Lung Metastasis Assay Research Articles

Targeting deubiquitinating enzyme USP26 by microRNA-203 regulates Snail1\u2019s pro-metastatic functions in esophageal cancer

BackgroundEsophageal cancer is one of the most common cancers worldwide with poor prognosis and high mortality. The transcription factor SNAI1, encoding Snail1, is important for metastatic progression in esophageal cancer whereas the microRNA (miRNA)-203 has been shown to function as an inhibitor of metastasis in EC. The Snail1 protein is stabilized in EC partially by the deubiquitinating enzyme USP26; however, how USP26 is regulated is not completely known.MethodsExpression of SNAI1 and USP26 messenger RNA (mRNA) and miR-203 was performed in datasets within The Cancer Genome Atlas and Gene Expression Omnibus, respectively. Expression of Snail1 and USP26 protein and miR-203 was determined in the normal esophageal cell line HET-1A and EC cell lines Kyse150 and TE-1 using western blot and quantitative polymerase chain reaction, respectively. TargetScan was used for in situ prediction of miR-203 targets and in vitro heterologous reporter assays using the wild-type and miR-203 seed mutant of the 3′ Untranslated region (UTR) of USP26 were used to investigate whether USP26 is a target of miR-203. Effects of increasing miR-203 using MIR203A/5P mimic on USP26 and Snail1 in the HET-1A, Kyse150 and TE-1 cell lines were performed using western blot and cycloheximide-based protein stability analysis. Effects of modulating miR-203 in Kyse150 and TE-1 cell lines on in vitro pro-metastatic effects were analyzed by invasion assay, scratch wound-healing assay, and chemosensitivity to 5-fluoruracil (5-FU). In vivo lung metastasis assay was used to study the effect of modulating miR-203 in Kyse150 cells.ResultsSNAI1 mRNA and HSA/MIR203 was higher and lower, respectively, in EC patients compared to tumor-adjacent normal tissues. No changes in expression of USP26 mRNA were observed in these datasets. MIR/203 expression was downregulated whereas protein expression of both Snail1 and USP26 were higher in EC cell lines Kyse150 and TE-1 compared to normal esophageal cell line HET-1A. USP26 was predicted as a potential target of miR-203 by TargetScan Release 2.0. Reporter assays confirmed USP26 as a target of miR-203 in the EC cell lines. Transfection of EC cell lines with MIR203 mimic decreased USP26 protein expression and Snail1 protein stability indicating the ability of miR-203 to regulate Snail1 protein levels via USP26. Exogenous increase in miR-203 in the EC cell lines significantly inhibited Snail-1 mediated in vitro pro-metastatic function of invasion, wound-healing, and increased chemosensitivity to 5-FU. Finally, overexpression of miR-203 inhibited in vivo lung metastasis of Kyse150 cells, which was reversed following overexpression of USP26, indicating a direct role of miR-203-mediated regulation of USP26 in metastatic progression of EC.ConclusionsCumulatively, these results establish an important mechanism by which decrease in miR-203 expression potentiates metastatic progression in EC via USP26-mediated stabilization of Snail1. Hence, miR-203 can serve as a biomarker of metastasis in EC and is a potential target for therapeutic intervention in EC.

Fucosyltransferase 4 shapes oncogenic glycoproteome to drive metastasis of lung adenocarcinoma.

BackgroundAberrant fucosylation plays a critical role in lung cancer progression. Nevertheless, the key fucosyltransferase with prognostic significance in lung cancer patients, the enzyme's intracellular targets, and complex molecular mechanisms underlying lung cancer metastasis remain incompletely understood.MethodsWe performed a large-scale transcriptome-clinical correlation to identify major fucosyltransferases with significant prognostic values. Invasion, migration, cell adhesion assays were performed using lung cancer cells subject to genetic manipulation of FUT4 levels. Genome-wide RNA-seq and immunoprecipitation-mass spectrometry were used to characterize major cellular processes driven by FUT4, as well as profiling its intracellular protein targets. We also performed lung homing and metastasis assays in mouse xenograft models to determine in vivo phenotypes of high FUT4-expressing cancer cells.FindingsWe show that FUT4 is associated with poor overall survival in lung adenocarcinoma patients. High FUT4 expression promotes lung cancer invasion, migration, epithelial-to-mesenchymal transition, and cell adhesion. FUT4-mediated aberrant fucosylation markedly activates multiple cellular processes, including membrane trafficking, cell cycle, and major oncogenic signaling pathways. The effects are independent of receptor tyrosine kinase mutations. Notably, genetic depletion of FUT4 or targeting FUT4-driven pathways diminishes lung colonization and distant metastases of lung cancer cells in mouse xenograft models.InterpretationWe propose that FUT4 can be a prognostic predictor and therapeutic target in lung cancer metastasis. Our data provide a scientific basis for a potential therapeutic strategy using targeted therapy in a subset of patients with high FUT4-expressing tumors with no targetable mutations.

