哺乳动物雷帕霉素靶蛋白小分子干扰RNA对食管鳞癌细胞中mTOR-p70S6K信号通路及裸鼠移植瘤生长的影响

目的 检测哺乳动物雷帕霉素靶蛋白(mTOR)小分子干扰RNA(siRNA)对食管鳞癌中mTOR-p70S6K信号通路的阻断作用以及对裸鼠移植瘤生长的影响.方法 以食管鳞癌细胞株EC9706为研究对象,采用siRNA干扰、逆转录聚合酶链反应(RT-PCR)、Western blot、流式细胞术及CCK-8等方法,观察mTOR-p70S6K信号通路被阻断后,通路中各因子蛋白及mRNA表达的变化,及其对细胞增殖和凋亡的影响.进行裸鼠成瘤实验,观察mTOR siRNA对肿瘤生长的影响.结果 与未转染细胞相比,转染mTOR siRNA的细胞中mTOR和磷酸化p70S6K的表达水平降低(P<0.05),而pTOS6K的表达水平升高(P<0.05).转染mTOR siRNA后,细胞凋亡增加,细胞增殖能力降低,且对顺铂的敏感性增高,细胞被阻滞在G,期(P<0.05).mTOR siRNA能明显抑制裸鼠移植瘤的生长,siRNA组和siRNA+顺铂组的肿瘤抑制率分别为50.9%和62.3%.结论 mTOR siRNA能有效抑制mTOR-pTOS6K信号通路,抑制细胞增殖,促进细胞凋亡,在裸鼠体内能显著抑制肿瘤的生长.mTOR-pVOSSK信号通路在食管鳞癌发生发展中起重要作用.     

哺乳动物雷帕霉素靶蛋白小分子干扰RNA对食管鳞癌细胞中mTOR-p70S6K信号通路及裸鼠移植瘤生长的影响

目的 检测哺乳动物雷帕霉素靶蛋白(mTOR)小分子干扰RNA(siRNA)对食管鳞癌中mTOR-p70S6K信号通路的阻断作用以及对裸鼠移植瘤生长的影响.方法 以食管鳞癌细胞株EC9706为研究对象,采用siRNA干扰、逆转录聚合酶链反应(RT-PCR)、Western blot、流式细胞术及CCK-8等方法,观察mTOR-p70S6K信号通路被阻断后,通路中各因子蛋白及mRNA表达的变化,及其对细胞增殖和凋亡的影响.进行裸鼠成瘤实验,观察mTOR siRNA对肿瘤生长的影响.结果 与未转染细胞相比,转染mTOR siRNA的细胞中mTOR和磷酸化p70S6K的表达水平降低(P<0.05),而pTOS6K的表达水平升高(P<0.05).转染mTOR siRNA后,细胞凋亡增加,细胞增殖能力降低,且对顺铂的敏感性增高,细胞被阻滞在G,期(P<0.05).mTOR siRNA能明显抑制裸鼠移植瘤的生长,siRNA组和siRNA+顺铂组的肿瘤抑制率分别为50.9%和62.3%.结论 mTOR siRNA能有效抑制mTOR-pTOS6K信号通路,抑制细胞增殖,促进细胞凋亡,在裸鼠体内能显著抑制肿瘤的生长.mTOR-pVOSSK信号通路在食管鳞癌发生发展中起重要作用.

Abstract：

Objective To investigate the effects of mTOR siRNA on mTOR-p70S6K signaling pathway in esophageal squamous cell carcinoma ( ESCC) cells in vitro, and growth and apoptosis in transplanted tumor in nude mice. Methods mTOR siRNA was transfected into ESCC cell line EC9706 cells. The expressions of factors of the mTOR/p70S6K signaling pathway were detected by RT-PCR and Western blot. DNA contents and cell apoptosis were determined by flow cytometry, and cell proliferation was measured by CCK-8 assay. The effects of mTOR siRNA on the transplanted tumor growth were assessed in nude mice. Results The levels of mTOR and p-p70S6K were significantly decreased ( P < 0.05 ) while the level of p70S6K was increased (P<0.05) in the cells transfected with mTOR siRNA, compared with that in untransfected cells and cells transfected with control siRNA. After being interfered by mTOR siRNA, the number of apoptotic cells was increased, cell proliferation became slower and cell cycle was arrested in G, phase compared with that in control cells. Also, mTOR siRNA inhibited the growth of transplanted tumor in vivo. Conclusions mTOR siRNA can effectively interfere in mTOR-p70S6K signaling pathway, induce cell apoptosis and inhibit cell proliferation and tumor growth, suggesting that mTOR-p70S6K signaling pathway plays an important role in the carcinogenesis and development of esophageal squamous cell carcinoma.     

