DUSP1/MKP1 promotes angiogenesis, invasion and metastasis in non-small cell lung cancer
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Moncho-Amor V
PROVIDER: S-ECPF-GEOD-19592 | biostudies-other | 2011 Feb
REPOSITORIES: biostudies-other
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Project description:DUSP1 is involved in different cellular pathways including cancer cell proliferation, angiogenesis, invasion and resistance to chemotherapy. To understand more about the cellular responses regulated by DUSP1 in NSCLC cells, we interfered DUSP1 expression in the NSCLC cell line H460 and studied the changes in gene expression differentially regulated by this phosphatase. Keywords: Expression profiling by array in cells with genetic modification The human non small lung cancer cell line H460 used in this study was from ATCC (American Type Culture Collection). The cell line was maintained in RPMI (Gibco, Invitrogen) supplemented with 10% bovine serum and transfected by Lipofectamine Plus Reagent from Life Technologies as directed by the manufacturer. The DUSP1 pSuperRetro-derived vectors were constructed as described (Chattoppadhyay et al., 2006). Clones expressing siRNA for DUSP1 were selected for their ability to grow in the presence of puromycin and kept for selection. Stable transfection was confirmed by Western blotting. RNA was extracted and quality control was checked before hybridization on Affymetrix microarrays.
2010-12-09 | E-GEOD-19592 | biostudies-arrayexpress
Project description:DUSP1 is involved in different cellular pathways including cancer cell proliferation, angiogenesis, invasion and resistance to chemotherapy. To understand more about the cellular responses regulated by DUSP1 in NSCLC cells, we interfered DUSP1 expression in the NSCLC cell line H460 and studied the changes in gene expression differentially regulated by this phosphatase. Keywords: Expression profiling by array in cells with genetic modification
2010-12-09 | GSE19592 | GEO
Project description:CD47 is overexpressed in many human cancers, its level positively correlates with tumor invasion and metastasis. However, it is largely unknown whether CD47 overexpression drives metastasis and how CD47 lead to tumor metastasis in non-small cell lung cancer (NSCLC). In this study, we analyzed NSCLC specimens and cell lines, and revealed that CD47 is expressed at a higher level than in tumor-free control samples. Furthermore, increased CD47 expression correlated with clinical staging, lymph node metastasis and distant metastasis. In order to understand the molecular mechanisms underlying CD47 functions, we applied both gain-of-function and loss-of-function approaches in cell lines. The siRNA-mediated downregulation of CD47 inhibited cell invasion and metastasis in vitro, while the overexpression of CD47 by plasmid transfection generated opposite effects. In vivo, CD47-specific shRNA significantly reduced tumor growth and metastasis. On the molecular level, the expression of CD47 correlated with that of Cdc42, both in cell lines and NSCLC specimens. The inhibition of Cdc42 attenuates the invasion and metastasis of CD47-overexpressing cells. These results indicate that Cdc42 is a downstream mediator of CD47-promoted metastasis. Our findings provide first evidence that CD47 is an adverse prognostic factor for disease progression and metastasis, and a promising therapeutic target for NSCLC.
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Project description:BackgroundAberrant microRNA expression has been implicated in metastasis of cancers. MiR-661 accelerates proliferation and invasion of breast cancer and ovarian cancer, while impedes that of glioma. Its role in non small cell lung cancer (NSCLC) and underlying mechanism are worthy elucidation.MethodsExpression of miR-661 was measured with real-time PCR in both NSCLC tissues and cell lines. The effects of miR-661 on migration, invasion and metastasis capacity of NSCLC were evaluated using wound healing, transwell assay and animal models. Dual reporter luciferase assay and complementary experiments were performed to validate RB1 as a direct target of miR-661 for participation in the progression of NSCLC.ResultsMiR-661 was upregulated in NSCLC tissues as compared to paired adjacent tissues and associated with shorter overall survival. Furthermore, miR-661 promoted proliferation, migration and metastasis of NSCLC. Then, we identified RB1 as a direct target of miR-661 through which miR-661 affected EMT process and metastasis of NSCLC. RB1 interacted with E2F1 and both could mediate EMT process in NSCLC.ConclusionMiR-661 promotes metastasis of NSCLC through RB/E2F1 signaling and EMT events, thus may serves as a negative prognostic factor and possible target for treatment of NSCLC patient.
