Thioredoxin 1 mediates TGF-β-induced epithelial-mesenchymal transition in salivary adenoid cystic carcinoma

Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of salivary adenoid cystic carcinoma (SACC) which is characterized by wide local infiltration, perineural spread, a propensity to local recurrence and late distant metastasis. Our recent studies have disclosed that TGF-β is a crucial factor for EMT in metastatic SACC. In this study, we further uncovered small redox protein thioredoxin 1 (TXN) as a critical mediator of TGF-β induced EMT. Immunohistochemistry analysis revealed significantly higher expressions of TXN, thioredoxin reductase 1 (TXNRD1) and N-cadherin, and lower expression of E-cadherin in human metastatic SACC compared to non-metastatic SACC tissues. Consistently, cultured SACC cells with stable TXN overexpression had decreased E-cadherin and increased N-cadherin as well as Snail and Slug expressions. The enhanced migration and invasion potential of these cells was abrogated by Akt or TXNRD1 inhibitors. Expression of N-cadherin and Akt p-Akt decreased, whereas E-cadherin expression increased in a BBSKE (TXNRD1 inhibitor)-dose-dependent manner. In a xenograft mouse model, TXN overexpression facilitated the metastatic potential of SACC-83 cells to the lung. Our results indicate that TXN plays a key role in SACC invasion and metastasis through the modulation of TGF-β-Akt/GSK-3β on EMT. TXN could be a potential therapeutic target for SACC.     

Gab2通过GSK-3β/Snail信号通路促进乳腺癌的上皮-间质转化

背景与目的：越来越多的证据显示,Grb2协同结合蛋白2(Grb2 binding protein-2,Gab2)与肿瘤的侵袭转移相关,但Gab2与乳腺癌上皮-间质转化(epithelial-mesenchymal transition,EMT)的关系尚不清楚。本研究旨在探讨Gab2对乳腺癌EMT标志物的影响,明确Gab2在乳腺癌侵袭和转移中的作用机制。方法：采用免疫组织化学染色法检测80例乳腺癌组织中Gab2及EMT标记物上皮性钙黏着蛋白(E-cadherin)、波形蛋白(vimentin)的表达情况并分析其相关性,用蛋白[质]印迹法(Western blot)检测乳腺组织Gab2的表达情况,采用小干扰RNA(siRNA)技术降低乳腺癌细胞系MDA-MB-231中Gab2的表达,采用划痕实验检测表皮生长因子(epithelial growth factor,EGF)刺激后转染细胞的侵袭能力变化,用Western blot检测敲低Gab2后MDA-MB-231细胞中E-cadherin及vimentin的表达情况,同时检测p-GSK-3β的表达情况、转录因子Snail转核情况。结果：Gab2在乳腺癌组织中的表达与E-cadherin的表达呈负相关,而与vimentin的表达呈正相关(P<0.05);乳腺癌组织中Gab2的表达量明显高于正常乳腺组织;siRNA质粒转染后,SiGab2/MDA-MB-231细胞组中Gab2蛋白的表达量明显降低,结果显示转染成功,划痕实验显示细胞的侵袭能力减弱,表明Gab2影响乳腺癌细胞系的侵袭能力;敲低Gab2后,MDA-MB-231细胞中的E-cadherin的表达明显升高,而vimentin的表达明显降低;GSK-3β的磷酸化受到抑制,而Snail在敲低Gab2的细胞核中的表达明显下调。结论：Gab2可以通过GSK-3β/Snail信号通路促进乳腺癌的EMT,从而促进乳腺癌的侵袭和转移。     

转录因子Snail调控上皮-间质转型及对肿瘤转移的逆转作用

背景与目的:研究表明转录因子Snail调控上皮-间质转型(epithelialtomesenchymaltransition,EMT)与肿瘤转移有一定的相关性。本研究通过观察Snail促进EMT以及反义Snail对肿瘤细胞EMT的逆转,明确Snail在肿瘤转移中的作用。方法:分别以SnailcDNA转染MDCK细胞,反义Snail转染MDA-MB231细胞。Westernblot法检测上皮性标记基因上皮钙粘附素(E-cadherin)、β-连环素(β-catenin)、角蛋白18(Cytokeratin18)与间质性标记基因纤粘蛋白(Fibronectin)及转移相关基因基质金属蛋白酶-2(MMP-2)、RhoA蛋白的改变;细胞划痕实验、Boyden小室体外侵袭实验反映细胞转移潜能的变化。结果:转染SnailcDNA的MDCK细胞,上皮标记基因E-cadherin、β-catenin、Cytokeratin18蛋白表达下调,而间质及转移相关基因Fibronectin、MMP-2、RhoA蛋白表达增强(P<0.05);细胞体外运动力与侵袭力增加(P<0.05);反义Snail转染MDA-MB231细胞后,其实验结果与经SnailcDNA处理后的MDCK细胞相反。结论:Snail能促进正常上皮细胞发生EMT,拮抗肿瘤细胞Snail的表达可逆转EMT表型、降低肿瘤转移潜能。     

Complete loss of PTEN expression as a possible early prognostic marker for prostate cancer metastasis

