Downregulation of integrin-linked kinase inhibits epithelial-to-mesenchymal transition and metastasis in bladder cancer cells

Integrin-linked kinase (ILK) is a multifunctional serine/threonine kinase in cytoplasm. Recent studies showed that cancer patients with increased ILK expression had low survival, poor prognosis and increased metastasis. Although the causes of ILK overexpression remain to be fully elucidated, accumulating evidence suggests that its oncogenic capacity derives from its regulation of several downstream targets that provide cells with signals that promote proliferation, survival and migration. However, the mechanisms underlying tumor metastasis by ILK is still not fully understood. Epithelial–mesenchymal transition (EMT) is a critical event of cancer cells that triggers invasion and metastasis. We recently reported that knockdown of ILK inhibited the growth and induced apoptosis in human bladder cancer cells. Therefore, we postulate that ILK might involve in EMT. Here we further investigate the function of ILK with RNA interference in bladder cancer cells. Knockdown of ILK impeded an EMT with low Vimentin, Snail, Slug and Twist as well as high E-cadherin expression in vivo and vitro. In addition, we found that knockdown of ILK inhibited cell proliferation, migration and invasion as well as changed cell morphology, adhesion and rearranged cytoskeleton in vitro. We also demonstrated that ILK siRNA inhibited phosphorylation of downstream signaling targets Akt and GSK3β, increased expression of nm23-H1, as well as reduced expression of MMP-2 and MMP-9 in vivo and vitro. Furthermore, downregulation of ILK could increase expression of Ribonuclease inhibitor (RI), an important acidic cytoplasmic protein with many functions. Finally, the effects of ILK siRNA on bladder cancer cell phenotype and invasiveness translate into suppression for tumorigenesis and metastasis in vivo. Taken together, our findings highlight that ILK signaling pathway plays a novel role in the development of bladder cancer through regulating EMT. ILK could be a promising diagnostic marker and therapeutic target for bladder cancer.     

Starvation-induced autophagy promotes the invasion and migration of human bladder cancer cells via TGF-β1/Smad3-mediated epithelial-mesenchymal transition activation

The biological characteristics of bladder cancer include enhanced invasion and migration, which are the main causes of death in patients. Starvation is a typical feature of the bladder cancer microenvironment and can induce autophagy. Autophagy has an important relationship with the invasion and migration of tumors. However, the role of autophagy in the invasion and migration of bladder cancer cells remains unclear. Hence, the aim of the current study was to clarify this role and underlying mechanism. In this study, we found that starvation enhanced the epithelial-mesenchymal transition (EMT)-mediated invasion and migration of T24 and 5637 cells while inducing autophagy. The inhibition of autophagy with chloroquine (CQ) or 3-methyladenine (3MA) decreased EMT-mediated invasion and migration. In addition, the expression of transforming growth factor 1 (TGF-β1) and phosphorylated Smad3 (p-Smad3) increased after starvation. The inhibition of autophagy with CQ or 3MA also decreased the expression of TGF-β1 and p-Smad3. The inhibitor of TGF-β receptor sb431542 also inhibited the invasion, migration, and EMT of T24 and 5637 cells during starvation. Furthermore, recombinant TGF-β1 induced autophagy and inhibition of the TGF-β/Smad signaling pathway with sb431542 suppressed autophagy. In summary, our results suggested that autophagy promotes the invasion and migration of bladder cancer cells by inducing EMT through the TGF-β1/Smad3 signaling pathway. Moreover, autophagy and TGF-β1 can form a positive feedback loop to synergistically promote invasion and migration. Thus, our findings may provide a theoretical basis for the prevention of invasion and migration in bladder cancer.     

Butein, a tetrahydroxychalcone, inhibits nuclear factor (NF)-kappaB and NF-kappaB-regulated gene expression through direct inhibition of IkappaBalpha kinase beta on cysteine 179 residue

Although butein (3,4,2',4'-tetrahydroxychalcone) is known to exhibit anti-inflammatory, anti-cancer, and anti-fibrogenic activities, very little is known about its mechanism of action. Because numerous effects modulated by butein can be linked to interference with the NF-kappaB pathway, we investigated in detail the effect of this chalcone on NF-kappaB activity. As examined by DNA binding, we found that butein suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation in a dose- and time-dependent manner; suppressed the NF-kappaB activation induced by various inflammatory agents and carcinogens; and inhibited the NF-kappaB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-beta. We also found that butein blocked the phosphorylation and degradation of IkappaBalpha by inhibiting IkappaBalpha kinase (IKK) activation. We found the inactivation of IKK by butein was direct and involved cysteine residue 179. This correlated with the suppression of phosphorylation and the nuclear translocation of p65. In this study, butein also inhibited the expression of the NF-kappaB-regulated gene products involved in anti-apoptosis (IAP2, Bcl-2, and Bcl-xL), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). Suppression of these gene products correlated with enhancement of the apoptosis induced by TNF and chemotherapeutic agents; and inhibition of cytokine-induced cellular invasion. Overall, our results indicated that antitumor and anti-inflammatory activities previously assigned to butein may be mediated in part through the direct inhibition of IKK, leading to the suppression of the NF-kappaB activation pathway.     

