The Roles Of MTOR/p70S6K Pathway And Its Blockade Strategies In Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most frequentlydiagnosed cancers in developing countries, especially in China. However, theunderstanding of etiology and mode of carcinogenesis of this disease is still lacking.Although therapy strategies have been improved, the prognosis of patients with ESCCis still poor. Moreover, cells of ESCC are known to develop resistance tochemotherapeutic drugs, thus resulting in a dramatic decrease in the 5-year survivalrate for ESCC. Obviously, a better understanding of the molecular mechanisms incarcinogenesis and progression of ESCC helps to improve the prognosis of patientswith ESCC.The mammalian target of rapamycin (mTOR) that is an evolutionarily conservedserine-threonine kinase of a 289-kDa in length belongs to the PIKK[phosphoinositide 3-kinase (PI3K)-related kinase] family, mTOR signaling pathway isfrequently activated in human cancers. In mammals, the two best-characterized targetsof mTOR are the ribosomal S6 kinases S6K1 and S6K2, and the eukaryotic initiationfactor(eIF4E)-binding protein 1(4E-BP1). mTOR activation leads tophosphorylations of S6K1/2 and 4EBP1. The latter releases from the cap-dependenttranslation initiation factor eIF4E, ultimately resulting in enhanced translation fromsubset of genes required for cell growth. These two events lead to an increase inribosomal biogenesis and the selective translation of special mRNA populations. mTOR has recently been recognized as an important and attractive therapeutictarget for cancer therapy. The potential applications of mTOR inhibitors for treatingvarious cancers including renal, prostate, breast, pancreatic, and lung cancers, etc,have been actively studied both preclinically and clinically. However, themTOR/p70S6K signaling pathway in ESCC has not been investigated so far, whichimpel the authors to investigate the role of mTOR/p70S6K signaling pathway forfinding a novel target for the anticancer drugs in ESCC. In the present study, weinvestigated the activated mTOR and its major downstream members in ESCC celllines EC9706 and Eca109, as well as the changes of mRNA and protein expressionlevels, cell cycle, and apoptosis in the ESCC cells treated with rapamycin and smallinterference RNA (siRNA) against mTOR (mTOR-siRNA). Furthemore, weinvestigated the inhibitory effects of rapamycin and mTOR-siRNA on transplantabletumor growth in nude mice.The results showed that mTOR/p70S6K signaling pathway was constitutivelyactivated in the ESCC cell lines. Furthermore, rapamycin could specially blockmTOR-mediated signaling pathway, and mTOR-siRNA not only inhibited the pathway,arrested cells to G₀/G₁ phase and induced apoptosis, but also increased the sensitivity ofEC9706 cells to rapamycin and cisplatin. In vivo experiments revealed that rapamycinand mTOR-siRNA could effectively inhibit the growth of transplantable tumor, andcombination of mTOR-siRNA and rapamycin had a collaborative inhibition effect ontumor growth. Finally, eukaryotic expression vectors pcDNA3.1-PTEN containing thecloned wild-type phosphatase and tensin homolog deleted from chromosome 10 (PTEN)gene from human placenta tissue were transfected to EC 9706 cells using lipofectamine.The cells with stable expression of the PTEN gene were screened by G418.Methods1. mTOR signaling pathway constitutively activated in ESCCExpressions of the mTOR protein and its downstream factors in poorlydifferentiated ESCC cell line EC9706 and well differentiated ESCC cell line Eca109 as well as human normal esophageal and esophageal cancer tissues were investigatedusing immunocytochemistry or immunohistochemisty, and Western blotting. Besides,expression of the mTOR mRNA and its downstream factors in ESCC cell lines wereinvestigated by RT-PCR.2. The status of proliferation and anti-apoptosis when mTOR pathway wasblockaded in EC9706 cells2.1 Proliferation and mobility of EC9706 cellsAfter EC9706 cells were treated with rapamycin, mTOR-siRNA and cisplatinrespectively, cell proliferation was detected by CCK-8. The effects of rapamycin oncell