on different Dukes stage and paired normal colon mucosas/mesentery lymph node metastases, distant organ metastases, which represent different stages of colon cancer progression. 2. In serial sections, expression of Kindlin-1 and proliferative marker Ki-67 was examined by immunohistochemistry to analyze their correlation. 3. GST pull-down assay and co-immunoprecipitation were used to examine the interaction of Kindlin-1 with Integrinβ1. Colocalization of Kindlin-1 and Integrinβ1 in SW480 cells and colon cancer tissues was detected by double immunofluorescence staining. 4. Genetic manipulation was performed to up-regulate or knock down expression of Kindlin-lin colon cancer cells and followed by cell adhesion, cell migration, cell invasion and MTT assays to test Integrin mediated cell adhesion and migration, cell invasion and proliferation respectively. 5. After Genetic manipulation on colon cancer cells, Western blot was performed to examine the regulatory effects of Kindlin-1 on key proteins in Wnt/β-catenin pathway. Luciferase reporter gene assay was used to test affection of Kindlin-1 on Wnt/β-catenin target transcriptional factor Tcf by calculating ratio of Topflash/Fopflash.
Results
1. Kindlin-1 expression increases with colon cancer progression: in normal colon mucosa, Kindlin-1 immunoactivity was either absent or barely detectable in progenitor cells at the bottom of normal mucosa crypts. Kindlin-1 expression was nearly negative in benign polyp but began to increase in adenomas with atypical hyperplasia. Interestingly, an even stronger expression of Kindlin-1 was found in primary colorectal carcinoma and the strongest in metastasis cancers (p＜0.0001). In colon cancers on different Dukes stage, Kindlin-1 expression in colon cancer on Dukes A was lower than that in colon cancer on Dukes B, Dukes C and Dukes D (p＜0.05). In addition, Kindlin-1 was weakly expressed in mucinous adenocarcinomas compared with that in adenocarcinomas (p＜0.0001). 2. Kindlin-1 expression was positively correlated with that of proliferative marker Ki-67 (p＜0.001) and MTT assay showed that SW480 cells transfected with Flag-Kindlin-1 proliferate much more quickly than those transfected with empty vector Flag (p＜0.05). 3. GST pull down assay showed GST-Integrinβ1 tail pulled down exogenous Flag-Kindlin-1 transfected into COS-7 cells and co-immunoprecipitation showed endogenous Kindlin-1 interacted with endogenous Integrin 131 in colon cell lines. Consistent with this, Kindlin-1 and Integrinβ1 were found colocalized at focal adhesion and lamellipodia in SW480 cells and at the invasive front of human colon cancer tissues. 4. Transfection of Flag-Kindlin-1 into SW480 cells enhanced Integrin mediated cell adhesion (p＜0.05) and migration (p＜0.001) on Collagen typeⅠwhile knocking down of Kindlin-1 by siRNA in SW480 cells impaired cell adhesion (p＜0.05) and migration (p＜0.01). In addition, overexpression of Kindlin-1 in SW480 cell by gene transfection promoted cell invasion on Matrigel (p＜0.01). 5. Knocking down of Kindlin-1 (p＜0.01) in HCT116 cells down regulated phosphorylated GSK-3β(p＜0.05), which enhanced activity of GSK-3βand thereby down regulatedβ-catenin (p＜0.01) and its down stream proteins including cyclin D1 (p＜0.01) and Snail (p＜0.001) simultaneously. In SW480 cells, similar results were obtained by knocking down Kindlin-1 expression with another two siRNAs. Along with the downregulation of Snail, E-cadherin is re-expressed in SW480 cells. On the other hand, transfection of Kindlin-1 into SW480 cells up-regulated Wnt/β-catenin downstream proteins c-myc (p＜0.05), cyclin D1 (p＜0.01) and Snail (p＜0.05). Luciferase assay showed that Topflash/Fopflash ratio was much higher in HCT116 cells transfected with Flag-Kindlin-1 than in those tansfected with empty Flag vector (p＜0.05), which suggested that Kindlin-1 activate Wnt/β-catenin-Tcf/Lef pathway.
Conclusion
1. Kindlin-1 expression increases with human colon cancer progression.
2. Kindlin-1 interacts with Integrinβ1 and enhances Integrin-mediated colon cancer cell adhesion, migration and invasion.