哺乳动物雷帕霉素靶蛋白小分子干扰RNA对食管鳞癌细胞中mTOR-p70S6K信号通路及裸鼠移植瘤生长的影响

目的 检测哺乳动物雷帕霉素靶蛋白(mTOR)小分子干扰RNA(siRNA)对食管鳞癌中mTOR-p70S6K信号通路的阻断作用以及对裸鼠移植瘤生长的影响.方法 以食管鳞癌细胞株EC9706为研究对象,采用siRNA干扰、逆转录聚合酶链反应(RT-PCR)、Western blot、流式细胞术及CCK-8等方法,观察mTOR-p70S6K信号通路被阻断后,通路中各因子蛋白及mRNA表达的变化,及其对细胞增殖和凋亡的影响.进行裸鼠成瘤实验,观察mTOR siRNA对肿瘤生长的影响.结果 与未转染细胞相比,转染mTOR siRNA的细胞中mTOR和磷酸化p70S6K的表达水平降低(P<0.05),而pTOS6K的表达水平升高(P<0.05).转染mTOR siRNA后,细胞凋亡增加,细胞增殖能力降低,且对顺铂的敏感性增高,细胞被阻滞在G,期(P<0.05).mTOR siRNA能明显抑制裸鼠移植瘤的生长,siRNA组和siRNA+顺铂组的肿瘤抑制率分别为50.9%和62.3%.结论 mTOR siRNA能有效抑制mTOR-pTOS6K信号通路,抑制细胞增殖,促进细胞凋亡,在裸鼠体内能显著抑制肿瘤的生长.mTOR-pVOSSK信号通路在食管鳞癌发生发展中起重要作用.     

mTOR/p70S6K Signal transduction pathway contributes to osteosarcoma progression and patients’ prognosis

The mTOR/p70S6K signal transduction pathway plays a key role in the regulation of cancer cells’ survival and proliferation. However, its roles in osteosarcoma, which is one of the most rapidly growing sarcomas, remain unknown. This study investigated for the first time the correlation between the mTOR/p70S6K signal transduction pathway in human osteosarcoma and patients’ prognosis. The expression patterns of mTOR and p70S6K in paraffin-embedded specimens gathered from 65 patients with primary osteosarcoma were detected by the method of immunohistochemistry using antibodies against mTOR and p70S6K. Kaplan–Meier survival and Cox regression analyses were performed to evaluate the prognosis of patients. Immunostaining revealed that the mTOR/p70S6K signal transduction pathway is activated in human osteosarcoma. Additionally, positive expression of mTOR and p70S6K proteins was significantly correlated with surgical stage, metastasis pattern and percentage of dead cells of osteosarcoma. Moreover, in univariate analysis, surgical stage, metastasis pattern and percentage of dead cells, mTOR and p70S6K expression showed significant influence on overall survival (OS) and disease-free survival (DFS). In multivariate analysis, surgical stage (IIA vs. IIB/III), metastasis pattern (without vs. with), percentage of dead cells (≥90 vs. <90%), mTOR expression pattern (negative vs. positive) and p70S6K expression pattern (negative vs. positive) were significant for DFS and OS. Our results demonstrate the correlation of mTOR and p70S6K expression patterns with the oncological progression of osteosarcoma patients, suggesting the prognostic significance of the mTOR/p70S6K signal transduction pathway in osteosarcoma patients, which may lay a foundation for making further investigations on the mTOR/p70S6K signal transduction pathway as a potential target for osteosarcoma therapy.     