Hypoxia-induced miR-3677-3p promotes the proliferation, migration and invasion of hepatocellular carcinoma cells by suppressing SIRT5.

Hepatocellular carcinoma (HCC), with life‐threatening malignant behaviours, often develops distant metastases and is the fourth most common primary cancer in the world, having taken millions of lives in Asian countries such as China. The novel miR‐3677‐3p is involved in a high‐expression‐related poor prognosis in HCC tissues and cell lines, indicating oncogenesis functions in vitro and in vivo. Initially, we confirmed the inhibition of proliferation, migration and invasion in miR‐3677‐3p knock‐down MHCC‐97H and SMMC‐7721 cell lines, which are well known for their high degree of invasiveness. Then, we reversed the functional experiments in the low‐miR‐3677‐3p‐expression Hep3B cell line via overexpressing miR‐3677‐3p. In nude mice xenograft and lung metastasis assays, we found suppressor behaviours, smaller nodules and low density of organ spread, after injection of cells transfected with shRNA‐miR‐3677‐3p. A combination of databases (Starbase, TargetScan and MiRgator) illustrated miR‐3677‐3p targets, and it was shown to suppress the expression of SIRT5 in a dual‐luciferase reporter system. To clarify the conclusions of previous ambiguous research, we up‐regulated SIRT5 in Hep3B cells, and rescue tests were established for confirmation that miR‐3677‐3p suppresses SIRT5 to enhance the migration and invasion of HCC. Interestingly, we discovered hypoxia‐induced miR‐3677‐3p up‐regulation benefited HCC malignancy and invasiveness. In conclusion, the overexpression of miR‐3677‐3p mediated SIRT5 inhibition, which could increase proliferation, migration and invasion of HCC in hypoxic microenvironments.

Enhanced monoacylglycerol lipolysis by ABHD6 promotes NSCLC pathogenesis.

BackgroundTumor cells display metabolic changes that correlate with malignancy, including an elevated hydrolysis of monoacylglycerol (MAG) in various cancer types. However, evidence is absent for the relationship between MAG lipolysis and NSCLC.MethodsMAG hydrolase activity assay, migration, invasion, proliferation, lipids quantification, and transactivation assays were performed in vitro. Tumor xenograft studies and lung metastasis assays were examined in vivo. The correlations of MAGL/ABHD6 expression in cancerous tissues with the clinicopathological characteristics and survival of NSCLC patients were validated.FindingsABHD6 functions as the primary MAG lipase and an oncogene in NSCLC. MAG hydrolase activities were more than 11-fold higher in cancerous lung tissues than in paired non-cancerous tissues derived from NSCLC patients. ABHD6, instead of MAGL, was significantly associated with advanced tumor node metastasis (TNM) stage (HR, 1.382; P = 0.004) and had a negative impact on the overall survival of NSCLC patients (P = 0.001). ABHD6 silencing reduced migration and invasion of NSCLC cells in vitro as well as metastatic seeding and tumor growth in vivo. Conversely, ectopic overexpression of ABHD6 provoked the pathogenic potential. ABHD6 blockade significantly induced intracellular MAG accumulation which activated PPARα/γ signaling and inhibited cancer pathophysiology.InterpretationThe present study provide evidence for a previously uncovered pro-oncogenic function of ABHD6 in NSCLC, with the outlined metabolic mechanisms shedding light on new potential strategies for anticancer therapy.FundThis work was supported by the Project for Major New Drug Innovation and Development (2015ZX09501010 and 2018ZX09711001-002-003).

MiR-106b-5p contributes to the lung metastasis of breast cancer via targeting CNN1 and regulating Rho/ROCK1 pathway.

Objectives: Breast cancer has been the second most prevalent and fatal malignancy due to its frequent metastasis to other organs. We aim to study the effects of a key miRNA-mRNA signaling in breast cancer.Results: CNN1 was identified as the key gene in breast cancer by the bioinformatics analysis, and the downregulation of CNN1 in breast cancer tissues and cell lines was observed. Upregulating CNN1 inhibited cell survival, migration, invasion, and adhesion, but enhanced cell apoptosis. miR-106b-5p not only bound to CNN1 mRNA 3’UTR, but also promoted lung metastasis in vivo. Besides, the miR-106b-5p mimic enhanced breast cancer canceration by targeting CNN1 and activating Rho/ROCK1 signaling pathway.Conclusion: Overall, our results proved that miR-106b-5p promoted the metastasis of breast cancer by suppressing CNN1 and activating Rho/ROCK1 pathway.Methods: Bioinformatics analysis was performed to select the key gene in breast cancer. The overexpression and knockdown of Calponin 1 (CNN1) in breast cancer cell lines were performed to conduct cell viability, migrating, invasion, proliferation, adhesion, and apoptosis experiments. To identify the role of miR-106b-5p and Rho/ROCK1 in CNN1-induced breast cancer, a dual-luciferase assay, tumor lung metastasis assay, transcript half-life assay, and Rho/ROCK1 inhibition assay were performed.