3. Kindlin-1 activates Wnt/β-catenin pathway by inhibiting GSK-3βactivity and up-regulates its down steam protein c-myc and cyclin D1 expressions, therefore promoting cell prolifetation.
4. Kindlin-1 up-regulates Snail and down-regulates E-cadherin expression through either activating Wnt/β-catenin pathway or inhibiting GSK-3βthat can phosphorylate Snail directly and lead to its degradation by ubquitin-mediated pathway, thus contributing to colon cancer EMT.
Kindlin家族蛋白包括Kindlin-1、Kindlin-2、Kindlin-3，得名来源于一种常染色体隐性遗传性皮肤病—Kindler综合症。Kindler综合症临床表现为：皮肤的光敏感、皱缩、异常色素沉着、脆性增加、肢端大庖，少数病例伴有出血性肠炎以及直乙状结肠腐蚀、溃烂并覆盖有假膜。随后研究表明导致Kindler综合症的原因是由于编码Kindlin-1的新基因KIND1的功能丧失性突变从而导致Kindlin-1失去连接细胞外基质(ECM)与细胞骨架actin的功能而致。Kindlin家族蛋白的氨基酸序列高度同源，结构上都具有两个FERM((?)our pointone-(?)zrin-(?)adxin-(?)oesin)结构域，中间包含一个PH(pleckstrin homology)结构域。FERM结构域是把胞浆内蛋白质带到细胞膜的一个独特的结构域，含有FERM结构域的蛋白质是连接细胞膜糖蛋白和细胞骨架actin的桥梁，参与调节细胞的信号传导和细胞膜结构的完整性。PH结构域则广泛存在于和细胞膜相连的信号蛋白。由于这种独特的结构以及Kindlin-1被发现定位于角化细胞的粘附结构(focal adhesion)，预示着Kindlin-1和整合素的相互作用并调节整合素介导的信号传递、细胞的粘附以及运动。由于Kindlin-1的mRNA在70％的大肠癌病人和60％的肺癌病人中高度表达，特别是Kindlin-1的表达受转化生长因子TGF-β的上调，同时伴随细胞—细胞粘附连接(adhere junction)蛋白E-cadherin的下调以及E-cadherin的转录抑制因子Snail的表达上调，从而促进细胞发生上皮—间质转换(epithelial-mesenchymal transition，EMT)。因此本课题拟研究Kindlin-1在大肠癌演化过程中的表达以及在整合素介导的大肠癌细胞粘附、运动以及侵袭的影响，并探讨Kindlin-1参与大肠癌演化的可能机制。
方法
1、收集代表大肠癌演化过程中不同阶段的组织标本：正常粘膜、大肠增生性息肉、大肠腺瘤，不同Dukes分期大肠癌以及部分同一大肠癌病人相应的淋巴结转移癌，远处转移癌，用免疫组化法检测Kindlin-1的表达水平；2、在连续切片上用免疫组化检测Kindlin-1及核增殖标记物Ki-67的表达水平，分析二者的相关性；3、采用GST下拉实验以及免疫共沉淀实验验证Kindlin-1和整合素β1的相互作用，并采用免疫荧光双染色法检测二者是否在细胞粘附结构、片状伪足／丝状伪足(lamellipodia／fillopodia)以及大肠癌组织共定位；4、运用基因转染的手段，在大肠癌细胞中过度表达Kindlin-1，或者采用siRNA(smallinterference RNA)敲低Kindlin-1在大肠癌细胞中的表达水平，从获得性功能实验(gain of function)以及缺失性功能实验(loss of function)正反两个方面检测Kindlin-1对整合素介导的细胞粘附、运动以及Kindlin-1对大肠癌细胞侵袭以及增殖能力的影响；5、在用获得性功能实验以及缺失性功能实验检测Kindlin-1对大肠癌细胞生物学行为影响的同时，运用Western blot检测Kindlin-1对Wnt／β-catenin信号通路中关键蛋白表达的调节作用，并用Topflash／Fopflashluciferase报告基因检测Kindlin-1对Wnt／β-catenin信号通路下游转录因子TCF激活的影响，探讨Kindlin-1参与大肠癌演化的可能机制。
结果
1、Kindlin-1的表达水平随着大肠癌的演化过程逐渐升高：Kindlin-1在正常大肠粘膜表达阴性或者只在正常大肠干细胞存在的大肠腺管隐窝底部表达。在良性增生性息肉中Kindlin-1低表达或者阴性。在大肠腺瘤中，Kindlin-1表达开始增加，但表达仍低于原发灶大肠癌，在转移性大肠癌中表达进一步升高(p＜0.0001)。在不同Dukes分期的大肠癌中，Kindlin-1在早期的Dukes A期大肠癌中的表达低于处于进展期的Dukes B以及发生转移的Dukes C和Dukes D期大肠癌(p＜0.05)。另外，Kindlin-1在大肠粘液腺癌中的表达低于大肠腺癌(p＜0.0001)；2、Kindlin-1和核增殖标记物Ki-67在大肠癌中的表达呈正相关(p＜0.0001)，MTT增殖实验结果显示：与转染Flag空载体的SW480细胞相比，转染Flag-Kindlin-1的SW480细胞增殖能力增强(p＜0.05)；3、用GST下拉实验，发现GST-Integrinβ1 tail可以下拉外源性的Flag-Kindlin-1。在大肠癌细胞株中，用免疫共沉淀实验发现内源性的整合素β1可以和内源性的Kindlin-1相互作用并被结合有整合素β1抗体的珠子沉淀。