CLIC4参与上皮性卵巢癌肿瘤间质相关成纤维细胞的转化及其microRNAs调控
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摘要
【研究背景】
卵巢癌是妇科肿瘤中致死率最高的恶性肿瘤,因为在其诊断时多数已为晚期病例,虽然经过系统的手术、化学治疗,5年生存率仍不超过45%,一直被认为是一个“沉默杀手”。越来越多的学者认识到,肿瘤的生物学行为不仅取决于肿瘤细胞本身的特征,而且与肿瘤间质之间的相互作用密切相关。不同的实体肿瘤虽然实质和间质的比例不尽相同,但是一般肿瘤的间质成分占整个实体肿瘤的50-90%。间质成分毗邻于肿瘤细胞,受到肿瘤细胞旁分泌信号的调节,可以转化为肿瘤相关的间质细胞,因为其改变了细胞的表型、功能和肿瘤局部的微环境,可以促进肿瘤的增殖、浸润、转移和血管形成。肿瘤间质的肿瘤相关纤维母细胞是间质细胞中最主要的细胞成分,其主要来源包括:肿瘤局部的纤维母细胞、上皮间质转化、远处部位的纤维母细胞、骨髓祖细胞、干细胞等。其中,间质纤维母细胞是最主要的细胞成分,其不同于正常的纤维母细胞,因为表达已分化肌纤维母细胞表型标志α-SMA(α-smooth muscle actin),被称为肿瘤相关成纤维细胞或肌纤维母细胞(tumour-associated fibroblasts,myofibroblasts),但其分子转化机制尚不完全清楚。对肿瘤相关成纤维细胞转化激活机制的深入研究,将有助于靶向肿瘤间质治疗新策略的提出。
细胞内氯通道蛋白4(Chloride intracellular channel 4,CLIC4),是细胞内的一种氯通道,调节细胞内pH值和细胞体积。它不仅存在于细胞器膜上,而且可以以可溶性成分存在于细胞浆和细胞核内,作为一种通道调节剂或信号蛋白。有研究显示,TGF-β1可以刺激乳腺组织原位的纤维母细胞的CLIC4转录水平升高,更重要的是,CLIC4高表达于乳腺癌组织的间质肿瘤相关成纤维细胞,而正常组织间质中几乎无CLIC4表达。故我们推测CLIC4可能参与上皮性卵巢癌的间质肌纤维母细胞的转化。
MicroRNAs(miRNAs)是一类分布广泛的小的非编码蛋白质的RNAs,其功能是负性调控基因表达。在人类基因组中至少存在700个miRNAs(也有可能为1000个),约占人类基因的1-4%,预测他们能够调节至少三分之一的人类基因,这使得其成为最大的一类基因表达调控因子。miRNAs参与生命过程中几乎所有的重要过程,包括早期发育,细胞增殖,细胞凋亡和细胞分化等。肿瘤间质肌纤维母细胞转化过程中存在一系列表型和功能基因的表达改变,但参与此过程调控基因表达的miRNAs尚未见报道。
本研究中,我们首先检测上皮性卵巢癌患者组织中CLIC4的表达,并构建间质肌纤维母细胞转化的细胞模型,探讨是否CLIC4参与此转化过程及其在此过程中的作用,进一步探讨调控肿瘤间质肌纤维母细胞转化的miRNAs。
第一部分CLIC4介导卵巢癌肿瘤相关成纤维细胞转化
【目的】
来自肿瘤与间质相互作用的信号分子激活肿瘤间质肌纤维母细胞,其对肿瘤的进展起重要的作用。CLIC4可能参与间质纤维母细胞的激活,但其分子机制尚不清楚。本研究拟探讨CLIC4是否参与上皮性卵巢癌间质纤维母细胞向肌纤维母细胞的转化及其在此过程中的作用。
【方法】
1.应用免疫组织化学方法检测上皮性卵巢癌组织间质中CLIC4的表达。
2.卵巢癌细胞条件培养基(Conditioned medium,CM)或转化生长因子β1(Transforming growth factor-β1,TGF-β1)培养原代卵巢正常纤维母细胞或人纤维母细胞系构建卵巢癌肿瘤间质纤维母细胞向肌纤维母细胞转化的细胞模型,应用倒置显微镜观察细胞形态学改变和肌纤维母细胞转化细胞表型标志α-SMA表达上调,评价转化细胞模型成功构建。
3.应用肌纤维母细胞转化的细胞模型,证实CLIC4和活性氧产物(reactive oxygen species,ROS)参与此转化过程。
4.应用CLIC4特异性siRNA阻断CLIC4的表达,探讨其在肿瘤间质肌纤维母细胞转化中的作用。
5.应用CLIC4特异性siRNA阻断CLIC4的表达或氯离子通道抑制剂抑制CLIC4的功能、抗氧化剂阻断ROS水平上调,探讨它们对肿瘤间质肌纤维母细胞转化相关功能蛋白表达的抑制作用。
【结果】
1.上皮性卵巢癌组织中间质CLIC4的阳性表达率为97.22%,肌纤维母细胞表型标志物α-SMA的阳性表达率为94.44%,两者密切相关性(P＜0.05)。肿瘤间质CLIC4表达强度与患者的肿瘤分化程度和淋巴结转移密切相关,中、低分化者,有淋巴结转移者肿瘤组织间质CLIC4的表达明显高于高分化,无淋巴结转移者(P＜0.05)。
2.应用卵巢癌细胞CM或TGF-β1培养卵巢组织原代正常纤维母细胞或人纤维母细胞系MRC-5 48小时,细胞增大呈多角形,而无血清培养的纤维母细胞呈细长纺锤状,同时α-SMA的转录(P＜0.05)和蛋白表达水平显著增加,表明正常纤维母细胞向肌纤维母细胞转化。
3.应用卵巢癌细胞CM或TGF-β1培养卵巢组织原代正常纤维母细胞或人纤维母细胞系MRC-5 48小时,纤维母细胞CLIC4表达在转录(P＜0.05)和蛋白水平明显上调。
4.应用卵巢癌细胞CM或TGF-β1培养卵巢组织原代正常纤维母细胞或人纤维母细胞系MRC-5促进细胞内ROS的产生,应用抗氧化剂抑制ROS产生可以抑制CM或TGF-β1诱导的CLIC4和α-SMA上调(P＜0.05)。
5.应用CLIC4特异性siRNA能够有效阻断CLIC4的表达,抑制CM或TGF-β1诱导的α-SMA表达水平上调(P＜0.05)。
6.应用CLIC4特异性siRNA阻断CLIC4的表达或氯离子通道抑制剂抑制CLIC4的功能、抗氧化剂阻断ROS水平上调,可以抑制与肌纤维母转化相关促血管因子VEGFA和HGF mRNA水平表达29-80%(P＜0.05)。
【结论】
CLIC4参与卵巢癌肿瘤间质纤维母细胞向肌纤维母细胞转化。来自肿瘤的旁分泌信号TGF-β1刺激使纤维母细胞内ROS产生增加,进而促进CLIC4的表达上调,最终导致纤维母细胞向肌纤维母细胞转化,即肿瘤相关成纤维细胞的活化。阻断CLIC4和ROS具有靶向肿瘤间质的治疗前景。
第二部分microRNA-21参与肿瘤相关成纤维细胞转化
【目的】
肿瘤细胞分泌的TGF-β1诱导肿瘤间质肌纤维母细胞的转化表现为一系列表型和功能基因的表达改变。miRNAs作为重要的基因负性调节因子,目前参与肿瘤间质肌纤维母细胞转化的miRNAs尚未见报道。
【方法】
1.应用real time RT-PCR方法检测一系列候选参与肿瘤间质肌纤维母细胞转化过程相关的miRNAs的表达水平。
2.应用microRNA-21(miR-21)抑制物和类似物转染人纤维母细胞,探讨miRNA-21在肿瘤间质肌纤维母细胞的转化过程中的作用。
3.应用计算机软件预测肿瘤间质成纤维细胞转化过程中miR-21的靶向调控基因,应用构建包含miR-21靶基因调控程序性细胞死亡4基因(programmed cell death 4,PDCD4)3'-UTR的荧光素酶报告载体方法和应用转染或敲除miR-21后检测纤维母细胞中PDCD4表达水平的变化,确定PDCD4是否是miR-21在肌纤维母细胞转化过程中的靶向调节基因。
【结果】
1.应用卵巢癌细胞CM或TGF-β1培养人纤维母细胞系MRC-5,细胞内miR-21表达水平上调(P＜0.05),并且具有时间和剂量依赖性效应。
2.应用miR-21抑制物和类似物转染纤维母细胞MRC-5,miR-21抑制物特异性降调而其类似物特异性上调miR-21的水平(P＜0.05)。miR-21抑制物可以抑制CM或TGF-β1诱导的肿瘤间质肌纤维母细胞的转化过程中α-SMA等转化标志基因和功能基因的表达(P＜0.05)。
3.生物信息软件预测调控PDCD4的3'UTR与miR-21具有明显的相互作用“种子序列”。
4.构建含有miR-21潜在结合位点的PDCD4基因3'UTR荧光素酶报告载体,其与miR-21抑制剂或模仿剂共转染入纤维母细胞MRC-5,miR-21模仿剂增加miR-21的表达而抑制荧光素酶活性,而miR-21抑制剂显著增加荧光素酶活性(P＜0.05)。提示,在纤维细胞中miR-21能够结合到PDCD4基因的3'UTR。
5.应用特异性转染或沉默方法,miR-21模仿剂和miR-21抑制剂分别降低和升高纤维母细胞中PDCD4的蛋白表达,并且转染miR-21抑制剂可以拮抗TGF-β1诱导的miR-21内源性负性调节PDCD4表达的作用。
【结论】
miR-21参与卵巢癌细胞CM或TGF-β1诱导的肿瘤间质肌纤维母细胞转化是通过靶向调节PDCD4。
Background