G9a-mediated repression of CDH10 in hypoxia enhances breast tumour cell motility and associates with poor survival outcome.

Rationale: Epigenetic mechanisms are fundamental processes that can modulate gene expression, allowing cellular adaptation to environmental conditions. Hypoxia is an important factor known to initiate the metastatic cascade in cancer, activating cell motility and invasion by silencing cell adhesion genes. G9a is a histone methyltransferase previously shown to accumulate in hypoxic conditions. While its oncogenic activity has been previously reported, not much is known about the role G9a plays in the hypoxia-mediated metastatic cascade.Methods: The role of G9a in cell motility in hypoxic condition was determined by inhibiting G9a either by short-hairpin mediated knock down or pharmacologically using a small molecule inhibitor. Through gene expression profiling, we identified CDH10 to be an important G9a target that regulates breast cancer cell motility. Lung metastasis assay in mice was used to determine the physiological significance of G9a.Results: We demonstrate that, while hypoxia enhances breast cancer migratory capacity, blocking G9a severely reduces cellular motility under both normoxic and hypoxic conditions and prevents the hypoxia-mediated induction of cellular movement. Moreover, inhibition of G9a histone methyltransferase activity in mice using a specific small molecule inhibitor significantly reduced growth and colonisation of breast cancer cells in the lung. We identify the type-II cadherin CDH10 as being a novel hypoxia-dependent gene, directly repressed by G9a through histone methylation. CDH10 overexpression significantly reduces cellular movements in breast cancer cell lines and prevents the hypoxia-mediated increase in cell motility. In addition, we show that CDH10 expression is prognostic in breast cancer and that it is inversely correlated to EHMT2 (G9a) transcript levels in many tumor-types, including breast cancer.Conclusion: We propose that G9a promotes cellular motility during hypoxic stress through the silencing of the cell adhesion molecule CDH10 and we describe CDH10 as a novel prognostic biomarker for breast cancer.

Hypoxia-induced MFAP5 Promotes Tumor Migration and Invasion via AKT Pathway in Head and Neck Squamous Cell Carcinoma.

Objective: Microfibrillar-associated protein 5 (MFAP5) is highly expressed in many types of cancers. Our previous study has observed that overexpression of MFAP5 was correlated with lymph nodes metastasis and poor prognosis in head and neck squamous cell carcinoma (HNSCC), but the underlying mechanism is poorly understood.Materials and methods: The MFAP5 expression is detected under hypoxia condition. HNSCC cell lines are transfected with MFAP5-expressing lentivirus vector to establish stable overexpression model. Wound-healing, migration and invasion assay are used to determine the effect of MFAP5 on HNSCC and metastasis-related proteins are examined by Western blot. In vivo lung metastasis assays are conducted by the tail vein injection. In addition, immunohistochemistry is applied to analyze the correlation of MFAP5, hypoxia-induced factor-1 α (HIF-1α), and vimentin in 84 HNSCC patients' tissue samples.Results: Firstly, MFAP5 expression can be markedly induced under hypoxia condition in HNSCC cell lines. Cell lines with MFAP5 overexpression has a significant higher ability of migration and invasion. In addition, in vivo assay observes that overexpression of MFAP5 can promote tumor lung metastasis. Furthermore, MFAP5 facilitates this process by activating epithelial-mesenchymal transition (EMT) program via AKT pathway in HNSCC cell lines. The pro-metastatic effect of MFAP5 can be reversed by MK2206, an AKT phosphorylation inhibitor. Lastly, the positive correlation among HIF-1α, MFAP5 and vimentin from tissue samples and TCGA dataset are also observed in HNSCC.Conclusion: Our study demonstrates MFAP5 plays a critical role in hypoxia-induced EMT program via AKT pathway in HNSCC, which would be a very promising therapeutic target.

Fucosyltransferase 4 Shapes Oncogenic Glycoproteome to Drive Metastasis of Lung Adenocarcinoma