采用免疫荧光双染色，发现Kindlin-1和整合素β1共定位于SW480细胞的粘附结构、片状伪足以及大肠癌组织侵袭前缘(invasive front)的细胞；4、和转染Flag空载体的SW480细胞相比，转染Flag-Kindlin-1增强了细胞在Collagen I上的粘附能力(p＜0.05)以及运动能力(p＜0.001)，并且细胞侵袭穿透Matrigel的能力显著增强(p＜0.01)。而用Kindlin-1 siRNA敲低Kindlin-1在SW480细胞中的表达水平后，细胞在Collagen I上的粘附能力(p＜0.05)及运动能力(p＜0.01)均下降。5、用siRNA敲低HCT116细胞中Kindlin-1的表达水平后(p＜0.01)，同时也下调磷酸化GSK-3β的水平(p＜0.05)，增强GSK-3β的活性，导致β-catenin(p＜0.01)及其下游蛋白cyclin D1(p＜0.01)、Snail(p＜0.001)的下调。在SW480细胞中，用另外2个siRNA也获得相同的结果。并且在SW480细胞中，表达丢失的E-cadherin又重新表达。另一方面，在SW480细胞中过度表达Kindlin-1，可以上调Wnt／β-catenin的下游靶蛋白cyclin D1(p＜0.01)、c-myc(p＜0.05)以及Snail(p＜0.05)。在HCT116细胞中转染Flag-Kindlin-1后，Topflash／Fopflash比值明显高于转染Flag空载体的细胞(p＜0.05)，表明过度表达Kindlin-1激活Wnt／β-catenin信号通路，增强了Tcf的活性。
结论
1、Kindlin-1的表达随大肠癌的演化进程逐渐升高；
2、Kindlin-1和整合素β1相互作用，增强整合素介导的大肠癌细胞的粘附力以及运动、侵袭能力。
3、Kindlin-1和整合素相互作用，通过抑制GSK-3β的活性，激活Wnt／β-catenin信号通路，上调其下游蛋白c-myc、cyclin D1的表达，促进大肠癌细胞增殖。
4、Kindlin-1通过激活Wnt／β-catenin信号通路上调Snail表达或者抑制GSK-3β的活性直接抑制Snail的降解从而抑制E-cadherin的表达，促进大肠癌的EMT过程。
Background/Objective
Kindlins are a newly found protein family including Kindlin-1, Kindlin-2 and Kindlin-3. Loss of function mutation of KIND1 gene leads to a defect in actin-ECM linkage, which causes Kindler syndrome, an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Some special cases showed hemorrhagic colitis, erosions and ulcers with pseudomembranous on mucosa of the descending and sigmoid colon and the rectum. The Kindlin family proteins show high homology of their amino acid sequence and share the same protein structure that all of them have two FERM (Four point one-ezrin-radxin-moesin) domains interrupted with a PH (pleckstrin homology) domain. FERM domain is a conservative structure existing in proteins that connect the cytoplasmic domains of transmembrane proteins to the actin cytoskeleton and modulate cell signaling and cell membrane structures while PH domain is a feature of a number of cytoskeletal-associated and/or cell-signaling molecules. The special structure of Kindlin-1 and its localization at focal adhesion imply Kindlin-1 may interact with Integrins and modulate Integrin mediated cell adhesion and migraton. Interestingly, Kindlin-1 mRNA was found overexpressed in 70% of human cancers and 60% of lung cancer patients. What's more, Kindlin-1 can be upregulated by TGF-131 and suppress expression of E-cadherin, upregulating Snail expression simultaneously, which contributes to EMT (epithelial-mesenchymal transition). In our study, we will focus on Kindlin-1 expression during colon cancer progression and its role in Integrin mediated colon cancer cell adhesion, migration and invasion and elucidate the signaling pathway in which Kindlin-1 involves and contributes to colon cancer progression.