Epithelial ovarian cancer is the most lethal disease among gynaecologic malignancies.For years,it has been dubbed as a "silent killer"--causing no symptom till it is too advanced at the time of diagnosis.5-year survival for patients with advanced ovarian cancer improved little even with the treatment of optimal cytoreductive surgery and systemic combination chemotherapy.Increasing evidence has indicated that cancer development is facilitated by interaction between tumour cells and activated stromal cells.The tumour stroma(also referred to as "reactive stroma") is characterized by marked alterations in the phenotype and expression profile of fibroblast-like cells.These cells commonly expressα-SMA and thus are termed as myofibroblasts.Stromal fibroblasts are located at the tumour border near the invasion front,and play a crucial role in tumour progression.Myofibroblasts are recruited from different sources,including local fibroblast population,distant fibroblasts,EMT (epithelial-mesenchymal transition) and bone marrow progenitor cells or stem cells,during cancer development and the invasion progression. Although it has been shown that the conversion from fibroblasts to myofibroblasts constitutes the major source of myofibrblasts in tumour stroma,the molecular mechanism underlying fibroblast-to-myofibroblast transdifferentiation is still not fully understood.
Chloride intracellular channel 4(CLIC4),a chloride channel of intracellular organelles,regulates intracellular pH and cell volume. Besides its presence on the organelle membrane,CLIC4 exists in soluble form in the cytoplasm and nucleus acting as signalling protein or channel regulator.Transcription level of CLIC4 is up-regulated when fibroblasts transdifferentiate into myofibroblasts induced by TGF-β1,and more importantly CLIC4 is highly expressed in myofibroblasts of breast cancer. Hence,we hypothesized that CLIC4 might invlove in myofibroblast transdifferentiation in ovarian cancer stroma,but the pathway in which CLIC4 participated during the fibroblast-to-myofibroblast transition induced by TGF-β1 from ovarian cancer cells is still unknown.
MicroRNAs(miRNAs) are a class of endogenous,short (20-25-nucleotide),noncoding single-stranded RNA molecules, which negatively regulate gene expression through mRNAs degradation or translational inhibition of their target gene.miRNAs are involved in regulators of gene expression that control diverse physiological and pathological processes,such as development,cell differentiation, migration,proliferation and apoptosis,miRNAs represent a new layer of gene expression regulators at the transcript and translational levels,but the involvement of miRNAs and the roles of these miRNAs in TGF-β1-induced myofibroblasts differentiation in tumor-stroma interaction are uncertain.
PartⅠCLIC4 mediares TGF-β1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer
【Objective】
Stromal myofibroblasts,activated by crosstalk signaling between the tumour and stroma,play a critical role in tumour development and progression.Chloride intracellular channel 4(CLIC4) may be functionally import for tumour stromal fibroblast-to-myofibroblast transdifferentiaton,but the molecular mechanism of the process has not been addressed.This study was to investigate the role of CLIC4 in the myofibroblast transdifferentiaton of ovarian cancer stroma.
【Methods】
1.The expression of CLIC4 in ovarian cancer tissues was analyzed by immunohischemistry,
2.A cell model of fibroblast-to-myofibroblast transdifferentiaton was constructed,where ovarian primary fibroblasts or human fibroblasts MRC-5 cells were cultured with conditioned medium(CM) from ovarian cancer cells or transforming growth factor-β1(TGF-β1).Cellular morphologic change and the upregulation ofα-SMA expression were used to identify the differentiated myofibroblasts.
3.The cell model of myofibroblast transdifferentiaton was used to demonstrate whether CLIC4 and ROS participated in the fibroblast-to-myofibroblast transdifferentiation.