A novel role of ribonuclease inhibitor in regulation of epithelial-to-mesenchymal transition and ILK signaling pathway in bladder cancer cells

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein possibly involved in biological functions other than the inhibition of RNase A and angiogenin activities. We have previously shown that RI can inhibit growth and metastasis in some cancer cells. Epithelial-mesenchymal transition (EMT) is regarded as the beginning of invasion and metastasis and has been implicated in the metastasis of bladder cancer. We therefore postulate that RI regulates EMT of bladder cancer cells. We find that the over-expression of RI induces the up-regulation of E-cadherin, accompanied with the decreased expression of proteins associated with EMT, such as N-cadherin, Snail, Slug, vimentin and Twist and of matrix metalloprotein-2 (MMP-2), MMP-9 and Cyclin-D1, both in vitro and in vivo. The up-regulation of RI inhibits cell proliferation, migration and invasion, alters cell morphology and adhesion and leads to the rearrangement of the cytoskeleton in vitro. We also demonstrate that the up-regulation of RI can decrease the expression of integrin-linked kinase (ILK), a central component of signaling cascades controlling an array of biological processes. The over-expression of RI reduces the phosphorylation of the ILK downstream signaling targets p-Akt and p-GSK3β in T24 cells. We further find that bladder cancer with a high-metastasis capability shows higher vimentin, Snail, Slug and Twist and lower E-cadherin and RI expression in human clinical specimens. Finally, we provide evidence that the up-regulation of RI inhibits tumorigenesis and metastasis of bladder cancer in vivo. Thus, RI might play a novel role in the development of bladder cancer through regulating EMT and the ILK signaling pathway.     

Potential of butein,a tetrahydroxychalcone to obliterate cancer

BackgroundDespite the major advances made in the field of cancer biology, it still remains one of the most fatal diseases in the world. It is now well established that natural products are safe and efficacious and have high potential in the prevention and treatment of different diseases including cancer. Butein is one such compound which is now found to have anti-cancer properties against various malignancies.PurposeTo thoroughly review the literature available on the anti-cancer properties of butein against different cancers and its molecular targets.MethodsA thorough literature search has been done in PubMed for butein, its biological activities especially cancer and its molecular targets.ResultsOur search retrieved several reports on the various biological activities of butein in which around 43 articles reported that butein shows potential anti-proliferative effect against a wide range of neoplasms and the molecular target varies with cancer types. Most often it targets NF-κB and its downstream pathways. In addition, butein induces the expression of genes which mediate the cell death and apoptosis in cancer cells. It also inhibits tumor angiogenesis, invasion and metastasis in prostate, liver and bladder cancers through the inhibition of MMPs, VEGF etc. Moreover, it inhibits the overexpression of several proteins and enzymes such as STAT3, ERK, CXCR4, COX-2, Akt, EGFR, Ras etc. involved in tumorigenesis.ConclusionCollectively, all these findings suggest the enormous potential and efficacy of butein as a multitargeted chemotherapeutic, chemopreventive and chemosensitizing agent against a wide range of cancers with minimal or no adverse side effects.     

Crosstalk between ferroptosis and the epithelial-mesenchymal transition: Implications for inflammation and cancer therapy

Both genomic instability and the presence of chronic inflammation are involved in carcinogenesis and tumor progression. These alterations predispose the cancer cells to undergo metabolic reprogramming as well as the epithelial-mesenchymal transition (EMT). These pathways allow cancer cells to avoid apoptosis and stimulate tumor progression. EMT is an important early event in tumor cell invasion, which can be regulated through inflammatory signaling pathways. Cancer cells undergoing EMT are vulnerable to cell death by the process of ferroptosis. Ferroptosis is a form of regulated cell death involving iron-dependent lipid peroxidation, designed to maintain cellular homeostasis. Several reports have linked ferroptosis, inflammation, and cancer. Ferroptosis inhibitors and EMT inducers have been used to understand the anti-inflammatory and anticancer effects in experimental models. A better understanding of the crosstalk between ferroptosis and EMT, and the involvment of inflammatory mediators may accelerate the discovery of therapeutic strategies to eradicate cancer cells and overcome drug-resistance.     

