Changes In Epithelial-mesenchymal Transition Markers Research Articles

Linking of mPGES-1 and iNOS activates stem-like phenotype in EGFR-driven epithelial tumor cells

Inflammatory prostaglandin E-2 (PGE-2) favors cancer progression in epithelial tumors characterized by persistent oncogene input. However, its effects on tumor cell stemness are poorly understood at molecular level. Here we describe two epithelial tumor cells A431 and A459, originating from human lung and skin tumors, in which epithelial growth factor (EGF) induces sequential up-regulation of mPGES-1 and iNOS enzymes, producing an inflammatory intracellular milieu. We demonstrated that concerted action of EGF, mPGES-1 and iNOS causes sharp changes in cell phenotype demonstrated by acquisition of stem-cell features and activation of the epithelial-mesenchymal transition (EMT). When primed with EGF, epithelial tumor cells transfected with mPGES-1 or iNOS to ensure steady enzyme levels display major stem-like and EMT markers, such as reduction in E-cadherin with a concomitant rise in vimentin, ALDH-1, CD133 and ALDH activity. Tumorsphere studies with these cells show increased sphere number and size, enhanced migratory and clonogenic capacity and sharp changes in EMT markers, indicating activation of this process. The concerted action of the enzymes forms a well-orchestrated cascade where expression of iNOS depends on overexpression of mPGES-1. Indeed, we show that through its downstream effectors (PGE-2, PKA, PI3K/Akt), mPGES-1 recruits non-canonical transcription factors, thus facilitating iNOS production.In conclusion, we propose that the initial event leading to tumor stem-cell activation may be a leveraged intrinsic mechanism in which all players are either inherent constituents (EGF) or highly inducible proteins (mPGES-1, iNOS) of tumor cells. We suggest that incipient tumor aggressiveness may be moderated by reducing pivotal input of mPGES-1.

Shenqi detoxification granule combined with P311 inhibits epithelial-mesenchymal transition in renal fibrosis via TGF-β1-Smad-ILK pathway.

Shenqi detoxification granule (SDG), a traditional Chinese herbal formula, has been shown to have nephroprotective and anti-fibrotic activities in patients with chronic kidney disease (CKD). However, its mechanisms in renal fibrosis and the progression of CKD remain largely unknown. P311, a highly conserved 8-kDa intracellular protein, plays a key role in renal fibrosis by regulating epithelial-mesenchymal transition (EMT). Previously, we found P311 might be involved in the pathogenesis of renal fibrosis by inhibiting EMT via the TGF-β1-Smad-ILK pathway. We also found SDG combined with P311 could ameliorate renal fibrosis by regulating the expression of EMT markers. Here we further examined the effect and mechanism of SDG combined with P311 on TGF-β1-mediated EMT in a rat model of unilateral ureteral occlusion (UUO) renal fibrosis. After establishment of the UUO model successfully, the rats were gavaged with SDG daily and/or injected with recombinant adenovirus p311 (also called Ad-P311) through the tail vein each week for 4 weeks. Serum creatinine (Cr), blood urea nitrogen (BUN) and albumin (ALB) levels were tested to observe renal function, and hematoxylin eosin (HE) and Masson staining were performed to observe kidney histopathology. Furthermore, the expression of EMT markers (E-cadherin and α-smooth muscle actin (α-SMA)) and EMT-related molecules TGF-β1, pSmad2/3, Smad7 and ILK were observed using immunohistochemical staining and Western blot analysis. Treatment with SDG and P311 improved renal function and histopathological abnormalities, as well as reversing the changes of EMT markers and EMT-related molecules, which indicated SDG combined with P311 could attenuate renal fibrosis in UUO rats, and the underlying mechanism might involve TGF-β1-mediated EMT and the TGF-β1-Smad-ILK signaling pathway. Therefore, SDG might be a novel alternative therapy for treating renal fibrosis and delaying the progression of CKD. Furthermore, SDG combined with P311 might have a synergistic effect on attenuating renal fibrosis.

MDA-9/Syntenin (SDCBP) modulates small GTPases RhoA and Cdc42 via transforming growth factor β1 to enhance epithelial-mesenchymal transition in breast cancer.

