Klotho suppresses tumor progression via inhibiting PI3K/Akt/GSK3β/Snail signaling in renal cell carcinoma

Klotho is an anti‐aging protein predominantly expressed in renal tubular epithelial cells. Although Klotho was recently identified as a tumor suppressor gene in a variety of cancers, the potential role and molecular events for Klotho in renal cell carcinoma (RCC) remain obscure. In the present study, immunohistochemical staining in tissue microarrays containing 125 RCC samples showed that intratumoral Klotho levels were negatively correlated with tumor size, TNM stage and nuclear grade. The overall survival rate of RCC patients with high Klotho expression was significantly higher than that of patients with low Klotho expression. Functional analysis after gain and loss of Klotho expression revealed that Klotho blunted epithelial‐mesenchymal transition and cellular migration and invasion in RCC. Also, no alteration of α‐2,6‐sialidase activity was found after Klotho overexpression in RCC. The molecular signals for this phenomenon involved the Klotho‐mediated inhibition of PI3K/Akt/GSK3β/Snail pathway. Importantly, compared to localized RCC tissues, advanced RCC tissues exhibited low Klotho expression accompanied with high pAkt and Snail expression. These results indicate Klotho acts as a tumor suppressor by inhibiting PI3K/Akt/GSK3β/Snail signaling, thus suppressing epithelial‐mesenchymal transition and tumor migration and invasion during RCC progression. As a result, Klotho might be used as a potential therapy for advanced RCC.     

Hepatitis B virus X protein promotes hepatoma cell invasion and metastasis by stabilizing Snail protein

A high incidence of tumor recurrence and metastasis has been reported in hepatocellular carcinoma (HCC) patients with chronic hepatitis B virus (HBV) infection. Although the pathological relevance and significance of hepatitis B virus X protein (HBx) in HBV‐associated hepatocarcinogenesis attracted much attention in recent years, the role and molecular mechanism for HBx in hepatoma invasion and metastasis remains poorly understood. In the present study, we found that HBx expression could induce epithelial–mesenchymal transition in hepatoma and hepatic cells. This effect was shown due to stabilized Snail protein through activating the phosphatidylinositol 3‐kinase/protein kinase B/glycogen synthase kinase‐3β (PI3K/AKT/GSK‐3β) signal pathway by HBx expression. Functional studies revealed that HBx expression could enhance hepatoma cell migration and invasion in vitro. Moreover, stable HBx expression could also facilitate intrahepatic and distant lung metastasis of HCC in a nude mice tumor metastasis model in vivo. The correlation between increased PI3K/AKT/GSK‐3β signaling with elevated Snail protein level was also observed in HCC tumor tissues with intrahepatic metastasis or chronic HBV infection. These results revealed a novel function of HBx in promoting epithelial–mesenchymal transition through Snail protein stabilization by activating PI3K/AKT/GSK‐3β signaling, thus facilitating tumor invasion and metastasis during HCC progression. This could provide a putative molecular mechanism for tumor recurrence and metastasis in HBV‐associated HCC patients.     

DCLK1 promotes epithelial‐mesenchymal transition via the PI3K/Akt/NF‐κB pathway in colorectal cancer

Double cortin‐like kinase 1 (DCLK1) plays important roles during the epithelial‐mesenchymal transition (EMT) process in human colorectal cancer (CRC). However, the role of DCLK1 in regulating the EMT of CRC is still poorly understood. In this study, we report evidence that DCLK1 acts as a potent oncogene to drive its extremely malignant character of EMT in an NF‐κB‐dependent manner in CRC cells. Mechanistic investigations showed that DCLK1 induced the NF‐κBp65 subunit expression through the PI3K/Akt/Sp1 axis and activated NF‐κBp65 through the PI3K/Akt/IκBα pathway during the EMT of CRC cells. Moreover, we found that silencing the expression of DCLK1 inhibited the invasion and metastasis of CRC cells in vivo. Collectively, our findings identify DCLK1 as a pivotal regulator of an EMT axis in CRC, thus implicating DCLK1 as a potential therapeutic target for CRC metastasis.     