4.Specific CLIC4 siRNA was used to knockdown the CLIC4 expression, then to investigate the role of CLIC4 in the myofibroblast transdifferentiaton.
5.The agents,specific CLIC4 siRNA,chloride channel inhibitor and antioxidant,were used to investigate the roles in suppression of the functional gene expression related with the myofibroblasts conversion.
【Results】
1.The expression of CLIC4 in 97.22%of ovarian cancer stroma and correlated with the up-regulation of myofibroblast markerα-SMA,which was detected in 94.44%of patients(P＜0.05).The immunohischemistry data in different pathologic groups showed that the protein expression level of CLIC4 in ovarian cancer stroma which was in mid- or low-differentiated and accompanied by lymphatic metastasis was obviously higher than that which was in high-differentiated and nonaccompanied by lymphatic metastasis(P＜0.05).
2.In the presence of CM~(SKOV3) or TGF-β1,the fibroblasts,which displayed typical small spindle shape under serum-free condition, changed into bigger and polygonal cells,andα-SMA transcript and translational expression increased(P＜0.05),indicating fibroblast-to-myofibroblast conversion.
3.The expression of CLIC4 in the activated fibroblasts increased significantly in the presence of CM~(SKOV3) or 10 ng/ml TGF-β1 for 48 h both at the transcription(P＜0.05) and protein levels compared with that in serum-free medium control.
4.When cells were pretreated with the antioxidant NAC,which inhibited the generation of intracellular ROS,the up-regulation ofα-SMA and CLIC4 expression stimulated by TGF-β1 or CM~(SKOV3) was significantly suppressed(P＜0.05).
5.The up-regulation ofα-SMA expression induced by TGF-β1 or CM~(SKOV3) was significantly blocked in CLIC4 siRNA-transfected fibroblasts(P＜0.05).
6.Pre-treatment with CLIC4 siRNA to silence CLIC4 expression, chloride channel inhibitor IAA-94 to block function of CLIC4 or NAC to inhibit the generation of ROS lowered VEGF and HGF expression levels in the activated fibroblasts by 29-80%(P＜0.05).
【Conclusion】
These results suggest that ROS-initiated CLIC4 up-regulation is required for TGF-β1-induced fibroblast-to-myofibroblast transdifferentiaton in ovarian cancer,indicating inhibiting CLIC4 or ROS might have therapeutic potential targeting tumour stroma.
PartⅡMicroRNA-21 participates in TGF-β1-induced myofibroblasts differentiation in tumor stroma
【Objectire】
Stromal fibroblast-to-myofibroblast transdifferentiation induced by TGF-β1 in the microenvironment of tumor and stroma interaction via expression changes of multiple phenotypic and functional genes plays a critical role in the tumor progression.Up to now,the involvement of microRNAs(miRNAs) and the roles of these miRNAs in TGF-β1-induced myofibroblasts differentiation in tumor-stroma interaction are unclear.
【Methods】
1.Quantitative real time RT-PCR was used to measure the expression change of a range of microRNAs during the fibroblast-to-myofibroblast transdifferentiation induced by TGF-β1 or CM from cancer cells.
2.To determine the potential roles of miR-21 in TGF-β1-mediated gene regulation during myofibroblast conversion,miR-21 expression was regulated by miR-21 inhibitor and miR-21 mimic,respectively.
3.Computer analysis,luciferase report construction including the fragment of 3'-UTR of PDCD4(programmed cell death 4) mRNA with the putative miR-21 binding sequence and loss-of-function and gain-of-function experiments using miR-21 inhibitor and miR-21 mimic were used to predict and detect the target genes of miR-21 during the fibroblast-to-myofibroblasts transdifferentiation in ovarian cancer stroma.
【Results】
1.miR-21 was up-regulated in active fibroblasts after treatment with TGF-β1 or CM from ovarian cancer cells in time-dependent and dose-dependent manner(P＜0.05).
2.The expression of miR-21 was downregulated by miR-21 inhibitor and upregulated by miR-21 mimic in fibroblasts MRC-5.TGF-β1-induced myofibroblasts differentiation was inhibited by mir-21 inhibitor and promoted by miR-21 mimic(P＜0.05).
3.Computer analysis shows that PDCD4 might be a potential target gene of miR-21 based on existing the "seed sequence" of miR-21 in the PDCD4 3'-UTR.
4.The fragment of 3'-UTR of PDCD4 mRNA with the putative miR-21 binding sequence was cloned into a pGL3 vector at the downstream of the luciferase gene and co-transfected the pGL3 construct with miR-21 mimic or miR-21 inhibitor into MRC-5 fibroblasts using Lipofectamine 2000. The miR-21 mimic increased miR-21 expression and inhibited luciferase activity.On the contrary,the miR-21 inhibitor down-regulated miR-21 and sequentially up-regulated luciferase activity(P＜0.05).
5.Reduction of miR-21 by miR-21 inhibitor and enhanced expression of miR-21 by the miR-21 mimic increased or decreased PDCD4 protein expression,respectively.TGF-β1 stimulated miR-21 expression,then reduced PDCD4 expression in the translation level,however miR-21 inhibitor abolished the effect.
【Conclusion】
miR-21 participated in TGF-β1-induced gene regulation and functional modulation by targeting PDCD4 in stomal myofibroblasts differentiation.
