同源盒基因HOXB7在肺腺癌增殖与转移中的作用研究
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摘要
研究背景和目的
目前，肺癌发病率和病死率位于各类癌症之首。肺癌分为小细胞肺癌与非小细胞肺癌两大类，其中非小细胞肺癌中最常见的是肺腺癌、肺鳞癌和肺大细胞癌三个亚型。肺腺癌(Lung adencarcinoma，LAC)的发病在所有类型肺癌中占第一位，已经达到40％。大量临床资料显示，肺腺癌的高侵袭力和易转移性是导致临床治疗失败和预后不良的主要原因，约90%的肺腺癌病人死于癌细胞的多器官转移。迄今为止，肺腺癌发病的危险因素已被初步确定，但有关其快速侵袭与转移的详细机制尚不清楚。因此，发现特异性的肺腺癌治疗靶标，深入探明调控肺腺癌增殖与转移的相关机制并进行针对性干预，对提高肺癌病人的疗效十分重要。
研究证实，与细胞分化、发育相关的同源盒分子如Oct4、Nanog等参与肺癌侵袭转移的调节。而同属于同源盒基因的HOX(Homeobox genes)基因家族类似作用越来越受到关注。HOX基因家族共包括39个含高度保守同源结构域的基因，分为HOXA、HOXB、HOXC和HOXD四簇，分别位于第7、17、12、2号染色体。HOX基因家族主要负责调节脊椎类动物和人类胚胎器官发育。作为细胞增殖和分化的主控基因，其表达蛋白的作用靶标通常为膜蛋白受体、粘附分子、生长因子等与肿瘤进展、转移密切相关的基因家族。研究表明，一些在成体细胞时期本该关闭的HOX基因迷乱表达，参与肿瘤的发生发展，尤其是肿瘤发展的后期阶段。在肺癌、乳腺癌、结肠癌等多种肿瘤中可检测到HOX基因的异常表达。另外，研究发现在干细胞中，HOX基因家族也参与其自我更新、无限增殖等干细胞特性的调控。
其中，HOXB7是近年来研究的热点。HOXB7属于HOX基因家族B簇，位于第17号染色体，已被确证属于癌基因的范畴。胚胎发育时期，HOXB7是主要负责调控上皮包括肺上皮细胞增殖、分化、迁移的主控基因。目前，已有研究表明在结肠癌及口腔癌中，HOXB7表达异常增高，并与恶性肿瘤快速进展及预后不良相关。在乳腺癌中，异常高表达的HOXB7导致肿瘤细胞EMT (Epithelial-mesenchymal transition，EMT)并诱导肿瘤干细胞生成，进而影响肿瘤转移。但有关HOXB7基因在肺腺癌中的表达情况以及其是否参与调控肺腺癌肿瘤细胞恶性进展与转移的能力，目前尚未见文献报道。
本课题通过检测肺腺癌细胞系及病人肿瘤组织中HOXB7基因的表达情况，结合临床病理学变量，初步探讨HOXB7表达在肺腺癌中意义，并进一步利用RNA干扰使HOXB7基因沉默。我们发现HOXB7降低表达可导致肺腺癌细胞体外增殖减慢、细胞周期阻滞、细胞迁移及侵袭能力降低。皮下注射HOXB7降低表达的肺腺癌细胞的裸鼠比对照组更晚产生肿瘤，且肿瘤体积更小。腹腔注射HOXB7降低表达的肺腺癌细胞的裸鼠比对照组发生更少的器官转移。
综上所述，本研究从细胞和实验动物水平证明HOXB7是肺腺癌细胞增殖和转移能力的重要调控因子，可成为控制肺腺癌的恶性进展与转移的临床防治新的靶点。
研究方法
1.同源盒基因HOXB7在肺腺癌细胞系及病人组织中的表达与意义
①从ATCC购买正常肺支气管腺上皮细胞系HBE以及肺腺癌细胞系A549、H1975、H1650、H322等，利用RT-PCR、Western blot等方法检测在各肺腺癌细胞系中HOXB7的mRNA及蛋白表达情况。②收集75例临床肺腺癌病人手术切除肿瘤组织标本及其癌旁正常组织标本，石蜡包埋，制作组织芯片。采用免疫组织化学染色方法，分析HOXB7蛋白表达与肺腺癌的临床分期、病理分级、远处转移能力、生存期等临床病理学变量的相关性。
2.同源盒基因HOXB7调控肺腺癌细胞体外增殖及体内成瘤能力
在A549细胞系中，采用逆转录病毒介导的标记有绿色荧光蛋白(GFP)的质粒载体进行HOXB7基因的RNA干扰，使HOXB7去表达，并进行以下研究：
①利用平板克隆形成实验，检测转染了空白质粒的对照组与HOXB7RNAi干扰组的肿瘤细胞体外增殖能力的差异。②利用流式细胞仪检测对照组与干扰组肿瘤细胞的细胞周期分布差异情况。③将对照组及干扰组肿瘤细胞分别皮下接种于NOD/SCID联合免疫缺陷鼠(6周龄)，每组5只，建立肺腺癌皮下移植瘤模型，每天记录肿瘤生长情况。30天后处死动物。利用公式[长*宽2]/2，计算移植瘤体积。将移植瘤取出，石蜡切片并进行免疫组化染色，观察各组间移植瘤组织的HOXB7及增殖标志如Ki-67蛋白的表达差异情况。
3.同源盒基因HOXB7调控肺腺癌细胞转移能力
①利用Western blot方法检测对照组与干扰组肿瘤细胞上皮细胞表面标志如E-钙粘蛋白及间质细胞表面标志如波形蛋白和纤连蛋白等蛋白表达差异情况。②利用免疫荧光染色方法检测对照组与干扰组肿瘤细胞上皮细胞表面标志如E-钙粘蛋白及间质细胞表面标志如波形蛋白和纤连蛋白等蛋白表达差异情况。③利用细胞划痕实验及transwell细胞迁移实验检测对照组与干扰组肿瘤细胞体外迁移能力的差异情况。④将对照组及干扰组肿瘤细胞分别腹腔注射于NOD/SCID联合免疫缺陷鼠(6周龄)，每组10只。35天后处死动物，观察腹腔脏器肿瘤转移发生的差异情况。
研究结果
1.人肺腺癌细胞系A549、H1975、H1650、H322中HOXB7的mRNA及蛋白表达水平明显高于人正常肺支气管上皮细胞系。其中A549细胞系的HOXB7表达水平高于其余几种肺腺癌肿瘤细胞系。
2.免疫组织化学染色结果显示，在75例配对组织样本中，有54例(72%)肺腺癌肿瘤组织呈现HOXB7阳性高表达，而仅有7例(9.3%)癌旁正常组织HOXB7表达阳性。进一步相关性检验显示： HOXB7蛋白表达水平与肿瘤T分期(P=0.028),肿瘤临床分级(P=0.029)及淋巴结转移(P=0.012)均呈显著的正相关性，而与肿瘤病理分级(P=0.146)无显著的相关性。另外，Kaplan–Meier生存分析显示肿瘤组织HOXB7蛋白表达水平与临床病人生存时间呈负相关，影响病人的预后。
3.用RNAi技术特异性沉默A549细胞HOXB7基因表达，与对照组细胞相比，干扰组细胞中的HOXB7蛋白表达明显受到抑制。平板克隆形成实验结果显示，干扰组细胞形成的集落克隆数明显少于(P<0.001)且小于对照组细胞。流式细胞仪分析细胞周期结果显示，干扰组细胞G1期细胞比例明显高于对照组细胞(P=0.006)，而S期细胞比例明显低于对照组细胞(P=0.002)，说明HOXB7通过影响G1/S细胞周期调控点，调控肺腺癌肿瘤细胞增殖能力。小鼠皮下移植瘤实验结果显示，干扰组小鼠皮下移植瘤体积小于对照组(P=0.007)，说明干扰组细胞体内成瘤能力明显降低。免疫组织化学染色观察移植瘤组织，发现干扰组组织的细胞增殖标志Ki-67表达低于对照组组织。
4.利用显微镜观察发现，在形态上，与对照组典型的纺锤形间质细胞形态相比，干扰组细胞向卵圆形的上皮细胞形态转变，并且聚集形成细胞簇。利用Western blot及免疫荧光染色方法检测发现，干扰组肿瘤细胞上皮细胞表面标志E-钙粘蛋白表达明显高于对照组，而间质细胞表面标志波形蛋白和纤连蛋白表达明显低于对照组。细胞划痕实验结果显示，单层细胞被划痕后24h及48h，干扰组细胞划痕修复能力明显低于对照组，说明干扰组细胞横向迁移能力明显降低。Transwell细胞迁移实验检测结果显示，细胞接种Transwell小室24h后，干扰组迁移至小室下层细胞数明显少于对照组(P=0.001)，说明干扰组细胞体外迁移能力明显降低。小鼠体内转移瘤模型结果发现，腹腔注射干扰组细胞的小鼠，腹腔积液少于对照组小鼠且器官转移发生率明显少于对照组小鼠。
结论
1.HOXB7在肺腺癌细胞系及病人肿瘤组织中异常高表达，且与肿瘤分期、分级及淋巴结转移等病理参数相关，说明其表达与肺腺癌进展及病人预后相关。提示HOXB7可能是肺腺癌的独立预后因子及疾病进展的标志。
2.HOXB7参与调控肺腺癌肿瘤细胞体外增殖和体内成瘤能力，控制肿瘤恶性进展。
3.HOXB7参与调控肺腺癌肿瘤细胞体外迁移能力，影响体内肿瘤转移。
4.进一步深入研究HOXB7对肺腺癌的作用机制，可为控制肺腺癌的恶性进展与转移的临床防治提供新的靶点。
Introduction
Lung cancer is one of the most frequent malignant tumors in the world, and theincidence of lung adenocarcinoma (LAC) is the first one in all types of lung cancer, havingbeen reached about40%.Many clinical datas indicated that fast progress, high rate ofrecurrence and metastasis ability of LAC is the main reason for the clinical treatment failureand poor prognosis of LAC patients, about90%of LAC patients died of multi-organmetastasis. So far, the risk factors for LAC incidence have been identified, but themechanisms about its rapid progression and metastasis are still unclear. Therefore, findingout the mechanisms of regulation of invasion and metastasis about LAC is very importantfor improving the survival rate of LAC patients.
Some studies have confirmed that some homeobox molecules such as Oct4, Nanog areinvolved in the regulation of lung cancer metastasis. And the homeobox gene family (HOX)have been paid more and more attention. HOX gene family is consists of39highlyconserved homeodomain genes,which are divided into four clusters HOXA, HOXB, HOXCand HOXD. HOX gene family is mainly responsible for the regulation of vertebrateanimals and human embryonic organ development. As the master genes for cellproliferation and differentiation, its target protein are usually some membrane proteinreceptors, adhesion molecules and growth factors.
Among all the HOX genes, HOXB7is an important gene during these years. HOXB7is a gene which belongs to the B clusters of HOX genes family and locates on chromosome17. During the period of embryonic development, HOXB7is primarily responsible for theregulation of proliferation,differentiation and migration of lung epithelial cells. Currently,some studies found that overexpression of HOXB7has been observed in colon cancer andoral cancer,which promotes the aberrant tumorigenesis and metastases.In breast cancer,abnormally high expression of HOXB7induced EMT and affected tumor metastasis. But there are no report on the expression of HOXB7gene in LAC, and whether its expressionparticipate in the regulation of progression and metastasis of LAC cells.
In this study, we frist detected the expression of HOXB7in surgical specimens fromLAC patients and LAC cell lines.Then we compared its expression with the clinicalpathological variables and found out the significance of HOXB7expression in LAC.Futhermore,we used the RNA interference silence HOXB7gene expression and investigated therole of HOXB7in regulation of progression and metastasis of LAC through a series ofexperiment in vivo and in vitro.
Methods
1.The expression of HOXB7in surgical specimens of LAC patients and LAC celllines,and its correlation with clinical pathological variables.
①A total of75pairs of primary LAC tumor tissues and corresponding normal lungepithelium tissues were obtained from the LAC patients who underwent a surgical resection.All the samples were used to construct a tissue microarray.Using Immunohistochemicalstaining analyze HOXB7protein expression and its correlation with clinical pathologicalvariables.②Using RT-PCR and western blot detected the expression of HOXB7in normallung bronchial epithelial cell line HBE and4LAC cell lines A549, H1975, H1650, H322.
2. Investigate the role of HOXB7in regulation of proliferation capacity of LAC cellsin vitro and in vivo.
In A549cells, silenced HOXB7gene expression with retrovirus-mediated RNAinterference and do the following research:①Using colony formation assay to detect thedifferences of proliferation capacity between the control group and HOXB7RNAi group.②Using flow cytometry assay to check the differences of cell cycle distribution betweenthe control group and HOXB7RNAi group.③Control group and HOXB7RNAi groupcells were subcutaneously injected into6-week-old female nude mice (five mice eachgroup).All mice were kept under specific pathogen-free conditions.Tumor volumes weremeasured every3days and calculated as [larger diameter*(smaller diameter)2]/2. After30days, all the mice were sacrificed.Xenograft tumors were fixed, embedded and stained withhaematoxylin and eosin or immunohistochemistry stained with antibodies against HOXB7and Ki-67.
