Akt/Ezrin Tyr353/NF-?B pathway regulates EGF-induced EMT and metastasis in tongue squamous cell carcinoma.
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ABSTRACT:
SUBMITTER: Wang Y
PROVIDER: S-EPMC3915131 | biostudies-literature | 2014 Feb
REPOSITORIES: biostudies-literature
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Project description:Locoregional lymph nodes metastasis in oral tongue squamous cell carcinoma represents one of important and common prognostic factors for poor clinical outcome. The human Telomerase Reverse Transcriptase (hTERT) is one of key players in cancer metastasis and stemness, but its exact function in tongue squamous cell carcinoma remains unknown. Here, we aim to understand the role of hTERT by utilizing the CRISPR/Cas9 gene editing system to deplete hTERT in the SCC-15 cell line. Functional comparison of SCC-15 control and knockout cells (hTERT-/-) showed that loss of hTERT suppressed cell proliferation and migration/invasion. Furthermore, hTERT depletion significantly decreased tumorigenesis, including alterations in cellular morphology that areindicative for epithelial-mesenchymal transition (EMT). Mechanistically we demonstrated that the hTERT knockout attenuates NF-κB signaling via a negative feedback regulation in tumorprogression. From these results we propose a novel molecular mechanism of hTERT to promote SCC-15 invasion and metastasis via NF-κB activation. We conclude that targeting hTERT may represent a new therapeutic strategy to improve therapy and survival of tongue squamous cell carcinoma patients.
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Project description:Background: Malignant proliferation and cervical lymphatic metastasis restrict the prognosis of oral squamous cell carcinoma (OSCC). Erythropoietin-producing human hepatocellular B4 (EPHB4) regulates a series of tumour functions involving tumourigenesis, cancer cell attachment and metastasis. However, the mechanism of EphB4 regulating the malignant progression of OSCC has not been fully elucidated. Methods: EPHB4 expression was analysed in 65 OSCC samples and adjacent noncancerous tissues through immunohistochemistry (IHC). siRNA and overexpression plasmids were transfected into OSCC cells to modify EPHB4 expression, and then, regulatory functions were explored in vitro and in vivo. Co-immunoprecipitation (Co-IP) and mass spectrometry were applied to detect proteins interacting with EPHB4. Subsequently, protein stability assays and NF-κB pathway inhibition assays were used to verify the regulation of EPHB4, high-mobility group box 1 (HMGB1) and nuclear factor-κB (NF-κB) activation. Results: EPHB4 was found to be highly expressed in OSCC tissues, which was related to tumour stage and lymphatic metastasis and resulted in a poor prognosis. Cellular experiments and mouse tongue xenograft models further confirmed that high EPHB4 expression promoted the proliferation and metastasis of OSCC tumours. Mechanistically, co-IP and mass spectrometry studies indicated that EPHB4 could bind to HMGB1 and maintain HMGB1 stability. Downregulation of HMGB1 inhibited the proliferation and metastasis of OSCC cells and inhibited NF-κB phosphorylation activation but did not affect EPHB4 expression. Conclusion: This study revealed the mechanism by which EPHB4 promotes the proliferation and metastasis of OSCC by activating the HMGB1-mediated NF-κB signalling pathway, which can be exploited as a novel marker or therapeutic target to control metastasis and improve the survival rate of OSCC.
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Project description:miR-19a/b belong to the miR-17-92 family. We have demonstrated previously that miR-19a/b are overexpressed in glioma and glioma cell lines. However, the role of miR-19a/b in glioma remains unclear. In the present study, we aim to identify the biological function and molecular mechanism of miR-19a/b in glioma cell proliferation and epithelial-mesenchymal transition (EMT). Knocking down miR-19a/b in LN308 glioblastoma (GBM) cells with higher expression of miR-19a/b inhibits cell proliferation and invasion, induces apoptosis, and suppresses EMT by downregulating the expression of Akt, phosphorylated p-Akt, nuclear factor κB (NF-κB), Snail, N-cadherin, and Vimentin and upregulating E-cadherin in vitro and in vivo. Enhanced proliferation and EMT are also observed when miR-19a/b are transfected into SNB19 GBM cells, with lowered expression of miR-19a/b. miR-19a is more effective than miR-19b in the regulation of biological behavior of glioma cells. miR-19a/b modulate molecular events for the promotion of EMT via the Akt-NF-κB pathway. SEPT7 has been confirmed as the target gene of miR-19a/b. The effect of miR-19a/b on proliferation and EMT of glioma cells and the Akt-NF-κB pathway could be reversed by transfection with SEPT7. Our study strongly suggests that miR-19a/b play a significant role in glioma progression and EMT through regulating target gene-SEPT7 and the SEPT7-Akt-NF-κB pathway.