Background: Aberrant fucosylation plays a critical role in lung cancer progression. Nevertheless, the key fucosyltransferase with prognostic significance in lung cancer patients, the enzyme’s intracellular targets and complex molecular mechanisms underlying lung cancer metastasis remain incompletely understood. Methods: We performed a large-scale transcriptome-clinical correlation to identify major fucosyltransferases with significant prognostic values. Invasion, migration, cell adhesion assays were performed using lung cancer cells subject to genetic manipulation of FUT4 levels. Genome-wide RNA-seq and immunoprecipitation-mass spectrometry were used to characterize major cellular processes driven by FUT4, as well as profiling its intracellular protein targets. We also performed lung homing and metastasis assays in mouse xenograft models to determine in vivo phenotypes of high FUT4-expressing cancer cells. Findings: We show that FUT4 is associated with poor overall survival in lung adenocarcinoma patients. High FUT4 expression promotes lung cancer invasion, migration, epithelial-to-mesenchymal transition and cell adhesion. FUT4-mediated aberrant fucosylation markedly activates multiple cellular processes, including membrane trafficking, cell cycle, and major oncogenic signaling pathways. The pathway augmentation effects are independent of receptor tyrosine kinase mutations. Notably, genetic depletion of FUT4 or targeting FUT4-driven pathways diminishes lung colonization and distant metastases of lung cancer cells in mouse xenograft models. Interpretation: We propose that FUT4 can be a prognostic predictor and therapeutic target in lung cancer metastasis. Our data also offer a potential therapeutic strategy using targeted therapy in a subset of patients with high FUT4-expressing tumors with no targetable mutations. Funding Statement: Research supported by grants from Ministry of Science and Technology (MOST 107-2314-B-002 -241, MOST 105-2628-B-002 -040), and National Health Research Institutes (NHRIEX106-10610BC) in Taiwan. Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: The study on primary tumor tissues from lung cancer patients was approved by the Institutional Review Board (IRB) of National Taiwan University Hospital (Approval No.: 201701010RINB). Written informed consent was obtained from all participants prior to inclusion in the study. Every participant was assigned a number and the data were analyzed at the group level for patient de-identification. All mice experiments were performed in compliance with an experimental protocol (20160126) approved by the NTU College of Medicine Institutional Animal Care and Use Committee (IACUC).

Metastasis Suppressors NME1 and NME2 Promote Dynamin 2 Oligomerization and Regulate Tumor Cell Endocytosis, Motility, and Metastasis.

NM23 (NME) is a metastasis suppressor that significantly reduces metastasis without affecting primary tumor size, however, the precise molecular mechanisms are not completely understood. We examined the role of dynamin (DNM2), a GTPase regulating membrane scission of vesicles in endocytosis, in NME1 and NME2 regulation of tumor cell motility and metastasis. Overexpression of NMEs in MDA-MB-231T and MDA-MB-435 cancer cell lines increased endocytosis of transferrin and EGF receptors (TfR and EGFR) concurrent with motility and migration suppression. The internalized vesicles, costained with Rab5, had AP2 depleted from the cell surface and exhibited increased Rab5-GTP levels, consistent with endocytosis. Dynamin inhibitors Iminodyn-22 and Dynole-34-2, or shRNA-mediated downregulation of DNM2, impaired NME's ability to augment endocytosis or suppress tumor cell motility. In a lung metastasis assay, NME1 overexpression failed to significantly suppress metastasis in the DNM2 knockdown MDA-MB-231T cells. Using the EGF-EGFR signaling axis as a model in MDA-MB-231T cells, NME1 decreased pEGFR and pAkt expression in a DNM2-dependent manner, indicating the relevance of this interaction for downstream signaling. NME-DNM2 interaction was confirmed in two-way coimmunoprecipitations. Transfection of a NME1 site-directed mutant lacking histidine protein kinase activity but retaining nucleoside diphosphate kinase (NDPK) activity showed that the NDPK activity of NME was insufficient to promote endocytosis or inhibit EGFR signaling. We show that addition of NME1 or NME2 to DNM2 facilitates DNM2 oligomerization and increases GTPase activity, both required for vesicle scission. NME-DNM2 interaction may contribute to metastasis suppression by altering tumor endocytic and motility phenotypes. SIGNIFICANCE: NME1 suppresses metastasis via changes in tumor endocytosis and motility, mediated by dynamin (DNM2) GTPase activity.

Breast Cancer Stem Cells with Tumor- versus Metastasis-Initiating Capacities Are Modulated by TGFBR1 Inhibition.

SummaryCancer stem cells (CSCs) are defined by their ability to regenerate a tumor upon transplantation. However, it is not yet clear whether tumors contain a single CSC population or different subsets of cells with mixed capacities for initiating primary and secondary tumors. Using two different identification strategies, we studied the overlap between metastatic stem cells and tumor-initiating cells (TICs) in the MMTV-PyMT model. Our results show that in the MMTV-PyMT model, Lin−CD90−ALDHhigh cells retained a high tumor-initiating potential (TIP) in orthotopic transplants, in contrast to Lin−CD24+CD90+, which retained higher metastatic capacity. Interestingly, suppression of TGFβ signaling increased TIC numbers. We here describe the existence of distinct populations of CSCs with differing capacities to initiate tumors in the primary or the secondary site. Inhibiting TGFβ signaling shifts the balance toward the former, which may have unanticipated implications for the therapeutic use of TGFβ/TGFBR1 inhibitors.

