RACK1 promotes lung cancer cell growth via an MCM7/RACK1/ Akt signaling complex.
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ABSTRACT:
SUBMITTER: Fei L
PROVIDER: S-EPMC5522230 | biostudies-other | 2017 Jun
REPOSITORIES: biostudies-other
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Project description:BackgroundNon-small cell lung cancer (NSCLC) is the most common type of lung cancer and the most lethal tumour worldwide. Copine 1 (CPNE1) was identified as a novel oncogene in NSCLC in our previous study. However, its specific function and relative mechanisms remain poorly understood.MethodsThe biological role of CPNE1 and RACK1 in NSCLC was investigated using gene expression knockdown and overexpression, cell proliferation assays, clonogenic assays, and Transwell assays. The expression levels of CPNE1, RACK1 and other proteins were determined by western blot analysis. The relationship between CPNE1 and RACK1 was predicted and investigated by mass spectrometry analysis, immunofluorescence staining, and coimmunoprecipitation. NSCLC cells were treated with a combination of a MET inhibitor and gefitinib in vitro and in vivo.ResultsWe found that CPNE1 facilitates tumorigenesis in NSCLC by interacting with RACK1, which further induces activation of MET signaling. CPNE1 overexpression promoted cell proliferation, migration, invasion and MET signaling in NSCLC cells, whereas CPNE1 knockdown produced the opposite effects. In addition, the suppression of the enhancing effect of CPNE1 overexpression on tumorigenesis and MET signaling by knockdown of RACK1 was verified. Moreover, compared to single-agent treatment, dual blockade of MET and EGFR resulted in enhanced reductions in the tumour volume and downstream signaling in vivo.ConclusionsOur findings show that CPNE1 promotes tumorigenesis by interacting with RACK1 and activating MET signaling. The combination of a MET inhibitor with an EGFR-TKI attenuated tumour growth more significantly than either single-drug treatment. These findings may provide new insights into the biological function of CPNE1 and the development of novel therapeutic strategies for NSCLC. Video Abstract.
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Project description:Background: Lung cancer has the highest mortality rate among cancers worldwide, with non-small cell lung cancer (NSCLC) the most common type. Increasing evidence shows that PHB2 is highly expressed in other cancer types; however, the effects of PHB2 in NSCLC are currently poorly understood. Method: PHB2 expression and its clinical relevance in NSCLC tumor tissues were analyzed using a tissue microarray. The biological role of PHB2 in NSCLC was investigated in vitro and in vivo using immunohistochemistry and immunofluorescence staining, gene expression knockdown and overexpression, cell proliferation assay, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, wound healing assay, Transwell assay, western blot analysis, qRT-PCR, coimmunoprecipitation, and mass spectrometry analysis. Results: Our major finding is that PHB2 facilitates tumorigenesis in NSCLC by interacting with and stabilizing RACK1, which further induces activation of downstream tumor-promoting effectors. PHB2 was found to be overexpressed in NSCLC tumor tissues, and its expression was correlated with clinicopathological features. Furthermore, PHB2 overexpression promoted proliferation, migration, and invasion, whereas PHB2 knockdown enhanced apoptosis in NSCLC cells. The stimulating effect of PHB2 on tumorigenesis was also verified in vivo. In addition, PHB2 interacted with RACK1 and increased its expression through posttranslational modification, which further induced activation of the Akt and FAK pathways. Conclusions: Our results reveal the effects of PHB2 on tumorigenesis and its regulation of RACK1 and RACK1-associated proteins and downstream signaling in NSCLC. We believe that the crosstalk between PHB2 and RACK1 provides us with a great opportunity to design and develop novel therapeutic strategies for NSCLC.
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Project description:Approximately 90% of patients diagnosed with non-small cell lung cancer (NSCLC) die due to distant metastases. However, the complicated molecular and cellular mechanisms involved in lung cancer metastasis remain poorly understood. Copine III (CPNE3), a member of a Ca2+-dependent phospholipid-binding protein family, was identified as a novel metastasis-associated protein in NSCLC in our previous study, however, its function in metastasis remains unclear. Here, we found that CPNE3 was expressed at high levels in NSCLC tissues and advanced TNM stages and was significantly associated with poor prognosis. In addition, CPNE3 interacted with phosphorylated erb-b2 receptor tyrosine kinase 2 (pErbB2) and receptor of activated protein C kinase 1 (RACK1) and activated the focal adhesion (FA) signaling pathway in NSCLC cells. Moreover, knockdown of RACK1 inhibited cell motility in the CPNE3-overexpressed NSCLC cells. These findings offer mechanistic insights into the oncogenic roles of CPNE3 and the pivotal effects of CPNE3 as a biomarker and therapeutic target for NSCLC metastasis.