mobility were assessed by the wound healing assay.2.2 Western blotting and RT-PCRTotal proteins of EC9706 cells treated with rapamycin or mTOR-siRNA wererespectively abstracted and the expressions of roTOR, p70S6K, p-p70S6K, 4E-BP1,p-4E-BP1 and Akt were investigated by Western blotting. Total RNAs of EC9706cells treated with rapamycin or mTOR-siRNA were abstracted and the expressions ofmTOR, p70S6K and 4E-BP1 were investigated by RT-PCR.2.3 Analysis of cell cycle and apoptosisEffects of rapamycin and mTOR-siRNA on cell cycle and apoptosis wereassayed by flow cytometry, and effect of rapamycin on apoptosis was assayed byDNA ladder.2.4 In vivo experiments in nude miceTransplantable tumors were produced using EC9706 cells and the transfectedEC9706 cells with mTOR-siRNA, respectively, in nude mice. After the tumors weretreated with rapamycin or cisplatin, Apoptosis of transplantable tumor tissues fromnude mice was investigated by TUNEL. The expressions of factors in mTOR/p70S6Ksignaling pathway of the tumor tissues mentioned above were analyzed byimmunohistochemistry, Western blotting and RT-PCR, respectively.3. Vector pcDNA3.1-PTEN containing PTEN gene was transfected to EC9706 cells 3.1 Cloning and sequencing of the PTEN genePrimers for amplifying the intact PTEN fragment were designed according to thewild-type PTEN gene sequence on GenBank using Primer Primer5.0 software. ThePTEN gene was amplified from human placenta tissue and introduced into pMD 18-Tvector. The resulting pMD18-T- PTEN was transformed to E. coli JM109 and thenpositive colonies were picked out for sequencing. Results of sequencing were blastedwith the PTEN sequence on GenBank.3.2 Construction and transfection of vector pcDNA3.1-PTENThe recovered correct fragments were inserted into a eukaryotic expression vectorpcDNA3.1 to create a recombinant vector pcDNA3.1-PTEN, and then EC9706 cellswere transfected with the recombinant vector pcDNA3.1-PTEN.3.3 Screening and identification of the cells with stable expression of the PTEN geneEC9706 cells transfected with pcDNA3.1 and pcDNA3.1-PTEN were screenedusing G418. The growth of the screened cells was measured by growth curve, and themRNA and protein expressions of the PTEN gene were investigated by RT-PCR andWestern blotting, respectively, in cells transfected with and without pcDNA3.1-PTEN.4. Statistical analysisAll experiments results were from at least three separate experiments. The datawas performed by one-way analysis of variance using SPSS version13.0 (SPSS,Chicago, USA). Summary statistics were expressed as the means±the standarddeviations, except as otherwise stated. In all statistical analyses, a P value of＜0.05was considered statistically significant, and all P values were two-sided.Results1. mTOR signaling pathway constitutively activated in ESCC1.1 Expressions of mTOR and p70S6K proteinsResults of immunocytochemistry, immunohistochemistry and Western Blottingshowed that mTOR and p-p70S6K proteins were expressed in the nuclei andcytoplasm of the two ESCC cell lines. Expression of mTOR proteins in human normal esophageal tissue was much lower than that in esophageal squamous cell cancertissue.1.2 Expressions of mRNAs of mTOR, p70S6K and 4E-BP1The expressions of mTOR, p70S6K and 4E-BP1 mRNA were detected byRT-PCR in both ESCC cell lines. The expression level of mTOR mRNA was higher inEC9706 than Ecal09 cells, while expression levels of p70S6K and 4EoBP1 mRNAswere lower in EC9706 than Ecal09 cells.2. The status of proliferation and anti-apoptosis when mTOR pathway wasblockaded in EC9706 cells2.1 Effects of rapamycin and mTOR-siRNA on proliferation and motility of cellsThe results of CCK-8 dyeing showed that rapamycin inhibited the cellproliferation in a dose-and time-dependent manner. After cells were transfected bymTOR-siRNA, they became more sensitive to rapamycin and cisplatin. Additionally,the results of wound healing assay revealed that rapamycin inhibited the motility ofEC9706 cells.2.2 Effects of rapamycin and mTOR-siRNA on the protein and mRNA expressions offactors in mTOR signaling pathwayResults of Western blotting