TRAF4 Regulates Migration,Invasion, and Epithelial–Mesenchymal Transition via PI3K/AKT Signaling in Hepatocellular Carcinoma

Overexpression of the tumor necrosis factor receptor-associated factor 4 (TRAF4) has been detected in many cancer types and is considered to foster tumor progression. However, the role of TRAF4 in hepatocellular carcinoma (HCC) remains elusive. In this study, we found that TRAF4 was highly expressed in HCC cell lines and HCC tissues compared with normal liver cell lines and adjacent noncancerous tissues. TRAF4 overexpression in HCC tissues was correlated with tumor quantity and vascular invasion. In vitro studies showed that TRAF4 was associated with HCC cell migration and invasion. An in vivo study verified that TRAF4 overexpression facilitated metastasis in nude mice. In addition, overexpressed TRAF4 promoted the phosphorylation of Akt and induced Slug overexpression, leading to downregulated E-cadherin and upregulated vimentin, while silencing TRAF4 moderated the phosphorylation of Akt and repressed the expression of Slug, which resulted in upregulated E-cadherin and downregulated vimentin. These effects were inversed after pretreatment of the PI3K/Akt inhibitor LY294002 or overexpression of constitutively active Akt1. Our study demonstrated that TRAF4 was involved in promoting HCC cell migration and invasion. The process was induced by the EMT through activation of the PI3K/Akt signaling pathway.     

miR⁃203a⁃3p通过靶向LASP1介导Akt/GSK⁃3β/Snail信号通路调控肝癌细胞生物学行为

目的 研究miR?203a?3p对肝细胞癌（hepatocellular carcinoma,HCC）细胞生物学行为的影响及其相关分子机制。方法 将miR?203a?3p 模拟物（miR?203a?3p mimics）及阴性对照（miR?NC mimics）、LIM和SH3蛋白1（LIM and SH3 protein 1,LASP1）过表达质粒（pcDNA?LASP1）及阴性对照质粒（pcDNA?NC）分别转染至HepG2细胞。以qRT?PCR法检测细胞miR?203a?3p、LASP1 mRNA表达情况,CCK?8法和异体移植瘤实验分析细胞增殖能力,划痕实验检测细胞迁移能力,Transwell小室实验检测细胞侵袭能力,Annexin V?FITC/PI法检测细胞凋亡,双荧光素酶报告基因检测miR?203a?3p与LASP1的靶向关系,Western blot法检测细胞中LASP1、蛋白激酶B（protein kinase B,Akt）、磷酸化Akt（phosphorylated Akt,p?Akt）、糖原合成酶激酶3β（glycogen synthase kinase?3β,GSK?3β）、磷酸化GSK?3β（phosphorylated GSK?3β,p?GSK?3β）、Snail表达情况。结果 与miR?NC组相比,miR?203a?3p组HepG2细胞增殖活性、迁移率、侵袭数目、LASP1 mRNA和蛋白表达量、p?Akt/Akt和p?GSK?3β/GSK?3β比值、Snail蛋白表达量均显著降低,小鼠移植瘤体积和质量显著减少,细胞凋亡率显著升高（均P<0.01）。Targetscan软件预测显示,miR?203a?3p与LASP1 存在靶向关系;与LASP1?Wt+miR?NC组比较,LASP1?Wt+miR?203a?3p组相对荧光素酶活性显著下降（P<0.001）。与miR?203a?3p+pcDNA?NC组比较,miR?203a?3p+LASP1组HepG2细胞增殖活性、迁移率、侵袭数目、p?Akt/Akt和p?GSK?3β/GSK?3β比值、Snail蛋白表达量均显著升高,小鼠移植瘤体积和质量显著增加,细胞凋亡率显著降低（均P<0.01）。结论 miR?203a?3p可能通过靶向抑制LASP1表达调控Akt/GSK?3β/Snail信号通路活性,从而调控HCC细胞生物学行为。     