3. Investigate the role of HOXB7in regulation metastasis capacity of LAC cells invitro and in vivo.
①Using Western blot to detect the expression of epithelial markers such as E-cadherin and mesenchymal markers such as vimentin and Fibronectin between the controlgroup and HOXB7RNAi group.②Using immunofluorescence staining to detect theexpression of epithelial markers such as E-cadherin and mesenchymal markers such asvimentin and Fibronectin between the control group and HOXB7RNAi group.③Using thescratch wound healing and transwell migration assay to detect migration ability betweenthe control group and HOXB7RNAi group in vitro.④Control group and HOXB7RNAigroup cells were intraperitoneally (i.p.) injected into6-week-old female nude mice (10miceeach group). After35days, the mice were sacrificed and dissected. The metastatic statewere analyzed.
Results
1.mRNA and protein levels of HOXB7in all four LAC cell lines were significantlyhigher than in HBE cells.Among these cell lines, including A549, H1975,H1650, and H322,the highest expression of HOXB7protein was observed in A549cells.
2. Out of the75tissue samples, HOXB7was highly expressed in54(72.0%) tumortissues,but only in seven (9.3%) adjacent normal tissues. Increasing HOXB7expression wasobserved with the progression of the tumor. Statistical analysis revealed that HOXB7protein expression was correlated with the tumor status (P=0.028),nodal status (P=0.012),and the tumor stage (P=0.029) in LAC. Kaplan–Meier survival analysis showed thatincreased expression of HOXB7was associated with poor clinical outcomes in LACpatients.
3. Lentivirus encoding short hairpin RNAs(shRNA) targeting HOXB7was used toinfect A549cells, which has the highest expression of HOXB7of the four LAC cell lines.HOXB7RNAi group cells grew much slower and formed less and smaller colonies than thecontrol group cells. Flow cytometry analysis revealed that compared to the control groupcells, the number of HOXB7RNAi group cells at the G1phase was increased,whereas thatnumber at the S phase decreased,respectively. These results suggested that HOXB7plays acritical role in regulation of G1/S cell cycle checkpoint, which controls proliferation of LAC cells.
4.The size of tumors formed from HOXB7RNAi group cells was much smaller thanthe tumors formed from control group cells in nude mice at30days post-injection.IHCanalysis of the dissected xenograft tumors revealed that the expressions of HOXB7and Ki-67, a cellular marker for proliferation,in tumors form HOXB7RNAi group cells were lowerthan their expression in tumors form control group cells.
5. HOXB7RNAi group cells had a spindle-like mesenchymal morphology, controlgroup cells had a cobblestone-like epithelial morphology. HOXB7RNAi group cells alsohad a smaller cell size and formed a highly clustered pattern at48h after seeding. Theexpression of epithelial marker E-cadherin was increased, the expressions of mesenchymalmarkers fibronectin and vimentin were markedly reduced in HOXB7RNAi group cells. Thechanges of EMT markers were confirmed by Immunofluorescence staining followed byconfocal microscopy analysis.The HOXB7RNAi group cells healed the wound-scratchmuch slower than the control group cells. Less HOXB7RNAi group cells than controlgroup cells migrated from the upper chamber to the bottom surface of boyden chamber at24h post-seeding. These results suggested that HOXB7regulates the migratory ability ofLAC cells in vitro.
6. The mice injected (i.p.) with HOXB7RNAi group cells had much less ascitic fluidin the peritoneal cavity than the ones injected (i.p.) with control group cells. The incidencesof metastases of organs in mice injected with HOXB7RNAi group cells were much fewerthan that in the mice injected with control group cells.
Conclusion
1. HOXB7expression might play a critical role in regulation of malignantprogression and metastasis of LAC.
2. HOXB7plays a critical role in regulation of G1/S cell cycle checkpoint, whichcontrols proliferation of LAC cells in vitro and in vivo.
3. HOXB7regulates the migratory ability of LAC cells in vitro and in vivo.
4. HOXB7could be an independent prognostic factor for LAC and an additionalmarker for determination of LAC malignant progression.
目前，肺癌发病率和病死率位于各类癌症之首。肺癌分为小细胞肺癌与非小细胞肺癌两大类，其中非小细胞肺癌中最常见的是肺腺癌、肺鳞癌和肺大细胞癌三个亚型。肺腺癌(Lung adencarcinoma，LAC)的发病在所有类型肺癌中占第一位，已经达到40％。大量临床资料显示，肺腺癌的高侵袭力和易转移性是导致临床治疗失败和预后不良的主要原因，约90%的肺腺癌病人死于癌细胞的多器官转移。迄今为止，肺腺癌发病的危险因素已被初步确定，但有关其快速侵袭与转移的详细机制尚不清楚。因此，发现特异性的肺腺癌治疗靶标，深入探明调控肺腺癌增殖与转移的相关机制并进行针对性干预，对提高肺癌病人的疗效十分重要。
研究证实，与细胞分化、发育相关的同源盒分子如Oct4、Nanog等参与肺癌侵袭转移的调节。而同属于同源盒基因的HOX(Homeobox genes)基因家族类似作用越来越受到关注。HOX基因家族共包括39个含高度保守同源结构域的基因，分为HOXA、HOXB、HOXC和HOXD四簇，分别位于第7、17、12、2号染色体。HOX基因家族主要负责调节脊椎类动物和人类胚胎器官发育。作为细胞增殖和分化的主控基因，其表达蛋白的作用靶标通常为膜蛋白受体、粘附分子、生长因子等与肿瘤进展、转移密切相关的基因家族。研究表明，一些在成体细胞时期本该关闭的HOX基因迷乱表达，参与肿瘤的发生发展，尤其是肿瘤发展的后期阶段。在肺癌、乳腺癌、结肠癌等多种肿瘤中可检测到HOX基因的异常表达。另外，研究发现在干细胞中，HOX基因家族也参与其自我更新、无限增殖等干细胞特性的调控。
其中，HOXB7是近年来研究的热点。HOXB7属于HOX基因家族B簇，位于第17号染色体，已被确证属于癌基因的范畴。胚胎发育时期，HOXB7是主要负责调控上皮包括肺上皮细胞增殖、分化、迁移的主控基因。目前，已有研究表明在结肠癌及口腔癌中，HOXB7表达异常增高，并与恶性肿瘤快速进展及预后不良相关。在乳腺癌中，异常高表达的HOXB7导致肿瘤细胞EMT (Epithelial-mesenchymal transition，EMT)并诱导肿瘤干细胞生成，进而影响肿瘤转移。但有关HOXB7基因在肺腺癌中的表达情况以及其是否参与调控肺腺癌肿瘤细胞恶性进展与转移的能力，目前尚未见文献报道。
本课题通过检测肺腺癌细胞系及病人肿瘤组织中HOXB7基因的表达情况，结合临床病理学变量，初步探讨HOXB7表达在肺腺癌中意义，并进一步利用RNA干扰使HOXB7基因沉默。我们发现HOXB7降低表达可导致肺腺癌细胞体外增殖减慢、细胞周期阻滞、细胞迁移及侵袭能力降低。皮下注射HOXB7降低表达的肺腺癌细胞的裸鼠比对照组更晚产生肿瘤，且肿瘤体积更小。腹腔注射HOXB7降低表达的肺腺癌细胞的裸鼠比对照组发生更少的器官转移。
综上所述，本研究从细胞和实验动物水平证明HOXB7是肺腺癌细胞增殖和转移能力的重要调控因子，可成为控制肺腺癌的恶性进展与转移的临床防治新的靶点。
研究方法
1.同源盒基因HOXB7在肺腺癌细胞系及病人组织中的表达与意义
①从ATCC购买正常肺支气管腺上皮细胞系HBE以及肺腺癌细胞系A549、H1975、H1650、H322等，利用RT-PCR、Western blot等方法检测在各肺腺癌细胞系中HOXB7的mRNA及蛋白表达情况。②收集75例临床肺腺癌病人手术切除肿瘤组织标本及其癌旁正常组织标本，石蜡包埋，制作组织芯片。采用免疫组织化学染色方法，分析HOXB7蛋白表达与肺腺癌的临床分期、病理分级、远处转移能力、生存期等临床病理学变量的相关性。
2.同源盒基因HOXB7调控肺腺癌细胞体外增殖及体内成瘤能力
在A549细胞系中，采用逆转录病毒介导的标记有绿色荧光蛋白(GFP)的质粒载体进行HOXB7基因的RNA干扰，使HOXB7去表达，并进行以下研究：
①利用平板克隆形成实验，检测转染了空白质粒的对照组与HOXB7RNAi干扰组的肿瘤细胞体外增殖能力的差异。②利用流式细胞仪检测对照组与干扰组肿瘤细胞的细胞周期分布差异情况。③将对照组及干扰组肿瘤细胞分别皮下接种于NOD/SCID联合免疫缺陷鼠(6周龄)，每组5只，建立肺腺癌皮下移植瘤模型，每天记录肿瘤生长情况。30天后处死动物。利用公式[长*宽2]/2，计算移植瘤体积。将移植瘤取出，石蜡切片并进行免疫组化染色，观察各组间移植瘤组织的HOXB7及增殖标志如Ki-67蛋白的表达差异情况。
3.同源盒基因HOXB7调控肺腺癌细胞转移能力
①利用Western blot方法检测对照组与干扰组肿瘤细胞上皮细胞表面标志如E-钙粘蛋白及间质细胞表面标志如波形蛋白和纤连蛋白等蛋白表达差异情况。②利用免疫荧光染色方法检测对照组与干扰组肿瘤细胞上皮细胞表面标志如E-钙粘蛋白及间质细胞表面标志如波形蛋白和纤连蛋白等蛋白表达差异情况。③利用细胞划痕实验及transwell细胞迁移实验检测对照组与干扰组肿瘤细胞体外迁移能力的差异情况。④将对照组及干扰组肿瘤细胞分别腹腔注射于NOD/SCID联合免疫缺陷鼠(6周龄)，每组10只。35天后处死动物，观察腹腔脏器肿瘤转移发生的差异情况。
研究结果
1.人肺腺癌细胞系A549、H1975、H1650、H322中HOXB7的mRNA及蛋白表达水平明显高于人正常肺支气管上皮细胞系。其中A549细胞系的HOXB7表达水平高于其余几种肺腺癌肿瘤细胞系。
2.免疫组织化学染色结果显示，在75例配对组织样本中，有54例(72%)肺腺癌肿瘤组织呈现HOXB7阳性高表达，而仅有7例(9.3%)癌旁正常组织HOXB7表达阳性。进一步相关性检验显示： HOXB7蛋白表达水平与肿瘤T分期(P=0.028),肿瘤临床分级(P=0.029)及淋巴结转移(P=0.012)均呈显著的正相关性，而与肿瘤病理分级(P=0.146)无显著的相关性。另外，Kaplan–Meier生存分析显示肿瘤组织HOXB7蛋白表达水平与临床病人生存时间呈负相关，影响病人的预后。
3.用RNAi技术特异性沉默A549细胞HOXB7基因表达，与对照组细胞相比，干扰组细胞中的HOXB7蛋白表达明显受到抑制。平板克隆形成实验结果显示，干扰组细胞形成的集落克隆数明显少于(P<0.001)且小于对照组细胞。流式细胞仪分析细胞周期结果显示，干扰组细胞G1期细胞比例明显高于对照组细胞(P=0.006)，而S期细胞比例明显低于对照组细胞(P=0.002)，说明HOXB7通过影响G1/S细胞周期调控点，调控肺腺癌肿瘤细胞增殖能力。小鼠皮下移植瘤实验结果显示，干扰组小鼠皮下移植瘤体积小于对照组(P=0.007)，说明干扰组细胞体内成瘤能力明显降低。免疫组织化学染色观察移植瘤组织，发现干扰组组织的细胞增殖标志Ki-67表达低于对照组组织。
4.利用显微镜观察发现，在形态上，与对照组典型的纺锤形间质细胞形态相比，干扰组细胞向卵圆形的上皮细胞形态转变，并且聚集形成细胞簇。利用Western blot及免疫荧光染色方法检测发现，干扰组肿瘤细胞上皮细胞表面标志E-钙粘蛋白表达明显高于对照组，而间质细胞表面标志波形蛋白和纤连蛋白表达明显低于对照组。细胞划痕实验结果显示，单层细胞被划痕后24h及48h，干扰组细胞划痕修复能力明显低于对照组，说明干扰组细胞横向迁移能力明显降低。Transwell细胞迁移实验检测结果显示，细胞接种Transwell小室24h后，干扰组迁移至小室下层细胞数明显少于对照组(P=0.001)，说明干扰组细胞体外迁移能力明显降低。小鼠体内转移瘤模型结果发现，腹腔注射干扰组细胞的小鼠，腹腔积液少于对照组小鼠且器官转移发生率明显少于对照组小鼠。
结论
1.HOXB7在肺腺癌细胞系及病人肿瘤组织中异常高表达，且与肿瘤分期、分级及淋巴结转移等病理参数相关，说明其表达与肺腺癌进展及病人预后相关。提示HOXB7可能是肺腺癌的独立预后因子及疾病进展的标志。
2.HOXB7参与调控肺腺癌肿瘤细胞体外增殖和体内成瘤能力，控制肿瘤恶性进展。
3.HOXB7参与调控肺腺癌肿瘤细胞体外迁移能力，影响体内肿瘤转移。
4.进一步深入研究HOXB7对肺腺癌的作用机制，可为控制肺腺癌的恶性进展与转移的临床防治提供新的靶点。
Introduction
Lung cancer is one of the most frequent malignant tumors in the world, and theincidence of lung adenocarcinoma (LAC) is the first one in all types of lung cancer, havingbeen reached about40%.Many clinical datas indicated that fast progress, high rate ofrecurrence and metastasis ability of LAC is the main reason for the clinical treatment failureand poor prognosis of LAC patients, about90%of LAC patients died of multi-organmetastasis. So far, the risk factors for LAC incidence have been identified, but themechanisms about its rapid progression and metastasis are still unclear. Therefore, findingout the mechanisms of regulation of invasion and metastasis about LAC is very importantfor improving the survival rate of LAC patients.
