Inhibition of mTOR pathway attenuates migration and invasion of gallbladder cancer via EMT inhibition

Gallbladder cancer (GBC) is an aggressive disease in which epithelial-mesenchymal transition (EMT) plays a critical role. Whether inhibition of mTOR effects via EMT reversal in GBC remains unclear. Using genetic and pharmacologic inhibitions of mTOR, we investigated the changes of EMT levels in GBC cells. Expressions of EMT related genes were also studied. Migration and invasion assays were carried out and in vivo tumour metastasis mouse models were established. Circulating tumour DNA was quantified. We used EMT index (ratio of Vimentin/Ecadherin expression) to profile EMT levels. We found that inhibition of mTOR using shRNAs and rapamycin inhibited EMT in GBC-SD gallbladder cancer cells. Inhibition of mTOR inhibited EMT in GBC-SD cells in TGF-β-dependent manner, which was contributed majorly by mTORC2 inhibition. Rapamycin decreased invasiveness and migration of GBC-SD cells in vitro and in vivo. We have in the current study shown that rapamycin diminishes the ability of invasion and migration of GBC via inhibition of TGF-β-dependent EMT. Our findings contribute to the understanding of the carcinogenesis of GBC.     

Knockdown of Long Non-coding RNA HOTAIR Suppresses Tumor Invasion and Reverses Epithelial-mesenchymal Transition in Gastric Cancer

Background: Over-expression of long non-coding RNA HOTAIR has been reported in several types of cancer. Yet its involvement in gastric cancer (GC) has not been well understood. The aim of present study was to examine the expression pattern of HOTAIR in GC patients, then, explore its role in promoting cancer invasion and underlying molecular mechanism. Methods: The expression level of HOTAIR in the tumor specimens of GC patients was quantified by Realtime RT-PCR. The correlation between HOTAIR level and clinicopathological factors as well as prognosis was then examined. Down-regulation of HOTAIR by RNA interference was applied to investigate its roles in tumor invasiveness via the view of Epithelial-to-mesenchymal transition (EMT). Results: The expression level of HOTAIR in cancer tissues was higher than that in adjacent noncancerous tissues. Expression level of HOTAIR was significantly correlated with lymph node metastasis and TNM stage. Furthermore, high expression level of HOTAIR was a predictor of poor over-all survival in GC patients. In vitro, inhibition of HOTAIR in GC cells could reduce invasiveness, as well as the expression of MMP1 and MMP3. In addition, suppression of HOTAIR could reverse EMT process. Conclusions: HOTAIR could act as a potential predictor for over-all survival in patients with GC. Inhibition of HOTAIR could reduce invasiveness and reverse EMT process in GC cells, indicating the potential role of HOTAIR in GC diagnostics and therapeutics.     

MMP2/3 promote the growth and migration of laryngeal squamous cell carcinoma via PI3K/Akt-NF-κB-mediated epithelial–mesenchymal transformation

Laryngeal squamous cell carcinoma (LSCC) is an aggressive malignancy with metastatic potential and high mortality worldwide. Matrix metalloproteinases (MMPs) are a group of extracellular proteolytic enzymes. Although MMPs have been proved to be essential in the process of tumor migration and metastasis in most types of carcinomas, the expression and function of MMP2/3 in LSCC remain unknown. Here, we investigated the roles of MMP2/3 in LSCC and elucidated the underlying mechanism. We found that levels of MMP2/3 were significantly higher in clinical LSCC samples than in paired normal sample examined by real-time polymerase chain reaction and western blot assays. Moreover, overexpression of MMP2/3 promoted the proliferation and migration of LSCC cells by Cell Counting Kit-8, transwell, and scratch wound-healing assays in vitro. Furthermore, levels of epithelial cell markers (E-cadherin and occludin) were decreased following MMP2/3 overexpression, with increased levels of mesenchymal cell markers (N-cadherin and vimentin), suggesting the involvement of epithelial–mesenchymal transformation (EMT) process in MMP2/3-mediated LSCC cell migration. By using short hairpin RNA, knockdown of MMP2/3 expression inhibited the proliferation, migration, and EMT process in LSCC cells in vitro. More importantly, we confirmed that MMP2/3 promoted LSCC in the PI3K/Akt-NF-κB-dependent manner. This study provides insight into MMP2/3-mediated LSCC development and lays a foundation for potential pharmacological targets to LSCC.     