雷帕霉素对食管鳞癌细胞系EC9706的mTOR/p70S6K信号通路的影响

目的：观察雷帕霉素（rapamycin，rapa）对食管鳞癌细胞系EC9706的mTOR／p70S6K信号通路的影响。方法：采用免疫细胞化学证实mTOR／p70S6K信号通路的存在，然后通过DNALadder、RT—PCR、Westernblot及流式细胞术分别从DNA、RNA、蛋白及细胞水平研究rapa对细胞凋亡和信号通路的影响。结果：免疫细胞化学结果显示，在细胞核及细胞质中mTOR均呈阳性；rapa处理后有明显DNALadder产生，且mTOR的mRNA水平及蛋白水平下调。但是，mTOR下游的直接靶点p70S6K的mRNA水平及蛋白水平则升高，二者的变化程度均与rapa剂量的相关；流式细胞术检测结果表明，rapa可使细胞停滞于G1期。结论：食管鳞癌细胞系EC9706中存在mTOR／p70S6K信号通路并且处于激活状态，rapa能明显促进细胞凋亡并抑制该通路激活，从而间接抑制翻译的进行。     

Involvement of the Akt/mTOR pathway on EGF-induced cell transformation

Our previous study demonstrated that phosphatidylinositol 3-kinase (PI3K) is necessary for epidermal growth factor (EGF)-induced cell transformation in mouse epidermal JB6 cells. Akt and the mammalian target of rapamycin (mTOR) are regarded as PI3K downstream effectors. Therefore, in this study, we investigated the role of Akt and mTOR on EGF-induced cell transformation in JB6 cells using rapamycin, a specific mTOR inhibitor, and cells expressing dominant negative mutants of Akt1 (DNM-Akt1). We found that the treatment of cells with rapamycin inhibited EGF-induced cell transformation but only slightly inhibited JB6 cell proliferation at 72 h. Although LY294002, a PI3K inhibitor, attenuated EGF-induced activator protein 1 (AP-1) activation, treatment with rapamycin did not affect AP-1 activity. Treatment with rapamycin inhibited EGF-induced phosphorylation and activation of ribosomal p70 S6 protein kinase (p70 S6K), an mTOR downstream target, but had no effect on phosphorylation and activation of Akt. Rapamycin also had no effect on EGF-induced phosphorylation of extracellular signal-regulated protein kinases (ERKs). We showed that introduction of DNM-Akt1 into JB6 mouse epidermal Cl 41 (JB6 Cl 41) cells inhibits EGF-induced cell transformation without blocking cell proliferation. The expression of DNM-Akt1 also suppressed EGF-induced p70 S6K activation as well as Akt activation. These results indicated an involvement of the Akt/mTOR pathway in EGF-induced cell transformation in JB6 cells.     

Ornithine decarboxylase inhibition downregulates multiple pathways involved in the formation of precancerous lesions of esophageal squamous cell cancer

The high incidence and mortality of esophageal squamous cell cancer (ESCC) is a major health problem worldwide. Precancerous lesions of ESCC may either progress to cancer or revert to normal epithelium with appropriate interventions; the bidirectional instability of the precancerous lesions of ESCC provides opportunities for intervention. Reports suggest that the upregulation of ornithine decarboxylase (ODC) is closely related to carcinogenesis. In this study, we investigated whether ODC may act as a target for chemoprevention in ESCC. Immunohistochemistry (IHC) assays indicate that ODC expression is higher in esophageal precancerous lesions compared with normal tissue controls. Its overexpression promotes cell proliferation and transformation of normal esophageal epithelial cells, and its activity is increased after N-nitrosomethylbenzylamine (NMBA) induction in Shantou human embryonic esophageal cell line (SHEE) and human immortalized cells (Het1A) cells. In addition, p38 α, extracellular regulated kinase (ERK1/2) in the mitogen-activated protein kinase pathway and protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) pathways are activated in response to NMBA treatment. Difluoromethylornithine (DFMO) is an ODC inhibitor, which inhibits NMBA-induced activation of p38 α, ERK1/2 and AKT/mTOR/p70S6K pathways; this has been verified by Western blotting. DFMO was also found to suppress the development of esophageal precancerous lesions in an NMBA-induced rat model; IHC demonstrated p38 α, ERK1/2, and AKT/mTOR/p70S6K pathways to be downregulated in these rats. These findings indicate the mechanisms by which ODC inhibition suppresses the development of esophageal precancerous lesions by downregulating p38 α, ERK1/2, and AKT/mTOR/p70S6k signaling pathways, ODC may be a potential target for chemoprevention in ESCC.     

