| BACKGROUD AND OBJECTIVEEsophageal cancer is the eighth most common cancer and the sixth most commoncause of cancer deaths worldwide. The incidence of esophageal cancer varies greatly bygeographic location, Chinese accounts for more than half of the esophageal cancer patientsand related deaths in the world. Compared with the high incidence of Barrett’s associatedadenocarcinoma in Europe and the United States, the incidence of esophageal squamouscell carcinoma (ESCC) is prevalent in China. Despite the advances in therapy, ESCC isstill one of the most lethal malignancies in China, with an overall5-year survival rate of20%-30%after surgery. Disease relapase and metastasis are the main cause of poorprognosis of esophageal carcinoma. Metastasis is a complex procedure in which cancercells leave the original tumor site, migrate to other parts of the body via vasculature or bydirect extension and finally form tumorous lesions in the new site. Each of these complexcell-biological events are orchestrated by molecular pathways involving prometastasis andmetastasis-inhibiting genes. Discovery of metastasis-related genes and well knowledge ofits function mechanism in metastasis may help us to find molecular targets with therapeuticpotential.LMO1is a small zinc finger protein containing two proteininteracting LIM domainsbut little additional sequence. LMO1usually functions by nucleating the formation of newtranscriptional complexes and/or by disrupting existing transcriptional complexes tomodulate gene expression programmes. LMO1has important cellular roles in processesrelevant to cancer such as self-renewal, cell cycle regulation and metastasis. LMO1isinvolved in tumorigenesis of T-cell leukemia, acute lymphoblastic leukemia, T-cell acutelymphoblastic leukemia and precursor T-cell lymphoblastic lymphoma/leukemia. GWASon neuroblastoma patients demonstrates common polymorphisms at the LMO1locus arestrongly associated with susceptibility to developing neuroblastoma and its expression isrelated to poor prognosis. We can see that LMO1plays important role in tumorgenesis andmetastasis. This highlights the therapeutic potential of LMO1in cancers.Metastases spawned by carcinomas are formed following the completion of acomplex succession of cell-biological events-collectively termed the invasion-metastasis cascade. EMT plays great roles in multiple stage of tumor metastasis. EMT comprises acomplex series of reversible events that can lead to the loss of epithelial cell adhesion andthe induction of a mesenchymal phenotype. Thus, EMT is characterized by the loss ofepithelial differentiation markers including E-cadherin and the induction of mesenchymalmarkers such as vimentin, fibronectin and N-cadherin. There is good evidence that EMTgives rise to the dissemination of single carcinoma cells from the sites of the primarytumors. EMT is correlated with carcinogenesis, metastasis and poor prognosis in varioushuman cancers, including those of the esophagus. These findings demonstrate that thesuppression of EMT could be used as a potential target for treatment of cancer. Althoughmany non-LMO LIM-domain proteins have been found to be associated with EMT, thelinks between LMO proteins and EMT are less direct. LMO2is linked to E cadherinregulation and LMO4can enhance TGF-β signaling in epithelial cells. There’s still noreport about LMO1and EMT. However, LMO1interacts with GATA3, who can reverseEMT by down-regulation of E-cadherin. Therefore, LMO1may functions in EMT.TGF-β is a key regulator of EMT. TGF-β has a dual role during carcinogenesis. Initially itsuppresses tumorigenesis by inducing growth arrest and promoting apoptosis. However, inadvanced cancers, where TGF-β often is over-expressed, it promotes tumorigenesis andmetastases. The strong evidence for pro-tumorigenic and pro-metastatic actions of TGF-β,suggests that inhibitors of this signaling pathway may be useful to treat advanced cancer,whereas the tumor suppressor action of TGF-β complicates the use of TGF-β inhibitors forcancer treatment. Detail mechanism study on pro-tumorigenic and pro-metastatic actionsof TGF-β is important. The main mechanism TGF-β promotes progression and metastasis ofadvanced cancer is regulating EMT. TGF-β can promote esophageal cancer migration andmetastasis by promoting EMT. There’s report that LMO1is involved in TGF-βinducedapoptosis of the pit cells of the gastric epithelium. However, there’s still no study about theroles of LMO1in TGF-β related roles in advanced cancers.This study will focus on the role of LMO1in ESCC metastasis and its potential role inmodulating pro-tumorigenic and pro-metastatic actions of TGF-β by inducing EMT inESCC.METHODS1) LMO1expression of138primary ESCC samples with adjacent non-tumors esophageal tissues was detected and compared by IHC assay with anti-LMO1antibody.2) Lenti-virus expressing corresponding plasmids were applied