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Project description:PurposeNon-small cell lung cancer (NSCLC) is a leading cause of cancer death, and metastasis is a crucial determinant of increased cancer mortality. DDX24 has garnered increased attention due to its correlation with tumorigenesis and malignant progression. However, the correlation between DDX24 and NSCLC remains unclear.MethodsDDX24 expression in NSCLC tissues and survival rate of patients was analyzed using bioinformatic analysis. Transwell assays, wound-healing assays, and tail vein lung colonization models were employed to determine the role of DDX24 in migration and invasion in vitro and in vivo. We searched for DDX24-interacting proteins using co-immunoprecipitation followed by mass spectroscopy and verified the interaction. The influence of DDX24 on RPL5 expression and ubiquitination was examined using protein stability assays.ResultsDDX24 expression was upregulated in NSCLC cell lines and tumors of patients, particularly those with high tumor grades. A high DDX24 level was also correlated with a poor prognosis. DDX24 upregulation enhanced the migration and invasion ability of NSCLC cells, whereas its downregulation had the opposite effects. In vivo xenograft experiments confirmed that tumors with high DDX24 expression had higher metastatic abilities. The interaction between DDX24 and RPL5 promoted its ubiquitination and destabilized it.ConclusionsDDX24 acted as a pro-tumorigenic factor and promoted metastasis in NSCLC. DDX24 interacted with RPL5 to promote its ubiquitination and degradation. As a result, targeting DDX24/RPL5 axis may provide a novel potential therapeutic strategy for NSCLC.
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Project description:Non-small cell lung cancer (NSCLC) is a fatal disease, and its metastatic process is poorly understood. Although aberrant methylation is involved in tumor progression, the mechanisms underlying dynamic DNA methylation remain to be elucidated. It is significant to study the molecular mechanism of NSCLC metastasis and identify new biomarkers for NSCLC early diagnosis. Here, we performed MeDIP-seq and hMeDIP-seq analyses to detect the genes regulated by dynamic DNA methylation. Comparison of the 5mC and 5hmC sites revealed that the CD147 gene underwent active demethylation in NSCLC tissues compared with normal tissues, and this demethylation upregulated CD147 expression. Significantly high levels of CD147 expression and low levels of promoter methylation were observed in NSCLC tissues. Then, we identified the CD147 promoter as a target of KLF6, MeCP2, and DNMT3A. Treatment of cells with TGF-β triggered active demethylation involving loss of KLF6/MeCP2/DNMT3A and recruitment of Sp1, Tet1, TDG, and SMAD2/3 transcription complexes. A dCas9-SunTag-DNMAT3A-sgCD147-targeted methylation system was constructed to reverse CD147 expression. The targeted methylation system downregulated CD147 expression and inhibited NSCLC proliferation and metastasis in vitro and in vivo. Accordingly, we used cfDNA to detect the levels of CD147 methylation in NSCLC tissues and found that the CD147 methylation levels exhibited an inverse relationship with tumor size, lymphatic metastasis, and TNM stage. In conclusion, this study clarified the mechanism of active demethylation of CD147 and suggested that the targeted methylation of CD147 could inhibit NSCLC invasion and metastasis, providing a highly promising therapeutic target for NSCLC.
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Project description:BackgroundCRTC2 is highly expressed in lung cancer and contributes to lung cancer pathogenesis; however, whether CRTC2 promotes lung cancer metastasis remains unknown. In the present study, we investigated the role of CRTC2 in lung cancer metastasis in vitro.MethodsCRTC2 stable knockdown of lung cancer cell A549 was generated with small hairpin RNA and confirmed by quantitative reverse transcription-PCR and Western blot. Wound healing and invasion transwell assays were performed to explore migration and invasion activity, and Western blot was conducted to detect the expression of related proteins.ResultsSuppression of CRTC2 significantly inhibited A549 cell migration and invasion in vitro. Mechanistic studies showed that knockdown of CRTC2 greatly downregulated MMP2 and MMP9 expression. CRTC2 silencing remarkably suppressed epithelial-mesenchymal transition by modulating the expression of E-cadherin and vimentin. Furthermore, suppression of CRTC2 expression significantly reduced MAPK/c-Jun N-terminal kinase activity.ConclusionCRTC2 may promote A549 migration and invasion by modulation of c-Jun N-terminal kinase-mediated epithelial-mesenchymal transition and matrix metalloproteinase expression.
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Project description:Circular RNAs (circRNAs) are related to the progression of non-small cell lung cancer (NSCLC). However, the roles and mechanism of circ_0006988 are largely unknown. The levels of circ_0006988, Low-Density Lipoprotein Receptor Class A Domain Containing 3 (LDLRAD3), microRNA-491-5p (miR-491-5p), Mitogen-Activated Protein Kinase Kinase Kinase 3 (MAP3K3) were measured using quantitative real-time polymerase-chain reaction (qRT-PCR) and western blot assay. The characteristic of circ_0006988 was analyzed by RNase R assay and Actinomycin D assay. Functional analyses were processed by Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, flow cytometry analysis, transwell assay, wound-healing assay and tube formation assay. The interactions between circ_0006988 and miR-491-5p as well as miR-491-5p and MAP3K3 were analyzed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Murine xenograft model assay was processed to verify the function of circ_0006988 in vivo. Immunohistochemistry (IHC) assay was conducted to examine the level of Ki67. Circ_0006988 abundance was increased in NSCLC tissues and cells. Circ_0006988 silencing restrained NSCLC cell proliferation, migration, invasion and angiogenesis, and induced apoptosis. Circ_0006988 sponged miR-491-5p, which directly targeted MAP3K3. MiR-491-5p overexpression repressed NSCLC cell malignant behaviors. MiR-491-5p downregulation or MAP3K3 overexpression reversed the effect of circ_0006988 silencing on NSCLC cell progression. In addition, circ_0006988 knockdown reduced xenograft tumor growth. ssCirc_0006988 contributed to the development of NSCLC by miR-491-5p/MAP3K3 axis.