Some studies have confirmed that some homeobox molecules such as Oct4, Nanog areinvolved in the regulation of lung cancer metastasis. And the homeobox gene family (HOX)have been paid more and more attention. HOX gene family is consists of39highlyconserved homeodomain genes,which are divided into four clusters HOXA, HOXB, HOXCand HOXD. HOX gene family is mainly responsible for the regulation of vertebrateanimals and human embryonic organ development. As the master genes for cellproliferation and differentiation, its target protein are usually some membrane proteinreceptors, adhesion molecules and growth factors.
Among all the HOX genes, HOXB7is an important gene during these years. HOXB7is a gene which belongs to the B clusters of HOX genes family and locates on chromosome17. During the period of embryonic development, HOXB7is primarily responsible for theregulation of proliferation,differentiation and migration of lung epithelial cells. Currently,some studies found that overexpression of HOXB7has been observed in colon cancer andoral cancer,which promotes the aberrant tumorigenesis and metastases.In breast cancer,abnormally high expression of HOXB7induced EMT and affected tumor metastasis. But there are no report on the expression of HOXB7gene in LAC, and whether its expressionparticipate in the regulation of progression and metastasis of LAC cells.
In this study, we frist detected the expression of HOXB7in surgical specimens fromLAC patients and LAC cell lines.Then we compared its expression with the clinicalpathological variables and found out the significance of HOXB7expression in LAC.Futhermore,we used the RNA interference silence HOXB7gene expression and investigated therole of HOXB7in regulation of progression and metastasis of LAC through a series ofexperiment in vivo and in vitro.
Methods
1.The expression of HOXB7in surgical specimens of LAC patients and LAC celllines,and its correlation with clinical pathological variables.
①A total of75pairs of primary LAC tumor tissues and corresponding normal lungepithelium tissues were obtained from the LAC patients who underwent a surgical resection.All the samples were used to construct a tissue microarray.Using Immunohistochemicalstaining analyze HOXB7protein expression and its correlation with clinical pathologicalvariables.②Using RT-PCR and western blot detected the expression of HOXB7in normallung bronchial epithelial cell line HBE and4LAC cell lines A549, H1975, H1650, H322.
2. Investigate the role of HOXB7in regulation of proliferation capacity of LAC cellsin vitro and in vivo.
In A549cells, silenced HOXB7gene expression with retrovirus-mediated RNAinterference and do the following research:①Using colony formation assay to detect thedifferences of proliferation capacity between the control group and HOXB7RNAi group.②Using flow cytometry assay to check the differences of cell cycle distribution betweenthe control group and HOXB7RNAi group.③Control group and HOXB7RNAi groupcells were subcutaneously injected into6-week-old female nude mice (five mice eachgroup).All mice were kept under specific pathogen-free conditions.Tumor volumes weremeasured every3days and calculated as [larger diameter*(smaller diameter)2]/2. After30days, all the mice were sacrificed.Xenograft tumors were fixed, embedded and stained withhaematoxylin and eosin or immunohistochemistry stained with antibodies against HOXB7and Ki-67.
3. Investigate the role of HOXB7in regulation metastasis capacity of LAC cells invitro and in vivo.
①Using Western blot to detect the expression of epithelial markers such as E-cadherin and mesenchymal markers such as vimentin and Fibronectin between the controlgroup and HOXB7RNAi group.②Using immunofluorescence staining to detect theexpression of epithelial markers such as E-cadherin and mesenchymal markers such asvimentin and Fibronectin between the control group and HOXB7RNAi group.③Using thescratch wound healing and transwell migration assay to detect migration ability betweenthe control group and HOXB7RNAi group in vitro.④Control group and HOXB7RNAigroup cells were intraperitoneally (i.p.) injected into6-week-old female nude mice (10miceeach group). After35days, the mice were sacrificed and dissected. The metastatic statewere analyzed.
Results
1.mRNA and protein levels of HOXB7in all four LAC cell lines were significantlyhigher than in HBE cells.Among these cell lines, including A549, H1975,H1650, and H322,the highest expression of HOXB7protein was observed in A549cells.
2. Out of the75tissue samples, HOXB7was highly expressed in54(72.0%) tumortissues,but only in seven (9.3%) adjacent normal tissues. Increasing HOXB7expression wasobserved with the progression of the tumor. Statistical analysis revealed that HOXB7protein expression was correlated with the tumor status (P=0.028),nodal status (P=0.012),and the tumor stage (P=0.029) in LAC. Kaplan–Meier survival analysis showed thatincreased expression of HOXB7was associated with poor clinical outcomes in LACpatients.
3. Lentivirus encoding short hairpin RNAs(shRNA) targeting HOXB7was used toinfect A549cells, which has the highest expression of HOXB7of the four LAC cell lines.HOXB7RNAi group cells grew much slower and formed less and smaller colonies than thecontrol group cells. Flow cytometry analysis revealed that compared to the control groupcells, the number of HOXB7RNAi group cells at the G1phase was increased,whereas thatnumber at the S phase decreased,respectively. These results suggested that HOXB7plays acritical role in regulation of G1/S cell cycle checkpoint, which controls proliferation of LAC cells.
4.The size of tumors formed from HOXB7RNAi group cells was much smaller thanthe tumors formed from control group cells in nude mice at30days post-injection.IHCanalysis of the dissected xenograft tumors revealed that the expressions of HOXB7and Ki-67, a cellular marker for proliferation,in tumors form HOXB7RNAi group cells were lowerthan their expression in tumors form control group cells.
5. HOXB7RNAi group cells had a spindle-like mesenchymal morphology, controlgroup cells had a cobblestone-like epithelial morphology. HOXB7RNAi group cells alsohad a smaller cell size and formed a highly clustered pattern at48h after seeding. Theexpression of epithelial marker E-cadherin was increased, the expressions of mesenchymalmarkers fibronectin and vimentin were markedly reduced in HOXB7RNAi group cells. Thechanges of EMT markers were confirmed by Immunofluorescence staining followed byconfocal microscopy analysis.The HOXB7RNAi group cells healed the wound-scratchmuch slower than the control group cells. Less HOXB7RNAi group cells than controlgroup cells migrated from the upper chamber to the bottom surface of boyden chamber at24h post-seeding. These results suggested that HOXB7regulates the migratory ability ofLAC cells in vitro.
6. The mice injected (i.p.) with HOXB7RNAi group cells had much less ascitic fluidin the peritoneal cavity than the ones injected (i.p.) with control group cells. The incidencesof metastases of organs in mice injected with HOXB7RNAi group cells were much fewerthan that in the mice injected with control group cells.
Conclusion
1. HOXB7expression might play a critical role in regulation of malignantprogression and metastasis of LAC.
2. HOXB7plays a critical role in regulation of G1/S cell cycle checkpoint, whichcontrols proliferation of LAC cells in vitro and in vivo.
3. HOXB7regulates the migratory ability of LAC cells in vitro and in vivo.
4. HOXB7could be an independent prognostic factor for LAC and an additionalmarker for determination of LAC malignant progression.
引文
[1] Ettinger DS, Akerley W, Bepler G, Blum MG, et al.Non-small cell lung cancer.J NatlCompr Canc Netw.2010;8(7):740-801.
[2] Minna JD, Roth JA, Gazdar AF. Focus on lung cancer.Cancer Cell.2002;1:49-52.
[3] Raz DJ, Zell JA, Ou SHI, et al. Natural history of stage I non-small cell lung cancer:implications for early detection. Chest.2007;132:193-199.
[4] Thomas KW, Gould MK. Diagnosis and staging of non-small cell lung cancer. In:Basow DS, ed. UpToDate.Waltham, MA,2010.
[5] Iwatsuki M, Mimori K, Yokobori T, Ishi H, Beppu T, Nakamori S.Epithelial-mesenchymal transition in cancer development and its clinical significance.Cancer Sci.2010;101(2):293-9.
[6] Sendurai A. Mani, Wenjun Guo, Mai-Jing Liao, Elinor Ng. Eaton.The epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells.Cell.2008;133:704-715.
[7] Jenndahl LE,Isakson P,Baeckstrom D.c-erbB2-Induced epithelial-mesenchymaltransition in mammary epithelial cells is suppressed by cell-cell contact and initiatedprior to E-cadherin downregulation.Int J Oncol.2005;27:439-448.
[8] Thiery JP. Epithelial-mesenchymal transitions in development and pathologies.CurrOpin Cell Biol2003;15:740-746.
[9] Larue L, Bellacosa A. Epithelial-mesenchymal transition in development and cancer:role of phosphatidylinositol3kinase/Akt pathways.Oncogene.2005;24:7443-7454.
[10] Shih-Hwa Chiou, Mong-Lien Wang, Yu-Ting Chou.Coexpression of Oct4and NanogEnhances Malignancy in Lung Adenocarcinoma by Inducing Cancer Stem Cell-LikeProperties and Epithelial-Mesenchymal Transdifferentiation. Cancer Res.2010;70:10433-10444.
[11] Palma CS, Tannous MA, Malta TM, Russo EM, Covas DT, Pican o-Castro V.Forcedexpression of OCT4influences the expression of pluripotent genes in humanmesenchymal stem cells and fibroblasts.Genet Mol Res.2013;12(2):1054-60.
[12] Wellik, D. M. Hox genes and vertebrate axial pattern.Curr. Top. Dev. Biol.2009;88:257-278.
[13] Zacchetti, G., Duboule, D.&Zakany, J. Hox gene function in vertebrate gutmorphogenesis: the case of the caecum. Development.2007;134:3967-3973.
[14] Hoegg S, Meyer A Hox clusters as models for vertebrate genome evolution. TrendsGenet.2005;21:421-424.
[15] Iimura T, Pourquie′O,Hox genes in time and space during vertebrate body formation.Dev Growth Differ.2007;49:265-275.
[16] McIntyre DC, Rakshit S, Yallowitz AR, Loken L, Jeannotte L, Capecchi MR,WellikDM Hox patterning of the vertebrate rib cage.Development.2007;134:2981-2989.
[17] Scott MP A rational nomenclature for vertebrate homeobox (HOX) genes. NucleicAcids Res.1993;21:1687-1688.
[18] Wagner, G.P., C. Amemiya&F. Ruddle. Hox cluster duplications and the opportunityfor evolutionary novelties.Proc. Natl Acad.2003;100:14603-14606.
[19] Levine, M. Transcriptional enhancers in animal development and evolution. Curr Biol.2010;20: R754–R763.
[20] Heffer, A., J. Shultz&L. Pick. Surprising flexibility in a conserved Hox transcriptionfactor over550million years of evolution. Proc. Natl. Acad. Sci.2010;107:18040-18045.
[21] Huang, L., Pu, Y., Hepps, D., Danielpour, D.&Prins, G. S. Posterior Hox geneexpression and differential androgen regulation in the developing and adult rat prostatelobes. Endocrinology.2007;148:1235-1245.
[22] Karnoub, A. E. et al. Mesenchymal stem cells within tumour stroma promote breastcancer metastasis.Nature.2007;449:557-563.
[23] Cillo C, Cantile M, Faiella A et al. Homeobox genes in normal and malignant cells. JCell Physiol.2001;188(2):161-169.
[24] Waltregny, D., Alami, Y., Clausse, N., de Leval, J.&Castronovo, V. Overexpression ofthe homeobox gene HOXC8in human prostate cancer correlates with loss of tumordifferentiation. Prostate,2002;50:162-169.
[25] Cantile M, Schiavo G, Franco R, Cindolo L, Procino A, D'Armiento M, Facchini G,Terracciano L, Botti G, Cillo C.Expression of lumbosacral HOX genes, crucial inkidney organogenesis, is systematically deregulated in clear cell kidneycancers.Anticancer Drugs.2011;22(5):392-401.