Nucleocytoplasmic localization of p70 S6K1, but not of its isoforms p85 and p31, is regulated by TSC2/mTOR

The tuberous sclerosis complex gene 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway controls many cellular functions via phosphorylation of ribosomal protein S6 kinases (S6Ks). Alternative splicing and translation generate three S6K1 proteins. Although nuclear and cytoplasmic S6K targets are known, the nucleocytoplasmic localization of the S6K1 proteins has not been comparatively elucidated so far. We show that in primary fibroblasts p85 S6K1 is cytoplasmic, p70 can be found in both compartments and p31 is exclusively nuclear. As already known for p70 and p85, our data suggest that p31 is also a target of mTOR-mediated phosphorylation. Blocking mTOR kinase activity via rapamycin and its activation in TSC2(-/-) cells and via TSC2 small interfering RNAs revealed that it regulates the localization of p70, but not of p85 and p31. The mTOR-dependent phosphorylation of p70 S6K1 at T389 is essential for its nuclear localization and exclusively hyperphosphorylated p70 S6K1 can be found in the nucleus. We further demonstrate this mTOR-controlled p70 S6K1 localization to be growth factor dependent. During the cell-cycle phosphorylation and nuclear localization of p70 S6K1 occur in mid G1 phase. We report that the different S6K1 proteins exhibit different nucleocytoplasmic localizations and that the TSC2/mTOR cascade not only regulates p70 S6K1 activity, but also its localization. These findings provide new important insights into the temporal and spatial dynamics of TSC2/mTOR/S6K regulation.     

PI3K/AKT/mTOR pathway activation in primary and corresponding metastatic breast tumors after adjuvant endocrine therapy

Both preclinical and clinical data suggest that activation of the PI3K/AKT/mTOR pathway in response to hormonal therapy results in acquired endocrine therapy resistance. We evaluated differences in activation of the PI3K/AKT/mTOR pathway in estrogen receptor α (ERα) positive primary and corresponding metastatic breast cancer tissues using immunohistochemistry for downstream activated proteins, like phosphorylated mTOR (p‐mTOR), phosphorylated 4E Binding Protein 1 (p‐4EBP1) and phosphorylated p70S6K (p‐p70S6K). For p‐mTOR and p‐4EBP1, the proportion of immunostained tumor cells (0–100%) was scored. Cytoplasmic intensity (0–3) was assessed for p‐p70S6K. The difference between expression of these activated PI3K/AKT/mTOR proteins‐ in primary and metastatic tumor was calculated and tested for an association with adjuvant endocrine therapy. In patients who had received endocrine therapy (N = 34), p‐mTOR expression increased in metastatic tumor lesions compared to the primary tumor (median difference 45%), while in patients who had not received adjuvant endocrine therapy (N = 37), no difference was found. Similar results were observed for p‐4EBP1 and p‐p70S6K expression. In multivariate analyses, adjuvant endocrine therapy was significantly associated with an increase in p‐mTOR (p = 0.01), p‐4EBP1 (p = 0.03) and p‐p70S6K (p = 0.001), indicating that compensatory activation of the PI3K/AKT/mTOR pathway might indeed be a clinically relevant resistance mechanism resulting in acquired endocrine therapy resistance.     

Rapamycin provides a therapeutic option through inhibition of mTOR signaling in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a neoplasm of myeloid progenitor cells expressing Bcr-Abl fusion protein. However, some patients with CML are less likely to respond to imatinib, the inhibitor of Bcr-Abl kinase. Recent studies showed that mTOR pathway can increase responses to imatinib. The analysis of mTOR pathway in CML may provide new insights into possible targets of novel therapies. Therefore, we examined the expression of mTOR pathway molecules in bone marrow cells from CML patients and effect of rapamycin on K562 cells in?vitro. Western blot analysis showed the visibly higher phosphorylation of mTOR (70.6%), 4E-BP1 (76.5%) and p70S6K (73.5%) in bone marrow cells from CML patients. Moreover, treatment of CML cell line (K562) with rapamycin resulted in a decrease of phosphorylation of mTOR, 4E-BP1 and p70S6K. In?vitro, the cell viability in groups with rapamycin treatment displayed a significant decrease in a dose-dependent manner by MTT. The data presented an increase of G0/G1 phase cells and decrease of S phase cells after rapamycin treatment, and the decreased expression of cyclinD1, higher expression of p21 at mRNA level was also detected in K562 with rapamycin. Treatment with 20 nmol/l or more rapamycin could increase apoptotic cells, decrease expression of bcl-2 and activate caspase-3. In conclusion, the mTOR pathway might be involved in chronic myelogenous leukemia. Inhibition of mTOR pathway could interfer with cell proliferation and increase cell apoptosis in K562 cells. It suggested that mTOR might be an important therapeutic target for myelogenous leukemia.     