卵巢癌是妇科肿瘤中致死率最高的恶性肿瘤,因为在其诊断时多数已为晚期病例,虽然经过系统的手术、化学治疗,5年生存率仍不超过45%,一直被认为是一个“沉默杀手”。越来越多的学者认识到,肿瘤的生物学行为不仅取决于肿瘤细胞本身的特征,而且与肿瘤间质之间的相互作用密切相关。不同的实体肿瘤虽然实质和间质的比例不尽相同,但是一般肿瘤的间质成分占整个实体肿瘤的50-90%。间质成分毗邻于肿瘤细胞,受到肿瘤细胞旁分泌信号的调节,可以转化为肿瘤相关的间质细胞,因为其改变了细胞的表型、功能和肿瘤局部的微环境,可以促进肿瘤的增殖、浸润、转移和血管形成。肿瘤间质的肿瘤相关纤维母细胞是间质细胞中最主要的细胞成分,其主要来源包括:肿瘤局部的纤维母细胞、上皮间质转化、远处部位的纤维母细胞、骨髓祖细胞、干细胞等。其中,间质纤维母细胞是最主要的细胞成分,其不同于正常的纤维母细胞,因为表达已分化肌纤维母细胞表型标志α-SMA(α-smooth muscle actin),被称为肿瘤相关成纤维细胞或肌纤维母细胞(tumour-associated fibroblasts,myofibroblasts),但其分子转化机制尚不完全清楚。对肿瘤相关成纤维细胞转化激活机制的深入研究,将有助于靶向肿瘤间质治疗新策略的提出。
细胞内氯通道蛋白4(Chloride intracellular channel 4,CLIC4),是细胞内的一种氯通道,调节细胞内pH值和细胞体积。它不仅存在于细胞器膜上,而且可以以可溶性成分存在于细胞浆和细胞核内,作为一种通道调节剂或信号蛋白。有研究显示,TGF-β1可以刺激乳腺组织原位的纤维母细胞的CLIC4转录水平升高,更重要的是,CLIC4高表达于乳腺癌组织的间质肿瘤相关成纤维细胞,而正常组织间质中几乎无CLIC4表达。故我们推测CLIC4可能参与上皮性卵巢癌的间质肌纤维母细胞的转化。
MicroRNAs(miRNAs)是一类分布广泛的小的非编码蛋白质的RNAs,其功能是负性调控基因表达。在人类基因组中至少存在700个miRNAs(也有可能为1000个),约占人类基因的1-4%,预测他们能够调节至少三分之一的人类基因,这使得其成为最大的一类基因表达调控因子。miRNAs参与生命过程中几乎所有的重要过程,包括早期发育,细胞增殖,细胞凋亡和细胞分化等。肿瘤间质肌纤维母细胞转化过程中存在一系列表型和功能基因的表达改变,但参与此过程调控基因表达的miRNAs尚未见报道。
本研究中,我们首先检测上皮性卵巢癌患者组织中CLIC4的表达,并构建间质肌纤维母细胞转化的细胞模型,探讨是否CLIC4参与此转化过程及其在此过程中的作用,进一步探讨调控肿瘤间质肌纤维母细胞转化的miRNAs。
第一部分CLIC4介导卵巢癌肿瘤相关成纤维细胞转化
【目的】
来自肿瘤与间质相互作用的信号分子激活肿瘤间质肌纤维母细胞,其对肿瘤的进展起重要的作用。CLIC4可能参与间质纤维母细胞的激活,但其分子机制尚不清楚。本研究拟探讨CLIC4是否参与上皮性卵巢癌间质纤维母细胞向肌纤维母细胞的转化及其在此过程中的作用。
【方法】
1.应用免疫组织化学方法检测上皮性卵巢癌组织间质中CLIC4的表达。
2.卵巢癌细胞条件培养基(Conditioned medium,CM)或转化生长因子β1(Transforming growth factor-β1,TGF-β1)培养原代卵巢正常纤维母细胞或人纤维母细胞系构建卵巢癌肿瘤间质纤维母细胞向肌纤维母细胞转化的细胞模型,应用倒置显微镜观察细胞形态学改变和肌纤维母细胞转化细胞表型标志α-SMA表达上调,评价转化细胞模型成功构建。
3.应用肌纤维母细胞转化的细胞模型,证实CLIC4和活性氧产物(reactive oxygen species,ROS)参与此转化过程。
4.应用CLIC4特异性siRNA阻断CLIC4的表达,探讨其在肿瘤间质肌纤维母细胞转化中的作用。
5.应用CLIC4特异性siRNA阻断CLIC4的表达或氯离子通道抑制剂抑制CLIC4的功能、抗氧化剂阻断ROS水平上调,探讨它们对肿瘤间质肌纤维母细胞转化相关功能蛋白表达的抑制作用。
【结果】
1.上皮性卵巢癌组织中间质CLIC4的阳性表达率为97.22%,肌纤维母细胞表型标志物α-SMA的阳性表达率为94.44%,两者密切相关性(P＜0.05)。肿瘤间质CLIC4表达强度与患者的肿瘤分化程度和淋巴结转移密切相关,中、低分化者,有淋巴结转移者肿瘤组织间质CLIC4的表达明显高于高分化,无淋巴结转移者(P＜0.05)。
2.应用卵巢癌细胞CM或TGF-β1培养卵巢组织原代正常纤维母细胞或人纤维母细胞系MRC-5 48小时,细胞增大呈多角形,而无血清培养的纤维母细胞呈细长纺锤状,同时α-SMA的转录(P＜0.05)和蛋白表达水平显著增加,表明正常纤维母细胞向肌纤维母细胞转化。
3.应用卵巢癌细胞CM或TGF-β1培养卵巢组织原代正常纤维母细胞或人纤维母细胞系MRC-5 48小时,纤维母细胞CLIC4表达在转录(P＜0.05)和蛋白水平明显上调。
4.应用卵巢癌细胞CM或TGF-β1培养卵巢组织原代正常纤维母细胞或人纤维母细胞系MRC-5促进细胞内ROS的产生,应用抗氧化剂抑制ROS产生可以抑制CM或TGF-β1诱导的CLIC4和α-SMA上调(P＜0.05)。
5.应用CLIC4特异性siRNA能够有效阻断CLIC4的表达,抑制CM或TGF-β1诱导的α-SMA表达水平上调(P＜0.05)。
6.应用CLIC4特异性siRNA阻断CLIC4的表达或氯离子通道抑制剂抑制CLIC4的功能、抗氧化剂阻断ROS水平上调,可以抑制与肌纤维母转化相关促血管因子VEGFA和HGF mRNA水平表达29-80%(P＜0.05)。
【结论】
CLIC4参与卵巢癌肿瘤间质纤维母细胞向肌纤维母细胞转化。来自肿瘤的旁分泌信号TGF-β1刺激使纤维母细胞内ROS产生增加,进而促进CLIC4的表达上调,最终导致纤维母细胞向肌纤维母细胞转化,即肿瘤相关成纤维细胞的活化。阻断CLIC4和ROS具有靶向肿瘤间质的治疗前景。
第二部分microRNA-21参与肿瘤相关成纤维细胞转化
【目的】
肿瘤细胞分泌的TGF-β1诱导肿瘤间质肌纤维母细胞的转化表现为一系列表型和功能基因的表达改变。miRNAs作为重要的基因负性调节因子,目前参与肿瘤间质肌纤维母细胞转化的miRNAs尚未见报道。
【方法】
1.应用real time RT-PCR方法检测一系列候选参与肿瘤间质肌纤维母细胞转化过程相关的miRNAs的表达水平。
2.应用microRNA-21(miR-21)抑制物和类似物转染人纤维母细胞,探讨miRNA-21在肿瘤间质肌纤维母细胞的转化过程中的作用。
3.应用计算机软件预测肿瘤间质成纤维细胞转化过程中miR-21的靶向调控基因,应用构建包含miR-21靶基因调控程序性细胞死亡4基因(programmed cell death 4,PDCD4)3'-UTR的荧光素酶报告载体方法和应用转染或敲除miR-21后检测纤维母细胞中PDCD4表达水平的变化,确定PDCD4是否是miR-21在肌纤维母细胞转化过程中的靶向调节基因。
【结果】
1.应用卵巢癌细胞CM或TGF-β1培养人纤维母细胞系MRC-5,细胞内miR-21表达水平上调(P＜0.05),并且具有时间和剂量依赖性效应。
2.应用miR-21抑制物和类似物转染纤维母细胞MRC-5,miR-21抑制物特异性降调而其类似物特异性上调miR-21的水平(P＜0.05)。miR-21抑制物可以抑制CM或TGF-β1诱导的肿瘤间质肌纤维母细胞的转化过程中α-SMA等转化标志基因和功能基因的表达(P＜0.05)。
3.生物信息软件预测调控PDCD4的3'UTR与miR-21具有明显的相互作用“种子序列”。
4.构建含有miR-21潜在结合位点的PDCD4基因3'UTR荧光素酶报告载体,其与miR-21抑制剂或模仿剂共转染入纤维母细胞MRC-5,miR-21模仿剂增加miR-21的表达而抑制荧光素酶活性,而miR-21抑制剂显著增加荧光素酶活性(P＜0.05)。提示,在纤维细胞中miR-21能够结合到PDCD4基因的3'UTR。
5.应用特异性转染或沉默方法,miR-21模仿剂和miR-21抑制剂分别降低和升高纤维母细胞中PDCD4的蛋白表达,并且转染miR-21抑制剂可以拮抗TGF-β1诱导的miR-21内源性负性调节PDCD4表达的作用。
【结论】
miR-21参与卵巢癌细胞CM或TGF-β1诱导的肿瘤间质肌纤维母细胞转化是通过靶向调节PDCD4。
Background
Epithelial ovarian cancer is the most lethal disease among gynaecologic malignancies.For years,it has been dubbed as a "silent killer"--causing no symptom till it is too advanced at the time of diagnosis.5-year survival for patients with advanced ovarian cancer improved little even with the treatment of optimal cytoreductive surgery and systemic combination chemotherapy.Increasing evidence has indicated that cancer development is facilitated by interaction between tumour cells and activated stromal cells.The tumour stroma(also referred to as "reactive stroma") is characterized by marked alterations in the phenotype and expression profile of fibroblast-like cells.These cells commonly expressα-SMA and thus are termed as myofibroblasts.Stromal fibroblasts are located at the tumour border near the invasion front,and play a crucial role in tumour progression.Myofibroblasts are recruited from different sources,including local fibroblast population,distant fibroblasts,EMT (epithelial-mesenchymal transition) and bone marrow progenitor cells or stem cells,during cancer development and the invasion progression. Although it has been shown that the conversion from fibroblasts to myofibroblasts constitutes the major source of myofibrblasts in tumour stroma,the molecular mechanism underlying fibroblast-to-myofibroblast transdifferentiation is still not fully understood.