PCBP1 is an important mediator of TGF-β-induced epithelial to mesenchymal transition in gall bladder cancer cell line GBC-SD

Gall bladder carcinoma (GBC) is the seventh most common cancer across the globe and the most common malignancy of the biliary tract. Most GBC related deaths occur due to secondary progression and metastasis to distant organs. Epithelial–mesenchymal transition (EMT) is an important pre-requisite for tumor metastasis, however its mechanism in GBC has not yet been defined. Using the GBC-SD cell line, we have uncovered an important mediator, poly r(C) binding protein-1 (PCBP1), of transforming growth factor-beta (TGF-β)-induced EMT in GBC. Our results show that TGF-β treatment resulted in PCBP1 phosphorylation in accordance with similar observation in other model systems. We further showed through gain- and loss-of-function assays that PCBP1 expression levels regulate the capacity of GBC-SD cells to migrate and invade in vitro. Finally, our results showed that PCBP1 expression levels also regulate generation of CD44⁺CD24^? progenitor cell population in GBC-SD cells after TGF-β treatment. Cumulatively, our results indicate, pending further validation, that PCBP1 might be a prognostic marker for GBC metastasis.     

The microRNA cluster miR-214/miR-3120 prevents tumor cell switching from an epithelial to a mesenchymal-like phenotype and inhibits autophagy in gallbladder cancer

Tumor cells switch from an epithelial to a mesenchymal-like phenotype, which represents a key hallmark of human cancer metastasis, including gallbladder cancer (GBC). A large set of microRNAs (miRNAs/miRs) have been studied to elucidate their functions in initiating or inhibiting this phenotypic switching in GBC cells. In this paper, we attempted to identify the expression pattern of the miR-214/−3120 cluster and its mode of action in the context of GBC, with a specific focus being placed on their effects on EMT and autophagy in GBC cells. Human GBC cells GBC-SD were assayed for their migration, invasion, and autophagy using the Transwell chamber system, MDC staining, and transmission electron microscopy. The tumorigenicity and metastatic behavior of GBC-SD cells were tested in nude mice. The expression of EMT- and autophagy-specific markers (E-cadherin, N-cadherin, vimentin, ATG5, LC3II/LC3I, and Beclin1) was analyzed in cultured GBC-SD cells and in human GBC-SD xenografts. The E2F3 luciferase reporter activity in the presence of miR-214/−3120 was evaluated by a dual luciferase assay. The miR-214/−3120 was downregulated in GBC. Exogenous miR-214/−3120 inhibited the phenotypic switching of GBC cells from epithelial to mesenchymal, prevented autophagy, and suppressed the tumorigenicity and metastatic behavior of GBC-SD cells in vitro and in vivo. E2F3 was demonstrated to be the target gene of miR-214/−3120, and its knockdown in part mimicked the effect of miR-214/−3120 on the EMT, autophagy, tumorigenicity, and metastatic behavior of GBC-SD cells. These results demonstrated that the miR-214/−3120 cluster blocks the process of EMT and autophagy to limit GBC metastasis by repressing E2F3 expression.     

Oxymatrine induces apoptosis and inhibits invasion in Gallbladder carcinoma via PTEN/PI3K/AKT pathway

Oxymatrine extracted from Sophora flavescens Ait as a natural polyphenolic phytochemical has been demonstrated to exhibit anti-tumor effects on various cancers, including Gallbladder carcinoma (GBC). However, its underlying mechanisms of function are largely unknown in GBC cells. The present study is conducted to investigate the anti-tumor effects and the underlying mechanisms of oxymatrine on GBC cells in vitro and in vivo. The results showed that oxymatrine inhibited cell viability, metastatic ability and induced cell apoptosis in dose-dependent manners. Furthermore, we found that the expression of p-AKT, MMP-2, MMP-9 and the ratio of Bcl-2/Bax were significantly down-regulated, while the expression of PTEN was up-regulated in GBC cells. In addition, pretreatment with a specific PI3K/AKT activator (IGF-1) significantly antagonized the oxymatrine-mediated inhibition of GBC–SD cells. Subsequently, our in vivo studies showed that administration of oxymatrine induced a significant dose-dependent decrease in tumor growth. In conclusion, these findings indicated that the inhibition of cells proliferation, migration, invasion and the induction of apoptosis in response to oxymatrine in GBC cells, may function through the suppression of PTEN/PI3K/AKT pathway, which was considered as the vital signaling pathway in regulating tumorigenesis. These results suggested that oxymatrine might be a novel effective candidate as chemotherapeutic agent against GBC.     