AKt／mTOR 信号通路中 p70S6K1、4E －BPs 在促进肿瘤发生作用中的研究进展

当受细胞外生长因子等刺激作用后,可激活 PI3K／AKt／mTOR 信号通路,参与控制细胞的生长、增殖、生存、凋亡。AKt／mTOR 信号通路在肿瘤的发生发展过程中起着很重要的促进作用。AKt／mTOR 信号通路在肿瘤细胞中能够通过 p70S6K1和4E －BPs 的作用抑制细胞的凋亡,促进核糖体以及蛋白质的合成、促进肿瘤细胞的侵袭和转移、促进细胞周期进展、促进肿瘤血管的生成,进而促进肿瘤的发生发展。本文就AKt／mTOR 信号通路通过 p70S6K1和4E －BPs 促进肿瘤发生的机制进行综述。     

非小细胞肺癌组织mTOR/P70S6K表达临床意义的探讨

目的:分析在非小细胞肺癌(NSCLC)组织中mTOR/P70S6K/RPS6/eIF4G信号转导途径相关基因mTOR、P70S6K、RPS6、eIF4G的表达水平,探讨mTOR/P70s6K/RPS6/eIF4G基因在NSCIC组织中的表达及临床意义.方法:通过外科手术中获取65例NSCLC组织及30例癌旁组织标本,采用逆转录聚合酶链反应(RT-PCR)技术检测肺癌组织及癌旁组织中mTOR、P70S6K、RPS6和eIF4G基因的表达水平.结果:mTOR、P70S6K、RPS6和eIF4G基因在NSCLC组织中的表达水平分别为(0.54±0.36)%、(0.75±0.34)%、(0.82±0.43)%和(0.28±0.59)%,在癌旁组织中的表达水平分别为(0.28±0.21)%、(0.03±0.03)%、(0.07±0.07)%和(0.09±0.17)%,肺癌组织中的表达水平均显著高于癌旁组织.mTOR、P70S6K、RPS6和eIF4G基因的表达水平同NSCLC患者的年龄、性别、病理类型、分化程度及TNM分期没有明显关系.结论:NSCLC中mTOR/P70S6K/RPS6/eIF4G信号转导途径相关基因显著上调,说明该通路在NSCLC 中被激活,因而 mTOR/P70S6K/RPS6/eIF4G 信号转导途径异常活化可能与NSCLC 发病机制有关,可作为诊断NSCLC诊断的标志,且联合检测的结果更可靠.     

国产雷帕霉素对人淋巴瘤细胞Raji增殖的影响

目的探讨国产雷帕霉素（宜欣可）对人淋巴瘤细胞株Raji细胞体外生长及对mTOR/p 70S6K信号通路的影响。方法MTT法检测不同浓度（0、1、5、10、20、40、50、100 nmol/L）国产雷帕霉素作用不同时间（24、48、72 h）对Raji 细胞增殖的影响。光学显微镜观察Raji细胞形态学变化。流式细胞仪测定国产雷帕霉素对Raji细胞周期分布和凋亡的影响。Western blot 方法检测国产雷帕霉素处理前后对Raji 细胞mTOR、p70S6K、p-p70S6K蛋白的影响。结果国产雷帕霉素对Raji细胞增殖有明显的抑制作用（不同浓度P<0.01或P<0.05）,呈现明显的剂量-效应和时间-效应依赖关系。国产雷帕霉素明显抑制Raji细胞周期发展（P<0.05）,但没有发生明显的凋亡（P>0.05）。0、10、50、100 nmol/L国产雷帕霉作用于Raji细胞的mTOR、p-p70S6K,其蛋白量随药物浓度增大而降低（P<0.05）,p70S6K随药物浓度增大而升高（P<0.05）。结论人淋巴瘤细胞株Raji中存在mTOR/p70S6K信号通路激活状态,宜欣可可抑制该通路激活并通过阻滞细胞周期发展抑制Raji细胞增殖。     