CK2α通过PI3K/Akt/GSK-3β信号通路调控肺腺癌A549细胞的侵袭及迁移

背景与目的 肺癌已成为全球癌症死亡的首要原因,而侵袭和转移是导致肿瘤死亡的主要原因之一,蛋白激酶CK2是一种高度保守信使非依赖性丝氨酸苏氨酸蛋白激酶,其在各种肿瘤中高表达.本研究旨在探讨下调CK2α基因表达对肺腺癌A549细胞侵袭迁移的影响以及可能的机制.方法 构建pSilencerTM4.1-shCK2α-eGFP慢病毒表达载体,建立稳定干扰CK2α表达的A549细胞株.利用Transwell和Boyden小室实验检测干扰CK2α表达前后A549细胞的侵袭及迁移的能力.Western blot检测PI3K/Akt信号通路和上皮-间充质转化(mesenchymal-to-epithelial transition,EMT)相关蛋白的表达.结果 与对照组相比,干扰CK2α表达后肺腺癌A549细胞的侵袭及迁移能力明显下降,p-PTEN、Akt、p-Akt473、p-Akt308、p-PDK1、p-c-Raf、p-GSK-3p蛋白明显下调,PTEN蛋白表达水平显著上调.上皮-间充质转化的相关蛋白E-cadherin蛋白表达水平显著上调,而Vi-mentin、p-catenin、Snail蛋白表达水平显著下调,与侵袭转移相关蛋白的MMP2、MMP9表达水平显著下调.结论 CK2α可能通过PI3K/Akt/GSK-3p/Snail信号通路来调控上皮-间充质转化参与肺腺癌A549细胞的侵袭及迁移.     

XPA serves as an autophagy and apoptosis inducer by suppressing hepatocellular carcinoma in a PI3K/Akt/mTOR dependent manner

BackgroundTo explore the potential biological function of XPA (Xeroderma pigmentosum group A) in hepatic neoplasms and the underlying molecular mechanisms.MethodsLiver cells were used as experimental models to establish HCC (hepatocellular carcinoma) in vitro. Protein extractions were subjected to Western blotting to detect the proteins expression. The lentivirus transfection efficiency was confirmed by Western blot and RT-qPCR, Tunnel staining was used to detect apoptosis, and Transwell assays were used to observe cell migration and invasion. Cell proliferation was detected with colony formation and CCK-8 (cell counting kit-8) assays.ResultsXPA expression was obviously lower in HCC tissue and liver cancer cell lines. XPA overexpression induced autophagy and apoptosis by increasing LC3B II/I, Beclin1, cleaved-caspase-3, and Bax expression and decreasing p62 and Bcl2 protein levels. XPA also suppressed HCC EMT (Epithelial-Mesenchymal Transition) by increasing E-cadherin and decreasing N-cadherin and vimentin protein expression. Cell proliferation, migration and invasion in vivo were significantly inhibited by the overexpression of XPA, and p-PI3K, p-Akt, and p-mTOR expression were decreased in LV-XPA cells. In general, XPA inhibited HCC by inducing autophagy and apoptosis and by modulating the expression of PI3K/Akt/mTOR proteins.ConclusionsXPA overexpression was found to suppress HCC by inducing autophagy and apoptosis and repressing EMT and proliferation. Each of these effects may be involved in modulating the PI3K/Akt/mTOR signaling pathway.     

Radiation-enhanced hepatocellular carcinoma cell invasion with MMP-9 expression through PI3K/Akt/NF-kappaB signal transduction pathway