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Project description:Breast cancer metastasis is the primary cause of mortality of patients with breast cancer. The present study aimed to explore the role and underlying mechanisms of IGJ in the invasion and metastasis of breast cancer. The Cancer Genome Atlas database was utilized to analyze the differential gene expression profiles in patients with breast cancer with or without metastasis; the target gene, joining chain of multimeric IgA and IgM (JCHAIN, also known as IGJ, as referred to herein), with significant expression and with prognostic value was screened. The expression levels of IGJ in human breast cancer paired tissues and cell lines were detected using reverse transcription‑quantitative PCR and western blot analysis. IGJ differential expression was detected in paired human breast cancer tissues using immunohistochemistry. The role of IGJ in breast cancer was verified using CCK‑8, invasion and migration assays, and scratch tests in vivo and in vitro. Further exploration of the role and mechanism of IGJ in breast cancer was conducted through Gene Set Enrichment Analysis, Kyoto Encyclopedia of Genes and Genomes analysis, western blot analysis and immunofluorescence experiments. Through the analysis of gene expression profiles, it was found that IGJ was poorly expressed in patients with breast cancer with metastasis compared to patients with non‑metastatic breast cancer. The overexpression of IGJ was associated with an improved distant metastasis‑free survival and overall survival (OS). COX multivariate regression analysis demonstrated that IGJ was an independent prognostic factor for the OS and relapse‑free survival of patients with breast cancer. In comparison to healthy breast cancer adjacent tissues and cell lines, IGJ was poorly expressed in breast cancer tissues and cell lines (P<0.05). Further analyses indicated that the overexpression of IGJ suppressed the proliferation, invasion and metastasis of breast cancer cells in vivo and in vitro by inhibiting the occurrence of epithelial‑to‑mesenchymal transition (EMT) and suppressing the nuclear translocation of p65. Finally, rescue experiments indicated that IGJ restricted the proliferation and metastasis of breast cancer cells by regulating the NF‑κB signaling pathway. On the whole, the present study demonstrates that IGJ suppresses the invasion and metastasis of breast cancer by inhibiting both the occurrence of EMT and the NF‑κB signaling pathway. These findings may provide novel biomarkers and potential therapeutic targets for the treatment of metastatic breast cancer.
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Project description:Iroquois homeobox gene 5 (Irx5) is a highly conserved member of the Iroquois homeobox gene family. Members of this family play distinct and overlapping roles in normal embryonic cell patterning and development of malignancies. In this study, we observed that IRX5 was abnormally abundant in tongue squamous cell carcinoma (TSCC) tissues and cell lines. We used gain- and loss-of-function methods to overexpress and knockdown IRX5 expression in the TSCC cell line CAL27. Our results elucidated that elevated levels of IRX5 promoted proliferation, migration and invasion of TSCC cells, whereas stable or transient knockdown of IRX5 expression suppressed TSCC cell proliferation, migration and invasion. As a transcription factor, IRX5 performed this function by targeting osteopontin (OPN) promoter and activating the NF-κB pathway. Finally, studies in xenograft tumour model showed that IRX5 significantly enhanced OPN expression and promoted tumour growth. Taken together, our study elucidates a promotive effect of IRX5 in TSCC through the connection with OPN. These findings reveal the new molecular mechanism of TSCC, which may potentiate its use as a novel molecular therapy target for TSCC.
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Project description:G-protein-coupled receptors (GPCRs) signaling is critical to cell differentiation and activation. However, the function of GPCRs in osteoclast differentiation and activation remains unclear. We found that the G-protein coupled receptor 125 (GPCR 125) gene (Gpr125) gene was highly expressed in osteoclasts through RNA-sequencing technology, qRT-PCR, and Western blot analysis. We characterized the role of GPCR125 in osteoclast differentiation and activation by loss-of-function and gain-of-function methods in osteoclasts. Osteoclasts with lentivirus-mediated GPR125 silencing demonstrated a dramatic reduction in differentiation and impaired bone resorption function. In contrast, overexpression of Gpr125 in osteoclasts increased NFATC1 expression and enhanced osteoclast differentiation and enhanced osteoclast-mediated bone resorption. These results indicated that GPCR125 positively regulates osteoclast formation and function. Following receptor activator of nuclear factor kappa-Β ligand (RANKL) stimulation, the expression levels of MAPK signaling pathway proteins phosphorylated-ERK (p-ERK) and phosphorylated-p38 (p-p38) were significantly decreased in the Gpr125 knockdown (sh-GPR125) group compared to its control group. We also found that phosphorylated AKT (p-AKT) expression was downregulated, as well as nuclear factor kappa-B (NF-κB) signaling pathway protein phosphorylated-IKB alpha (p-IKBα). Our results demonstrated that GPCR125 positively regulates osteoclasts via RANKL-stimulated MAPK and AKT-NF-κB signaling pathways, and GPCR125 could potentially be utilized as a novel therapeutic target in bone related diseases including osteoporosis.