PLK4基因在人食管鳞癌组织中的表达及对癌细胞增殖、侵袭迁移影响的研究

背景与目的：作为调节细胞周期的蛋白激酶,polo样激酶4（polo-like kinase 4,PLK4）参与有丝分裂启动、中心体成熟、胞质分裂及DNA损伤检测等,在多种肿瘤中呈高表达,但PLK4是否参与食管鳞状细胞癌（esophageal squamous cell carcinoma,ESCC）的增殖和侵袭迁移尚不清楚。研究PLK4在ESCC细胞系和临床组织标本中的表达以及对癌细胞增殖、侵袭迁移的影响。方法：采用TRIzol提取细胞总RNA,反转录试剂盒合成cDNA,采用实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR）检测PLK4基因在正常食管上皮细胞Het-1A和ESCC细胞系TE-1、TE-8和TE-13中的mRNA表达水平。离心收集细胞后用RIPA裂解液提取细胞总蛋白,采用蛋白质印迹法（Western blot）检测正常食管上皮细胞Het-1A和ESCC细胞系TE-1、TE-8和TE-13中PLK4蛋白水平。选取2017年1月—2019年12月河南医学高等专科学校附属医院93例经病理组织学检查确诊为ESCC的患者癌组织及其配对的癌旁组织（距原发灶边缘5 cm以上）,临床组织用液氮速冻后加入RIPA裂解液研磨提取组织总蛋白,采用Western blot检测PLK4蛋白水平。绘制受试者工作特征曲线,分析PLK4表达与临床病理学参数之间的关系。利用siRNA干扰技术抑制TE-13细胞中PLK4的表达,设计并合成PLK4的siRNA干扰片段,采用Lipofectamine ^TM 2000转染细胞抑制PLK4在TE-13细胞中的表达,RTFQ-PCR实验和Western blot检测siRNA干扰片段对PLK表达的影响。PLK4在TE-13细胞中表达下调后,用细胞计数试剂盒-8（cell counting kit-8,CCK-8）实验和克隆形成实验检测细胞的增殖能力,transwell小室实验检测细胞的侵袭能力,划痕愈合实验检测细胞的迁移能力。Western blot实验检测PLK4下调后对mTOR/p70S6K信号转导通路中关键蛋白mTOR、p70S6K、p-mTOR ^Ser2448 和p-p70S6K ^{Thr421/Ser424} 表达的影响。结果：RTFQ-PCR和Western blot检测结果显示,PLK4基因在ESCC细胞系中的mRNA和蛋白表达水平显著高于正常食管上皮细胞（P＜0.05）。与癌旁正常组织相比,ESCC组织标本中PLK4的蛋白表达水平异常增高（P＜0.05）,绘制的受试者工作特征曲线的曲线下面积为0.841,95%CI为0.786~0.895,灵敏度为74.2%（69/93）,特异度为89.2%（83/93）（P＜0.000 1）。PLK4蛋白在ESCC组织中的表达水平与性别、年龄和肿瘤大小均无关（均P＞0.05）,但与分化程度、临床分期和淋巴结是否转移有关（均P＜0.05）。ESCC分化程度越低,PLK4高表达率越高,低分化程度的ESCC组织中PLK4高表达率为86.7%,Ⅲ~Ⅳ期患者ESCC组织中PLK4蛋白高表达率为92.3%。PLK4高表达与临床分期呈正相关（P＜0.05）。CCK-8和克隆形成实验结果显示,下调PLK4的表达可以显著抑制TE-13细胞的增殖（P＜0.05）,transwell小室实验和划痕实验结果显示,下调PLK4的表达可以显著抑制TE-13细胞的侵袭迁移能力（P＜0.05）。抑制PLK4的表达使TE-13细胞中mTOR和p70S6K蛋白的表达下降（P＜0.05）,且p-mTOR ^Ser2448 和p-p70S6K ^{Thr421/Ser424} 的表达下降（P＜0.05）。结论：PLK4在ESCC细胞和组织中均呈高表达,抑制PLK4的表达可以抑制ESCC细胞的增殖和侵袭迁移能力,PLK4可能通过mTOR/p70S6K信号转导通路促进ESCC细胞恶性进程。     

Activation of mammalian target of rapamycin pathway confers adverse outcome in nonsmall cell lung carcinoma

BACKGROUND:

Dysregulation of the mammalian target of rapamycin (mTOR) pathway has been shown to contribute to tumorigenesis. This study explored protein expression profiles of mTOR pathway and the relationship with prognosis in patients with nonsmall cell lung carcinoma (NSCLC).