Chloride intracellular channel 4(CLIC4),a chloride channel of intracellular organelles,regulates intracellular pH and cell volume. Besides its presence on the organelle membrane,CLIC4 exists in soluble form in the cytoplasm and nucleus acting as signalling protein or channel regulator.Transcription level of CLIC4 is up-regulated when fibroblasts transdifferentiate into myofibroblasts induced by TGF-β1,and more importantly CLIC4 is highly expressed in myofibroblasts of breast cancer. Hence,we hypothesized that CLIC4 might invlove in myofibroblast transdifferentiation in ovarian cancer stroma,but the pathway in which CLIC4 participated during the fibroblast-to-myofibroblast transition induced by TGF-β1 from ovarian cancer cells is still unknown.
MicroRNAs(miRNAs) are a class of endogenous,short (20-25-nucleotide),noncoding single-stranded RNA molecules, which negatively regulate gene expression through mRNAs degradation or translational inhibition of their target gene.miRNAs are involved in regulators of gene expression that control diverse physiological and pathological processes,such as development,cell differentiation, migration,proliferation and apoptosis,miRNAs represent a new layer of gene expression regulators at the transcript and translational levels,but the involvement of miRNAs and the roles of these miRNAs in TGF-β1-induced myofibroblasts differentiation in tumor-stroma interaction are uncertain.
PartⅠCLIC4 mediares TGF-β1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer
【Objective】
Stromal myofibroblasts,activated by crosstalk signaling between the tumour and stroma,play a critical role in tumour development and progression.Chloride intracellular channel 4(CLIC4) may be functionally import for tumour stromal fibroblast-to-myofibroblast transdifferentiaton,but the molecular mechanism of the process has not been addressed.This study was to investigate the role of CLIC4 in the myofibroblast transdifferentiaton of ovarian cancer stroma.
【Methods】
1.The expression of CLIC4 in ovarian cancer tissues was analyzed by immunohischemistry,
2.A cell model of fibroblast-to-myofibroblast transdifferentiaton was constructed,where ovarian primary fibroblasts or human fibroblasts MRC-5 cells were cultured with conditioned medium(CM) from ovarian cancer cells or transforming growth factor-β1(TGF-β1).Cellular morphologic change and the upregulation ofα-SMA expression were used to identify the differentiated myofibroblasts.
3.The cell model of myofibroblast transdifferentiaton was used to demonstrate whether CLIC4 and ROS participated in the fibroblast-to-myofibroblast transdifferentiation.
4.Specific CLIC4 siRNA was used to knockdown the CLIC4 expression, then to investigate the role of CLIC4 in the myofibroblast transdifferentiaton.
5.The agents,specific CLIC4 siRNA,chloride channel inhibitor and antioxidant,were used to investigate the roles in suppression of the functional gene expression related with the myofibroblasts conversion.
【Results】
1.The expression of CLIC4 in 97.22%of ovarian cancer stroma and correlated with the up-regulation of myofibroblast markerα-SMA,which was detected in 94.44%of patients(P＜0.05).The immunohischemistry data in different pathologic groups showed that the protein expression level of CLIC4 in ovarian cancer stroma which was in mid- or low-differentiated and accompanied by lymphatic metastasis was obviously higher than that which was in high-differentiated and nonaccompanied by lymphatic metastasis(P＜0.05).
2.In the presence of CM~(SKOV3) or TGF-β1,the fibroblasts,which displayed typical small spindle shape under serum-free condition, changed into bigger and polygonal cells,andα-SMA transcript and translational expression increased(P＜0.05),indicating fibroblast-to-myofibroblast conversion.
3.The expression of CLIC4 in the activated fibroblasts increased significantly in the presence of CM~(SKOV3) or 10 ng/ml TGF-β1 for 48 h both at the transcription(P＜0.05) and protein levels compared with that in serum-free medium control.
4.When cells were pretreated with the antioxidant NAC,which inhibited the generation of intracellular ROS,the up-regulation ofα-SMA and CLIC4 expression stimulated by TGF-β1 or CM~(SKOV3) was significantly suppressed(P＜0.05).
5.The up-regulation ofα-SMA expression induced by TGF-β1 or CM~(SKOV3) was significantly blocked in CLIC4 siRNA-transfected fibroblasts(P＜0.05).