TIPE1 suppresses invasion and migration through down‐regulating Wnt/β‐catenin pathway in gastric cancer

Epithelial–mesenchymal transition (EMT) plays an important role in the invasiveness and metastasis of gastric cancer. Therefore, identifying key molecules involved in EMT will provide new therapeutic strategy for treating patients with gastric cancer. TIPE1 is a newly identified member of the TIPE (TNFAIP8) family, and its contributions to progression and metastasis have not been evaluated. In this study, we found that the levels of TIPE1 were significantly reduced and inversely correlated with differentiation status and distant metastasis in primary gastric cancer tissues. We further observed overexpression of TIPE1 in aggressive gastric cancer cell lines decreased their metastatic properties both in vitro and in vivo as demonstrated by markedly inhibiting EMT and metastasis of gastric cancer cells in nude mice. Consistently, gene silencing of TIPE1 in well‐differentiated gastric cancer cell line (AGS) inhibited these processes. Mechanistically, we found that TIPE1‐medicated Wnt/β‐catenin signalling was one of the critical signal transduction pathways that link TIPE1 to EMT inhibition. Importantly, TIPE1 dramatically restrained the expression and activities of MMP2 and MMP9 which are demonstrated to promote tumour progression and are implicated in EMT. Collectively, these findings provide new evidence for a better understanding of the biological activities of TIPE1 in progression and metastasis of gastric cancer and suggest that TIPE1 may be an innovative diagnostic and therapeutic target of gastric cancer.     

NLRP3 inflammasome inhibition attenuates silica-induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells

Silicosis is an incurable and progressive lung disease characterized by chronic inflammation and fibroblasts accumulation. Studies have indicated a vital role for epithelial-mesenchymal transition (EMT) in fibroblasts accumulation. NLRP3 inflammasome is a critical mediator of inflammation in response to a wide range of stimuli (including silica particles), and plays an important role in many respiratory diseases. However, whether NLRP3 inflammasome regulates silica-induced EMT remains unknown. Our results showed that silica induced EMT in human bronchial epithelial cells (16HBE cells) in a dose- and time-dependent manner. Meanwhile, silica persistently activated NLRP3 inflammasome as indicated by continuously elevated extracellular levels of interleukin-1β (IL-1β) and IL-18. NLRP3 inflammasome inhibition by short hairpin RNA (shRNA)-mediated knockdown of NLRP3, selective inhibitor MCC950, and caspase-1 inhibitor Z-YVAD-FMK attenuated silica-induced EMT. Western blot analysis indicated that TAK1-MAPK-Snail/NF-κB pathway involved NLRP3 inflammasome-mediated EMT. Moreover, pirfenidone, a commercially and clinically available drug approved for treating idiopathic pulmonary fibrosis (IPF), effectively suppressed silica-induced EMT of 16HBE cells in line with NLRP3 inflammasome inhibition. Collectively, our results indicate that NLRP3 inflammasome is a promising target for blocking or retarding EMT-mediated fibrosis in pulmonary silicosis. On basis of this mechanism, pirfenidone might be a potential drug for the treatment of silicosis.     

NLK is a key regulator of proliferation and migration in gallbladder carcinoma cells

Gallbladder cancer (GBC) is one of the most lethal neoplasm and is the fifth most common malignancy of gastrointestinal tract. The prognosis of gallbladder cancer is extremely terrible partially due to metastasis. Thus, understanding the molecular pathways controlling metastasis of this lethal disease may provide new targets for targeted therapeutic approach. In this study, we investigated the function of nemo-like kinase (NLK) in GBC growth and migration. Lentivirus-mediated siRNA was employed to alleviate the expression level of NLK in GBC cell lines (GBC-SD and SGC-996). Real-time PCR and western-blot analysis demonstrated that both mRNA and protein levels of NLK in GBC-SD and SGC-996 cells were decreased after infection with NLK-siRNA-expressing lentivirus (Lv-shNLK). The proliferation and in vitro tumorigenesis (colony formation) ability as well as migration of GBC-SD and SGC-996 cells with low NLK expression decreased significantly. Our results suggested that NLK is a key regulator involved in proliferation and migration of GBC, and it could be used as a potential therapeutic target for GBC.     