METHODS:

The protein expression profiles of mTOR/phosphorylated (p‐)mTOR, phosphoinositide‐dependent kinase 1 (PDK1)/p‐PDK1, p‐Akt1, and P70 ribosomal protein S6 kinase (P70S6K)/p‐P70S6K were determined via immunohistochemical staining assay. The clinical prognostic values of both single and combined protein expression were investigated with univariate and multivariate survival analysis.

RESULTS:

Compared with normal lung tissues, the protein levels of mTOR/p‐mTOR, p‐Akt1 Ser473/Thr308, and P70S6K/p‐P70S6K were higher (all P < .05), whereas p‐PDK1 was lower (P < .05) in tumor tissues. p‐mTOR expression was associated with histological differentiation, histological type, lymph node invasion, and stage (all P < .05). Overall survival in NSCLC patients was significantly shorter in cases with positive phenotype for p‐mTOR, p‐PDK1, and p‐P70S6K (all P < .05). Subjects with coexpression of any 2 of p‐mTOR, p‐PDK1, p‐Akt1 Ser473, and p‐P70S6K demonstrated worse prognosis than those expressing no biomarker or any 1 biomarker alone (all P < .05). Multivariate analysis showed that the combination of p‐mTOR/p‐P70S6K is an independent prognostic factor in addition to tumor stage.

CONCLUSIONS:

This study provides clinical evidence that activated components of mTOR pathway, not total protein, are predictors of poor prognosis in NSCLC. Moreover, evaluating protein‐expression profiles of these molecules might be a new strategy for individual therapy in subjects with NSCLC. Cancer 2011;. © 2011 American Cancer Society.     

Fibronectin stimulates non-small cell lung carcinoma cell growth through activation of Akt/mammalian target of rapamycin/S6 kinase and inactivation of LKB1/AMP-activated protein kinase signal pathways

The Akt/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) pathway is considered a central regulator of protein synthesis and of cell proliferation, differentiation, and survival. However, the role of the Akt/mTOR/p70S6K pathway in lung carcinoma remains unknown. We previously showed that fibronectin, a matrix glycoprotein highly expressed in tobacco-related lung disease, stimulates non-small cell lung carcinoma (NSCLC) cell growth and survival. Herein, we explore the role of the Akt/mTOR/p70S6K pathway in fibronectin-induced NSCLC cell growth. We found that fibronectin stimulated the phosphorylation of Akt, an upstream inducer of mTOR, and induced the phosphorylation of p70S6K1 and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), two downstream targets of mTOR in NSCLC cells (H1792 and H1838), whereas it inhibited the phosphatase and tensin homologue deleted on chromosome 10, a tumor suppressor protein that antagonizes the phosphatidylinositol 3-kinase/Akt signal. In addition, treatment with fibronectin inhibited the mRNA and protein expression of LKB1 as well as the phosphorylation of AMP-activated protein kinase (AMPKalpha), both known to down-regulate mTOR. Rapamycin, an inhibitor of mTOR, blocked the fibronectin-induced phosphorylation of p70S6K and 4E-BP1. Akt small interfering RNA (siRNA) and an antibody against the fibronectin-binding integrin alpha5beta1 also blocked the p70S6K phosphorylation in response to fibronectin. In contrast, an inhibitor of extracellular signal-regulated kinase 1/2 (PD98095) had no effect on fibronectin-induced phosphorylation of p70S6K. Moreover, the combination of rapamycin and siRNA for Akt blocked fibronectin-induced cell proliferation. Taken together, these observations suggest that fibronectin-induced stimulation of NSCLC cell proliferation requires activation of the Akt/mTOR/p70S6K pathway and is associated with inhibition of LKB1/AMPK signaling.     