6.Pre-treatment with CLIC4 siRNA to silence CLIC4 expression, chloride channel inhibitor IAA-94 to block function of CLIC4 or NAC to inhibit the generation of ROS lowered VEGF and HGF expression levels in the activated fibroblasts by 29-80%(P＜0.05).
【Conclusion】
These results suggest that ROS-initiated CLIC4 up-regulation is required for TGF-β1-induced fibroblast-to-myofibroblast transdifferentiaton in ovarian cancer,indicating inhibiting CLIC4 or ROS might have therapeutic potential targeting tumour stroma.
PartⅡMicroRNA-21 participates in TGF-β1-induced myofibroblasts differentiation in tumor stroma
【Objectire】
Stromal fibroblast-to-myofibroblast transdifferentiation induced by TGF-β1 in the microenvironment of tumor and stroma interaction via expression changes of multiple phenotypic and functional genes plays a critical role in the tumor progression.Up to now,the involvement of microRNAs(miRNAs) and the roles of these miRNAs in TGF-β1-induced myofibroblasts differentiation in tumor-stroma interaction are unclear.
【Methods】
1.Quantitative real time RT-PCR was used to measure the expression change of a range of microRNAs during the fibroblast-to-myofibroblast transdifferentiation induced by TGF-β1 or CM from cancer cells.
2.To determine the potential roles of miR-21 in TGF-β1-mediated gene regulation during myofibroblast conversion,miR-21 expression was regulated by miR-21 inhibitor and miR-21 mimic,respectively.
3.Computer analysis,luciferase report construction including the fragment of 3'-UTR of PDCD4(programmed cell death 4) mRNA with the putative miR-21 binding sequence and loss-of-function and gain-of-function experiments using miR-21 inhibitor and miR-21 mimic were used to predict and detect the target genes of miR-21 during the fibroblast-to-myofibroblasts transdifferentiation in ovarian cancer stroma.
【Results】
1.miR-21 was up-regulated in active fibroblasts after treatment with TGF-β1 or CM from ovarian cancer cells in time-dependent and dose-dependent manner(P＜0.05).
2.The expression of miR-21 was downregulated by miR-21 inhibitor and upregulated by miR-21 mimic in fibroblasts MRC-5.TGF-β1-induced myofibroblasts differentiation was inhibited by mir-21 inhibitor and promoted by miR-21 mimic(P＜0.05).
3.Computer analysis shows that PDCD4 might be a potential target gene of miR-21 based on existing the "seed sequence" of miR-21 in the PDCD4 3'-UTR.
4.The fragment of 3'-UTR of PDCD4 mRNA with the putative miR-21 binding sequence was cloned into a pGL3 vector at the downstream of the luciferase gene and co-transfected the pGL3 construct with miR-21 mimic or miR-21 inhibitor into MRC-5 fibroblasts using Lipofectamine 2000. The miR-21 mimic increased miR-21 expression and inhibited luciferase activity.On the contrary,the miR-21 inhibitor down-regulated miR-21 and sequentially up-regulated luciferase activity(P＜0.05).
5.Reduction of miR-21 by miR-21 inhibitor and enhanced expression of miR-21 by the miR-21 mimic increased or decreased PDCD4 protein expression,respectively.TGF-β1 stimulated miR-21 expression,then reduced PDCD4 expression in the translation level,however miR-21 inhibitor abolished the effect.
【Conclusion】
miR-21 participated in TGF-β1-induced gene regulation and functional modulation by targeting PDCD4 in stomal myofibroblasts differentiation.
引文
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1. Jemal, A., et al., Cancer statistics, 2008. CA Cancer J Clin, 2008. 58(2): p.71-96.
2. Ozols, R.F., et al., Focus on epithelial ovarian cancer. Cancer Cell, 2004. 5(1):p. 19-24.
3. Levi, F., et al., Cancer mortality in Europe, 1995-1999, and an overview of trends since 1960. Int J Cancer, 2004. 110(2): p. 155-69.
4. Tlsty, T.D. and L.M. Coussens, Tumor stroma and regulation of cancer development. Annu Rev Pathol, 2006. 1: p. 119-50.
5. Mueller, M.M. and N.E. Fusenig, Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer, 2004. 4(11): p. 839-49.
6. Bhowmick, N.A., E.G. Neilson, and H.L. Moses, Stromal fibroblasts in cancer initiation and progression. Nature, 2004. 432(7015): p. 332-7.
7. Muerkoster, S.S., et al., Role of myofibroblasts in innate chemoresistance of pancreatic carcinoma—epigenetic downregulation of caspases. Int J Cancer,2008. 123(8): p. 1751-60.
8. Zalatnai, A., Molecular aspects of stromal-parenchymal interactions in malignant neoplasms. Curr Mol Med, 2006. 6(6): p. 685-93.
9. Orimo, A., et al., Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell, 2005. 121(3): p. 335-48.
10. Yang, F., D.W. Strand, and D.R. Rowley, Fibroblast growth factor-2 mediates transforming growth factor-beta action in prostate cancer reactive stroma.Oncogene, 2008. 27(4): p. 450-9.
11. Cat, B., et al., Enhancement of tumor invasion depends on transdifferentiation of skin fibroblasts mediated by reactive oxygen species. J Cell Sci, 2006.119(Pt 13): p. 2727-38.
12. Rocks, N., et al., ADAMTS-1 metalloproteinase promotes tumor development through the induction of a stromal reaction in vivo. Cancer Res, 2008. 68(22):p. 9541-50.
13. Verona, E.V., et al., Transforming growth factor-beta signaling in prostate stromal cells supports prostate carcinoma growth by up-regulating stromal genes related to tissue remodeling. Cancer Res, 2007. 67(12): p. 5737-46.
14. De Wever, O., et al., Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer, 2008. 123(10): p. 2229-38.
15. Marsh, D., et al., alpha vbeta 6 Integrin promotes the invasion of morphoeic basal cell carcinoma through stromal modulation. Cancer Res, 2008. 68(9): p.3295-303.
16. Denys, H., et al., Differential impact of TGF-beta and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion.Cancer Lett, 2008. 266(2): p. 263-74.
17. Shats, I., et al., Myocardin in tumor suppression and myofibroblast differentiation. Cell Cycle, 2007. 6(10): p. 1141-6.
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22. Radisky, D.C., P.A. Kenny, and M.J. Bissell, Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT? J Cell Biochem, 2007. 101(4): p. 830-9.
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25. Mishra, P.J., et al., Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells. Cancer Res, 2008. 68(11): p. 4331-9.
26. Jeon, E.S., et al., Cancer-derived lysophosphatidic acid stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells.Stem Cells, 2008. 26(3): p. 789-97.
27. Lederle, W., et al., Platelet-derived growth factor-BB controls epithelial tumor phenotype by differential growth factor regulation in stromal cells. Am J Pathol, 2006. 169(5): p. 1767-83.