This study is to investigate the molecular mechanism of radiation-enhanced cell invasiveness of hepatocellular carcinoma (HCC) correlating with clinical patients undergoing radiotherapy and subsequently developing metastasis. Three HCC cell lines (HepG2, Hep3B and Huh7) and normal hepatocyte cell line (CL-48) were irradiated with different doses. The effect of radiation on cell invasiveness was determined using the Boyden chamber assay. Radiation-enhanced invasion capability was evident in HCC cells but not in normal hepatocytes. Invasion was observed in gelatin-coated but not fibronectin-coated or type I collagen-coated membranes. Radiation upregulated matrix metalloproteinase-9 (MMP-9) mRNA level, MMP-9 protein level and MMP-9 activity. MMP-9 antisense oligonucleotides inhibited radiation-induced MMP-9 expression and thereby significantly inhibited radiation-induced HCC invasion. Furthermore, phosphatidylinositol 3-kinase (PI3K)/Akt chemical inhibitors LY294002 and wortmannin suppressed radiation-induced MMP-9 mRNA expression. Transient transfection with dominant-negative Akt plasmid also showed that the PI3K/Akt-signaling pathway was involved in this radiation-induced MMP-9 expression. Moreover, nuclear factor-kappaB (NF-kappaB) decoy oligodeoxynucleotide suppressed radiation enhanced MMP-9 promoter activity completely. PI3K/Akt chemical inhibitors inhibited radiation-induced NF-kappaB-driven luciferase promoter activity. Taken together, our results indicated that sublethal dose of radiation could enhance HCC cell invasiveness by MMP-9 expression through the PI3K/Akt/NF-kappaB signal transduction pathway.     

miR‐203 inhibits augmented proliferation and metastasis of hepatocellular carcinoma residual in the promoted regenerating liver

Liver resection is still the most commonly used therapeutic treatment for hepatocellular carcinoma (HCC), and liver regeneration promotes HCC growth in the regenerating liver. The high recurrence/metastasis of HCC is the main cause of death for HCC patients after liver resection. However, how the augmented growth and metastasis of residual HCC induced by the promoted liver regeneration following liver resection can be abolished remains unclear. In this study, a rat model with liver cirrhosis and diffused HCC was established by administration of diethylnitrosamine. Recombinant miR‐203 adenovirus was administered to induce hepatic miR‐203 overexpression and 30% partial hepatectomy (PH) followed. The effect of miR‐203 on the proliferation, invasion and metastasis of the residual HCC in the remnant cirrhotic liver with promoted regeneration was investigated. We found that the basic spontaneous regeneration of the non‐tumorous liver by 30% PH promoted proliferation, invasion and lung metastasis of the hepatic residual HCC. miR‐203 overexpression further promoted the regeneration of the non‐tumorous liver by upregulating Ki67 expression and enhancing IL‐6/SOCS3/STAT3 pro‐proliferative signals. Importantly, miR‐203 overexpression markedly inhibited the proliferation, invasion and metastasis of hepatic residual HCC through suppressing expression of Ki67, CAPNS1 and lung metastasis. Moreover, it was found that miR‐203 overexpression reversed the epithelial–mesenchymal transition induced by hepatectomy through targeting IL‐1β, Snail1 and Twist1. In conclusion, our results suggested that miR‐203 overexpression inhibited the augmented proliferation and lung metastasis of the residual HCC induced by the promoted liver regeneration following PH partly by regulating epithelial–mesenchymal transition.     

Inhibition of histamine receptor 3 suppresses glioblastoma tumor growth,invasion, and epithelial-to-mesenchymal transition

Histamine receptor 3 (H3R) is expressed in various tumors and correlated with malignancy and tumor proliferation. However, the role of H3R in tumor invasion and epithelial to mesenchymal transition (EMT) remains unknown. Here, we explored the H3R in the highly invasive glioblastoma (GBM) and U87MG cells. We found that H3R mRNA and protein levels were up-regulated in the GBM and glioma cell lines compared to normal brain tissue and astrocytes. In U87MG cell line, inhibition of H3R by siRNA or the antagonist ciproxifan (CPX) suppressed proliferation, invasiveness, and the expression of EMT activators (Snail, Slug and Twist). In addition, expression of epithelial markers (E-cadherin and ZO-1) was up-regulated and expression of mesenchymal markers (vimentin and N-cadherin) was down-regulated in vitro and in vivo in a xenograft model. In addition, we also showed that inhibition of H3R by siRNA or CPX inactivated the PI3K/Akt and MEK/ERK signaling pathways, while inhibition of Akt or ERK activity with antagonists or siRNAs suppressed H3R agonist (R)-(α)-(−)- methylhistamine dihydrobromide (RAMH) mediated invasion and reorganization of cadherin-household. In conclusion, overexpression of H3R is associated with glioma progression. Inhibition of H3R leads to suppressed invasion and EMT of GBM by inactivating the PI3K/Akt and MEK/ERK pathways in gliomas.     