[26] Grunert S, Jechlinger M, Beug H. Diverse cellular and molecular mechanismscontribute to epithelial plasticity and metastasis. Nat Rev Mol Cell Biol.2003;4:657-665.
[27] Sullivan JP, Minna JD, Shay JW.Evidence for self-renewing lung cancer stem cells andtheir Implications in tumor initiation, progression, and targeted therapy. CancerMetastasis Rev.2010;29(1):61-72.
[28] Meccia E, Bottero L, Felicetti F, Peschle C, Colombo MP, Carè A.HOXB7expressionis regulated by the transcription factors NF-Y, YY1, Sp1and USF-1. Biochim Biophys2003;15:1-9.
[29] Rubin E, Wu X, Zhu T, Cheung JC, Chen H, Lorincz A, Pandita RK, Sharma GG, HaHC,Gasson J, Hanakahi LA, Pandita TK, Sukumar S. A role for the HOXB7homeodomain protein in DNA repair.Cancer Res.2007;15;67(4):1527-35.
[30] Wu X, Chen H, Parker B, Rubin E, Zhu T, Lee JS, et al. HOXB7, a homeodomainprotein, is overexpressed in breast cancer and confers epithelial-mesenchymaltransition. Cancer Res.2006;66:9527–34.
[31] Care A, Silvani A, Meccia E, Mattia G, Stoppacciaro A,Parmiani G, Peschle C andColombo MP: HOXB7constitutively activates basic fibroblast growth factor inmelanomas. Mol Cell Biol.1996;16:4842-4851.
[32] Liao WT, Jiang D, Yuan J, et al. HOXB7as a prognostic factor and mediator ofcolorectal cancer progression. Clin Cancer Res.2011;17:3569–3578.
[33] Bitu CC, Carrera M, Lopes MA, Kowalski LP, Soares FA,Coletta RD. HOXB7expression is a prognostic factor for oral squamous cell carcinoma. Histopathology.2012;60:662–665.
[34] Cardoso, W. V. Transcription factors and pattern formation in the developing lung. Am.J. Physiol.1995;269:L429–L442.
[35] Zhai, Y. et al. Gene expression analysis of preinvasive and invasive cervical squamouscell carcinomas identifies HOXC10as a key mediator of invasion.Cancer Res.2007;67:10163–10172.
[36] Rauch, T. et al. Homeobox gene methylation in lung cancer studied by genome-wideanalysis with a microarray-based methylated CpG island recovery assay. Proc. NatlAcad.2007;104:5527–5532.
[37] Fanti, L. et al. The trithorax group and Pc group proteins are differentially involved inheterochromatin formation in Drosophila. Chromosoma,2008;117:25–39.
[38] Ghannam, G. et al. The oncogene Nup98-HOXA9induces gene transcription inmyeloid cells. J. Biol.Chem.2004;279:866–875.
[39] Miller, G. J. et al. Aberrant HOXC expression accompanies the malignant phenotypein human prostate. Cancer Res.2003;63:5879–5888.
[40] Zhang, X. et al. HOXC6and HOXC11increase transcription of S100beta gene inBrdU-induced in vitro differentiation of GOTO neuroblastoma cells into Schwanniancells. J. Cell. Mol. Med.2007;11:299–306.
[41] Calvo R, West J, Franklin W, Erickson P, Bemis L, Li E,Helfrich B, Bunn P, Roche J,Brambilla E, Rosell R, Gemmill RM and Drabkin HA: Altered HOX and WNT7Aexpression in human lung cancer. Proc Natl Acad Sci USA2000;97:12776-12781.
[42] Jerevall, P. L. et al. Exploring the two-gene ratio in breast cancer—independent rolesfor HOXB13and IL17BR in prediction of clinical outcome. Breast Cancer Res.2008;107:225–234.
[43] Ota T, Klausen C, Salamanca MC, Woo HL, Leung PC, Auersperg N. Expression andfunction of HOXA genes in normal and neoplastic ovarian epithelialcells.Differentiation.2009;77(2):162-71.
[44] Cillo, C., P. Barba, G. Freschi, G. Bucciarelli, M. C. Magli, and E.Boncinelli. HOXgene expression in normal and neoplastic human kidney. Int. J. Cancer.1992;51:892–897.
[45] Raman, V. et al. Compromised HOXA5function can limit p53expression in humanbreast tumours. Nature.2000;405:974–978.
[46] Chen H, Lee JS, Liang X, Zhang H, Zhu T, Zhang Z, et al. Hoxb7inhibits transgenicHER-2/neu-induced mouse mammary tumor onset but promotes progression and lungmetastasis. Cancer Res.2008;68:3637–44.
[47] Care A, Silvani A, Meccia E, Mattia G, Peschle C, Colombo MP.Transduction of theSkBr3breast carcinoma cell line with the HOXB7gene induces bFGF expression,increases cell proliferation and reduces growth factor dependence. Oncogene.1998;16:3285–9.
[48] Wei T,Wang M,Wang Mi,et a1.Relationship of sRANKL level and vascularcalcification score to cardiovascular events in maintenanee hemodialysispatients.Blood Purif.2009;28:342-345.
[49] Sang-Gu Hwang,Sung-Min Song,Jeong-Ran Kim,et al. Regulation of Type II CollagenExpression by Cyclin-Dependent Kinase6,Cyclin D1,and p21in ArticularChondrocytes.IUBMB Life,February.2007;59(2):90-98.
[50] Gordon A,Greenfield EM,Eastell R,et a1.Individual susceptibility to periprostheticosteolysis is associated with altered patterns of innate immune gene expression inresponse to pro-inflammatory stimuli.J Orthop Res.2010;28(9):1127-1135.
[51] Daniel C，Dufield J，Brunner T，et a1．Matrix metallopro-teirmse inhibitors causecell cycle arrest and apoptosis in glomerular mesangial cells.J Pharmaeol ExpTher.2001;297(1):57-68.
[52] Storti P, Donofrio G, Colla S, et al. HOXB7expression by myeloma cells regulatestheir pro-angiogenic properties in multiple myeloma patients. Leukemia.2011;25:527-537.
[53] Smith LM, Pontes O, Searle I, et al. An SNF2protein associated with nuclear RNAsilencing and the spread of a silencing signal between cells in Arabidopsis. PlantCell.2007;19(5):1507-1521.
[54] Shankar P, Manjunath N, Lieberman J. The prospect of silencingd isease using RNAinterference. JAMA.2005;293(11):1367-1373.
[55] Katayama K, Wada K, Nakajima A, et al.Nuclear receptors as targets for drugdevelopment: the role of nuclear receptors duringn eural stem cell proliferation anddifferentiation. J Pharmacol Sci.2005;97(2):171-176.
[56] Kiang AS, Palfi A, Ader M, et al. Toward a gene therapy for dominant disease:validation of an RNA interference-based mutation-independent approach. MolTher.2005;12(3):555-561.
[57] Nakamura M, Masutomi K, Kyo S, et al. Efficient inhibition of human telomerasereverse transcriptase expression by RNA interference sensitizes cancer cells to ionizingradiation and hemotherapy. Hum Gene Ther.2005;16(7):859-868.
[58] Mello CC, Conte D Jr. Revealing the world of RNA interference.Nature.2004;431(7006):338-342.
[59] Laura B. RNAi:silencing never sounded better. Nat Methods.2004;1(1):79-86.
[60] Westbrook TF, Stegmeier F, Elledge SJ. Dissecting cancerp athways andvulnerabilities with RNAi. Cold Spring Harb SympQuant Biol.2005;70:435-444.
[61] Ku G, McManus MT. Behind the scenes of a small RNA gene-silencing pathway. HumGene Ther.2008;19(1):17-26.
[62] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of shortinterfering RNAs in mammalian cells. Science.2002;296(5567):550-553.
[63] Usami Y, Satake S, Nakayama F, et al. Snail-associated epithelial-mesenchymaltransition promotes oesophageal squamous cell carcinoma motility and progression. JPathol.2008;215:330-339.
[64] Brabletz T, Hlubek F, Spaderna S, et al. Invasion and metastasis in colorectal cancer:pithelial-mesenchymaltransition, mesenchymalepithelial transition, stem cells andbeta-catenin. Cells Tissues Organs.2005;179:56-65.
[65] Trimboli AJ, Fukino K, de Bruin A, et al. Direct evidence for epithelial-mesenchymaltransitions in breast cancer. Cancer Res.2008;68:937-945.
[66] Matter K, Balda MS. Signalling to and from tight junctions.Nat Rev Mol Cell Biol.2003;4(3):225-36.
[67] Jean PT， Herve A， Ruby YJ， Huang and M， Angela Nieto. Epithelial-Mesenchymal Transitions in Development and Disease.Cell.2009;139(5):871-90.
[68] Bilder D. Epithelial polarity and proliferation control: links from the Drosophilaneoplastic tumor suppressors. Genes Dev.2004;18(16):1909-25.
[69] Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications forfibrosis. J Clin Invest.2003;112:1776-1784.
[70] Hay ED. An overview of epithelio-mesenchymal transformation[J].ActaAnat(Basel).1995;154(1):8-20.
[71] Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis[J]. J AmSoc Nephrol.2010;21(2):212-222.
[72] Rajasekaran SA, Huynh TP, Wolle DG, et al. Na, K-ATPase subunits as markers forepithelial-mesenchymal transition in cancer and fibrosis. Mol Cancer Ther.2010;9:151-1524.
[73] Valcourt U, Kowanetz M, Niimi H, et al. TGF-beta and the Smad signaling pathwaysupport transcriptomic reprogramming during epithelial-mesenchymal cell transition.Mol Biol Cell.2005;16:1987-2002.
[74] Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions Nat Rev MolCell Biol.2001;2:285-293.
[75] Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renalfibrosis. J Mol Med.2004;82:175-181.
[76] Chen L, Liu BC, Zhang XL, et al. Influence of connective tissue growth factorantisense oligonucleotide on angiotensin II-induced epithelial mesenchymal transitionin HK2cells. Acta Pharmacol Sin.2006;27:1029-1036.
[77] Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymaltransition during tumor progression. Current Opin Cell Biol.2005;17:548-558.
[78] Soini Y,Tuhkanen H,Sironen R,et al.Transcription factors zeb1,twist and snai1in breastcarcinoma［J］.BMC Cancer.2011;11:73.
[79] Nieto MA. The snail superfamily of zinc-finger transcription factors.Nat Rev Mol CellBiol.2002;3:155-166.
[80] Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumor progression: analliance against the epithelial phenotype? Nat Rev Cancer.2007;7:415-428.
[81] Hector Peinado,Amparo Cano.A hypoxic twist in metastasis ［J］.Nature Cell.Biology.2008;10(3):253-54.
[82] Kong B, Michalski CW, Hong X, et al. AZGP1is a tumor suppressor inpancreaticcancer inducing mesenchymal-to-epithelial transdifferentiation by inhibiting TGF-beta-mediated ERK signaling. Oncogene,2010.
[83] Pantuck AJ, An J, Liu H et al. NF-kappaB-dependent plasticity of the epithelial tomesenchymal transition induced by Von Hippel-Lindau inactivationin renal cellcarcinomas. Cancer Res.2010;70:752-761.
[84] Yu M, Smolen GA, Zhang J, et al. A developmentally regulated inducer of EMT, LBX1,contributes to breast cancer progression. Genes Dev.2009;23:1737-1742.
[85] Siegel PM, Massagué J. Cytostatic and apoptotic actions of TGF-β in homeostasis andcancer. Nat Rev Cance.2003;3:807-821.
[86] Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF–βfamily signaling. Nature,2003;425(6958):577-584.
[87] Moustakas A, Heldin CH. Non-Smad TGF-βsignals.J Cell Sci,2005;118:3573-3584.
[88] Bierie B,Moses HL.Transforming growth factor beta (TGF=β)and inflammation incancer.Cytokine Growth Factor Rev.2010;21(1):49-59.
[89] Gatza CE,Oh SY,Blobe GC.Roles for the type III TGF-β receptor in humancancer.Cellular Signal.2010;22(8):1163-1174.
[90] Gerard B, Tait L, Nangia-Makker P, et al.Rad6B acts downstream of Wnt signaling tostabilize β-catenin: Implications for a novel Wnt/β-catenin target. J MolSignal.2011;6:6.
[91] Grego-Bessa J, Diez J, Timmerman L, et al. Notch and epithelial-mesenchymaltransition in development and tumor progression: another turn of the screw. CellCycle.2004;3:718-721.
[92] Wang Z, Banerjee S, Li Y, et al. Down-regulation of notch-1inhibits invasion byinactivation of nuclear factor-kappaB, vascular endothelial growth factor, and matrixmetallopro-teinase-9in pancreatic cancer cells. Cancer Res.2006;66:2778-2784.
[93] Bonnomet A, Brysse A, Tachsidis A, et al. Epithelial-to-mesenchymal transitions andcirculating tumor cells. J Mammary Gland Biol Neoplasia.2010;15:261-273.
[94] Aktas B, Tewes M, Fehm T, et al. Stem cell and epithelial-mesenchymal transitionmarkers are frequently overexpressed in circulating tumor cells of metastatic breastcancer patients. Breast Cancer Res.2009;11: R46.