28. Suh, K.S., et al., CLIC4, skin homeostasis and cutaneous cancer: surprising connections. Mol Carcinog, 2007. 46(8): p. 599-604.
29. Ronnov-Jessen, L., et al., Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-betal -mediated conversion of fibroblasts to myofibroblasts. Am J Pathol, 2002. 161(2): p.471-80.
30. Littler, D.R., et al., Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4. FEBS J, 2005. 272(19): p.4996-5007.
31. Yang, K.L., et al., Inhibition of transforming growth factor-beta-induced liver fibrosis by a retinoic acid derivative via the suppression of Col 1A2 promoter activity. Biochem Biophys Res Commun, 2008. 373(2): p. 219-23.
32. Radisky, D.C., et al., Raclb and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature, 2005. 436(7047): p. 123-7.
33. Stuhlmann, D., et al., Paracrine effect of TGF-betal on downregulation of gap junctional intercellular communication between human dermal fibroblasts.Biochem Biophys Res Commun, 2004. 319(2): p. 321-6.
34. Olumi, A.F., et al., Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res, 1999. 59(19): p. 5002-11.
35. Walter-Yohrling, J., et al., Myofibroblasts enable invasion of endothelial cells into three-dimensional tumor cell clusters: a novel in vitro tumor model.Cancer Chemother Pharmacol, 2003. 52(4): p. 263-9.
36. Mazzocca, A., et al., A secreted form of ADAM9 promotes carcinoma invasion through tumor-stromal interactions. Cancer Res, 2005. 65(11): p. 4728-38.
37. Samoszuk, M., J. Tan, and G. Chorn, Clonogenic growth of human breast cancer cells co-cultured in direct contact with serum-activated fibroblasts.Breast Cancer Res, 2005. 7(3): p. R274-83.
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42. Bohman, S., et al., Proteomic analysis of vascular endothelial growth factor-induced endothelial cell differentiation reveals a role for chloride intracellular channel 4 (CLIC4) in tubular morphogenesis. J Biol Chem, 2005.280(51): p. 42397-404.
43. Ulmasov, B., et al., Chloride intracellular channel protein-4 functions in angiogenesis by supporting acidification of vacuoles along the intracellular tubulogenic pathway. Am J Pathol, 2009. 174(3): p. 1084-96.
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2. Ozols, R.F., et al., Focus on epithelial ovarian cancer. Cancer Cell, 2004. 5(1):p. 19-24.
3. Levi, F., et al., Cancer mortality in Europe, 1995-1999, and an overview of trends since 1960. Int J Cancer, 2004. 110(2): p. 155-69.
4. Tlsty, T.D. and L.M. Coussens, Tumor stroma and regulation of cancer development. Annu Rev Pathol, 2006. 1: p. 119-50.
5. Mueller, M.M. and N.E. Fusenig, Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer, 2004. 4(11): p. 839-49.
6. Bhowmick, N.A., E.G. Neilson, and H.L. Moses, Stromal fibroblasts in cancer initiation and progression. Nature, 2004. 432(7015): p. 332-7.
7. Muerkoster, S.S., et al., Role of myofibroblasts in innate chemoresistance of pancreatic carcinoma--epigenetic downregulation of caspases. Int J Cancer,2008. 123(8): p. 1751-60.
8. Zalatnai, A., Molecular aspects of stromal-parenchymal interactions in malignant neoplasms. Curr Mol Med, 2006. 6(6): p. 685-93.
9. Orimo, A., et al., Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell, 2005. 121(3): p. 335-48.
10. Yang, F., D.W. Strand, and D.R. Rowley, Fibroblast growth factor-2 mediates transforming growth factor-beta action in prostate cancer reactive stroma.Oncogene, 2008. 27(4): p. 450-9.
11. Cat, B., et al., Enhancement of tumor invasion depends on transdifferentiation of skin fibroblasts mediated by reactive oxygen species. J Cell Sci, 2006.119(Pt 13): p. 2727-38.
12. Rocks, N., et al., ADAMTS-1 metalloproteinase promotes tumor development through the induction of a stromal reaction in vivo. Cancer Res, 2008. 68(22):p. 9541-50.
13. Verona, E.V., et al., Transforming growth factor-beta signaling in prostate stromal cells supports prostate carcinoma growth by up-regulating stromal genes related to tissue remodeling. Cancer Res, 2007. 67(12): p. 5737-46.
14. De Wever, O., et al., Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer, 2008. 123(10): p. 2229-38.
15. Marsh, D., et al., alpha vbeta 6 Integrin promotes the invasion of morphoeic basal cell carcinoma through stromal modulation. Cancer Res, 2008. 68(9): p.3295-303.
16. Denys, H., et al., Differential impact of TGF-beta and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion.Cancer Lett, 2008. 266(2): p. 263-74.
17. Shats, I., et al., Myocardin in tumor suppression and myofibroblast differentiation. Cell Cycle, 2007. 6(10): p. 1141-6.
18. Nazareth, M.R., et al., Characterization of human lung tumor-associated fibroblasts and their ability to modulate the activation of tumor-associated T cells. J Immunol, 2007. 178(9): p. 5552-62.
19. Shao, J., et al., Roles of myofibroblasts in prostaglandin E2-stimulated intestinal epithelial proliferation and angiogenesis. Cancer Res, 2006. 66(2):p. 846-55.
20. Chaffer, C.L., et al., Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis: role of fibroblast growth factor receptor-2. Cancer Res,2006. 66(23): p. 11271-8.
21. Varro, A., et al., Increased gastric expression of MMP-7 in hypergastrinemia and significance for epithelial-mesenchymal signaling. Am J Physiol Gastrointest Liver Physiol, 2007. 292(4): p. G1133-40.
22. Radisky, D.C., P.A. Kenny, and M.J. Bissell, Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT? J Cell Biochem, 2007. 101(4):p.830-9.
23.Granot,D.,et al.,In vivo imaging of the systemic recruitment of fibroblasts to the angiogenic rim of ovarian carcinoma tumors.Cancer Res,2007.67(19):p.9180-9.
24.Guo,X.,et al.,Stromal fibroblasts activated by tumor cells promote angiogenesis in mouse gastric cancer.J Biol Chem,2008.283(28):p.19864-71.
25.Mishra,P.J.,et al.,Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells.Cancer Res,2008.68(11):p.4331-9.
26.Jeon,E.S.,et al.,Cancer-derived lysophosphatidic acid stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells.Stem Cells,2008.26(3):p.789-97.
27.Lederle,W.,et al.,Platelet-derived growth factor-BB controls epithelial tumor phenotype by differential growth factor regulation in stromal cells.Am J Pathol,2006.169(5):p,1767-83.
28.Suh,K.S.,et al.,CLIC4,skin homeostasis and cutaneous cancer:surprising connections.Mol Carcinog,2007.46(8):p.599-604.