Platelet‐type 12‐lipoxygenase induces MMP9 expression and cellular invasion via activation of PI3K/Akt/NF‐κB

Prostate cancer is the most frequently diagnosed cancer and the second leading cause of death in males in the United States. Using human prostate cancer specimens, the authors have previously shown that elevated expression levels of 12‐lipoxygenase (12‐LOX) occurred more frequently in advanced stage, high‐grade prostate cancer, suggesting that 12‐LOX expression is associated with carcinoma progression and invasion. Previous reports from their group and others have shown that 12‐LOX is a positive modulator of invasion and metastasis; however, the mechanism remains unclear. In this work, a new link between 12‐LOX and the matrix metalloproteinase 9 (MMP9) in prostate cancer angiogenesis is reported. This study demonstrated that overexpression of 12‐LOX in prostate cancer PC‐3 cells resulted in elevated expression of MMP9 mRNA, protein and secretion. Exogenous addition of 12(S)‐hydroxy eicosatetraenoic acid, the sole and stable end product of arachidonic acid metabolism by 12‐LOX, is able to increase MMP9 expression in wild‐type PC‐3 cells. Furthermore, using pharmacological and genetic inhibition approaches, it was found that 12‐LOX activates phosphoinositol 3 kinase (PI3K)/Akt, which results in nuclear factor‐kappa B (NF‐κB)‐driven MMP9 expression, ensuing in enhanced chemoattraction of endothelial cells. Specific inhibitors of 12‐LOX, PI3K or NF‐κB inhibited MMP9 expression in 12‐LOX‐expressing PC‐3 cells and resulted in the blockade of the migratory ability of endothelial cells. In summary, the authors have identified a new pathway by which overexpression of 12‐LOX in prostate cancer cells leads to augmented production of MMP9 via activation of PI3K/Akt/NF‐κB signaling. The role of 12‐LOX‐mediated MMP9 secretion in endothelial cell migration may account for the proangiogenic function of 12‐LOX in prostate cancer.     

Human immunodeficiency virus Tat‐TIP30 interaction promotes metastasis by enhancing the nuclear translocation of Snail in lung cancer cell lines

Lung cancer patients with human immunodeficiency virus (HIV) have a poorer prognosis than do patients without HIV infection. HIV1 Tat is a secreted viral protein that penetrates the plasma membrane and interacts with a number of proteins in non‐HIV‐infected cells. The loss of function of Tat‐interacting protein 30 (TIP30) has been linked to metastasis in non‐small cell lung cancer (NSCLC). However, it is unknown how the interaction of HIV1 Tat with TIP30 regulates the metastasis of NSCLC cells. In this study, the overexpression of TIP30 decreased tumor growth factor‐β‐induced epithelial‐to‐mesenchymal transition (EMT) and invasion of NSCLC cells, whereas the knockdown of TIP30 promoted EMT, invasion and stemness. Exposure to recombinant HIV1 Tat proteins promoted EMT and invasion. A mechanistic study showed that the interaction of HIV1 Tat with TIP30 blocked the binding of TIP30 to importin‐β, which is required for the nuclear translocation of Snail. Indeed, the loss of TIP30 promoted the nuclear translocation of Snail. In vivo studies demonstrated that the overexpression of TIP30 inhibited the metastasis of NSCLC cells. In contrast, the coexpression of HIV1 Tat and TIP30 diminished the inhibitory effect of TIP30 on metastasis. Immunohistochemistry confirmed that TIP30 overexpression reduced the nuclear localization of Snail, whereas the coexpression of HIV1 Tat and TIP30 increased nuclear Snail in metastatic tumors. In conclusion, the binding of HIV1 Tat to TIP30 enhanced EMT and metastasis by regulating the nuclear translocation of Snail. Targeting Tat‐interacting proteins may be a potential therapeutic strategy to prevent metastasis in NSCLC patients with HIV infection.