Establishment of a Chinese bladder cancer cell line (T921) with high metastatic activity

Muscle-invasive bladder cancer is prone to metastasis without a standard organ preference. The current cell lines used to study bladder cancer have primarily been derived from individuals in Western populations, and no human bladder cancer cell line has been established from the Chinese population. A bladder cancer cell line was derived from a female Chinese patient with muscle-invasive bladder cancer, and these cells were then xenografted into the bladders of three nude mice. Five weeks later, these mice were killed to observe local invasion and distant metastasis. The metastatic tumors were also removed and analyzed to assess the metastatic mechanism. This bladder cancer cell line, named T921, was successfully established, as evidenced by karyotype and immunohistochemistry analyses. Multi-organ metastases were observed in all three of the nude mice 5 wk after the orthotopic transfer of the cell line. In addition, epithelial–mesenchymal transition (EMT)-related genes were involved in the tumor metastases. The T921 bladder cancer cell line was successfully established, and EMT was observed to play a role in bladder cancer metastasis.     

Enrichment of cancer stem-like cells by the induction of epithelial-mesenchymal transition using lentiviral vector carrying E-cadherin shRNA in HT29 cell line

A better understanding of cancer stem cells (CSCs) may facilitate the prevention and treatment of cancers. Epithelial-mesenchymal transition (EMT) is a process activated during invasion and metastasis of tumors. EMT induction in normal and tumor cells makes them more resistant to chemotherapy. E-cadherin is a membrane protein and plays a role in tumor invasion, metastasis, and prognosis. Downregulation of E-cadherin is a hallmark of EMT. Here, we created a model of cancer stem-like cells enrichment via EMT induction using E-cadherin downregulation in HT29 cell line using a lentiviral vector carrying shRNA. We aimed to evaluate cancer and anti-CSC chemotherapeutics screening. The markers of EMT and CSCs were assessed and compared with control cells using flow cytometry, real-time PCR, immunocytochemistry, western blot, migration assay, invasion assay, and colony formation assay. The transduced cells showed a mesenchymal morphology. High levels of EMT-related proteins were also expressed. These results confirmed that the transduced cells underwent EMT. In addition, we observed an increased population of E-cadherin-downregulated HT29 cell line among the cells expressing colon CSC markers (CD133⁺ and CD44⁺) after EMT induction. E-cadherin-downregulated cells were morphologically like mesenchymal cells, and the number of CD133⁺- and CD44⁺-cells (CSC-like cells) increased. These cells can be used as stable models to study cancer cells and screening of antitumor therapeutics.     

Loss of the Keratin Cytoskeleton Is Not Sufficient to Induce Epithelial Mesenchymal Transition in a Novel KRAS Driven Sporadic Lung Cancer Mouse Model

Epithelial-to-mesenchymal transition (EMT), the phenotypical change of cells from an epithelial to a mesenchymal type, is thought to be a key event in invasion and metastasis of adenocarcinomas. These changes involve loss of keratin expression as well as loss of cell polarity and adhesion. We here aimed to determine whether the loss of keratin expression itself drives increased invasion and metastasis in adenocarcinomas and whether keratin loss leads to the phenotypic changes associated with EMT. Therefore, we employed a recently described murine model in which conditional deletion of the Keratin cluster II by Cre-recombinase leads to the loss of the entire keratinmultiprotein family. These mice were crossed into a newly generated Cre-recombinase inducible KRAS-driven murine lung cancer model to examine the effect of keratin loss on morphology, invasion and metastasis as well as expression of EMT related genes in the resulting tumors. We here clearly show that loss of a functional keratin cytoskeleton did not significantly alter tumor morphology or biology in terms of invasion, metastasis, proliferation or tumor burden and did not lead to induction of EMT. Further, tumor cells did not induce synchronously expression of vimentin, which is often seen in EMT, to compensate for keratin loss. In summary, our data suggest that changes in cell shape and migration that underlie EMT are dependent on changes in signaling pathways that cause secondary changes in keratin expression and organization. Thus, we conclude that loss of the keratin cytoskeleton per se is not sufficient to causally drive EMT in this tumor model.     