[95] Wallerand H, Robert G, Pasticier G, et al. The epithelial-mesenchymal transition-inducing factor TWIST is an attractive target in advanced and/or metastatic bladderand prostate cancers. Urol Oncol.2010;28:473-479.
[96] Zhang Jun,Zhu Zheng,JI Jan,et a1.Transcription fact or Spl expression in gastric cancerand its relationship to long-term prognosis.World J Gastroenterol.2005;11(15):2213-2217.
[97] Srokaic, Naglerb, Bowdeng.Membrane-type1matrix metalloproteinase regulated bySpl through the differential activation AKT, JNK,and ERK pathways in humprostatetumor cells. Neoplasia.2007;9(5):406-417.
[98] You H L, Eng, Hsu, et a1. PKC-Spl signaling pathway induces early differentiation ofhuman keratinocytes through upregulation of TSGIO1.Cell Signal.2007;19:1201-1211.
[1] Bridges, C. B. Current maps of the location of the mutant genes of DrosophilaMelanogaster. Proc. NatlAcad.1921,127–132.
[2] Cardoso, W. V. Transcription factors and pattern formation in the developing lung.Am. J. Physiol.1995,269:L429–L442.
[3] Dolle, P., Izpisua-Belmonte, J.Brown, J.Tickle, C.&Duboule, D. HOX genes and themorphogenesis of the vertebrate limb. Prog. Clin. Biol. Res.1993,383A:11–20.
[4] Wellik, D. M. HOX genes and vertebrate axial pattern.Curr. Top. Dev. Biol.2009,88:257–278.
[5] Perez-Cabrera, A., Kofman-Alfaro, S.&Zenteno, J. C.Mutational analysis ofHOXD13and HOXA13genes in the triphalangeal thumb-brachyectrodactylysyndrome. J. Orthop. Res.2002,20:899–901.
[6] Simpson, Genetics of the female reproductive ducts. Am. J. Med. Genet.1999,89:224–239.
[7] Shen, W. F., Krishnan, K., Lawrence, H. J.&Largman,C. The HOX homeodomainproteins block CBP histone acetyltransferase activity. Mol. Cell. Biol.2001,21:7509–7522.
[8] Zacchetti, G., Duboule, D.&Zakany, J. HOX gene function in vertebrate gutmorphogenesis: the case of the caecum. Development.2007,134:3967–3973.
[9] Mortlock, D. P.&Innis, J. W. Mutation of HOXA13in hand-foot-genital syndrome.Nature Genet.1997,15:179–180.
[10] Suemori H,Noguchi S.HOXC cluster genes are dispensable for overall body plan ofmouse embryonic development［J］.Dev Biol,2000,220(2):333.
[11] Kmita M,Duboule D.Organizing axes in time and space:25years of collinear tinkering［J］.2003,301(5631):331
[12] Mark M,Rijli FM,Chambon P.Homeobox genes in embry-ogenesis and pathogenesis［J］.Pediatr Res,1997,42(4):421
[13] Pearson, J. C., D. Lemons&W. McGinnis. Modulating HOX gene functions duringanimal body patterning. Nat. Rev. Genet.2005,6:893–904.
[14] Janssen, R.&W.G. Damen. The ten HOX genes of the millipede Glomeris marginata.Dev. Genes. Evol.2006,216:451–465.
[15] Heffer, A., J. Shultz&L. Pick..Surprising flexibility in a conserved HOXtranscription factor over550million years of evolution. Proc. Natl. Acad.2010,107:18040–18045.
[16] Wasik, B.R., D.J. Rose&A.P. Moczek. Beetle horns are regulated by the HOX gene,Sex combs reduced, in a species-and sex-specific manner. Evol. Dev.2010,12:353–362.
[17] Bach C，Buhl S，Mueller D，et al.Leukemogenic transformation by HOXA clustergenes［J］.Blood.2010,115(14):2910-2918.
[18] Zhang, X. et al. HOXC6and HOXC11increase transcription of S100beta gene inBrdU-induced in vitro differentiation of GOTO neuroblastoma cells into Schwanniancells. J. Cell. Mol. Med.2007,11:299–306.
[19] Abate-Shen, C. Deregulated homeobox gene expression in cancer: cause orconsequence? Nature Rev.2002,2:777–785.
[20] Takahashi, O. et al. Dysregulated expression of HOXand ParaHOX genes in humanesophageal squamous cell carcinoma. Oncol. Rep.2007,17:753–760.
[21] Jung, C. et al. HOXB13is downregulated in colorectal cancer to confer TCF4-mediated transactivation. Br. J.2005,92:2233–2239.
[22] Roser Calvo, et al.Altered HOX and WNT7A expression in human lung cancer.PNAS.2000,97:12776–12781.
[23] Wang, Z. et al. The prognostic biomarkers HOXB13,IL17BR, and CHDH areregulated by estrogen in breast cancer. Clin. Cancer Res.2007,13:6327–6334.
[24] Chu MC,Selam FB,Taylor HS.HOXA10regulates P53expression and matrigelinvasion in human breast［J］.Cancer Biol Ther,2004,6(3):568.
[25] Armstrong, S. A., Golub, T. R.&Korsmeyer, S. J.MLL-rearranged leukemias:insights from gene expression profiling. Semin. Hematol.2003,40:268–273.
[26] Golub, T. R. et al. Molecular classification of cancer:class discovery and classprediction by gene expression monitoring. Science,1999,286:531–537.
[27] Ghannam, G. et al. The oncogene Nup98-HOXA9induces gene transcription inmyeloid cells. J. Biol.Chem.2004,279:866–875.
[28] Lawrence, H. J. et al. Mice bearing a targeted interruption of the homeobox geneHOXA9have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood.1997,89:1922–1930.
[29] Hershko, A. Y., Kafri, T., Fainsod, A.&Razin, A.Methylation of HOXA5andHOXB5and its relevance to expression during mouse development.Gene,2003,302:65–72.
[30] Fanti, L. et al. The trithorax group and Pc group proteins are differentially involved inheterochromatin formation in Drosophila. Chromosoma.2008,117:25–39.
[31] Hanson, R. D. et al. Mammalian Trithorax and polycomb-group homologues areantagonistic regulators of homeotic development. Proc. Natl Acad.1999,96:14372–14377.
[32]32Rauch, T. et al. Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay. Proc.Natl Acad. Sci.2007,104:5527–5532.
[33] MLL-rearranged T-lineage and B-precursor acute leukemias: dominance of HOXdysregulation. Blood.2003,102:262–268.
[34] Armstrong, S. A., Golub, T. R.&Korsmeyer, S. J.MLL-rearranged leukemias:insights from gene expression profiling. Semin. Hematol.2003,40:268–273.
[35] Golub, T. R. et al. Molecular classification of cancer:class discovery and classprediction by gene expression monitoring. Science.1999,286:531–537.
[36] Faber, J. et al. HOXA9is required for survival in human MLL-rearranged acuteleukemias. Blood.2009,113:2375–2385.
[37] Crickmore, M. A., Ranade, V.&Mann, R. S. Regulation of Ubx expression byepigenetic enhancer silencing in response to Ubx levels and genetic variation. PLoSGenet.2009,5:e1000633.
[38] Kannan, R., Berger, C., Myneni, S., Technau, G. M.&Shashidhara, L. S. Abdominal-A mediated repression of Cyclin E expression during cell-fate specification in theDrosophila central nervous system. Mech. ev.2009,127:137–145.
[39] Karnoub, A. E. et al. Mesenchymal stem cells within tumour stroma promote breastcancer metastasis.Nature.2007,449:557–563.
[40] Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc frommouse and human fibroblasts. Nature Biotechnol.2008,26:101–106.
[1] Usami Y, Satake S, Nakayama F, et al. Snail-associated epithelialmesenchymaltransition promotes oesophageal squamous cell carcinoma motility and progression. JPathol.2008,215:330-339.
[2] Brabletz T, Hlubek F, Spaderna S, et al. Invasion and metastasis in colorectal cancer:pithelial-mesenchymaltransition, mesenchymalepithelial transition, stem cells andbeta-catenin. Cells Tissues Organs.2005,179:56-65.
[3] Gallo D, Ferlini C, Scambia G, et al. The epithelial-mesenchymal transition andtheestrogen-signaling in ovarian cancer. Curr Drug Targets.2010,11:474-481.
[4] Trimboli AJ, Fukino K, de Bruin A, et al. Direct evidence for epithelial-mesenchymaltransitions in breast cancer. Cancer Res.2008,68:937-945.
[5] Knust E. Regulation of epithelial cell shape and polarity by cell-cell adhesion (Review).Mol Membr Biol.2002,19(2):113-20.
[6] Matter K, Balda MS. Signalling to and from tight junctions.Nat Rev Mol Cell Biol2003;4(3):225-36.
[7] Matter K, Aijaz S, Tsapara A, Balda MS. Mammalian tight junctions in the regulationof epithelial differentiation and proliferation. Curr Opin Cell Biol.2005,17(5):453-8.
[8] Jean PT， Herve A， Ruby YJ， Huang and M， Angela Nieto. Epithelial-Mesenchymal Transitions in Development and Disease［J］.Cell.2009,139(5):871-90.
[9] Takanori Tsuji，Soichiro Ibaragi，Guo fu Hu.Epithelial-Mesenchymal Transition andCell Cooperativity in Metastasis［J］.Cancer Res,2009,69(18):7135-39.
[10] Bilder D. Epithelial polarity and proliferation control: links from the Drosophilaneoplastic tumor suppressors. Genes Dev.2004,18(16):1909-25.
[11] Moustakas A, Heldin CH. Signaling networks guiding epithelial-mesenchymaltransitions during embryogenesis and cancer progression.Cancer Sci.2007,98:1512–1520.
[12] Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications forfibrosis. J Clin Invest.2003,112:1776-1784.
[13] Klymkowsky MW, Savagner P. Epithelial-mesenchymal transition: a Cancerresearcher's conceptual friend and foe[J]. Am J Pathol.2009,174(5):1588-1593.
[14] Hay ED. An overview of epithelio-mesenchymal transformation[J].ActaAnat(Basel).1995,154(1):8-20.
[15] Thiery JP, Acloque H, Huang RY, et al. Epithelial-mesenchymal transitions indevelopment and disease[J]. Cell.2009,139(5):871-890.
[16] Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis[J]. J AmSoc Nephrol.2010.21(2):212-222.
[17] Moustakas A, Heldin CH. Signaling networks guiding epithelial-mesenchymaltransitions during embryogenesis and cancer progression.Cancer Sci.2007,98:1512-1520.
[18] Rajasekaran SA, Huynh TP, Wolle DG, et al. Na, K-ATPase subunits as markers forepithelial-mesenchymal transition in cancer and fibrosis. Mol Cancer Ther.2010,9:151-1524.
[19] Valcourt U, Kowanetz M, Niimi H, et al. TGF-beta and the Smad signaling pathwaysupport transcriptomic reprogramming during epithelial-mesenchymal cell transition.Mol Biol Cell.2005,16:1987-2002.
[20] Fuchs BC, Fujii T, Dorfman JD, et al. Epithelial-to-mesenchymal transition andintegrin-linked kinase mediate sensitivity to epidermal growth factor receptorinhibition in human hepatoma cells. Cancer Res.2008,68:2391-2399.
[21] Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions Nat Rev MolCell Biol.2001,2:285-293.
[22] Savagner P. The epithelial-mesenchymal transition (EMT) phenomenon[J]. Ann Oncol.2010,21:89-92.
[23] Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renalfibrosis. J Mol Med.2004,82:175-181.
[24] Xie L, Law BK, Chytil AM, et al. Activation of the Erk pathway is required for TGF-β1-induced EMT in vitro. Neoplasia.2004,6:603-610.
[25] Chen L, Liu BC, Zhang XL, et al. Influence of connective tissue growth factorantisense oligonucleotide on angiotensin II-induced epithelial mesenchymal transitionin HK2cells. Acta Pharmacol Sin.2006,27:1029-1036.
[26] Nelson WJ, Nusse R. Convergence of Wnt, b-catenin, and cadherin pathways.Science.2004,303:1483-1487.
[27] Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymaltransition during tumor progression. Current Opin Cell Biol.2005,17:548-558.
[28] Soini Y,Tuhkanen H,Sironen R,et al.Transcription factors zeb1,twist and snai1in breastcarcinoma［J］.BMC Cancer,2011,11:73．
[29] Nieto MA. The snail superfamily of zinc-finger transcription factors.Nat Rev Mol CellBiol.2002,3:155-166.
[30] Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumor progression: analliance against the epithelial phenotype? Nat Rev Cancer.2007,7:415-428.
[31] Hector Peinado,Amparo Cano.A hypoxic twist in metastasis ［J］.Nature Cell.Biology.2008,10(3):253-54.
[32] Kong B, Michalski CW, Hong X, et al. AZGP1is a tumor suppressor inpancreaticcancer inducing mesenchymal-to-epithelial transdifferentiation by inhibiting TGF-beta-mediated ERK signaling. Oncogene,2010.