Scutellarin inhibits proliferation and invasion of hepatocellular carcinoma cells via down‐regulation of JAK2/STAT3 pathway

The prognosis of hepatocellular carcinoma (HCC) is poor because of high incidence of recurrence and metastasis. JAK/STAT signalling pathway regulates cell proliferation, apoptosis, differentiation and migration and epithelial‐mesenchymal transition (EMT) is also considered to contribute to invasion and metastasis of epithelial malignant tumours. Scutellarin is an active component found in many traditional Chinese herbs and has been regularly used in anti‐inflammatory and antitumour medicine. This study aimed to identify the effect of scutellarin and its possible mechanism of action in HCC cells. Proliferation, colony‐forming, apoptosis and cell migration assays were used to examine the effect of scutellarin on HCC cells. Quantitative real‐time PCR and Western blotting were performed to study the molecular mechanisms of action of scutellarin. Light and electron microscopy and immunofluorescence analysis were performed to study the effect of scutellarin on cellular mechanics. We show that scutellarin potentially suppresses invasiveness of HepG2 and MHCC97‐H cells in vitro by remodelling their cytoskeleton. The molecular mechanism behind it might be the inhibition of the EMT process, which could be attributed to the down‐regulation of the JAK2/STAT3 pathway. These findings may provide new clinical ideas for the treatment of liver cancer.     

OLFM4 depletion sensitizes gallbladder cancer cells to cisplatin through the ARL6IP1/caspase-3 axis

BackgroundGallbladder cancer (GBC) is a highly lethal malignancy that carries an extremely poor prognosis due to its chemoresistant nature. Cisplatin (CDDP) is a first-line chemotherapeutic for GBC; however, patients experienced no benefit when treated with CDDP alone. The underlying mechanisms of CDDP resistance in GBC remain largely unknown.MethodsAgilent mRNA microarray analysis was performed between paired GBC and paracarcinoma to explore differentially expressed genes that might underlie drug resistance. Gene Set Enrichment Analysis (GSEA) was employed to identify key genes mediating CDDP resistance in GBC, and immunohistochemistry was performed to validate protein expression and test correlations with clinicopathological features. In vitro and in vivo functional assays were performed to investigate the proteins’ roles in CDDP resistance.ResultsOlfactomedin 4 (OLFM4) was differentially expressed between GBC and paracarcinoma and had the highest rank metric score in the GSEA. OLFM4 expression was increasingly upregulated from chronic cholecystitis to GBC in clinical tissue samples, and OLFM4 depletion decreased GBC cell proliferation and invasion. Interestingly, downregulation of OLFM4 reduced ARL6IP1 (antiapoptotic factor) expression and sensitized GBC cells to CDDP both in vitro and in vivo. The evidence indicated that CDDP could significantly increase Bax and Bad expression and activate caspase-3 cascade in OLFM4-depleted GBC cells through ARL6IP1. Clinically, lower OLFM4 expression was associated with good prognosis of GBC patients.ConclusionsOur results suggest that OLFM4 is an essential gene that contributes to GBC chemoresistance and could serve as a prognostic biomarker for GBC. Importantly, OLFM4 could be a potential chemotherapeutic target.     

新型精胺氧化酶抑制剂SI-4650对人卵巢癌SKVO-3细胞增殖和上皮细胞间质化的影响*

目的: 研究新型精胺氧化酶(SMO)小分子抑制剂SI-4650对人卵巢癌SKVO-3细胞增殖和上皮细胞间质化影响及其分子机制。方法: 体外培养SKVO-3细胞,以未加药作为对照组,30、60 μmol/L SI-4650处理48 h细胞为实验组,每组设3个复孔,检测SI-4650对SKVO-3细胞内SMO的酶活性及多胺含量、酶促反应产物活性氧水平的影响,对细胞增值、周期、线粒体膜电位的作用,以及对细胞凋亡、侵袭和迁移能力的影响,同时检测凋亡相关蛋白Bax、Bcl-2、Caspase3以及上皮细胞间质化(EMT)相关蛋白E-Cad、N-Cad、Vimentin、MMP-2、MMP-9表达。结果: 与对照组相比,实验组SKVO-3细胞中SI-4650浓度增加,明显抑制SKVO-3细胞内SMO酶活性(P＜0.01)、减少SMO酶促反应产物活性氧水平(P＜0.01)、降低细胞内总多胺含量(P＜0.01)。SI-4650高效抑制SKVO-3细胞生长(P＜0.01),实验组细胞生长抑制率分别为32.27%、47.31%;将SKVO-3细胞阻滞在S期(P＜0.01),实验组细胞 Bax表达和Caspase3活化剪切增加,Bcl-2表达减少,线粒体膜电位下降,凋亡细胞比率增加至31.41%、43.51%;同时,实验组E-cad表达显著增加,N-cad、Vimentin表达均明显减少,合成分泌MMP-2、MMP-9减少,干扰了SKVO3细胞EMT进程,降低肿瘤细胞侵袭、迁移能力(P＜0.01)。结论: SI-4650对人卵巢癌SKVO-3 细胞具有杀伤和抑制肿瘤细胞侵袭、迁移的抗肿瘤活性,其机制可能与干扰多胺代谢、诱导细胞S周期阻滞和线粒体途径细胞凋亡以及干扰细胞EMT进程相关。     