Activation of mammalian target of rapamycin signaling pathway contributes to tumor cell survival in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of chimeric nucleophosmin-ALK. Previously, nucleophosmin-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. In this study, we hypothesized that the mammalian target of rapamycin (mTOR) pathway, which functions downstream of AKT, mediates the oncogenic effects of activated PI3K/AKT in ALK+ ALCL. Here, we provide evidence that mTOR signaling phosphoproteins, including mTOR, eukaryotic initiation factor 4E-binding protein-1, p70S6K, and ribosomal protein S6, are highly phosphorylated in ALK+ ALCL cell lines and tumors. We also show that AKT activation contributes to mTOR phosphorylation, at least in part, as forced expression of constitutively active AKT by myristoylated AKT adenovirus results in increased phosphorylation of mTOR and its downstream effectors. Conversely, inhibition of AKT expression or activity results in decreased mTOR phosphorylation. In addition, pharmacologic inhibition of PI3K/AKT down-regulates the activation of the mTOR signaling pathway. We also show that inhibition of mTOR with rapamycin, as well as silencing mTOR gene product expression using mTOR-specific small interfering RNA, decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in ALK+ ALCL cells. Cell cycle arrest was associated with modulation of G(1)-S-phase regulators, including the cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Apoptosis following inhibition of mTOR expression or function was associated with down-regulation of antiapoptotic proteins, including c-FLIP, MCL-1, and BCL-2. These findings suggest that the mTOR pathway contributes to nucleophosmin-ALK/PI3K/AKT-mediated tumorigenesis and that inhibition of mTOR represents a potential therapeutic strategy in ALK+ ALCL.     

Methods to identify molecular expression of mTOR pathway: a rationale approach to stratify patients affected by clear cell renal cell carcinoma for more likely response to mTOR inhibitors

Since target therapy with mTOR inhibitors plays an important role in the current management of clear cell renal cell carcinoma (RCC), there is an increasing demand for predictive biomarkers, which may help to select patients that are most likely to benefit from personalized treatment. When dealing with formalin-fixed paraffin-embedded (FFPE) cancer tissue specimens, several techniques may be used to identify potential molecular markers, yielding different outcome in terms of accuracy. We sought to investigate and compare the capability of three main techniques to detect molecules performing an active function in mTOR pathway in RCC. Immunohistochemistry (IHC), Western blot (WB) and immunofluorescence (IF) analyses were performed on FFPE RCC tissue specimens from 16 patients by using the following mTOR pathway-related: mTOR (Ser235/236), phospho-mTOR (p-mTOR/Ser2448), phospho-p70S6k (p-p70S6k/Thr389), both monoclonal and polyclonal, phospho-S6Rb (p-S6Rb) and phospho-4EBP1 (p-4EBP1/Thr37/46). No single molecule was simultaneously revealed by all three techniques. Only p-p70S6k was detected by two methods (IHC and IF) using a monoclonal antibody. The other molecules were detected exclusively by one technique, as follows: p-mTOR and polyclonal p-p70S6K by IHC, p70S6K, p-S6Rb and p-4EBP1 by WB, and, finally, mTOR by IF. We found significant differences in detecting mTOR pathway-related active biomarkers by using three common techniques such as IHC, WB and IF on RCC samples. Such results have important implications in terms of predictive biomarker testing, and need to be related to clinical end-points such as responsiveness to targeted drugs by prospective studies.     

Longitudinal inhibition of PI3K/Akt/mTOR signaling by LY294002 and rapamycin induces growth arrest of adult T-cell leukemia cells

This study found that phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling was activated in human T-cell lymphotropic virus type I (HTLV-1)-infected leukemia cells. Rapamycin (1-100 nM, 48h), the inhibitor of mTOR and its analog RAD001 (1-100 nM, 48 h)-induced growth inhibition and G0/G1 cell cycle arrest of these cells in association with de-phosphorylation of p70S6K and 4E-BP-1, although IC50 was not achieved. Paradoxically, rapamycin-stimulated phosphorylation of Akt at Ser473. Blockade of Akt signaling by the PI3K inhibitor LY294002 (1-20 microM, 48 h) also resulted in the growth inhibition and G0/G1 cell cycle arrest of HTLV-1-infected cells, with IC50 ranging from 5 to 20muM, and it caused de-phosphorylation of p70S6K and 4E-BP-1. Of note, when rapamycin was combined with LY294002, rapamycin-induced phosphorylation of Akt was blocked, and the ability of rapamycin to induce growth arrest of HTLV-1-infected T-cells and suppress the p-p70S6K and p-4E-BP-1 proteins was potentiated. Moreover, both LY294002 and rapamycin down-regulated the levels of c-Myc and cyclin D1 proteins in these cells, and their combination further decreased levels of these cell cycle-regulating proteins. Taken together, longitudinal inhibition of PI3K/Akt/mTOR signaling represents a promising treatment strategy for individuals with adult T-cell leukemia.