Epithelial-mesenchymal transition (EMT) is one of the decisive steps regulating cancer invasion and metastasis. However, the molecular mechanisms underlying this transition require further clarification. MDA-9/syntenin (SDCBP) expression is elevated in breast cancer patient samples as well as cultured breast cancer cells. Silencing expression of MDA-9 in mesenchymal metastatic breast cancer cells triggered a change in cell morphology in both 2D- and 3D-cultures to a more epithelial-like phenotype, along with changes in EMT markers, cytoskeletal rearrangement and decreased invasion. Conversely, over expressing MDA-9 in epithelial non-metastatic breast cancer cells instigated a change in morphology to a more mesenchymal phenotype with corresponding changes in EMT markers, cytoskeletal rearrangement and an increase in invasion. We also found that MDA-9 upregulated active levels of known modulators of EMT, the small GTPases RhoA and Cdc42, via TGFβ1. Reintroducing TGFβ1 in MDA-9 silenced cells restored active RhoA and cdc42 levels, modulated cytoskeletal rearrangement and increased invasion. We further determined that MDA-9 interacts with TGFβ1 via its PDZ1 domain. Finally, in vivo studies demonstrated that silencing the expression of MDA-9 resulted in decreased lung metastasis and TGFβ1 re-expression partially restored lung metastases. Our findings provide evidence for the relevance of MDA-9 in mediating EMT in breast cancer and support the potential of MDA-9 as a therapeutic target against metastatic disease.

WNT5A signaling impairs breast cancer cell migration and invasion via mechanisms independent of the epithelial-mesenchymal transition.

BackgroundWNT5A (-/-) mammary tissue has been shown to exhibit increased ductal elongation, suggesting elevated mammary cell migration. Increased epithelial cell migration/invasion has often but not always been linked to the epithelial-mesenchymal transition (EMT). In the current study, we investigated the loss of WNT5A in HB2 human mammary epithelial cells and hypothesized that this loss increased their invasion via the EMT. Based on these results, we postulated that suppression of breast cancer cell migration and invasion by WNT5A is due to EMT reversal.MethodsWNT5A was transiently knocked down using specific siRNAs, whereas WNT5A signaling was induced in MDA-MB468 and MDA-MB231 breast cancer cells by stably transfecting cells with WNT5A or treating them with recombinant WNT5A (rWNT5A). Changes in EMT markers, CD44, pAKT and AKT expression were assessed using Western blotting and immunofluorescence. The physiological relevance of altered WNT5A signaling was assessed using migration and invasion assays.ResultsWNT5A knockdown in HB2 mammary epithelial cells resulted in EMT-like changes and increased invasiveness, and these changes were partially reversed by the addition of rWNT5A. These data suggest that WNT5A might inhibit breast cancer cell migration and invasion by a similar EMT reversal. Contrary to our expectations, we did not observe any changes in the EMT status of breast cancer cells, either after treatment with rWNT5A or stable transfection with a WNT5A plasmid, despite the parallel WNT5A-induced inhibition of migration and invasion. Instead, we found that WNT5A signaling impaired CD44 expression and its downstream signaling via AKT. Moreover, knocking down CD44 in breast cancer cells using siRNA impaired cell migration and invasion.ConclusionsWNT5A bi-directionally regulates EMT in mammary epithelial cells, thereby affecting their migration and invasion. However, the ability of WNT5A to inhibit breast cancer cell migration and invasion is an EMT-independent mechanism that, at least in part, can be explained by decreased CD44 expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0421-0) contains supplementary material, which is available to authorized users.

Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition.

Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR.

Impact by pancreatic stellate cells on epithelial-mesenchymal transition and pancreatic cancer cell invasion: Adding a third dimension in vitro

Pancreatic cancer is associated with a highly abundant stroma and low-grade inflammation. In the local tumour microenvironment, elevated glucose levels, the presence of tumour-associated stellate cells and macrophages are hypothesised to promote the tumour progression and invasion. The present study investigated the influence by the microenvironment on pancreatic cancer cell invasion in vitro. After co-culture with tumour-associated pancreatic stellate cells (TPSCs), pancreatic cancer cells displayed up to 8-fold reduction in levels of epithelial-mesenchymal transition (EMT) markers E-cadherin and ZO-1, while β-catenin and vimentin levels were increased. A 3D organotypic model showed that TPSCs stimulated pancreatic cancer cell invasion, both as single cell (PANC-1) and cohort (MIAPaCa-2) invasion. The combined presence of TPSCs and M2-like macrophages induced invasion of the non-invasive BxPC-3 cells. High glucose conditions further enhanced changes in EMT markers as well as the cancer cell invasion. In summary, co-culture with TPSCs induced molecular changes associated with EMT in pancreatic cancer cells, regardless of differentiation status, and the organotypic model demonstrated the influence of microenvironmental factors, such as glucose, stellate cells and macrophages, on pancreatic cancer cell invasion.