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Project description:BackgroundThe primary issue arising from prostate cancer (PCa) is its high prevalence to metastasize to bone, which severely affects the quality of life and survival time of PCa patients. miR-210-3p is a well-documented oncogenic miRNA implicated in various aspects of cancer development, progression and metastasis. However, the clinical significance and biological roles of miR-210-3p in PCa bone metastasis remain obscure.MethodsmiR-210-3p expression was evaluated by real-time PCR in 68 bone metastatic and 81 non-bone metastatic PCa tissues. The biological roles of miR-210-3p in the bone metastasis of PCa were investigated both in vitro by EMT and Transwell assays, and in vivo using a mouse model of left cardiac ventricle inoculation. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to discern and examine the relationship between miR-210-3p and its potential targets. RT-PCR was performed to identify the underlying mechanism of miR-210-3p overexpression in bone metastasis of PCa. Clinical correlation of miR-210-3p with its targets was examined in human PCa and metastatic bone tissues.ResultsmiR-210-3p expression is elevated in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Overexpression of miR-210-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Upregulating miR-210-3p enhances, while silencing miR-210-3p represses the EMT, invasion and migration of PCa cells in vitro. Importantly, silencing miR-210-3p significantly inhibits bone metastasis of PC-3 cells in vivo. Our results further demonstrate that miR-210-3p maintains the sustained activation of NF-κB signaling via targeting negative regulators of NF-κB signaling (TNF-α Induced Protein 3 Interacting Protein 1) TNIP1 and (Suppressor Of Cytokine Signaling 1) SOCS1, resulting in EMT, invasion, migration and bone metastasis of PCa cells. Moreover, our results further indicate that recurrent gains (amplification) contribute to miR-210-3p overexpression in a small number of PCa patients. The clinical correlation of miR-210-3p with SOCS1, TNIP1 and NF-κB signaling activity is verified in PCa tissues.ConclusionOur findings unravel a novel mechanism for constitutive activation of NF-κB signaling pathway in the bone metastasis of PCa, supporting a functional and clinical significance of epigenetic events in bone metastasis of PCa.
| S-EPMC5504657 | biostudies-literature
Project description:microRNAs(miRNAs) can regulate epithelial-mesenchymal transition (EMT) through transcription factors, however, little is known whether EMT transcription factors can modulate miRNAs and further induce EMT and cancer metastasis. Here we show that overexpression of Snail and Slug leads to a mesenchymal phenotype and morphology and enhances cell invasion along with stem cell properties in squamous cell carcinoma of oral tongue (OTSCC) cells. Repression of miR-101 expression by Snail and Slug is essential for Snail/Slug-induced malignant phenotypes. The suppression of miR-101 subsequently activates EZH2, the sole histone methyltransferase, inducing EMT, migration and invasion of OTSCC cells. Importantly, co-overexpression of Slug and Snail correlates with poor survival and elevated EZH2 expression in two independent patient cohorts of OTSCC specimens. These findings defined a Snail and Slug/miR-101/EZH2 pathway as a novel regulatory axis of EMT-mediated-microRNA signaling.
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Project description:BackgroundTumor metastasis is driven by malignant cells and stromal cell components of the tumor microenvironment. Cancer stem cells (CSCs) are thought to be responsible for metastasis by altering the tumor microenvironment. Epithelial-mesenchymal transition (EMT) processes contribute to specific stages of the metastatic cascade, promoted by cytokines and chemokines secreted by stromal cell components in the tumor microenvironment. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine ligand 21(CCL21), to mediate metastasis in some cancer cells lines. This study investigated the role of CCL21/CCR7 in promoting EMT and metastasis of cluster of differentiation 133+ (CD133+) pancreatic cancer stem-like cells.MethodsPanc-1, AsPC-1, and MIA PaCa-2 pancreatic cancer cells were selected because of their aggressive invasive potentials. CCR7 expression levels were examined in total, CD133+ and CD133- cell fractions by Immunofluorescence analysis and real time-quantitative polymerase chain reaction (RT-qPCR). The role of CCL21/CCR7 in mediating metastasis and survival of CD133+ pancreatic cancer stem-like cells was detected by Transwell assays and flow cytometry, respectively. EMT and lymph node metastasis related markers (E-cadherin, N- cadherin, LYVE-1) were analyzed by western blot. CCR7 expression levels were analyzed by immunohistochemical staining and RT-qPCR in resected tumor tissues, metastatic lymph nodes, normal lymph nodes and adjacent normal tissues from patients with pancreatic carcinoma.ResultsCCR7 expression was significantly increased in CD133+ pancreatic cancer stem-like cells, resected pancreatic cancer tissues, and metastatic lymph nodes, compared with CD133- cancer cells, adjacent normal tissues and normal lymph nodes, respectively. CCL21/CCR7 promoted metastasis and survival of CD133+ pancreatic cancer stem-like cells and regulated CD133+ pancreatic cancer stem-like cells metastasis by modulating EMT and Erk/NF-κB pathway.ConclusionsThese results indicate a specific role for CCL21/CCR7 in promoting EMT and metastasis in CD133+ pancreatic cancer stem-like cells. Furthermore the data also indicated the potential importance of developing therapeutic strategies targeting cancer stem-like cells and CCL21/CCR7 for reducing metastasis.
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