Extracellular Signal Regulated Kinase 5 Promotes Cell Migration, Invasion and Lung Metastasis Through a FAK-Dependent Manner

Background: ERK5 has been demonstrated to be related to survival, growth, and differentiation of cancer cells. However, the function of ERK5 signaling in cancer metastasis has not been fully elucidated. This study was designed to ascertain the involvement of ERK5 signaling in lung cancer and melanoma metastasis process. Methods: The involvement of ERK5 in cancer metastasis was assessed on various cancer cell lines by wound-healing, transwell, immunofluorescence, and in vivo melanoma lung metastasis assays. High-content proteomics screen in combination with MetacoreTM GeneGo pathway analysis were performed to identify key signaling pathways regulated by ERK5, which is associated with cancer metastasis. Findings: Our data showed that ERK5 expression is abundant in human lung cancer samples, where elevated ERK5 expression related to the acquisition of more metastatic and invasive potential. Importantly, we observed a significant correlation between ERK5 activity and FAK expression and its phosphorylation at Ser910 site. Mechanistically, ERK5 increased the expression of transcription factor USF1, which could transcriptionally up-regulate FAK expression, resulting in FAK signaling activation to promote cell migration. We also provided evidence that the phosphorylation of Ser910 of FAK was due to ERK5 but not ERK1/2, and suggested a role for Ser910 in the control of cell motility. In addition, ERK5 had more targets other than FAK to regulate epithelial-to-mesenchymal transition and cell motility in cancer cells. Interpretation: Our findings uncover a cancer metastasis-promoting role of ERK5 and provide the rationale for targeting ERK5 as a potential therapeutic approach. Funding Statement: Chinese National Natural Sciences Foundation (81773099, 81570790). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: Animal welfare and experimental procedures were performed strictly in accordance with high standard animal welfare and other related ethical regulations approved by Nanjing University.

ANKHD1 is required for SMYD3 to promote tumor metastasis in hepatocellular carcinoma

AbsractBackgroundTumor metastasis is the major reason for poor prognosis of hepatocellular carcinoma (HCC) patients after hepatic resection. SMYD3 has been demonstrated to promote liver tumor metastasis in mice. However, the detailed molecular mechanism is still largely unknown.MethodsThe effect of SMYD3 on invasiveness and metastasis of HCC was analyzed by immunohistochemistry, migration assay, invasion assay, wound healing assay and in vivo lung metastasis assay. Mass spectrometry analysis was conducted using proteins pulled down by H3K4me3 antibody in SMYD3-overexpressing cells. Luciferase reporter, chromatin immunoprecipitation, Electrophoretic mobility shift assay were used to measure the regulation of SLUG transcription by SMYD3-ANKHD1. In addition, the role of SMYD3-ANKHD1 in determining clinical outcomes for HCC patients was investigated by immunohistochemistry in 243 HCC tissues.ResultsSMYD3 was an independent prognostic factor of HCC and promoted migration and invasion of human HCC cells. ANKHD1 was identified by mass spectrometry as a co-regulator with SMYD3. ANKHD1 interacted with H3K4me3 when cells were overexpressing SMYD3. The pro-migratory and pro-invasive effects of SMYD3 were attenuated when ANKHD1 was knocked down by siRNA. Furthermore, we found that SMYD3 bound and activated the SLUG gene promoter in a manner associated with elevating H3K4me3, H3K9Ac and H3K14Ac. Knockdown of ANKHD1 could attenuate the SMYD3-dependent activation of Slug expression. We further detected the expression of SMYD3 and ANKHD1 in 243 HCC patients and found that patients with positive coexpression of SMYD3 and ANKHD1 (SMYD3+ANKHD1+) had the shortest overall and recurrence-free survival.ConclusionOur findings provide a novel molecular mechanism for the SMYD3-regulated HCC migration and metastasis, and indicates that SMYD3-ANKHD1 may be a potential target for treating HCC.

Euxanthone Impairs the Metastatic Potential of Osteosarcoma by Reducing COX-2 Expression.

Osteosarcoma (OS) is one of the most common malignancies of bone. This study was aimed to explore the anti-metastatic effect of euxanthone on OS. Adhesion assay and Transwell assay were used to examine the effect of euxanthone on adhesion, migration and invasion of OS cells. COX-2-over-expressing plasmid was applied to transfect OS cells to assess whether COX-2 affects the anti-metastatic function of euxanthone. PDCD4 knockdown and miR-21 mimic were applied to assess whether euxanthone suppresses the transactivation of c-jun via modulating miR-21-PDCD4 signaling. The effect of euxanthone in vivo was also examined by lung metastasis assay. Euxanthone, a xanthone derivative extracted from Polygala caudata, has been found to exhibit anti-neoplastic activities. In present study, our results showed that euxanthone suppressed cell adhesion, migration, and invasion in OS cells. Our experimental data also showed that repression of COX-2 by euxanthone mediated its anti-metastatic activities. Moreover, our findings revealed that euxanthone modulated the COX-2 expression through the miR-21/PDCD4/c-jun signaling pathway. The anti-metastatic activities of euxanthone were also validated in a pulmonary metastasis model. Taken together, our results highlighted the potential of euxanthone to be used in the treatment of OS. Anat Rec, 302:1399-1408, 2019. © 2018 Wiley Periodicals, Inc.