The EGF/IGF growth factors are potent mitogens that regulate cell proliferation and cell survival and are involved in prostate cancer development. Using laser microdissection technology and real-time PCR, together with immunohistochemistry, we have explored the growth factor and integrin dependent PI3-kinase/PTEN/Akt signalling pathway in prostate cell lines and tumour samples by analysing EGF-R, IGF1-R, ILK, beta3 integrin, PTEN and p-Akt protein expression. We provide evidence that loss of PTEN expression rather than upregulated EGF/IGF1 receptor expression was responsible for increased p-Akt in neoplastic prostate cells. We therefore compared PTEN expression in patient biopsies at first time diagnosis recruited prospectively (Study I, 112 patients) and patients with confirmed metastasis recruited retrospectively from the Luxembourg cancer registry (Study II, 42 patients). In Study I, loss of PTEN expression at first time diagnosis was found in 26 of 112 patients (23%). In Study II, 25 of the 42 patients (59%) with lymph node metastasis had complete loss of PTEN expression in both the neoplastic glands of the prostate and the invasive prostate cancer cells in the lymph node, and of these 13 (52%) exhibited already loss of PTEN expression at first diagnosis. These findings demonstrate that loss of PTEN expression is an important factor in progression towards metastatic disease and could potentially serve as an early prognostic marker for prostate cancer metastasis.     

alpha(v) integrins regulate cell proliferation through integrin-linked kinase (ILK) in ovarian cancer cells

Integrins regulate both adhesion and signaling processes involved in proliferation and survival. alpha(v)beta(3) and alpha(v)beta(5) integrins have been shown to mediate cell adhesion and migration. Here we used human ovarian cancer cell lines (IGROV1, SKOV-3) that express alpha(v)beta(3) and alpha(v)beta(5) to study their role in cell proliferation and the signaling pathways involved. We found that alpha(v) integrins regulate cell proliferation through activation of integrin-linked kinase (ILK). An anti-alpha(v)-blocking antibody specifically inhibits the growth of IGROV1 and SKOV-3. The inhibition of cell proliferation involves alpha(v)beta(3) in IGROV1 cells, and both alpha(v)beta(3) and alpha(v)beta(5) in SKOV-3 cells. The reduced growth rate induced by alpha(v) integrin blockade is linked in both cell lines to G1/S cell cycle arrest. alpha(v) integrin blockade by neutralizing antibody as well as cyclic-RGD peptide caused an inhibition of ILK activity and phosphorylation of PKB/Akt on serine-473 but not on threonine-308, and was accompanied by an increase in p27(Kip1) expression. Overexpression of wild-type ILK rescued the phosphorylation of PKB/Akt on serine-473 in cells treated with anti-alpha(v) antibody. Inhibition of ILK by a pharmacological inhibitor results in inhibition of cell proliferation, PKB/Akt phosphorylation and increase of p27(Kip1). These results demonstrate that alpha(v) integrins regulate ovarian cancer cell proliferation through ILK.     

Arrest of human lung fibroblasts in G2 phase after irradiation is regulated by converging phosphatidylinositol-3 kinase and beta1-integrin signaling in vitro

PURPOSE: Cell-matrix interactions might confer cellular radioresistance in vitro. As a function of radiation, the impact of fibronectin (FN) and phosphatidylinositol-3 kinase (PI3K)-related signaling on survival, the cell cycle, and the beta1-integrin signaling kinases integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt), and glycogen synthase kinase-3beta (GSK-3beta) was examined in normal lung fibroblasts in vitro. METHODS AND MATERIALS: Normal human CCD32 lung fibroblasts grown on polystyrene, FN, or poly-L-lysine were irradiated with 0-8 Gy. Colony forming assays, flow cytometric DNA analysis, immunoblotting (Chk1, Chk2, Cdc25C, Cdk1, 14-3-3, p53, p21), and protein kinase assays (ILK, PKBalpha/Akt, GSK-3beta) were performed with or without PI3K inhibition using LY294002 or wortmannin. RESULTS: FN significantly elevated clonogenic survival of CCD32 cells after irradiation compared with polystyrene or poly-L-lysine. FN improved accumulation of irradiated cells in G(2)/M (60%) compared with polystyrene (43%). LY294002 prevented radiation-dependent G(2) blockage on polystyrene; on FN, G(2) arrest was only slightly reduced. Radiation- and PI3K inhibition-related changes in expression and phosphorylation of the various cell cycle proteins tested correlated with the cell cycle data acquired. The kinase activities of ILK, PKBalpha/Akt, and GSK-3beta were strongly induced by irradiation on polystyrene, but not on FN, which was a result of a FN-mediated increase of basal kinase activities. In contrast to polystyrene, FN enabled radiation-dependent induction of ILK and GSK-3beta in a PI3K-independent manner. CONCLUSION: The data indicate a tight convergence of cell-matrix and cell-growth factor interactions that seem to optimize the cellular responsiveness to ionizing radiation in terms of survival and G(2) arrest. ILK, PKBalpha/Akt, and GSK-3beta involved in integrin signaling were uncovered as new molecular factors within the cellular radiation response. Our findings might also provide insight into normal tissue effects and cellular radioresistance.     