to up-regulate ordown-regulate expression of LMO1by infecting ESCC cell lines. We performedsoft-agar colony formation, wound-healing assay, transwell cell migration assayand Matrigel cell invasion assay to study roles of LMO1on ESCC cellsmetastasis in Vitro. Spontaneous metastasis assay of KYSE150and TE11, tailvein injection lung metastasis assay of KYSE30on nude mice were applied toevaluate the effect of LMO1on ESCC cells metastasis in Vivo.3) Real-time PCR, Western Blotting and immunofluorescence were applied to testEMT marker changes in cells with up-regulated or down-regulated LMO1expression in Vitro. IHC with EMT markers were performed on tumors formed byESCC cells overexpressing LMO1percutaneously injected to nude mice, to findwhether LMO1induces EMT in ESCC in Vivo.4) We treated ESCC cells with TGF-β. And then real-time PCR and Western Blottingwere applied to test EMT marker changes and LMO1expression to find whetherLMO1expression was elevated in TGF-β induced EMT. We applied SiRNA todown-regulate expression of Smad2, Smad3, Smad4and SP1, downstreammolecular of TGF-β, and then treated ESCC cells with TGF-β. And then real-timePCR was applied to test LMO1expression and EMT marker changes to find howTGF-β modulates LMO1expression.5) We applied ShRNA to down-regulate expression of LMO1, and then treatedESCC cells with TGF-β. At last, real-time PCR was performed to test LMO1expression and EMT marker changes, and transwell cell migration assay wereapplied to test whether down-regulate LMO1expression can affect the pro-EMTand pro-metastatic roles of TGF-β in ESCC cells.RESULTS1) LMO1expressed on cell nucleus. LMO1expression was positive in50.7%（70/138）peri-tumor tissues vs86.9%（120/138） in tumor tissues. There was asignificant positive correlation of LMO1expression between esophageal tumortissues and peri-tumor tissues (p<0.001). 2) Up-regulation of LMO1enhanced cell migration ability in the wound-healingassay. Up-regulation of LMO1elevated cell migration ability to3.1folds forKYSE30and2.9folds for KYSE150, and increase cell invasion ability to1.5folds for KYSE30and2.7folds for KYSE150. Down-regulation of LMO1inTE11reduced cell migration ability to31%for LV-shLMO1-a and19%forLV-shLMO1-b, and lowered invasion ability to22%for LV-shLMO1-a and19%for LV-shLMO1-b. In Vivo metastasis assay on nude mice revealed up-regulationof LMO1increased metastasis ability to3.8folds for KYSE30and4.98folds forKYSE150. Down-regulation of LMO1in TE11reduced spontaneous lungmetastases to51.6%for LV-shLMO1-a and31.6%for LV-shLMO1-b,spontaneous liver metastases to37.2%for LV-shLMO1-a and23.7%forLV-shLMO1-b In Vivo.3) ESCC cells over-expressing LMO1exhibited a morphological change fromcobblestone-like shape to spindle-like morphology, accompanied by decreasedE-cadherin and increased N-cadherin expression. Down-regulation of LMO1inTE11induced a morphological change from spindle-like to cobblestone-likeshape, accompanied by increased E-cadherin and decreased N-cadherinexpression. H&E staining of nude mice tumor showed over-expression of LMO1exhibit a morphological change from cobblestone-like to spindle-like shape. Inconsistent, IHC staining of nude mice tumors showed an obvious E-cadherindecrease.4) TGF-β stimulation induced classical EMT morphological change, as wasconfirmed by E-cadherin decrease and N-cadherin increase both on mRNA andprotein level. Accompanied with the EMT, LMO1expression was also increasedby TGF-β stimulation, in a concentration and time dependent manner.Down-regulation of Smad2, Smad3and Smad4, but not SP1, greatly inhibited theTGF-β induced LMO1increase and EMT.5) Down-regulation of LMO1in KYSE30greatly reversed the TGF-β stimulationinduced EMT marker changes. TGF-β stimulation significantly increased cellmigration ability. However, Down-regulation of LMO1expression reversed thepro-migratory effect of TGF-β to a great degree. CONCLUSION1) LMO1expression increased in ESCC tumor tissues compared with peri-tumortissues. And LMO1was positively correlated with tumor metastasis. LMO1greatly increased cell migration and invasion ability of ESCC cells in Vitro andenhance metastasis ability of ESCC cells in Vivo. Therefore, LMO1enhancedmigration and metastasis of ESCC.2) LMO1over-expression induced classical EMT morphology change andE-cadherin decrease, which means LMO1promote tumor metastasis by inducingEMT.3) TGF-β stimulation induced EMT was accompanied by LMO1expression.However, interference of Smad2, Smad3and Smad4greatly inhibit TGF-βstimulation induced LMO1elevation. Hence LMO1was a transcriptional targetof TGF-β onthe Smad-dependent signaling pathway.4) Silence of LMO1expression greatly reversed TGF-β induced EMT and migrationability enhancement. This suggested that LMO1played a central role incoordinating TGF-β induced EMT and pro-migratory effects in ESCC. |
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