Methods
1. Immunohistochemistry was performed to examine Kindlin-1 expression in benign polyps, adenomas, primary colon cancers on different Dukes stage and paired normal colon mucosas/mesentery lymph node metastases, distant organ metastases, which represent different stages of colon cancer progression. 2. In serial sections, expression of Kindlin-1 and proliferative marker Ki-67 was examined by immunohistochemistry to analyze their correlation. 3. GST pull-down assay and co-immunoprecipitation were used to examine the interaction of Kindlin-1 with Integrinβ1. Colocalization of Kindlin-1 and Integrinβ1 in SW480 cells and colon cancer tissues was detected by double immunofluorescence staining. 4. Genetic manipulation was performed to up-regulate or knock down expression of Kindlin-lin colon cancer cells and followed by cell adhesion, cell migration, cell invasion and MTT assays to test Integrin mediated cell adhesion and migration, cell invasion and proliferation respectively. 5. After Genetic manipulation on colon cancer cells, Western blot was performed to examine the regulatory effects of Kindlin-1 on key proteins in Wnt/β-catenin pathway. Luciferase reporter gene assay was used to test affection of Kindlin-1 on Wnt/β-catenin target transcriptional factor Tcf by calculating ratio of Topflash/Fopflash.
Results
1. Kindlin-1 expression increases with colon cancer progression: in normal colon mucosa, Kindlin-1 immunoactivity was either absent or barely detectable in progenitor cells at the bottom of normal mucosa crypts. Kindlin-1 expression was nearly negative in benign polyp but began to increase in adenomas with atypical hyperplasia. Interestingly, an even stronger expression of Kindlin-1 was found in primary colorectal carcinoma and the strongest in metastasis cancers (p＜0.0001). In colon cancers on different Dukes stage, Kindlin-1 expression in colon cancer on Dukes A was lower than that in colon cancer on Dukes B, Dukes C and Dukes D (p＜0.05). In addition, Kindlin-1 was weakly expressed in mucinous adenocarcinomas compared with that in adenocarcinomas (p＜0.0001). 2. Kindlin-1 expression was positively correlated with that of proliferative marker Ki-67 (p＜0.001) and MTT assay showed that SW480 cells transfected with Flag-Kindlin-1 proliferate much more quickly than those transfected with empty vector Flag (p＜0.05). 3. GST pull down assay showed GST-Integrinβ1 tail pulled down exogenous Flag-Kindlin-1 transfected into COS-7 cells and co-immunoprecipitation showed endogenous Kindlin-1 interacted with endogenous Integrin 131 in colon cell lines. Consistent with this, Kindlin-1 and Integrinβ1 were found colocalized at focal adhesion and lamellipodia in SW480 cells and at the invasive front of human colon cancer tissues. 4. Transfection of Flag-Kindlin-1 into SW480 cells enhanced Integrin mediated cell adhesion (p＜0.05) and migration (p＜0.001) on Collagen typeⅠwhile knocking down of Kindlin-1 by siRNA in SW480 cells impaired cell adhesion (p＜0.05) and migration (p＜0.01). In addition, overexpression of Kindlin-1 in SW480 cell by gene transfection promoted cell invasion on Matrigel (p＜0.01). 5. Knocking down of Kindlin-1 (p＜0.01) in HCT116 cells down regulated phosphorylated GSK-3β(p＜0.05), which enhanced activity of GSK-3βand thereby down regulatedβ-catenin (p＜0.01) and its down stream proteins including cyclin D1 (p＜0.01) and Snail (p＜0.001) simultaneously. In SW480 cells, similar results were obtained by knocking down Kindlin-1 expression with another two siRNAs. Along with the downregulation of Snail, E-cadherin is re-expressed in SW480 cells. On the other hand, transfection of Kindlin-1 into SW480 cells up-regulated Wnt/β-catenin downstream proteins c-myc (p＜0.05), cyclin D1 (p＜0.01) and Snail (p＜0.05). Luciferase assay showed that Topflash/Fopflash ratio was much higher in HCT116 cells transfected with Flag-Kindlin-1 than in those tansfected with empty Flag vector (p＜0.05), which suggested that Kindlin-1 activate Wnt/β-catenin-Tcf/Lef pathway.