[33] Pantuck AJ, An J, Liu H et al. NF-kappaB-dependent plasticity of the epithelial tomesenchymal transition induced by Von Hippel-Lindau inactivationin renal cellcarcinomas. Cancer Res.2010,70:752-761.
[34] Yu M, Smolen GA, Zhang J, et al. A developmentally regulated inducer of EMT, LBX1,contributes to breast cancer progression. Genes Dev.2009,23:1737-1742.
[35] Siegel PM, Massagué J. Cytostatic and apoptotic actions of TGF-β in homeostasis andcancer. Nat Rev Cance.2003,3:807-821.
[36] Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF–βfamily signaling[J]. Nature.2003,425(6958):577-584.
[37] Moustakas A, Heldin CH. Non-Smad TGF-βsignals[J]. J Cell Sci.2005,118:3573-3584.
[38] Bierie B,Moses HL.Transforming growth factor beta (TGF=β)and inflammation incancer［J］.Cytokine Growth Factor Rev.2010,21(1):49-59．
[39] Gatza CE,Oh SY,Blobe GC.Roles for the type III TGF-βreceptor in human cancer［J］.Cellular Signal.2010,22(8):1163-1174．
[40] Cicchini C, Laudadio I, Citarella F, et al. TGFβ-induced EMT requires focal adhesionkinase (FAK) signaling. Exp Cell Res.2007,314:143-152.
[41] MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanismsand diseases. Dev Cell.2009,17:9-26.
[42] Gerard B, Tait L, Nangia-Makker P, et al.Rad6B acts downstream of Wnt signaling tostabilize β-catenin: Implications for a novel Wnt/β-catenin target[J]. J MolSignal.2011,6:6.
[43] Xu P, Yu SZ, Jiang RC, et al. Differential expression of Notch family members inastrocytomas and medulloblastomas. Pathol Oncol Res.2009,15:703-710.
[44] Fan X, Mikolaenko I, Elhassan I, et al. Notch1and Notch2have opposite effects onembryonal brain tumor growth. Cancer Res.2004,64:7787-7793.
[45] Grego-Bessa J, Diez J, Timmerman L, et al. Notch and epithelial-mesenchymaltransition in development and tumor progression: another turn of the screw. CellCycle.2004,3:718-721.
[46] Timmerman LA, Grego-Bessa J, Raya A, et al. Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation.Genes Dev.2003,18:99-115.
[47] Wang Z, Banerjee S, Li Y, et al. Down-regulation of notch-1inhibits invasion byinactivation of nuclear factor-kappaB, vascular endothelial growth factor, and matrixmetallopro-teinase-9in pancreatic cancer cells. Cancer Res.2006,66:2778-2784.
[48] Bonnomet A, Brysse A, Tachsidis A, et al. Epithelial-to-mesenchymal transitions andcirculating tumor cells. J Mammary Gland Biol Neoplasia.2010,15:261-273.
[49] Moody SE, Perez D, Pan TC, et al. The transcriptional repressor Snail promotesmammary tumor recurrence. Cancer Cell.2005,8:197-209.
[50] Aktas B, Tewes M, Fehm T, et al. Stem cell and epithelial-mesenchymal transitionmarkers are frequently overexpressed in circulating tumor cells of metastatic breastcancer patients. Breast Cancer Res.2009,11: R46.
[51] Yang MH, Chen CL, Chau GY, et al. Comprehensive analysis of the independent effectof twist and snail in promoting metastasis of hepatocellular carcinoma. Hepatology.2009,50:1464-1474.
[52] Wang Y, Xue TC, Xie XY, et al. Relationship between epithelial-mesenchymaltransition and lung metastasis in hepatocellular carcinoma.Chin J Surg.2008,46:1624-1627.
[53] McConkey DJ, Choi W, Marquis L, et al. Role of epithelial-to-mesenchymal transition(EMT) in drug sensitivity and metastasis in bladder cancer. Cancer Metastasis Rev.2009,28:335-344.
[54] Wallerand H, Robert G, Pasticier G, et al. The epithelial-mesenchymal transition-inducing factor TWIST is an attractive target in advanced and/or metastatic bladderand prostate cancers. Urol Oncol.2010,28:473-479.
[2] Minna JD, Roth JA, Gazdar AF. Focus on lung cancer.Cancer Cell.2002;1:49-52.
[3] Raz DJ, Zell JA, Ou SHI, et al. Natural history of stage I non-small cell lung cancer:implications for early detection. Chest.2007;132:193-199.
[4] Thomas KW, Gould MK. Diagnosis and staging of non-small cell lung cancer. In:Basow DS, ed. UpToDate.Waltham, MA,2010.
[5] Iwatsuki M, Mimori K, Yokobori T, Ishi H, Beppu T, Nakamori S.Epithelial-mesenchymal transition in cancer development and its clinical significance.Cancer Sci.2010;101(2):293-9.
[6] Sendurai A. Mani, Wenjun Guo, Mai-Jing Liao, Elinor Ng. Eaton.The epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells.Cell.2008;133:704-715.
[7] Jenndahl LE,Isakson P,Baeckstrom D.c-erbB2-Induced epithelial-mesenchymaltransition in mammary epithelial cells is suppressed by cell-cell contact and initiatedprior to E-cadherin downregulation.Int J Oncol.2005;27:439-448.
[8] Thiery JP. Epithelial-mesenchymal transitions in development and pathologies.CurrOpin Cell Biol2003;15:740-746.
[9] Larue L, Bellacosa A. Epithelial-mesenchymal transition in development and cancer:role of phosphatidylinositol3kinase/Akt pathways.Oncogene.2005;24:7443-7454.
[10] Shih-Hwa Chiou, Mong-Lien Wang, Yu-Ting Chou.Coexpression of Oct4and NanogEnhances Malignancy in Lung Adenocarcinoma by Inducing Cancer Stem Cell-LikeProperties and Epithelial-Mesenchymal Transdifferentiation. Cancer Res.2010;70:10433-10444.
[11] Palma CS, Tannous MA, Malta TM, Russo EM, Covas DT, Pican o-Castro V.Forcedexpression of OCT4influences the expression of pluripotent genes in humanmesenchymal stem cells and fibroblasts.Genet Mol Res.2013;12(2):1054-60.
[12] Wellik, D. M. Hox genes and vertebrate axial pattern.Curr. Top. Dev. Biol.2009;88:257-278.
[13] Zacchetti, G., Duboule, D.&Zakany, J. Hox gene function in vertebrate gutmorphogenesis: the case of the caecum. Development.2007;134:3967-3973.
[14] Hoegg S, Meyer A Hox clusters as models for vertebrate genome evolution. TrendsGenet.2005;21:421-424.
[15] Iimura T, Pourquie′O,Hox genes in time and space during vertebrate body formation.Dev Growth Differ.2007;49:265-275.
[16] McIntyre DC, Rakshit S, Yallowitz AR, Loken L, Jeannotte L, Capecchi MR,WellikDM Hox patterning of the vertebrate rib cage.Development.2007;134:2981-2989.
[17] Scott MP A rational nomenclature for vertebrate homeobox (HOX) genes. NucleicAcids Res.1993;21:1687-1688.
[18] Wagner, G.P., C. Amemiya&F. Ruddle. Hox cluster duplications and the opportunityfor evolutionary novelties.Proc. Natl Acad.2003;100:14603-14606.
[19] Levine, M. Transcriptional enhancers in animal development and evolution. Curr Biol.2010;20: R754–R763.
[20] Heffer, A., J. Shultz&L. Pick. Surprising flexibility in a conserved Hox transcriptionfactor over550million years of evolution. Proc. Natl. Acad. Sci.2010;107:18040-18045.
[21] Huang, L., Pu, Y., Hepps, D., Danielpour, D.&Prins, G. S. Posterior Hox geneexpression and differential androgen regulation in the developing and adult rat prostatelobes. Endocrinology.2007;148:1235-1245.
[22] Karnoub, A. E. et al. Mesenchymal stem cells within tumour stroma promote breastcancer metastasis.Nature.2007;449:557-563.
[23] Cillo C, Cantile M, Faiella A et al. Homeobox genes in normal and malignant cells. JCell Physiol.2001;188(2):161-169.
[24] Waltregny, D., Alami, Y., Clausse, N., de Leval, J.&Castronovo, V. Overexpression ofthe homeobox gene HOXC8in human prostate cancer correlates with loss of tumordifferentiation. Prostate,2002;50:162-169.
[25] Cantile M, Schiavo G, Franco R, Cindolo L, Procino A, D'Armiento M, Facchini G,Terracciano L, Botti G, Cillo C.Expression of lumbosacral HOX genes, crucial inkidney organogenesis, is systematically deregulated in clear cell kidneycancers.Anticancer Drugs.2011;22(5):392-401.
[26] Grunert S, Jechlinger M, Beug H. Diverse cellular and molecular mechanismscontribute to epithelial plasticity and metastasis. Nat Rev Mol Cell Biol.2003;4:657-665.
[27] Sullivan JP, Minna JD, Shay JW.Evidence for self-renewing lung cancer stem cells andtheir Implications in tumor initiation, progression, and targeted therapy. CancerMetastasis Rev.2010;29(1):61-72.
[28] Meccia E, Bottero L, Felicetti F, Peschle C, Colombo MP, Carè A.HOXB7expressionis regulated by the transcription factors NF-Y, YY1, Sp1and USF-1. Biochim Biophys2003;15:1-9.
[29] Rubin E, Wu X, Zhu T, Cheung JC, Chen H, Lorincz A, Pandita RK, Sharma GG, HaHC,Gasson J, Hanakahi LA, Pandita TK, Sukumar S. A role for the HOXB7homeodomain protein in DNA repair.Cancer Res.2007;15;67(4):1527-35.
[30] Wu X, Chen H, Parker B, Rubin E, Zhu T, Lee JS, et al. HOXB7, a homeodomainprotein, is overexpressed in breast cancer and confers epithelial-mesenchymaltransition. Cancer Res.2006;66:9527–34.
[31] Care A, Silvani A, Meccia E, Mattia G, Stoppacciaro A,Parmiani G, Peschle C andColombo MP: HOXB7constitutively activates basic fibroblast growth factor inmelanomas. Mol Cell Biol.1996;16:4842-4851.
[32] Liao WT, Jiang D, Yuan J, et al. HOXB7as a prognostic factor and mediator ofcolorectal cancer progression. Clin Cancer Res.2011;17:3569–3578.
[33] Bitu CC, Carrera M, Lopes MA, Kowalski LP, Soares FA,Coletta RD. HOXB7expression is a prognostic factor for oral squamous cell carcinoma. Histopathology.2012;60:662–665.
[34] Cardoso, W. V. Transcription factors and pattern formation in the developing lung. Am.J. Physiol.1995;269:L429–L442.
[35] Zhai, Y. et al. Gene expression analysis of preinvasive and invasive cervical squamouscell carcinomas identifies HOXC10as a key mediator of invasion.Cancer Res.2007;67:10163–10172.
[36] Rauch, T. et al. Homeobox gene methylation in lung cancer studied by genome-wideanalysis with a microarray-based methylated CpG island recovery assay. Proc. NatlAcad.2007;104:5527–5532.
[37] Fanti, L. et al. The trithorax group and Pc group proteins are differentially involved inheterochromatin formation in Drosophila. Chromosoma,2008;117:25–39.
[38] Ghannam, G. et al. The oncogene Nup98-HOXA9induces gene transcription inmyeloid cells. J. Biol.Chem.2004;279:866–875.
[39] Miller, G. J. et al. Aberrant HOXC expression accompanies the malignant phenotypein human prostate. Cancer Res.2003;63:5879–5888.
[40] Zhang, X. et al. HOXC6and HOXC11increase transcription of S100beta gene inBrdU-induced in vitro differentiation of GOTO neuroblastoma cells into Schwanniancells. J. Cell. Mol. Med.2007;11:299–306.
[41] Calvo R, West J, Franklin W, Erickson P, Bemis L, Li E,Helfrich B, Bunn P, Roche J,Brambilla E, Rosell R, Gemmill RM and Drabkin HA: Altered HOX and WNT7Aexpression in human lung cancer. Proc Natl Acad Sci USA2000;97:12776-12781.
[42] Jerevall, P. L. et al. Exploring the two-gene ratio in breast cancer—independent rolesfor HOXB13and IL17BR in prediction of clinical outcome. Breast Cancer Res.2008;107:225–234.
[43] Ota T, Klausen C, Salamanca MC, Woo HL, Leung PC, Auersperg N. Expression andfunction of HOXA genes in normal and neoplastic ovarian epithelialcells.Differentiation.2009;77(2):162-71.
[44] Cillo, C., P. Barba, G. Freschi, G. Bucciarelli, M. C. Magli, and E.Boncinelli. HOXgene expression in normal and neoplastic human kidney. Int. J. Cancer.1992;51:892–897.
[45] Raman, V. et al. Compromised HOXA5function can limit p53expression in humanbreast tumours. Nature.2000;405:974–978.