29.Ronnov-Jessen,L.,et al.,Differential expression of a chloride intracellular channel gene,CLIC4,in transforming growth factor-betal-mediated conversion of fibroblasts to myofibroblasts.Am J Pathol,2002.161(2):p.471-80.
30.Littler,D.R.,et al.,Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4.FEBS J,2005.272(19):p.4996-5007.
31.Yang,K.L.,et al.,Inhibition of transforming growth factor-beta-induced liver fibrosis by a retinoic acid derivative via the suppression of Col 1A2 promoter activity.Biochem Biophys Res Commun,2008.373(2):p,219-23.
32.Radisky,D.C.,et al.,Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability.Nature,2005.436(7047):p. 123-7.
33. Stuhlmann, D., et al., Paracrine effect of TGF-betal on downregulation of gap junctional intercellular communication between human dermal fibroblasts.Biochem Biophys Res Commun, 2004. 319(2): p. 321-6.
34. Olumi, A.F., et al., Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res, 1999. 59(19):p. 5002-11.
35. Walter-Yohrling, J., et al., Myofibroblasts enable invasion of endothelial cells into three-dimensional tumor cell clusters: a novel in vitro tumor model.Cancer Chemother Pharmacol, 2003. 52(4): p. 263-9.
36. Mazzocca, A., et al., A secreted form of ADAM9 promotes carcinoma invasion through tumor-stromal interactions. Cancer Res, 2005. 65(11): p.4728-38.
37. Samoszuk, M., J. Tan, and G. Chorn, Clonogenic growth of human breast cancer cells co-cultured in direct contact with serum-activated fibroblasts. Breast Cancer Res, 2005. 7(3): p. R274-83.
38. Suh, K.S., et al., Reciprocal modifications of CLIC4 in tumor epithelium and stroma mark malignant progression of multiple human cancers. Clin Cancer Res, 2007. 13(1): p. 121-31.
39. Suginta, W., et al., Chloride intracellular channel protein CLIC4 (p64Hl) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms. Biochem J, 2001. 359(Pt 1): p. 55-64.
40. Zeng, Y.X., X.S. Zhang, and Q. Liu, [Molecular target therapy: a milestone on the road for curing cancer]. Ai Zheng, 2008. 27(8): p. 785-7.
41. Mahadevan, D. and D.D. Von Hoff, Tumor-stroma interactions in pancreatic ductal adenocarcinoma. Mol Cancer Ther, 2007. 6(4): p. 1 186-97.
42. Bohman, S., et al., Proteomic analysis of vascular endothelial growth factor-induced endothelial cell differentiation reveals a role for chloride intracellular channel 4 (CLIC4) in tubular morphogenesis. J Biol Chem, 2005.280(51): p. 42397-404.
43. Ulmasov, B., et al., Chloride intracellular channel protein-4 functions in angiogenesis by supporting acidification of vacuoles along the intracellular tubulogenic pathway. Am J Pathol, 2009. 174(3): p. 1084-96.
44. Tung, J.J., et al., Chloride intracellular channel 4 is involved in endothelial proliferation and morphogenesis in vitro. Angiogenesis, 2009.
1. Jemal, A., et al., Cancer statistics, 2008. CA Cancer J Clin, 2008. 58(2): p.71-96.
2. Ozols, R.F., et al., Focus on epithelial ovarian cancer. Cancer Cell, 2004. 5(1):p. 19-24.
3. Levi, F., et al., Cancer mortality in Europe, 1995-1999, and an overview of trends since 1960. Int J Cancer, 2004. 110(2): p. 155-69.
4. Tlsty, T.D. and L.M. Coussens, Tumor stroma and regulation of cancer development. Annu Rev Pathol, 2006. 1: p. 119-50.
5. Mueller, M.M. and N.E. Fusenig, Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer, 2004. 4(11): p. 839-49.
6. Bhowmick, N.A., E.G. Neilson, and H.L. Moses, Stromal fibroblasts in cancer initiation and progression. Nature, 2004. 432(7015): p. 332-7.
7. Muerkoster, S.S., et al., Role of myofibroblasts in innate chemoresistance of pancreatic carcinoma—epigenetic downregulation of caspases. Int J Cancer,2008. 123(8): p. 1751-60.
8. Zalatnai, A., Molecular aspects of stromal-parenchymal interactions in malignant neoplasms. Curr Mol Med, 2006. 6(6): p. 685-93.
9. Orimo, A., et al., Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell, 2005. 121(3): p. 335-48.
10. Yang, F., D.W. Strand, and D.R. Rowley, Fibroblast growth factor-2 mediates transforming growth factor-beta action in prostate cancer reactive stroma.Oncogene, 2008. 27(4): p. 450-9.
11. Cat, B., et al., Enhancement of tumor invasion depends on transdifferentiation of skin fibroblasts mediated by reactive oxygen species. J Cell Sci, 2006.119(Pt 13): p. 2727-38.
12. Rocks, N., et al., ADAMTS-1 metalloproteinase promotes tumor development through the induction of a stromal reaction in vivo. Cancer Res, 2008. 68(22):p. 9541-50.
13. Verona, E.V., et al., Transforming growth factor-beta signaling in prostate stromal cells supports prostate carcinoma growth by up-regulating stromal genes related to tissue remodeling. Cancer Res, 2007. 67(12): p. 5737-46.
14. De Wever, O., et al., Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer, 2008. 123(10): p. 2229-38.
15. Marsh, D., et al., alpha vbeta 6 Integrin promotes the invasion of morphoeic basal cell carcinoma through stromal modulation. Cancer Res, 2008. 68(9): p.3295-303.
16. Denys, H., et al., Differential impact of TGF-beta and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion.Cancer Lett, 2008. 266(2): p. 263-74.
17. Shats, I., et al., Myocardin in tumor suppression and myofibroblast differentiation. Cell Cycle, 2007. 6(10): p. 1141-6.
18. Nazareth, M.R., et al., Characterization of human lung tumor-associated fibroblasts and their ability to modulate the activation of tumor-associated T cells. J Immunol, 2007. 178(9): p. 5552-62.
19. Shao, J., et al., Roles of myofibroblasts in prostaglandin E2-stimulated intestinal epithelial proliferation and angiogenesis. Cancer Res, 2006. 66(2):p. 846-55.
20. Chaffer, C.L., et al., Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis: role of fibroblast growth factor receptor-2. Cancer Res,2006. 66(23): p. 11271-8.
21. Varro, A., et al., Increased gastric expression of MMP-7 in hypergastrinemia and significance for epithelial-mesenchymal signaling. Am J Physiol Gastrointest Liver Physiol, 2007. 292(4): p. G1133-40.
22. Radisky, D.C., P.A. Kenny, and M.J. Bissell, Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT? J Cell Biochem, 2007. 101(4): p. 830-9.
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