Hypoxia-inducible factor 1α-induced epithelial-mesenchymal transition of endometrial epithelial cells may contribute to the development of endometriosis.

How does hypoxia promote growth of lesions in the development of endometriosis? Hypoxia induces the epithelial-mesenchymal transition (EMT) of endometrial cells, resulting in changes in cellular characteristics, which may be a prerequisite for the establishment of endometriotic lesions. Up-regulated hypoxia-inducible factor 1α (HIF-1α) has recently been found in ectopic endometrial lesions. There is increasing evidence that EMT, in which epithelial cells acquire mesenchymal and migratory properties, may also play a role in the establishment of the disease. EMT induced by HIF-1α has been reported to play a role in the development of many tumor types. We investigated expression changes of N-cadherin, E-cadherin, β-catenin, HIF-1α and vimentin using immunohistochemistry in normal, eutopic and ectopic endometria. Endometrial tissues from 96 additional females without related pathology were collected, and these tissues were subjected to subsequent primary cell culture for further experiments. The expression of N-cadherin, E-cadherin, β-catenin, HIF-1α and vimentin in 20 normal, 21 eutopic and 21 ectopic endometrial samples was assessed by immunohistochemistry. Human primary endometrial epithelial cells were isolated from 96 normal endometrial tissues. Times for hypoxia treatment for western blot analysis were 1, 2, 4 and 8 h, for transwell experiments was 48 h. Ishikawa cells were used instead of primary endometrial epithelial cells for transfection and part of the transwell experiments. The impact of hypoxia on invasion was evaluated by transwell assays. Overexpression and inhibition of HIF-1α were achieved by transfection of pG/CMV/HIF-1α/IRES/EGFP and MCS-shHIF-1α-EGFP-IRES plasmids separately. The effect of hypoxia, overexpression and knockdown of HIF-1α on hypoxia-induced changes of EMT markers and β-catenin were analyzed by western blot and reverse transcriptase polymerase chain reaction (RT-PCR). Overexpression of HIF-1α and changes associated with EMT were observed in normal, eutopic and ectopic endometrial tissues. Primary cultured human endometrial epithelial cells responded to hypoxia with classic EMT changes (fibroblastoid phenotype, increased expression of snail family zinc finger 1, β-catenin and mesenchymal markers, and decreased expression of E-cadherin) and increased invasiveness. The decreased invasiveness of Ishikawa cells by knockdown of HIF-1α was observed under hypoxic conditions. While up-regulation of HIF-1α induced changes characteristic of EMT, down-regulation of HIF-1α had the opposite effect. Statistical significance was defined as P < 0.05. A weakness of this study is the relatively small sample size for immunohistochemistry. Ishikawa cells were used instead of primary endometrial epithelial cells for transfection and part of the transwell experiments. Hypoxia-stabilized HIF-1α may play an important role in the invasion of endometrial cells in ectopic endometrial lesions, and it may induce EMT in the development of endometriosis. HIF-1α may be a new and important target for the endometriosis treatment. This study was funded by the National Nature Science Foundation of China (grant number 81170545 and 81471439). The authors declare no competing interests.

Overexpression of angiotensin II type 1 receptor in breast cancer cells induces epithelial–mesenchymal transition and promotes tumor growth and angiogenesis

The angiotensin II type I receptor (AGTR1) has been implicated in diverse aspects of human disease, from the regulation of blood pressure and cardiovascular homeostasis to cancer progression. We sought to investigate the role of AGTR1 in cell proliferation, epithelial–mesenchymal transition (EMT), migration, invasion, angiogenesis and tumor growth in the breast cancer cell line MCF7. Stable overexpression of AGTR1 was associated with accelerated cell proliferation, concomitant with increased expression of survival factors including poly(ADP-ribose) polymerase (PARP) and X-linked inhibitor of apoptosis (XIAP), as well as extracellular signal-regulated kinase (ERK) activation. AGTR1-overexpressing MCF7 cells were more aggressive than their parent line, with significantly increased activity in migration and invasion assays. These observations were associated with changes in EMT markers, including reduced E-cadherin expression and increased p-Smad3, Smad4 and Snail levels. Treatment with the AGTR1 antagonist losartan attenuated these effects. AGTR1 overexpression also accelerated tumor growth and increased Ki-67 expression in a xenograft model. This was associated with increased tumor angiogenesis, as evidenced by a significant increase in microvessels in the intratumoral and peritumoral areas, and enhanced tumor invasion, with the latter response associated with increased EMT marker expression and matrix metallopeptidase 9 (MMP-9) upregulation. In vivo administration of losartan significantly reduced both tumor growth and angiogenesis. Our findings suggest that AGTR1 plays a significant role in tumor aggressiveness, and its inhibition may have therapeutic implications.