Thymoquinone Suppresses the Proliferation,Migration and Invasiveness through Regulating ROS,Autophagic Flux and miR-877-5p in Human Bladder Carcinoma Cells

Bladder carcinoma is among the top 10 most frequently diagnosed cancer types in the world. As a phytochemical active metabolic, thymoquinone (TQ) is extracted from seeds of Nigella sativa, possessing various biological properties in a wide range of diseases. Moreover, the outstanding anti-cancer effect of TQ is attracting increasing attentions. In certain circumstances, moderate autophagy is regarded to facilitate the adaptation of malignant cells to different stressors. Conversely, closely linked with the mitochondrial membrane potential (MMP) loss, the upregulation of intracellular reactive oxygen species (ROS) is reported to activate the cell apoptosis in many cancer types. Furthermore, the vital effects of microRNAs in the pathological processes of cancer cells have also been confirmed by previous studies. The present research confirms that TQ restrains the viability, proliferation, migration and invasion through activating caspase-dependent apoptosis in bladder carcinoma cells, which is mediated by TQ induced ROS increase in bladder carcinoma cells. Furthermore, TQ is proved to block the fusion of autophagosomes and lysosomes, causing the accumulation of autophagosomes and subsequent cell apoptosis. In addition, TQ is also found to initiate the miR-877-5p/PD-L1 axis, which suppresses the epithelial mesenchymal transition (EMT) and invasion of bladder carcinoma cells. Taken together, TQ induces the apoptosis through upregulating ROS level and impairing autophagic flux, and inhibiting the EMT and cell invasion via activating the miR-877-5p/PD-L1 axis in bladder carcinoma cells.     

Long noncoding RNA AC073284.4 suppresses epithelial–mesenchymal transition by sponging miR-18b-5p in paclitaxel-resistant breast cancer cells

Breast cancer (BC) is the most prevalent malignant cancer in the world, is the leading cause of cancer-related death female. Recently, there is accumulating evidence that long noncoding RNAs (lncRNAs) might as an important role in the progression of BC. (epithelial-mesenchymal transition (EMT) is considered to play a vital role in tumor cells migration and invasion. Nevertheless, the entire biological mechanisms and functions of lncRNAs in tumor migration, invasion, and EMT remain uncertain. In the present research, we observed that the expression of lncRNA AC073284.4 was downregulated in BC paclitaxel-resistant (PR) cells (MCF-7/PR) and tissues. Bioinformatics analysis predicted that miR-18b-5p was a direct target of AC073284.4, which has been validated by dual-luciferase reporter gene assay. We further proved that AC073284.4 could directly bind to miR-18b-5p and relieve the suppression for dedicator of cytokinesis protein 4 (DOCK4). Furthermore, the underlying functional experiments demonstrated that AC073284.4 might sponge miR-18b-5p to attenuate the invasion, metastasis, and EMT of BC cell through upregulating DOCK4 expression. In summary, AC073284.4 might serve as a competing endogenous RNA (ceRNA) in BC progression via modulating miR-18b-5p/DOCK4 axis, which weakens EMT and migration of BC. These results suggesting that AC073284.4 might function as a potential novel diagnostic biomarker in the progression of BC.     

Alpha B‐crystallin promotes the invasion and metastasis of gastric cancer via NF‐κB‐induced epithelial‐mesenchymal transition

Alpha B‐crystallin (CRYAB) is overexpressed in a variety of cancers. However, little is known about its specific function and regulatory mechanism in gastric cancer. Here, we first explore the role of CRYAB in gastric cancer progression and metastasis. The expression of CRYAB was determined by western blot and immunohistochemistry in gastric cancer tissues. Besides, methods including stably transfected against CRYAB into gastric cancer cells, western blot, migration and invasion assays in vitro and metastasis assay in vivo were also conducted. The expression of CRYAB is up‐regulated in gastric cancer tissues compared with matched normal tissues. High expression level of CRYAB is closely correlated with cancer metastasis and shorter survival time in patients with gastric cancer. Additionally, CRYAB silencing significantly suppresses epithelial‐mesenchymal transition (EMT), migration and invasion of gastric cancer cells in vitro and in vivo, whereas CRYAB overexpression dramatically reverses these events. Mechanically, CRYAB facilitates gastric cancer cells invasion and metastasis via nuclear factor‐κ‐gene binding (NF‐κB)‐regulated EMT. These findings suggest that CRYAB expression predicts a poor prognosis in patients with gastric cancer. Besides, CRYAB contributes to gastric cancer cells migration and invasion via EMT, mediated by the NF‐κB signalling pathway, thus possibly providing a novel therapeutic target for gastric cancer.