[46] Chen H, Lee JS, Liang X, Zhang H, Zhu T, Zhang Z, et al. Hoxb7inhibits transgenicHER-2/neu-induced mouse mammary tumor onset but promotes progression and lungmetastasis. Cancer Res.2008;68:3637–44.
[47] Care A, Silvani A, Meccia E, Mattia G, Peschle C, Colombo MP.Transduction of theSkBr3breast carcinoma cell line with the HOXB7gene induces bFGF expression,increases cell proliferation and reduces growth factor dependence. Oncogene.1998;16:3285–9.
[48] Wei T,Wang M,Wang Mi,et a1.Relationship of sRANKL level and vascularcalcification score to cardiovascular events in maintenanee hemodialysispatients.Blood Purif.2009;28:342-345.
[49] Sang-Gu Hwang,Sung-Min Song,Jeong-Ran Kim,et al. Regulation of Type II CollagenExpression by Cyclin-Dependent Kinase6,Cyclin D1,and p21in ArticularChondrocytes.IUBMB Life,February.2007;59(2):90-98.
[50] Gordon A,Greenfield EM,Eastell R,et a1.Individual susceptibility to periprostheticosteolysis is associated with altered patterns of innate immune gene expression inresponse to pro-inflammatory stimuli.J Orthop Res.2010;28(9):1127-1135.
[51] Daniel C，Dufield J，Brunner T，et a1．Matrix metallopro-teirmse inhibitors causecell cycle arrest and apoptosis in glomerular mesangial cells.J Pharmaeol ExpTher.2001;297(1):57-68.
[52] Storti P, Donofrio G, Colla S, et al. HOXB7expression by myeloma cells regulatestheir pro-angiogenic properties in multiple myeloma patients. Leukemia.2011;25:527-537.
[53] Smith LM, Pontes O, Searle I, et al. An SNF2protein associated with nuclear RNAsilencing and the spread of a silencing signal between cells in Arabidopsis. PlantCell.2007;19(5):1507-1521.
[54] Shankar P, Manjunath N, Lieberman J. The prospect of silencingd isease using RNAinterference. JAMA.2005;293(11):1367-1373.
[55] Katayama K, Wada K, Nakajima A, et al.Nuclear receptors as targets for drugdevelopment: the role of nuclear receptors duringn eural stem cell proliferation anddifferentiation. J Pharmacol Sci.2005;97(2):171-176.
[56] Kiang AS, Palfi A, Ader M, et al. Toward a gene therapy for dominant disease:validation of an RNA interference-based mutation-independent approach. MolTher.2005;12(3):555-561.
[57] Nakamura M, Masutomi K, Kyo S, et al. Efficient inhibition of human telomerasereverse transcriptase expression by RNA interference sensitizes cancer cells to ionizingradiation and hemotherapy. Hum Gene Ther.2005;16(7):859-868.
[58] Mello CC, Conte D Jr. Revealing the world of RNA interference.Nature.2004;431(7006):338-342.
[59] Laura B. RNAi:silencing never sounded better. Nat Methods.2004;1(1):79-86.
[60] Westbrook TF, Stegmeier F, Elledge SJ. Dissecting cancerp athways andvulnerabilities with RNAi. Cold Spring Harb SympQuant Biol.2005;70:435-444.
[61] Ku G, McManus MT. Behind the scenes of a small RNA gene-silencing pathway. HumGene Ther.2008;19(1):17-26.
[62] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of shortinterfering RNAs in mammalian cells. Science.2002;296(5567):550-553.
[63] Usami Y, Satake S, Nakayama F, et al. Snail-associated epithelial-mesenchymaltransition promotes oesophageal squamous cell carcinoma motility and progression. JPathol.2008;215:330-339.
[64] Brabletz T, Hlubek F, Spaderna S, et al. Invasion and metastasis in colorectal cancer:pithelial-mesenchymaltransition, mesenchymalepithelial transition, stem cells andbeta-catenin. Cells Tissues Organs.2005;179:56-65.
[65] Trimboli AJ, Fukino K, de Bruin A, et al. Direct evidence for epithelial-mesenchymaltransitions in breast cancer. Cancer Res.2008;68:937-945.
[66] Matter K, Balda MS. Signalling to and from tight junctions.Nat Rev Mol Cell Biol.2003;4(3):225-36.
[67] Jean PT， Herve A， Ruby YJ， Huang and M， Angela Nieto. Epithelial-Mesenchymal Transitions in Development and Disease.Cell.2009;139(5):871-90.
[68] Bilder D. Epithelial polarity and proliferation control: links from the Drosophilaneoplastic tumor suppressors. Genes Dev.2004;18(16):1909-25.
[69] Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications forfibrosis. J Clin Invest.2003;112:1776-1784.
[70] Hay ED. An overview of epithelio-mesenchymal transformation[J].ActaAnat(Basel).1995;154(1):8-20.
[71] Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis[J]. J AmSoc Nephrol.2010;21(2):212-222.
[72] Rajasekaran SA, Huynh TP, Wolle DG, et al. Na, K-ATPase subunits as markers forepithelial-mesenchymal transition in cancer and fibrosis. Mol Cancer Ther.2010;9:151-1524.
[73] Valcourt U, Kowanetz M, Niimi H, et al. TGF-beta and the Smad signaling pathwaysupport transcriptomic reprogramming during epithelial-mesenchymal cell transition.Mol Biol Cell.2005;16:1987-2002.
[74] Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions Nat Rev MolCell Biol.2001;2:285-293.
[75] Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renalfibrosis. J Mol Med.2004;82:175-181.
[76] Chen L, Liu BC, Zhang XL, et al. Influence of connective tissue growth factorantisense oligonucleotide on angiotensin II-induced epithelial mesenchymal transitionin HK2cells. Acta Pharmacol Sin.2006;27:1029-1036.
[77] Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymaltransition during tumor progression. Current Opin Cell Biol.2005;17:548-558.
[78] Soini Y,Tuhkanen H,Sironen R,et al.Transcription factors zeb1,twist and snai1in breastcarcinoma［J］.BMC Cancer.2011;11:73.
[79] Nieto MA. The snail superfamily of zinc-finger transcription factors.Nat Rev Mol CellBiol.2002;3:155-166.
[80] Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumor progression: analliance against the epithelial phenotype? Nat Rev Cancer.2007;7:415-428.
[81] Hector Peinado,Amparo Cano.A hypoxic twist in metastasis ［J］.Nature Cell.Biology.2008;10(3):253-54.
[82] Kong B, Michalski CW, Hong X, et al. AZGP1is a tumor suppressor inpancreaticcancer inducing mesenchymal-to-epithelial transdifferentiation by inhibiting TGF-beta-mediated ERK signaling. Oncogene,2010.
[83] Pantuck AJ, An J, Liu H et al. NF-kappaB-dependent plasticity of the epithelial tomesenchymal transition induced by Von Hippel-Lindau inactivationin renal cellcarcinomas. Cancer Res.2010;70:752-761.
[84] Yu M, Smolen GA, Zhang J, et al. A developmentally regulated inducer of EMT, LBX1,contributes to breast cancer progression. Genes Dev.2009;23:1737-1742.
[85] Siegel PM, Massagué J. Cytostatic and apoptotic actions of TGF-β in homeostasis andcancer. Nat Rev Cance.2003;3:807-821.
[86] Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF–βfamily signaling. Nature,2003;425(6958):577-584.
[87] Moustakas A, Heldin CH. Non-Smad TGF-βsignals.J Cell Sci,2005;118:3573-3584.
[88] Bierie B,Moses HL.Transforming growth factor beta (TGF=β)and inflammation incancer.Cytokine Growth Factor Rev.2010;21(1):49-59.
[89] Gatza CE,Oh SY,Blobe GC.Roles for the type III TGF-β receptor in humancancer.Cellular Signal.2010;22(8):1163-1174.
[90] Gerard B, Tait L, Nangia-Makker P, et al.Rad6B acts downstream of Wnt signaling tostabilize β-catenin: Implications for a novel Wnt/β-catenin target. J MolSignal.2011;6:6.
[91] Grego-Bessa J, Diez J, Timmerman L, et al. Notch and epithelial-mesenchymaltransition in development and tumor progression: another turn of the screw. CellCycle.2004;3:718-721.
[92] Wang Z, Banerjee S, Li Y, et al. Down-regulation of notch-1inhibits invasion byinactivation of nuclear factor-kappaB, vascular endothelial growth factor, and matrixmetallopro-teinase-9in pancreatic cancer cells. Cancer Res.2006;66:2778-2784.
[93] Bonnomet A, Brysse A, Tachsidis A, et al. Epithelial-to-mesenchymal transitions andcirculating tumor cells. J Mammary Gland Biol Neoplasia.2010;15:261-273.
[94] Aktas B, Tewes M, Fehm T, et al. Stem cell and epithelial-mesenchymal transitionmarkers are frequently overexpressed in circulating tumor cells of metastatic breastcancer patients. Breast Cancer Res.2009;11: R46.
[95] Wallerand H, Robert G, Pasticier G, et al. The epithelial-mesenchymal transition-inducing factor TWIST is an attractive target in advanced and/or metastatic bladderand prostate cancers. Urol Oncol.2010;28:473-479.
[96] Zhang Jun,Zhu Zheng,JI Jan,et a1.Transcription fact or Spl expression in gastric cancerand its relationship to long-term prognosis.World J Gastroenterol.2005;11(15):2213-2217.
[97] Srokaic, Naglerb, Bowdeng.Membrane-type1matrix metalloproteinase regulated bySpl through the differential activation AKT, JNK,and ERK pathways in humprostatetumor cells. Neoplasia.2007;9(5):406-417.
[98] You H L, Eng, Hsu, et a1. PKC-Spl signaling pathway induces early differentiation ofhuman keratinocytes through upregulation of TSGIO1.Cell Signal.2007;19:1201-1211.
[1] Bridges, C. B. Current maps of the location of the mutant genes of DrosophilaMelanogaster. Proc. NatlAcad.1921,127–132.
[2] Cardoso, W. V. Transcription factors and pattern formation in the developing lung.Am. J. Physiol.1995,269:L429–L442.
[3] Dolle, P., Izpisua-Belmonte, J.Brown, J.Tickle, C.&Duboule, D. HOX genes and themorphogenesis of the vertebrate limb. Prog. Clin. Biol. Res.1993,383A:11–20.
[4] Wellik, D. M. HOX genes and vertebrate axial pattern.Curr. Top. Dev. Biol.2009,88:257–278.
[5] Perez-Cabrera, A., Kofman-Alfaro, S.&Zenteno, J. C.Mutational analysis ofHOXD13and HOXA13genes in the triphalangeal thumb-brachyectrodactylysyndrome. J. Orthop. Res.2002,20:899–901.
[6] Simpson, Genetics of the female reproductive ducts. Am. J. Med. Genet.1999,89:224–239.
[7] Shen, W. F., Krishnan, K., Lawrence, H. J.&Largman,C. The HOX homeodomainproteins block CBP histone acetyltransferase activity. Mol. Cell. Biol.2001,21:7509–7522.
[8] Zacchetti, G., Duboule, D.&Zakany, J. HOX gene function in vertebrate gutmorphogenesis: the case of the caecum. Development.2007,134:3967–3973.
[9] Mortlock, D. P.&Innis, J. W. Mutation of HOXA13in hand-foot-genital syndrome.Nature Genet.1997,15:179–180.
[10] Suemori H,Noguchi S.HOXC cluster genes are dispensable for overall body plan ofmouse embryonic development［J］.Dev Biol,2000,220(2):333.
[11] Kmita M,Duboule D.Organizing axes in time and space:25years of collinear tinkering［J］.2003,301(5631):331
[12] Mark M,Rijli FM,Chambon P.Homeobox genes in embry-ogenesis and pathogenesis［J］.Pediatr Res,1997,42(4):421
[13] Pearson, J. C., D. Lemons&W. McGinnis. Modulating HOX gene functions duringanimal body patterning. Nat. Rev. Genet.2005,6:893–904.
[14] Janssen, R.&W.G. Damen. The ten HOX genes of the millipede Glomeris marginata.Dev. Genes. Evol.2006,216:451–465.
[15] Heffer, A., J. Shultz&L. Pick..Surprising flexibility in a conserved HOXtranscription factor over550million years of evolution. Proc. Natl. Acad.2010,107:18040–18045.
[16] Wasik, B.R., D.J. Rose&A.P. Moczek. Beetle horns are regulated by the HOX gene,Sex combs reduced, in a species-and sex-specific manner. Evol. Dev.2010,12:353–362.
[17] Bach C，Buhl S，Mueller D，et al.Leukemogenic transformation by HOXA clustergenes［J］.Blood.2010,115(14):2910-2918.
[18] Zhang, X. et al. HOXC6and HOXC11increase transcription of S100beta gene inBrdU-induced in vitro differentiation of GOTO neuroblastoma cells into Schwanniancells. J. Cell. Mol. Med.2007,11:299–306.
[19] Abate-Shen, C. Deregulated homeobox gene expression in cancer: cause orconsequence? Nature Rev.2002,2:777–785.