ARHGAP9 suppresses the migration and invasion of hepatocellular carcinoma cells through up-regulating FOXJ2/E-cadherin

Rho GTPase activating protein 9 (ARHGAP9), a member of RhoGAP family, has been identified as a RhoGAP for Cdc42 and Rac1. Here, we aimed to clarify the expression and functional role of ARHGAP9 in hepatocellular carcinoma (HCC). By analyzing TCGA (The Cancer Genome Atlas) LIHC (liver hepatocellular carcinoma) database, we found that ARHGAP9 expression was lower in HCC tissues than in normal liver tissues, and that patients with ARHGAP9 lower expression had a significant shorter overall survival time than those with ARHGAP9 higher expression. Cell counting kit-8 (CCK-8), transwell assays and in vivo experimental lung metastasis assay revealed that ARHGAP9 overexpression could inhibit HCC cell proliferation, migration and invasion, as well as HCC lung metastases. By next-generation RNA-sequencing, we identified that a transcription factor, Forkhead Box J2 (FOXJ2), was significantly induced by ARHGAP9 overexpression in HepG2 cells. Ectopic expression of FOXJ2 in HCC cell lines also exerted inhibitory effects on cell migration and invasion. Moreover, the inhibitory effects of ARHGAP9 on HCC cell migration and invasion was significantly attenuated by FOXJ2 knockdown. Luciferase reporter assay demonstrated that ARHGAP9 enhanced the transcription of E-cadherin (CDH1) via FOXJ2. Chromatin immunoprecipitation (ChIP) assay demonstrated that FOXJ2 modulated the transcription of E-cadherin (CDH1) by directly binding to its promoter. Furthermore, Pearson’s correlation analysis indicated that the mRNA levels of ARHGAP9 in HCC tissues were positively correlated with the mRNA levels of FOXJ2 and CDH1. These data clearly show that ARHGAP9/FOXJ2 inhibit cell migration and invasion during HCC development via inducing the transcription of CDH1.

Effect of Slug-Mediated Down-Regulation of E-Cadherin on Invasiveness and Metastasis of Anaplastic Thyroid Cancer Cells.

BackgroundSlug has been found to promote migration and invasion of many cancer cells, including anaplastic thyroid cancer (ATC). Thus, targeting Slug expression could provide new approaches for the treatment of patients with ATC.Material/MethodsSmall interfering RNA (siRNA) targeting Slug (Slug siRNA) was used to construct clonal derivatives in the metastatic ATC SW1736 cells. Slug cDNA transfection was used to restore the Slug expression in the Slug siRNA-transfected SW1736 cells (Slug siRNA/SW1736). E-cadherin siRNA was used to inhibit E-cadherin expression in the Slug siRNA/SW1736 cells. The SW1736 cell migration, invasion, and signaling pathway was analyzed in vitro.Furthermore, the stable Slug siRNA-transfected SW1736 clones were used for the lung metastasis assay in an in vivo mouse model.ResultsTargeting Slug expression in SW1736 cells showed a 45% decrease in migration and an 85% decrease in invasiveness in vitro. Knockdown of E-cadherin by E-cadherin siRNA transfection or Slug overexpression by Slug cDNA transfection restored the invasive and migrative ability in SW1736 cells. In addition, we found an 80% decrease in the number of macroscopic lung metastases nodes of mice by in vivo analysis. Western blot assay showed that Slug expression was inhibited and E-cadherin expression was increased in the Slug siRNA-transfected tumors.ConclusionsTargeting Slug signaling pathway is effective in preventing lung metastasis in ATC.

BCL2L10 inhibits growth and metastasis of hepatocellular carcinoma both in vitro and in vivo.

BCL2L10 is an apoptosis-related member of the BCL-2 protein family. The role of BCL2L10 in the pathogenesis of hepatocellular carcinoma (HCC) is poorly understood. This study was aimed to investigate the function and underlying mechanisms of BCL2L10 in HCC. BCL2L10 expression in human HCC and corresponding adjacent normal tissues was investigated by quantitative real-time PCR and Western blot. The biological functions of BCL2L10 in HCC cell lines were determined by cell viability, colony formation, cell apoptosis, cell cycle, and cell metastasis assays, and in vivo by tumorigenicity and lung metastasis assays in nude mice. Human cancer pathway PCR array was employed to explore the genes regulated by BCL2L10 in HCC. BCL2L10 was down-regulated in human HCC tissues compared to their adjacent non-tumor tissues. Ectopic expression of BCL2L10 in HepG2 and Huh7 cells suppressed cell growth as evidenced by cell viability and colony formation assay, and induced cell apoptosis. HCC cells transfected with BCL2L10 revealed an increased cell proportion arrested at G2/M phase, concomitant with a reduction in the cell proportion in S-phase as compared with control cells. Additional, BCL2L10 repressed cell migration and angiogenesis. Over-expression of BCL2L10 also restrained the tumorigenecity and lung metastasis capacity in nude mice. The activation of JAK-STAT3 signaling was suppressed by BCL2L10 in HCC. BCL2L10 was down-regulated in human HCC tissues compared to adjacent normal tissues. BCL2L10 suppressed HCC progression through inhibiting cell growth and metastasis. Thus, BCL2L10 functions as a tumor-suppressor in HCC. © 2016 Wiley Periodicals, Inc.