Conclusion
1. Kindlin-1 expression increases with human colon cancer progression.
2. Kindlin-1 interacts with Integrinβ1 and enhances Integrin-mediated colon cancer cell adhesion, migration and invasion.
3. Kindlin-1 activates Wnt/β-catenin pathway by inhibiting GSK-3βactivity and up-regulates its down steam protein c-myc and cyclin D1 expressions, therefore promoting cell prolifetation.
4. Kindlin-1 up-regulates Snail and down-regulates E-cadherin expression through either activating Wnt/β-catenin pathway or inhibiting GSK-3βthat can phosphorylate Snail directly and lead to its degradation by ubquitin-mediated pathway, thus contributing to colon cancer EMT.
引文
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2. Sadler E, Klausegger A et al. Novel KIND1 gene mutation in Kindler syndrome with severe gastrointestinal tract involvement. Arch Dermatol.2006; 142(12):1619-24.
3. Siegel DH, Ashton GH et al. Loss of Kindlin-1, a Human Homolog of the caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome. Am J Hum Genet. 2003; 73:174-187
4. Ashton GH, McLean WH et al. Recurrent mutations in Kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin fragility and photosensitivity disorder, Kindler syndrome. J Invest Dermatol. 2004;122 (1):78-83.
5. Has C, Wessagowit V et al. Molecular basis of Kindler syndrome in Italy: novel and recurrent Alu/Alu recombination, splice site, nonsense, and frameshift mutations in the KIND1 gene. J Invest Dermatol. 2006; 126(8):1776-83.
6. Hamada K, Shimizu T, Matsui T, Tsukita S, Hakoshima T. Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain. EMBO J.2001; 1; 19 (17):4449-62
7. Chishti AH, et al. The FERM domain: a unique module involved in the linkage of cytoplasmic proteins to the membrane. Trends Biochem Sci. 1998; 23:281-282.
8. Maffucci T, Falasca M. Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide-protein co-operative mechanism. FEBS Lett .2001; 506:173-179
9. Lemmon MA, Ferguson KM, Abrams CS. Pleckstrin homology domains and the cytoskeleton. FEBS Lett 2002; 513:71-76
10. Itoh T, Takenawa T. Phosphoinositide-binding domains: functional units for temporal and spatial regulation of intracellular signalling. Cell Signal. 2002; 14:733-743
11. Weinstein EJ, et al. URP1: a member of a novel family of PH and FERM domain-containing membrane-associated proteins is significantly over-expressed in lung and colon carcinomas. Biochim Biophys Acta. 2003; 1637:207-216.
12. Kloeker S, et al .The Kindler syndrome protein is regulated by transforming growth factor-p and involved in Integrin-mediated adhesion. J Biol Chem. 2004; 279:6824-6833,
13. Peinado H, Quintanilla M et al. Transforming growth factor β1 induces Snail transcription factor in epithelial cell lines. J Biol Chem. 2003; 278(23):21113-23
14. Jemal A, Siegel R et al. Cancer statistics. CA Cancer J Clin. 2007;57(1):43-66
15. You WC, Jin F, et al. Rapid increase in colorectal cancer rates in urban Shanghai, 1972-97, in relation to dietary changes. J Cancer Epidemiol Prev. 2002; 7:143-6.
16. Goel HL, Languino LR. Integrin signaling in cancer. Cancer Treat Res. 2004; 119:15-31,
17. Hood JD, Cheresh DA. Role of Integrins in cell invasion and migration. Nat Rev Cancer. 2002;2(2):91-100. .
18. Arnaout MA, et al. Integrin structure, allostery, and bidirectional signaling. Annu Rev Cell Dev Biol. 2005; 21: 381-410.
19. Van der Flier, A. & Sonnenberg, A. Function and interactions of Integrins. Cell Tissue Res. 2001; 305, 285-298.
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