A supercritical-CO2 extract of Ganoderma lucidum spores inhibits cholangiocarcinoma cell migration by reversing the epithelial–mesenchymal transition

BackgroundGanoderma lucidum (G. lucidum) is an oriental medical mushroom that has been widely used in Asian countries for centuries to prevent and treat different diseases, including cancer. Hypothesis/PurposeThe objective of this study was to investigate the effect of A supercritical-CO2 extract of G. lucidum spores on the transforming growth factor beta 1 (TGF-β1)-induced epithelial–mesenchymal transition (EMT) of cholangiocarcinoma cells. Study designThis was an in vitro study with human cholangiocarcinoma TFK-1 cells treated with varying concentrations of G. lucidum. MethodsA supercritical-CO2 extract of G. lucidum spores (GLE) was obtained from completely sporoderm-broken germinating G. lucidum spores by supercritical fluid carbon dioxide (SCF-CO2) extraction. GLE pre-incubated with human cholangiocarcinoma TFK-1 cells prior to TGF-β1 treatment (2ng/ml) for 48h. Changes in EMT markers were analyzed by western blotting and immunofluorescence. The formation of F-actin stress fibers was assessed via immunostaining with phalloidin and examined using confocal microscopy. Additionally, the effect of the GLE on TGF-β1-induced migration was investigated by a Boyden chamber assay. ResultsTGF-β1-induced reduction in E-cadherin expression was associated with a loss of epithelial morphology and cell–cell contact. Concomitant increases in N-cadherin and Fibronectin were evident in predominantly elongated fibroblast-like cells. The GLE suppressed the TGF-β1-induced morphological changes and the changes in cadherin expression, and also inhibited the formation of F-actin stress fibers, which are a hallmark of EMT. The GLE also inhibited TGF-β1-induced migration of TFK-1 cells. ConclusionOur findings provide new evidence that GLE suppress cholangiocarcinoma migration in vitro through inhibition of TGF-β1-induced EMT. The GLE may be clinically applied in the prevention and/or treatment of cancer metastasis.

Cardiotoxin III Inhibits Hepatocyte Growth Factor-Induced Epithelial-Mesenchymal Transition and Suppresses Invasion of MDA-MB-231 Cells.

The epithelial-mesenchymal transition (EMT) is the first step required for breast cancer to initiate metastasis. In this study, hepatocyte growth factor (HGF) was used as a metastatic inducer of MDA-MB-231 cells. Cardiotoxin III (CTX III) inhibited HGF-induced morphological changes and upregulation of E-cadherin with the concomitant decrease in N-cadherin and Vimentin protein levels, resulting in inhibition of cell migration and invasion. CTX III-induced downregulation of transcription factors, Snail, Twist, and Slug, in MDA-MB-231 cells. CTX III suppressed c-Met phosphorylation and downstream activation of phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2. The c-Met specific inhibitor PHA665752 attenuated ERK1/2 and Akt phosphorylation, cell migration and invasion, as well as the expressional changes of EMT markers induced by HGF. Taken together, our data suggest that CTX III suppresses HGF/c-Met-induced cell migration and invasion by reversing EMT, which involves the inactivation of the HGF/c-Met-mediated ERK1/2 and PI3K/Akt pathways in MDA-MB-231 cells.

Allelic Variants in Arhgef11 via the Rho-Rock Pathway Are Linked to Epithelial-Mesenchymal Transition and Contributes to Kidney Injury in the Dahl Salt-Sensitive Rat.