Dioxin receptor regulates aldehyde dehydrogenase to block melanoma tumorigenesis and metastasis.

BackgroundThe dioxin (AhR) receptor can have oncogenic or tumor suppressor activities depending on the phenotype of the target cell. We have shown that AhR knockdown promotes melanoma primary tumorigenesis and lung metastasis in the mouse and that human metastatic melanomas had reduced AhR levels with respect to benign nevi.MethodsMouse melanoma B16F10 cells were engineered by retroviral transduction to stably downregulate AhR expression, Aldh1a1 expression or both. They were characterized for Aldh1a1 activity, stem cell markers and migration and invasion in vitro. Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging.ResultsDepletion of aldehyde dehydrogenase 1a1 (Aldh1a1) impairs the pro-tumorigenic and pro-metastatic advantage of melanoma cells lacking AhR expression (sh-AhR). Thus, Aldh1a1 knockdown in sh-AhR cells (sh-AhR + sh-Aldh1a1) diminished their migration and invasion potentials and blocked tumor growth and metastasis to the lungs in immunocompetent AhR+/+ recipient mice. However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo. Aldh1a1 knockdown reduced the high levels of CD133+/CD29+/CD44+ cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells. Interestingly, Sox2 increased Aldh1a1 expression in sh-AhR but not in sh-AhR + sh-Aldh1a1 cells, suggesting that Aldh1a1 and Sox2 may be co-regulated in melanoma cells. In vivo imaging revealed that mice inoculated with AhR + Aldh1a1 knockdown cells had reduced tumor burden and enhanced survival than those receiving Aldh1a1-expressing sh-AhR cells.ConclusionsAldh1a1 overactivation in an AhR-deficient background enhances melanoma progression. Since AhR may antagonize the protumoral effects of Aldh1a1, the AhRlow-Aldh1a1high phenotype could be indicative of bad outcome in melanoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0419-9) contains supplementary material, which is available to authorized users.

Abstract 5204: Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor

Abstract Migration and adhesion are two critical processes highly related to metastasic dissemination and all trans-retinoic acid (ATRA) is known to diminished migration and metalloprotease (MMP) secretion in breast cancer cell lines. Our objective was to evaluate the effect of activating each retinoic acid receptor (RAR) isotype in adhesion, migration and MMPs secretion using two hormone-independent murine cell lines: LM38-LP y 4T1. First we determined that both lines express the different RAR isotypes with the exception of RARβ in 4T1 cell line. Cells were treated for 96h with AM580 (RARα agonist, 200nM), AC55649 (RARβ agonist, 2μM), BMS961 (RARγ agonist, 50nM) or vehicle (DMSO). The migratory potential was evaluated by a “wound healing” assay. LM38-LP cell line reduced its migratory capacity in response to AM580 and AC55649 (AM580: 108.0±24.3μm, AC55649: 114.1±12.8μm vs. vehicle: 186.9±31.0μm, p<0.05). An inverse effect was observed in 4T1 cells which increased its migratory potential after the treatment with AM580 (378.9±32.0μm vs. vehicle 302.2±31.9μm, p<0.05). The same agonist treatments were used to obtain conditioned media in order to evaluate soluble MMPs activity by zymography. AM580, AC55649 and BMS961 pre-treatments decreased soluble MMP2 activity in LM38-LP cells (0.53±0.03au (arbitrary units), 0.47±0.01 au, and 0.75±0.04 au respectively, fold change vs. control). Conversely, AM580 pre-treatment increased MMP2 and MMP9 in 4T1 cells (1.57±0.10 au and 2.04±0.26 au respectively, fold change vs. control). Regarding adhesion assay, after the above mentioned treatments cells were detached, incubated 2h at 37°C in order to recover cell surface proteins and finally seeded. After 2h incubation, non-adherent cells were washed out with PBS and adherent cells were fixed, stained and quantified by densitometry. We observed that AM580 and AC55649 diminished LM38-LP adhesive capacity (0.51±0.02 au, 0.83±0.00 au respectively, fold change vs. control) while AC 55649 increased this parameter in 4T1 cells (1.28±0.06 au fold change vs. control). Finally we performed an experimental lung metastasis assay using LM38-LP cells. Cells where treated with the different agonist during 144h and then inoculated into the tail vein of syngeneic mice. Lungs were removed 21 days later and number of superficial lung metastasis was determined. AC55649 treatment induced an increase in the metastatic potential (Md [Rg]: 143, [86-314] vs. control Md [Rg]: [21.5, 2-73], p<0.05). In conclusion, the activation of RARα and RARβ isotypes leads to opposite responses in different cell lines. We hypothesized that the differences in RARβ expression between this two cell lines should be responsible of this effect. Surprisingly, the activation of RARβ increased the metastatic potential of LM38-LP cells in spite of the negative regulation of parameters associated with the metastatic process. Citation Format: Carolina Flumian, Damián E. Berardi, Stefano M. Cirigliano, María I. Díaz Bessone, Elisa D. Bal de Kier Joffé, Alejandro J. Urtreger, Laura B. Todaro. Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5204. doi:10.1158/1538-7445.AM2015-5204