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Project description:BACKGROUND:Brain metastasis (BM) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). Recent studies demonstrated that microRNA-330-3p (miR-330-3p) was involved in NSCLC brain metastasis (BM). However, the exact parts played by miR-330-3p in BM of NSCLC remain unknown. Discovery and development of biomarkers and elucidation of the mechanism underlying BM in NSCLC is critical for effective prophylactic interventions. Here, we evaluated the expression and biological effects of miR-330-3p in NSCLC cells and explored the underlying mechanism of miR-330-3p in promoting cell migration and invasion in NSCLC. METHODS:Stable over-expression and knockdown of miR-330-3p in NSCLC cells was constructed with lentivirus. Expression levels of miR-330-3p in NSCLC cells were quantified by quantitive real-time PCR (qRT-PCR). The effects of miR-330-3p on NSCLC cells were investigated using assays of cell viability, migration, invasion, cell cycle, apoptosis, western blotting, immunohistochemical, and immunofluorescence staining. A xenograft nude mouse model and in situ brain metastasis model were used to observe tumor growth and brain metastasis. The potential target of miR-330-3p in NSCLC cells was explored using the luciferase reporter assay, qRT-PCR, and western blotting. The miR-330-3p targets were identified using bioinformatics analysis and verified by luciferase reporter assay. The correlation between GRIA3 and DNA methyltransferase (DNMT) 1 and DNMT3A was tested by RT-PCR, western blotting, and co-immunoprecipitation (IP). RESULTS:miR-330-3p was significantly up-regulated in NSCLC cell lines. MTT assay, transwell migration, and invasion assays showed that miR-330-3p promoted the growth, migration, and invasion of NSCLC cells in vitro and induced tumor growth and metastasis in vivo. Luciferase reporter assays showed that GRIA3 was a target of miR-330-3p. qRT-PCR and western blotting exhibited that miR-330-3p promoted the growth, invasion, and migration of NSCLC cells by activating mitogen-activated protein kinase (MAPK)/extracellular-regulated protein kinases (ERK) signaling pathway. Furthermore, miR-330-3p up-regulated the total DNA methylation in NSCLC cells, and co-IP-demonstrated GRIA3 was directly related with DNMT1 and DNMT3A. CONCLUSIONS:miR-330-3p promoted the progression of NSCLC and might be a potential target for the further research of NSCLC brain metastasis.
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Project description:Purpose: TIAM2 (T-cell lymphoma invasion and metastasis 2), a RAC1 guanine nucleotide exchange factor, plays crucial roles in human cancer cells. Its homolog, TIAM1, has been reported to promote the migration and invasion of cancer cells through regulating the functions of cancer associated fibroblasts (CAFs). However, the functions of TIAM2 in CAFs have not been investigated. In this study, we explored how fibroblast TIAM2 influences the migration and invasion of lung cancer cells. Methods: We cultured primary lung CAFs and adjacent normal lung fibroblasts (NFs) from 12 non-small cell lung cancer (NSCLC) patients. RT-PCR and western blot were used to compare TIAM2 levels between CAFs and NFs. Two co-culture systems were designed, in which cancer cells were directly co-cultured with fibroblasts and indirectly co-cultured with conditional medium (CM) from fibroblasts. Subsequently, the wound healing and transwell tests were conducted to assess the migration and invasion ability of fibroblasts and co-cultured cancer cells. Finally, cytokine antibody arrays were used to screen differentially secreted cytokines in the CM. Results: The expression levels of TIAM2 were significantly higher in CAFs than NFs, and TIAM2-silenced fibroblasts showed decreased migration and invasion ability. In the direct co-culture system, the migration and invasion of cancer cells were retarded when co-culturing with TIAM2-silenced fibroblasts, and the expression levels of EMT-related genes also changed in cancer cells. Decreased migration and invasion of cancer cells were also observed when culturing with the CM from TIAM2-silenced fibroblasts. In addition, the cytokine antibody arrays revealed that Osteoprotegerin (OPG) was significantly decreased in the CM of TIAM2-silenced fibroblasts. This result suggested that OPG might be one of the main cytokines contributing to the migration and invasion of cancer cells in co-culture systems. Conclusion: Our results suggest that fibroblast TIAM2 promotes the invasion and migration of lung cancer cell, and OPG might be one of the main cytokines contributing to this pro-cancer process.
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