Kruppel-like Factors 3 Regulates Migration and Invasion of Gastric Cancer Cells Through NF-κB Pathway.

Context: The poorly understood regulatory mechanisms impede gastric cancer therapy. Kruppel-like factors (KLFs) are associated with the development of various tumors, The studies on the role of the KLF transcription factor 13 (KLF13) in gastric cancer progression haven't been studied. Objective: The current study aimed to investigate the role of KLF13 in the migration and invasion of gastric cancer and the regulatory mechanism of KLF13 in gastric cancer progression. Design: The research team performed a laboratory study. Setting: The study took place at the Zengcheng District People's Hospital of Guangzhou in Zengcheng, China. Participants: In addition to using normal gastric cells, GES1, and seven gastric cancer cell lines, the research team compared the fresh, gastric cancer tissues (T) and paired, adjacent, noncancerous gastric tissues (ANT) from eight patients undergoing surgical resection at the hospital. The research team also downloaded the data for 33 gastric cancer tissues and adjacent, normal gastric tissues from the Cancer Genome Atlas' TCGA database. Intervention: The research team used: (1) short hairpin RNAs (shRNAs) to knock down KLF13, (2) wound healing and transwell invasion analyses to determine the effects of KLF13 on the migration and invasion of gastric cancer, and (3) a Luciferase reporter assay to determine the effects of KLF13 on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity. Results: KLF13 was upregulated in gastric cancer cells and tissues, and the patients with a high KLF13 expression had poor outcome. Downregulation of KLF13 significantly inhibited the migration and invasion of gastric cancer cells. Mechanistically, downregulation of KLF13 significantly inhibited NF-κB activity, and its targets such as: (1) snail family transcriptional repressor 1 (SNAI1 or Snail), (2) snail family transcriptional repressor 2 (SNAI2 or Slug), (3) zinc finger e-box binding homeobox 1 (ZEB1), (4) Smad interacting protein 1 (Sip1), (5) twist family basic helix-loop-helix (BHLH) transcription factor (Twist), (6) matrix metallopeptidase 2 (MMP2), and (7) MMP9. Tumor necrosis factor alpha (TNF-α) can activate NF-κB. Treating with TNF-α can reverse the effects of KLF13 downregulation on migration and invasion, confirming that KLF13 promotes the migration and invasion of gastric cancer cells through activating the NF-κB pathway. Conclusions: KLF13 promoted the migration and invasion of gastric cancer cells through activating the NF-κB pathway, providing a new target for gastric cancer therapy.

Functional Alterations of Multidrug Resistance-Associated Proteins 2 and 5, and Breast Cancer Resistance Protein upon Snail-Induced Epithelial-Mesenchymal Transition in HCC827 Cells.

Our previous report indicated that Snail-induced epithelial-mesenchymal transition (EMT) enhanced P-glycoprotein (P-gp) function and drug resistance to P-gp substrate anticancer drug in a human non-small cell lung cancer (NSCLC) cell line, HCC827. Our objective is to evaluate the changes in the mRNA and protein expression levels and the functions of multidrug resistance-associated protein (MRP) 2, MRP5 and breast cancer resistance protein (BCRP). Snail-expressing HCC827 cells showed increased mRNA levels of Snail and a mesenchymal marker vimentin, and decreased mRNA levels of an epithelial marker E-cadherin after transduction, indicating that Snail had induced EMT consistent with our previous reports. The mRNA level of MRP2 was significantly decreased, while that of MRP5 remained unchanged, in Snail-expressing cells. The expression levels of MRP2 and MRP5 proteins in whole-cell homogenate were unchanged in Snail-expressing cells, but MRP5 protein showed significantly increased membrane localization. Snail-transduction increased the efflux transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDCF), a substrate of MRP2, 3 and 5. This increase was blocked by MK571, which inhibits MRP1, 2, and 5. Toxicity of cisplatin, a substrate of MRP2 and 5, was significantly decreased in Snail-expressing cells. BCRP mRNA and protein levels were both decreased in Snail-expressing cells, which showed an increase in the intracellular accumulation of 7-ethyl-10-hydroxycamptothecin (SN-38), a BCRP substrate, resulting in reduced viability. These results suggested that MRP5 function appears to be increased via an increase in membrane localization, whereas the BCRP function is decreased via a decrease in the expression level in HCC827 cells with Snail-induced EMT.

Competitive Effect of Overexpressed C-terminal of Snail-1 (CSnail) in Control of the Growth and Metastasis of Melanoma Cells.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) plays a role in the invasion and metastasis of cancer cells. During this phenomenon, Snail can promote tumor progression by upregulating mesenchymal factors and downregulating the expression of pro-apoptotic proteins. OBJECTIVE: Therefore, interventions on the expression rate of Snails may show beneficial therapeutic applications. METHODS: In this study, the C-terminal region of Snail1, capable of binding to E-box genomic sequences, was subcloned into the pAAV-IRES-EGFP backbone to make complete AAV-CSnail viral particles. B16F10 as a metastatic melanoma cell line, with a null expression of wild type TP53 was transduced by AAV-CSnail. Moreover, the transduced cells were analyzed for in vitro expression of apoptosis, migration, and EMT-related genes, and in vivo inhibition of metastasis. RESULTS: In more than 80% of the AAV-CSnail transduced cells, the CSnail gene expression competitively reduced the wild-type Snail functionality and consequently lowered the mRNA expression level of EMT-related genes. Furthermore, the transcription level of cell cycle inhibitory factor p21 and pro-apoptotic factors were promoted. The scratch test showed a decrease in the migration ability of AAV-CSnail transduced group compared to control. Finally, metastasis of cancer cells to lung tissue in the AAV-CSnail-treated B16F10 melanoma mouse model was significantly reduced, pointing out to prevention of EMT by the competitive inhibitory effect of CSnail on Snail1 and increased apoptosis of B16F10 cells. CONCLUSION: The capability of this successful competition in reducing the growth, invasion, and metastasis of melanoma cells indicates that gene therapy is a promising strategy for the control of the growth and metastasis of cancer cells.

A Truncated Snail1 Transcription Factor Alters the Expression of Essential EMT Markers and Suppresses Tumor Cell Migration in a Human Lung Cancer Cell Line.

BACKGROUND: Epithelial-to-Mesenchymal Transition (EMT) is necessary for metastasis. Zinc- finger domain-containing transcription factors, especially Snail1, bind to E-box motifs and play a crucial role in the induction and regulation of EMT. OBJECTIVE: We hypothesized if C-terminal region of Snail1 (CSnail1) may competitively bind to E-box and block cancer metastasis. METHODS: The CSnail1 gene coding sequence was inserted into the pIRES2-EGFP vector. Following transfection of A549 cells with the designed construct, EMT was induced with TGF-ß1 and the expression of essential EMT markers was evaluated by real-time PCR and immunoblotting. We also monitored cell migration. RESULTS: CSnail1 inhibited TGF-ß1-induced N-cadherin and vimentin mRNA expression and increased ß-catenin expression in transfected TGF-ß1-treated A549 cells. A similar finding was obtained in western blotting. CSnail1 also blocked the migration of transfected cells in the scratch test. CONCLUSION: Transfection of A549 cells with CSnail1 alters the expression of essential EMT markers and consequently suppresses tumor cell migration. These findings confirm the capability of CSnail1 in EMT blocking and in parallel to current patents could be applied as a novel strategy in the prevention of metastasis.