Previously, genetic analyses identified that variants in Arhgef11 may influence kidney injury in the Dahl salt-sensitive (S) rat, a model of hypertensive chronic kidney disease. To understand the potential mechanism by which altered expression and/or protein differences in Arhgef11 could play a role in kidney injury, stably transduced Arhgef11 knockdown cell lines as well as primary cultures of proximal tubule cells were studied. Genetic knockdown of Arhgef11 in HEK293 and NRK resulted in reduced RhoA activity, decreased activation of Rho-ROCK pathway, and less stress fiber formation versus control, similar to what was observed by pharmacological inhibition (fasudil). Primary proximal tubule cells (PTC) cultured from the S exhibited increased expression of Arhgef11, increased RhoA activity, and up regulation of Rho-ROCK signaling compared to control (small congenic). The cells were also more prone (versus control) to TGFβ-1 induced epithelial-mesenchymal transition (EMT), a hallmark feature of the development of renal interstitial fibrosis, and characterized by development of spindle shape morphology, gene expression changes in EMT markers (Col1a3, Mmp9, Bmp7, and Ocln) and increased expression of N-Cadherin and Vimentin. S derived PTC demonstrated a decreased ability to uptake FITC-albumin compared to the small congenic in vitro, which was confirmed by assessment of albumin re-uptake in vivo by infusion of FITC-albumin and immunofluorescence imaging. In summary, these studies suggest that genetic variants in the S form of Arhgef11 via increased expression and/or protein activity play a role in promoting kidney injury in the S rat through changes in cell morphology (Rho-Rock and/or EMT) that impact the function of tubule cells.

ERK1/2 Blockade Prevents Epithelial–Mesenchymal Transition in Lung Cancer Cells and Promotes Their Sensitivity to EGFR Inhibition

Overcoming cellular mechanisms of de novo and acquired resistance to drug therapy remains a central challenge in the clinical management of many cancers, including non-small cell lung cancer (NSCLC). Although much work has linked the epithelial-mesenchymal transition (EMT) in cancer cells to the emergence of drug resistance, it is less clear where tractable routes may exist to reverse or inhibit EMT as a strategy for drug sensitization. Here, we demonstrate that extracellular signal-regulated kinase (ERK) 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) signaling plays a key role in directing the mesenchymal character of NSCLC cells and that blocking ERK signaling is sufficient to heighten therapeutic responses to EGF receptor (EGFR) inhibitors. MEK1/2 (MAPKK1/2) inhibition promoted an epithelial phenotype in NSCLC cells, preventing induction of EMT by exogenous TGF-β. Moreover, in cells exhibiting de novo or acquired resistance to the EGFR inhibitor gefitinib, MEK inhibition enhanced the sensitivity to gefitinib and slowed cell migration. These effects only occurred, however, if MEK was inhibited for a period sufficient to trigger changes in EMT marker expression. Consistent with these findings, changes in EMT phenotypes and markers were also induced by the expression of mutant KRAS in a MEK-dependent manner. Our results suggest that prolonged exposure to MEK or ERK inhibitors may not only restrain EMT but also overcome naïve or acquired resistance of NSCLC to EGFR-targeted therapy in the clinic.

Pharmacological inhibition of platelet-tumor cell cross-talk prevents platelet-induced overexpression of cyclooxygenase-2 in HT29 human colon carcinoma cells.

Cyclooxygenase (COX)-2-derived prostanoids can influence several processes that are linked to carcinogenesis. We aimed to address the hypothesis that platelets contribute to aberrant COX-2 expression in HT29 colon carcinoma cells and to reveal the role of platelet-induced COX-2 on the expression of proteins involved in malignancy and marker genes of epithelial-mesenchymal transition (EMT). Human platelets cocultured with HT29 cells rapidly adhered to cancer cells and induced COX-2 mRNA expression, but not protein synthesis, which required the late release of platelet-derived growth factor and COX-2 mRNA stabilization. Platelet-induced COX-2-dependent prostaglandin E2 (PGE2) synthesis in HT29 cells was involved in the downregulation of p21(WAF1/CIP1) and the upregulation of cyclinB1 since these effects were prevented by rofecoxib (a selective COX-2 inhibitor) and rescued by exogenous PGE2. Galectin-3, which is highly expressed in HT29 cells, is unique among galectins because it contains a collagen-like domain. Thus, we studied the role of galectin-3 and platelet collagen receptors in platelet-induced COX-2 overexpression. Inhibitors of galectin-3 function (β-lactose, a dominant-negative form of galectin-3, Gal-3C, and anti-galectin-3 antibody M3/38) or collagen receptor-mediated platelet adhesion (revacept, a dimeric platelet collagen receptor GPVI-Fc) prevented aberrant COX-2 expression. Inhibition of platelet-cancer cell interaction by revacept was more effective than rofecoxib in preventing platelet-induced mRNA changes of EMT markers, suggesting that direct cell-cell contact and aberrant COX-2 expression synergistically induced gene expression modifications associated with EMT. In conclusion, our findings provide the rationale for testing blockers of collagen binding sites, such as revacept, and galectin-3 inhibitors in the prevention of colon cancer metastasis in animal models, followed by studies in patients.