FGF9/FGFR1 promotes cell proliferation,epithelial-mesenchymal transition,M2 macrophage infiltration and liver metastasis of lung cancer

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis.     

Role of Bmi-1 in Regulation of Ionizing Irradiation-Induced Epithelial-Mesenchymal Transition and Migration of Breast Cancer Cells

Radiotherapy is a widely used treatment for cancer. However, recent studies suggest that ionizing radiation (IR) can promote tumor invasion and metastasis. Bmi-1, a member of the polycomb group protein family, has been observed as a regulator of oxidative stress and promotes metastasis in some tumors. But, its potential role in the metastasis induced by IR of breast cancer has not been explored. In our study, we found that increased levels of Bmi-1 were correlated to EMT of breast cancer cells. Through analyzing the EMT state and metastasis of breast cancer induced by IR, we found the metastatic potential of breast cancer cells can either be inhibited or accelerated by IR following a time-dependent pattern. Silencing Bmi-1 completely abolished the ability of the IR to alter, reduce or increase, the migration of breast cancer cells. Also, when Bmi-1 was knocked down, the effect of inhibition of PI3K/AKT signaling on EMT affected by IR was blocked. These results suggest that Bmi-1 is a key gene in regulation of EMT and migration of breast cancer cells induced by IR through activation of PI3K/AKT signaling; therefore, Bmi-1 could be a new target for inhibiting metastasis caused by IR.     

Snail is required for transforming growth factor-beta-induced epithelial-mesenchymal transition by activating PI3 kinase/Akt signal pathway

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. We have previously shown that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and more specifically, on signaling via Smad3. In this report, we suggest phosphatidylinositol 3-OH kinase (PI3K)/Akt signaling is also necessary for TGF-beta-induced EMT in lens epithelial cells by showing that LY294002, an inhibitor of the p110 catalytic subunit of PI3K, blocked the expression of alpha-smooth muscle actin (alpha-SMA) and morphological changes. We also identify Snail as an effector of TGF-beta-induced EMT. Snail has been shown to be a mediator of EMT during metastasis of cancer. We show that Snail is an immediate-early response gene for TGF-beta and the proximal Snail promoter is activated by TGF-beta through the action of Smad2, 3, and 4. We show that antisense inhibition of Snail expression blocks TGF-beta-induced EMT and furthermore Akt activation. All of these findings suggest that Snail participates in TGF-beta-induced EMT by acting upstream of Akt activation.     

激活FXR抑制结肠癌细胞浸润转移及MMP-7的表达

目的:探讨法尼酯X受体(FXR)特异性激动剂GW4064抑制结肠癌细胞浸润转移的机制。方法:在体外培养人结肠癌细胞HT-29,应用GW4064作用于结肠癌细胞,以四唑氮蓝还原法(MTT)检测细胞活性的变化。用transwell小室研究结肠癌细胞的迁移及浸润。用RT-PCR检测FXRm RNA及MMP-7mRNA表达的变化,用western blot检测FXR及MMP-7蛋白表达的变化。结果:MTT结果显示GW4064作用于人结直肠HT-29细胞的生长抑制率呈浓度依赖性;transwell小室结果显示GW4064抑制结肠癌细胞的浸润转移,与对照组相比,差异具有统计学意义(P0.05),RT-PCR及Western blot显示GW4064促进FXR m RNA及蛋白表达,抑制MMP-7mRNA及蛋白的表达,与对照组相比差异有统计学意义(P0.05)。结论:GW4064抑制结肠癌细胞的生长及转移,上调HT-29细胞FXR m RNA及蛋白的表达,下调HT-29细胞MMP-7 m RNA及蛋白的表达。FXR被激活后抑制结肠癌细胞转移,MMP-7可能是其作用通路之一。