[20] Takahashi, O. et al. Dysregulated expression of HOXand ParaHOX genes in humanesophageal squamous cell carcinoma. Oncol. Rep.2007,17:753–760.
[21] Jung, C. et al. HOXB13is downregulated in colorectal cancer to confer TCF4-mediated transactivation. Br. J.2005,92:2233–2239.
[22] Roser Calvo, et al.Altered HOX and WNT7A expression in human lung cancer.PNAS.2000,97:12776–12781.
[23] Wang, Z. et al. The prognostic biomarkers HOXB13,IL17BR, and CHDH areregulated by estrogen in breast cancer. Clin. Cancer Res.2007,13:6327–6334.
[24] Chu MC,Selam FB,Taylor HS.HOXA10regulates P53expression and matrigelinvasion in human breast［J］.Cancer Biol Ther,2004,6(3):568.
[25] Armstrong, S. A., Golub, T. R.&Korsmeyer, S. J.MLL-rearranged leukemias:insights from gene expression profiling. Semin. Hematol.2003,40:268–273.
[26] Golub, T. R. et al. Molecular classification of cancer:class discovery and classprediction by gene expression monitoring. Science,1999,286:531–537.
[27] Ghannam, G. et al. The oncogene Nup98-HOXA9induces gene transcription inmyeloid cells. J. Biol.Chem.2004,279:866–875.
[28] Lawrence, H. J. et al. Mice bearing a targeted interruption of the homeobox geneHOXA9have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood.1997,89:1922–1930.
[29] Hershko, A. Y., Kafri, T., Fainsod, A.&Razin, A.Methylation of HOXA5andHOXB5and its relevance to expression during mouse development.Gene,2003,302:65–72.
[30] Fanti, L. et al. The trithorax group and Pc group proteins are differentially involved inheterochromatin formation in Drosophila. Chromosoma.2008,117:25–39.
[31] Hanson, R. D. et al. Mammalian Trithorax and polycomb-group homologues areantagonistic regulators of homeotic development. Proc. Natl Acad.1999,96:14372–14377.
[32]32Rauch, T. et al. Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay. Proc.Natl Acad. Sci.2007,104:5527–5532.
[33] MLL-rearranged T-lineage and B-precursor acute leukemias: dominance of HOXdysregulation. Blood.2003,102:262–268.
[34] Armstrong, S. A., Golub, T. R.&Korsmeyer, S. J.MLL-rearranged leukemias:insights from gene expression profiling. Semin. Hematol.2003,40:268–273.
[35] Golub, T. R. et al. Molecular classification of cancer:class discovery and classprediction by gene expression monitoring. Science.1999,286:531–537.
[36] Faber, J. et al. HOXA9is required for survival in human MLL-rearranged acuteleukemias. Blood.2009,113:2375–2385.
[37] Crickmore, M. A., Ranade, V.&Mann, R. S. Regulation of Ubx expression byepigenetic enhancer silencing in response to Ubx levels and genetic variation. PLoSGenet.2009,5:e1000633.
[38] Kannan, R., Berger, C., Myneni, S., Technau, G. M.&Shashidhara, L. S. Abdominal-A mediated repression of Cyclin E expression during cell-fate specification in theDrosophila central nervous system. Mech. ev.2009,127:137–145.
[39] Karnoub, A. E. et al. Mesenchymal stem cells within tumour stroma promote breastcancer metastasis.Nature.2007,449:557–563.
[40] Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc frommouse and human fibroblasts. Nature Biotechnol.2008,26:101–106.
[1] Usami Y, Satake S, Nakayama F, et al. Snail-associated epithelialmesenchymaltransition promotes oesophageal squamous cell carcinoma motility and progression. JPathol.2008,215:330-339.
[2] Brabletz T, Hlubek F, Spaderna S, et al. Invasion and metastasis in colorectal cancer:pithelial-mesenchymaltransition, mesenchymalepithelial transition, stem cells andbeta-catenin. Cells Tissues Organs.2005,179:56-65.
[3] Gallo D, Ferlini C, Scambia G, et al. The epithelial-mesenchymal transition andtheestrogen-signaling in ovarian cancer. Curr Drug Targets.2010,11:474-481.
[4] Trimboli AJ, Fukino K, de Bruin A, et al. Direct evidence for epithelial-mesenchymaltransitions in breast cancer. Cancer Res.2008,68:937-945.
[5] Knust E. Regulation of epithelial cell shape and polarity by cell-cell adhesion (Review).Mol Membr Biol.2002,19(2):113-20.
[6] Matter K, Balda MS. Signalling to and from tight junctions.Nat Rev Mol Cell Biol2003;4(3):225-36.
[7] Matter K, Aijaz S, Tsapara A, Balda MS. Mammalian tight junctions in the regulationof epithelial differentiation and proliferation. Curr Opin Cell Biol.2005,17(5):453-8.
[8] Jean PT， Herve A， Ruby YJ， Huang and M， Angela Nieto. Epithelial-Mesenchymal Transitions in Development and Disease［J］.Cell.2009,139(5):871-90.
[9] Takanori Tsuji，Soichiro Ibaragi，Guo fu Hu.Epithelial-Mesenchymal Transition andCell Cooperativity in Metastasis［J］.Cancer Res,2009,69(18):7135-39.
[10] Bilder D. Epithelial polarity and proliferation control: links from the Drosophilaneoplastic tumor suppressors. Genes Dev.2004,18(16):1909-25.
[11] Moustakas A, Heldin CH. Signaling networks guiding epithelial-mesenchymaltransitions during embryogenesis and cancer progression.Cancer Sci.2007,98:1512–1520.
[12] Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications forfibrosis. J Clin Invest.2003,112:1776-1784.
[13] Klymkowsky MW, Savagner P. Epithelial-mesenchymal transition: a Cancerresearcher's conceptual friend and foe[J]. Am J Pathol.2009,174(5):1588-1593.
[14] Hay ED. An overview of epithelio-mesenchymal transformation[J].ActaAnat(Basel).1995,154(1):8-20.
[15] Thiery JP, Acloque H, Huang RY, et al. Epithelial-mesenchymal transitions indevelopment and disease[J]. Cell.2009,139(5):871-890.
[16] Liu Y. New insights into epithelial-mesenchymal transition in kidney fibrosis[J]. J AmSoc Nephrol.2010.21(2):212-222.
[17] Moustakas A, Heldin CH. Signaling networks guiding epithelial-mesenchymaltransitions during embryogenesis and cancer progression.Cancer Sci.2007,98:1512-1520.
[18] Rajasekaran SA, Huynh TP, Wolle DG, et al. Na, K-ATPase subunits as markers forepithelial-mesenchymal transition in cancer and fibrosis. Mol Cancer Ther.2010,9:151-1524.
[19] Valcourt U, Kowanetz M, Niimi H, et al. TGF-beta and the Smad signaling pathwaysupport transcriptomic reprogramming during epithelial-mesenchymal cell transition.Mol Biol Cell.2005,16:1987-2002.
[20] Fuchs BC, Fujii T, Dorfman JD, et al. Epithelial-to-mesenchymal transition andintegrin-linked kinase mediate sensitivity to epidermal growth factor receptorinhibition in human hepatoma cells. Cancer Res.2008,68:2391-2399.
[21] Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions Nat Rev MolCell Biol.2001,2:285-293.
[22] Savagner P. The epithelial-mesenchymal transition (EMT) phenomenon[J]. Ann Oncol.2010,21:89-92.
[23] Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renalfibrosis. J Mol Med.2004,82:175-181.
[24] Xie L, Law BK, Chytil AM, et al. Activation of the Erk pathway is required for TGF-β1-induced EMT in vitro. Neoplasia.2004,6:603-610.
[25] Chen L, Liu BC, Zhang XL, et al. Influence of connective tissue growth factorantisense oligonucleotide on angiotensin II-induced epithelial mesenchymal transitionin HK2cells. Acta Pharmacol Sin.2006,27:1029-1036.
[26] Nelson WJ, Nusse R. Convergence of Wnt, b-catenin, and cadherin pathways.Science.2004,303:1483-1487.
[27] Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymaltransition during tumor progression. Current Opin Cell Biol.2005,17:548-558.
[28] Soini Y,Tuhkanen H,Sironen R,et al.Transcription factors zeb1,twist and snai1in breastcarcinoma［J］.BMC Cancer,2011,11:73．
[29] Nieto MA. The snail superfamily of zinc-finger transcription factors.Nat Rev Mol CellBiol.2002,3:155-166.
[30] Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumor progression: analliance against the epithelial phenotype? Nat Rev Cancer.2007,7:415-428.
[31] Hector Peinado,Amparo Cano.A hypoxic twist in metastasis ［J］.Nature Cell.Biology.2008,10(3):253-54.
[32] Kong B, Michalski CW, Hong X, et al. AZGP1is a tumor suppressor inpancreaticcancer inducing mesenchymal-to-epithelial transdifferentiation by inhibiting TGF-beta-mediated ERK signaling. Oncogene,2010.
[33] Pantuck AJ, An J, Liu H et al. NF-kappaB-dependent plasticity of the epithelial tomesenchymal transition induced by Von Hippel-Lindau inactivationin renal cellcarcinomas. Cancer Res.2010,70:752-761.
[34] Yu M, Smolen GA, Zhang J, et al. A developmentally regulated inducer of EMT, LBX1,contributes to breast cancer progression. Genes Dev.2009,23:1737-1742.
[35] Siegel PM, Massagué J. Cytostatic and apoptotic actions of TGF-β in homeostasis andcancer. Nat Rev Cance.2003,3:807-821.
[36] Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF–βfamily signaling[J]. Nature.2003,425(6958):577-584.
[37] Moustakas A, Heldin CH. Non-Smad TGF-βsignals[J]. J Cell Sci.2005,118:3573-3584.
[38] Bierie B,Moses HL.Transforming growth factor beta (TGF=β)and inflammation incancer［J］.Cytokine Growth Factor Rev.2010,21(1):49-59．
[39] Gatza CE,Oh SY,Blobe GC.Roles for the type III TGF-βreceptor in human cancer［J］.Cellular Signal.2010,22(8):1163-1174．
[40] Cicchini C, Laudadio I, Citarella F, et al. TGFβ-induced EMT requires focal adhesionkinase (FAK) signaling. Exp Cell Res.2007,314:143-152.
[41] MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanismsand diseases. Dev Cell.2009,17:9-26.
[42] Gerard B, Tait L, Nangia-Makker P, et al.Rad6B acts downstream of Wnt signaling tostabilize β-catenin: Implications for a novel Wnt/β-catenin target[J]. J MolSignal.2011,6:6.
[43] Xu P, Yu SZ, Jiang RC, et al. Differential expression of Notch family members inastrocytomas and medulloblastomas. Pathol Oncol Res.2009,15:703-710.
[44] Fan X, Mikolaenko I, Elhassan I, et al. Notch1and Notch2have opposite effects onembryonal brain tumor growth. Cancer Res.2004,64:7787-7793.
[45] Grego-Bessa J, Diez J, Timmerman L, et al. Notch and epithelial-mesenchymaltransition in development and tumor progression: another turn of the screw. CellCycle.2004,3:718-721.
[46] Timmerman LA, Grego-Bessa J, Raya A, et al. Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation.Genes Dev.2003,18:99-115.
[47] Wang Z, Banerjee S, Li Y, et al. Down-regulation of notch-1inhibits invasion byinactivation of nuclear factor-kappaB, vascular endothelial growth factor, and matrixmetallopro-teinase-9in pancreatic cancer cells. Cancer Res.2006,66:2778-2784.
[48] Bonnomet A, Brysse A, Tachsidis A, et al. Epithelial-to-mesenchymal transitions andcirculating tumor cells. J Mammary Gland Biol Neoplasia.2010,15:261-273.
[49] Moody SE, Perez D, Pan TC, et al. The transcriptional repressor Snail promotesmammary tumor recurrence. Cancer Cell.2005,8:197-209.
[50] Aktas B, Tewes M, Fehm T, et al. Stem cell and epithelial-mesenchymal transitionmarkers are frequently overexpressed in circulating tumor cells of metastatic breastcancer patients. Breast Cancer Res.2009,11: R46.
[51] Yang MH, Chen CL, Chau GY, et al. Comprehensive analysis of the independent effectof twist and snail in promoting metastasis of hepatocellular carcinoma. Hepatology.2009,50:1464-1474.
[52] Wang Y, Xue TC, Xie XY, et al. Relationship between epithelial-mesenchymaltransition and lung metastasis in hepatocellular carcinoma.Chin J Surg.2008,46:1624-1627.
[53] McConkey DJ, Choi W, Marquis L, et al. Role of epithelial-to-mesenchymal transition(EMT) in drug sensitivity and metastasis in bladder cancer. Cancer Metastasis Rev.2009,28:335-344.
[54] Wallerand H, Robert G, Pasticier G, et al. The epithelial-mesenchymal transition-inducing factor TWIST is an attractive target in advanced and/or metastatic bladderand prostate cancers. Urol Oncol.2010,28:473-479.