Clinical significance of altering epithelial–mesenchymal transition in metastatic lymph nodes of gastric cancer

Background

The E-cadherin, N-cadherin, and Snail genes are epithelial–mesenchymal transition (EMT)-inducible genes. Previous studies demonstrated that the expression of EMT markers in the primary tumor sites of gastric cancer correlates with tumor progression and prognosis. However, the clinical significance of the expression of these EMT markers in metastatic lymph nodes remains unclear. In the present study, we investigated the expression of these EMT markers in the primary tumor sites and metastatic lymph nodes.

Methods

Immunohistochemistry was used to investigate the expression of E-cadherin, N-cadherin, and Snail in 89 primary tumors and 511 metastatic lymph nodes obtained from patients with gastric cancer.

Results

The weak expression of E-cadherin in tumors and lymph nodes increased with more lymph node metastasis and in more undifferentiated tumors. The strong expression of N-cadherin in lymph nodes correlated with more lymph nodes metastasis, an advanced stage, and poor prognosis. The weak expression of Snail in tumors correlated with lymphatic invasion. The strong expression of Snail in lymph nodes correlated with more lymph node metastasis and an advanced stage. The strong expression of Snail in tumors and its weak expression in lymph nodes correlated with more lymph node metastasis, an advanced stage, and poor prognosis.

Conclusions

The expression of N-cadherin in metastatic lymph nodes is useful for predicting the prognosis of patients with gastric cancer. The Snail switch—namely, the positive-to-negative conversion of the Snail status—between primary tumors and lymph node metastasis may be important for confirming EMT and mesenchymal–epithelial transition.

     

Interleukin-1alpha enhances integrin alpha(6)beta(1) expression and metastatic capability of human pancreatic cancer

OBJECTIVE: To investigate the mechanisms of metastasis formation in metastatic human pancreatic cancer, we examined the enhancement in integrin expression, and adherence to and invasiveness into extracellular matrix (ECM) proteins of human pancreatic cancer cells after exposure to interleukin (IL)-1alpha. METHODS: Expression of IL-1 receptor type I (IL-1RI) and alterations in integrin subunits by IL-1alpha were examined by flow-cytometric analysis and by cellular enzyme-linked immunosorbent assay in four human pancreatic cancer cell lines (BxPC-3, PaCa-2, PANC-1, and SW1990), respectively. In addition, assays of cancer cell adhesion and invasion to ECM proteins were performed to investigate whether increased integrin expression affected the adhesive and invasive interaction between cancer cells and the putative integrin ECM ligands. Furthermore, immunohistochemistry was used to assess integrins and IL-1R1 expression in pancreatic tissues. RESULTS: In metastatic cancer cells, expression of the alpha(6) subunit was enhanced by IL-1alpha treatment. While metastatic cancer cells exhibited preferential adherence to and invasion into laminin, these properties were enhanced by IL-1alpha. The alpha(6) subunit and IL-1RI were strongly expressed in pancreatic tissues from pancreatic cancer patients with liver metastasis. CONCLUSIONS: In pancreatic cancer, IL-1alpha enhanced alpha(6)beta(1)-integrin expression, probably via increased IL-1RI levels. Our results indicated that alpha(6)beta(1)-integrin and IL-1RI expression may play important roles in metastasis formation.     

Epithelial-mesenchymal transition in cervical cancer: correlation with tumor progression, epidermal growth factor receptor overexpression, and snail up-regulation

PURPOSE: Acquisition of epithelial-mesenchymal transition (EMT) by primary carcinoma cells is associated with disrupted epithelial integrity, local invasion, and ultimately metastasis. Little is known about the existence and function of EMT in cervical cancer. This study aims to investigate the regulation of EMT in cervical squamous cell carcinoma. EXPERIMENTAL DESIGN: We investigated the molecular events of EMT in surgical specimens, which present the progression of cervical carcinoma. Two cervical cancer cell lines and the primary culture of normal cervical epithelia were used to study the regulatory mechanisms of EMT. RESULTS: The chronic epidermal growth factor (EGF) treatment induces the elongation of cell shape, increases cell scattering, and enhances cell invasion. EGF treatment down-regulates E-cadherin and up-regulates vimentin in cervical cancer cells. These characteristics are consistent with the morphologic changes, molecular events, and functional significance of EMT. EGF receptor (EGFR) signaling inactivates glycogen synthase kinase-3beta, which results in the nuclear accumulation of up-regulated Snail and then leads to EMT program. alpha(5)beta(1) integrin signaling and extracellular matrix fibronectin can modulate EGF-induced EMT. Importantly, the immunofluorescent stainings of surgical specimens indicate that cervical carcinoma progression is accompanied by EGFR overexpression, which is in parallel with decreased E-cadherin and increased vimentin. Up-regulation and nuclear accumulation of Snail correlate with EMT program in tumor tissues. CONCLUSION: EGF cooperates with alpha(5)beta(1) integrin signaling to induce EMT in cervical cancer cells via up-regulated Snail. Blockade of EGFR activity or expression may provide a potential target for the treatment of cervical cancer progression.