Abstract 5195: Shh/Gli signaling pathway promotes tumor metastasis and lymphangiogenesis via activation of PI3K/Akt and Erk pathway in gastric cancer

Abstract The activation of Sonic hedgehog (Shh)/Gli signaling is involved in the progression and invasion of various tumors types, including gastric carcinoma. Epithelial-mesenchymal transition (EMT) and matrix metalloproteinases (MMPs) have been implicated in facilitating the invasion and metastatsis. Herein, we investigated the impact of phosphoinositide 3-kinase (PI3K)/Akt and Erk pathway and MMPs activity on the Shh/Gli-mediated EMT and invasion of gastric cancer cells. We found that overexpression of Shh significantly enhanced cellular motility/invasiveness, lymphangiogenesis, and metastatic ability of tumor cells in gastric cancer cell lines and mice. Stimulation of Shh in gastric cancer cells also induced a full EMT process (characterized by Snail induction and E-cadherin down-regulation) and expression and activity of MMP-9. Meanwhile, blockade of Shh/Gli signaling by Cyclopamine (a Shh signaling inhibitor), anti-Shh neutralizing antibodies, or Gli siRNA inhibited Shh-induced motility/invasiveness and lymphangiogenesis in gastric cancer by subsequently restoring these changes of EMT markers and activity of MMP-9. The phosphorylation of Akt and Erk was also enhanced by treatment with Shh, but not cyclopamine, anti-Shh neutralizing antibodies, or Gli siRNA. Blockade of the Akt and MEK kinase using kinase-dead Akt, LY294002, or PD98059 significantly inhibited the Shh-induced EMT, activity of MMP-9, lymphangiogenesis, and metastasis in tumor cell lines and mice over-expressing Shh. Furthermore, knock-down of MMP-9 by its siRNA results in an decrease in invasiveness of gastric cancer cells treatment with Shh. Immunohistochemistry on gastric tumor biopsies showed that the levels of Gli, E-cadherin, MMP-9, D2-40 (lymphangiogenic factor), and phospho-Akt expression were enhanced in cases of metastatic gastric cancer than in cases of primary gastric cancer. Moreover, the strong correlation between Gli and E-cadherin, MMP-9, D2-40, phospho-Akt, or phospho-Erk expression was also observed in lymph node metastasis specimens. These data indicate that Shh/Gli signaling pathway promotes tumor metastasis in gastric cancer through activation of PI3K/Akt and Erk pathway followed by up-regulation of EMT, MMP-9, and lymphangiogenesis, which results in the stimulation of invasiveness of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5195.

BMP2 accelerates the motility and invasiveness of gastric cancer cells via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway

Up-regulation of bone morphogenetic proteins (BMPs) and their receptors by tumor is an important hallmark in cancer progression, as it contributes through autocrine and paracrine mechanisms to tumor development, invasion, and metastasis. Generally, increased motility and invasion are positively correlated with the epithelial-mesenchymal transition (EMT). The purpose of the present study was to determine whether BMP-2 signaling to induce gastric cancer cells to undergo EMT-mediated invasion might pass through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Herein we showed that gastric cancer cell lines express all the components of BMP-2 signaling, albeit to different extents. Moreover, an increased concentration of BMP-2 strongly enhanced motility and invasiveness in gastric cancer cells, whereas no increase was observed in cells treated with either Noggin (a BMP-2 inhibitor) or BMP-2 blocking antibodies. The stimulation of BMP-2 in gastric cancer cells induces a full EMT characterized by Snail induction, E-cadherin delocalization and down-regulation, and up-regulation of mesenchymal and invasiveness markers. Furthermore, blockade of BMP-2 signaling by Noggin or BMP-2 blocking antibodies also restored these changes in EMT markers. In addition, phosphorylation of Akt was also enhanced by treatment with BMP-2, but not Noggin or BMP-2 blocking antibodies. Pretreatment of gastric cancer cells with PI-3 kinase/Akt kinase inhibitor (kinase-dead Akt [DN-Akt], Akt siRNA, or LY294002) significantly inhibited BMP-2-induced EMT and invasiveness. Overall, our studies suggest that BMP-2 promotes motility and invasion of gastric cancer cells by activating PI-3 kinase/Akt and that targeting of this signaling pathway may provide therapeutic opportunities in preventing metastasis mediated by BMP-2.