demonstrated that rapamycin and mTOR-siRNAdownregulated the protein expression of mTOR and inhibited the phosphorylation ofp70S6K and 4E-BP1, but not of Akt. The results of RT-PCR showed that rapamycinand mTOR-siRNA downregulated the mRNA expression of mTOR and upregulatedthe mRNA expression of p70S6K, mTOR-siRNA had the biggest inhibitory effect at72h.2.3 Effects of rapamycin and mTOR-siRNA on cell cycle and apoptosisRapamycin restrained EC9706 cells at G₁ phase of cell cycle in a dose-dependentmanner and mTOR-siRNA had similar effect on cell cycle to rapamycin, while it hadbetter effects when they were combined. On the other hand, both rapamycin andmTOR-siRNA induced apoptosis of cells and combination use of them had asynergistic effect. 2.4 Effects of rapamycin and mTOR-siRNA on transplantable tumor in nude miceAs indicated in the growth curve of tumors, rapamycin and cisplatin inhibited thegrowth of transplantable tumors and had better effect when they were combined,compared to control group, mTOR-siRNA also inhibited the growth of transplantabletumors, and increased the sensitivity of EC9706 cells to rapamycin and cisplatin.Combined use of mTOR-siRNA and rapamycin had the best effect on growth oftransplantable tumors.The results of TUNEL demonstrated that rapamycin and mTOR-siRNAobviously induced apoptosis of EC9706 cells and had better effects when they werecombined. There were positive signals of mTOR, p-p70S6K and p-4E-BP1 in EC9706cells, but became weak after the cells were treated with rapamycin andmTOR-siRNA.3. Construction of eukaryotic expression vector pcDNA3.1-PTEN and itstransfection to EC9706 cells3.1 Cloning and identification of the PTEN geneThe PTEN gene fragments were amplified by one step RT-PCR and the sequencewas the same as the PTEN sequence on GenBank.3.2 Screening and identification of the cells with stable expression of the PTEN geneThe cells with stable expression of the PTEN gene were screened using G418. Asshown in the growth curve, proliferation of the EC9706 cells transfected withpcDNA3.1-PTEN was slower than that of control cells and cells transfected withpcDNA3.1. Expression levels of mRNA and protein of the PTEN gene were higher inEC9706 cells transfected with pcDNA3.1-PTEN compared to control cells.Conclusion1. mTOR/p70S6K signaling pathway is activated in the two ESCC cell lines, EC9706and Eca109, and mRNA and protein levels of mTOR in poorly differentiatedEC9706 are higher than in well differentiated Eca109 cells(P＜0.05), indicating thatthe activated status of mTOR /p70S6K signaling pathway is related to the differentiation of cells, and activated mTOR /p70S6K signaling pathway isimportant to ESCC cell growth, proliferation and anti-apoptosis.2. rapamycin can rapidly inhibit mRNA and protein expressions of mTOR and thephosphorylation of its major downstream effectors, p70S6K and eukaryoticinitiation factor 4E(eIF4E) binding protein 1(4E-BP1) in EC9706 cells, butrapamycin has no effect on Akt, an upstream factor of mTOR. These findingssuggest that mTOR /p70S6K signaling pathway may be an important moleculartarget for ESCC therapy, mTOR-siRNA not only has similar inhibition effects onmTOR/p70S6K pathway to rapamycin, but increases sensitivity of EC9706 cells tocisplatin and rapamycin. Besides, both rapamycin and mTOR-siRNA arrest cells inthe G0/G1 phase and induce apoptosis of EC9706 cells.3. Rapamycin can effectively inhibit the growth of transplantable tumor in nude micethrough inducing apoptosis of tumor cells, and combination of rapamycin andcisplatin has a collaborative inhibition effect on the tumor growth, mTOR-siRNAhas similar effects to rapamycin on the growth of transplantable tumor and increasessensitivity of the tumor cells to cisplatin and rapamycin in nude mice. Bothrapamycin and mTOR-siRNA specially block mTOR/p70S6K signaling pathway,downregulate the expression of mTOR and inhibit the phosphorylation of p70S6Kand 4E-BP1.4. A eukaryotic expression vector pcDNA3.1-PTEN containing the PTEN gene issuccessfully constructed. The PTEN gene is stably expressed in cells transfectedwith pcDNA3.1-PTEN.