Abstract 193: Epigenetic regulation of Notch1 signaling-driven melanoma progression

Abstract Objectives. Aberrant Notch signaling promotes melanoma progression, yet the underlying molecular mechanism remains largely unknown. We hypothesized a potential involvement of epigenetic mechanism in this process and sought to identify Notch-regulated miRNAs and their functional targets. Methods. Notch-regulated miRNAs were identified by examining miRNA profiles in response to Notch activation/inhibition in human melanoma cells using RT2-miRNAArray. The role of specific miRNA in mediating the oncogenic effect of the Notch signaling was studied by testing whether alteration of miRNA expression could reverse the Notch's effects on melanoma cell growth, survival and colony-formation in vitro, and lung metastasis in vivo. The targets of the Notch-regulated miRNA were identified by searching miRNA target database and validated by immunoblot. The roles of miRNA targets in mediating the Notch's oncogenic effects were further assessed by in vitro colony-formation and in vivo lung metastasis assays. Results. Notch activation down-regulated mi143 in melanoma cells. Reconstituted expression of mi143 antagonized Notch1's oncogenic effect. The anti-oncogenic effect of mi143 is mediated by inhibiting Notch1-induced expression of FGF-1, uPA and uPAR since expression of these targets was up-regulated by the Notch1 activation while down-regulated by reconstituted overexpression of mi143. Supplementation of exogenous FGF-1 or reconstituted expression of uPA/uPAR, but not uPA alone, reversed the suppressive effect of mir143 on Notch-enhanced melanoma cells colony formation in soft-agar or lung metastasis in SCID mice. Conclusions. Notch activation up-regulates expression of FGF-1, uPA and uPAR through down-regulating mi143 expression in melanoma cells. mi143 is responsible for mediating aberrant Notch signaling-driven melanoma progression. The effect of down-regulated mi143 is transmitted through elevated FGF-1 and enhanced uPA-uPAR signaling activity, but not enzyme activity of uPA. This study unveils novel Notch1/mi143/FGF-1 and Notch1/mi143/uPA-uPAR cascades and uncovers an epigenetic mechanism underlying the oncogenic effect of Notch signaling, and provides potential novel targets for melanoma prognosis and therapy. Note: This abstract was not presented at the meeting. Citation Format: Ling Yin, Qinghua Huang, Alan S. Livingstone, Omaida C. Velazquez, Zhao-Jun Liu. Epigenetic regulation of Notch1 signaling-driven melanoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 193. doi:10.1158/1538-7445.AM2015-193

Germ line knockout of IGFBP-3 reveals influences of the gene on mammary gland neoplasia.

Insulin-like growth factor binding protein-3 (IGFBP-3) is an important carrier protein for insulin-like growth factors (IGFs) in the circulation. IGFBP-3 antagonizes the growth-promoting and anti-apoptotic activities of IGFs in experimental systems, but in certain contexts can increase IGF bioactivity, probably by increasing its half-life. The goal of this study was to investigate the role of IGFBP-3 in breast carcinogenesis and breast cancer metastasis. In the first part of the study, we exposed IGFBP-3 knockout and wild-type female mice to dimethylbenz[a]anthracene (DMBA) and followed them for appearance of primary tumors for up to 13 months. In the second part, mice of each genotype received an IV injection of 4T1 mammary carcinoma cells and then lung nodules were counted. Our results show that IGFBP-3 knockout mice developed breast tumors significantly earlier than the wild-type (13.9 ± 1.1 versus 22.5 ± 3.3 weeks, respectively, P = 0.0144), suggesting tumor suppression activity of IGFBP-3. In tumors of IGFBP-3 knockout mice, levels of phospho-AKT(Ser473) were increased compared to wild-type mice. The lung metastasis assay showed significantly more and larger lung nodules in IGFBP-3 knockout mice than in wild-type mice. While we observed increased levels of IGFBP-5 protein in the IGFBP-3 knockout mice, our findings suggest that this was not sufficient to completely compensate for the absence of IGFBP-3. Even though knockout of IGFBP-3 is associated with only a subtle phenotype under control conditions, our results reveal that loss of this gene has measurable effects on breast carcinogenesis and breast cancer metastasis.