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Project description:The contractile protein MYH9 (non-muscle myosin IIA) is an actin-binding protein that plays a fundamental role in cell adhesion, migration, and division. However, its distinct role in colorectal cancer (CRC) still remains unidentified. In this study, we detected significant MYH9 overexpression in CRC samples compared with paired normal tissues using western blotting and tissue microarray immunohistochemistry (IHC). Moreover, analysis of patient clinical information demonstrated that MYH9 overexpression was strongly correlated with lymph node metastasis and poor overall survival. Endogenous overexpression of MYH9 enhanced the ability of cell proliferation and migration in vitro, and accelerated CRC growth in mouse models. Silencing of MYH9 revealed repressive effects on CRC cells in vitro and in vivo. Furthermore, primary biomechanics that involved MAPK/AKT signaling mediated epithelial-mesenchymal transition (EMT) was uncovered underlying MYH9 dependent cell behaviors. Collectively, our data showed that MYH9 significantly promoted tumorigenesis by regulating MAPK/AKT signaling, and was remarkably correlated with poor prognosis in CRC. MYH9 may thus be a novel biomarker and drug target in the diagnosis and treatment of CRC.
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Project description:Non-small-cell lung cancer (NSCLC) is a deadly disease due to lack of effective diagnosis biomarker and therapeutic target. Much effort has been made in defining gene defects in NSCLC, but its full molecular pathogenesis remains unexplored. Here, we found RACK1 (receptor of activated kinase 1) was elevated in most NSCLC, and its expression level correlated with key pathological characteristics including tumor differentiation, stage, and metastasis. In addition, RACK1 activated sonic hedgehog signaling pathway by interacting with and activating Smoothened to mediate Gli1-dependent transcription in NSCLC cells. And silencing RACK1 dramatically inhibited in vivo tumor growth and metastasis by blocking the sonic hedgehog signaling pathway. These results suggest that RACK1 represents a new promising diagnosis biomarker and therapeutic target for NSCLC.
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Project description:BACKGROUND:miRNAs regulate a multitude of cellular processes and their aberrant regulation is linked to human cancer. However, the role of miR-425-5p in lung cancer (LCa) is still largely unclear. Here, we explored the role of miR-425-5p during LCa tumorigenesis. METHODS:Cell proliferation was evaluated by cell counting Kit-8 and colony formation assay. Western blot and real-time PCR were accordingly used to detect the relevant proteins, miRNA and gene expression. Luciferase reporter assays were used to illustrate the interaction between miR-425-5p and PTEN. RESULTS:We demonstrate that miR-425-5p is overexpressed in LCa tissue and enhances the proliferative and colony formation capacity of the LCa cell lines A549 and NCI-H1299. Through predictive binding assays, PTEN was identified as a direct gene target and its exogenous expression inhibited the pro-cancer effects of miR-425-5p. Through its ability to down-regulate PTEN, miR-425-5p activated the PI3K/AKT axis. CONCLUSION:We conclude that miR-425-5p promotes LCa tumorigenesis through PTEN/PI3K/AKT signaling.
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Project description:Current knowledge of TMEM17, a recently identified protein of the transmembrane (TMEM) family, is limited, especially with respect to its expression and biological functions in malignant tumors. This study analyzed TMEM17 expression in invasive breast cancer tissue and breast cell lines and its relevance to clinicopathological factors, and investigated the mechanisms underlying the biological effects of TMEM17 on breast cancer cells.TMEM17 protein expression was determined in 20 freshly harvested specimens (tumor and paired normal tissues) by Western blotting. Immunohistochemical analysis was performed to determine the expression and subcellular localization of TMEM17 in samples from 167 patients (mean age, 49 years) diagnosed with invasive ductal carcinoma (38 with triple-negative breast cancer; 129 with non-triple-negative breast cancer) who underwent complete resection in the First Affiliated Hospital of China Medical University between 2011 and 2013. Furthermore, TMEM17 was knocked down by small interfering RNAs in breast cancer cell lines.TMEM17 was found to be significantly upregulated in breast cancer tissues compared to the corresponding normal breast tissues by Western blotting (p=0.015). Immunohistochemical analysis revealed that TMEM was significantly upregulated in invasive breast cancer cells compared to adjacent normal breast duct glandular epithelial cells (10.78% vs 76.05%, p<0.001), and its expression was closely related to the patient's T-stage (p=0.022), advanced TNM stages (p=0.007), and lymph node metastasis (p=0.012). After TMEM17 knockdown or overexpression in breast cancer cell lines, TMEM17 upregulated p-AKT, p-GSK3?, active ?-catenin, and Snail, and downstream target proteins c-myc and cyclin D1, and downregulated E-cadherin, resulting in increased cancer cell proliferation, invasion, and migration. These effects were reversed by the AKT inhibitor LY294002.Our results indicate that TMEM17 is upregulated in breast cancer tissues and can promote malignant progression of breast cancer cells by activating the AKT/GSK3? signaling pathway.
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Project description:Background:A number of JmjC domain-containing histone demethylases have been identified and biochemically characterized in mammalian models and humans. JMJD2A is a transcriptional co-factor and enzyme that catalyzes the demethylation of histone H3 lysine 9 and 36 (H3K9 and H3K36). Here in this study, we reported the role of JMJD2A in human glioma. Methods:Quantitative real-time PCR and western blot were performed to analyzed JMJD2A expression in glioma. Log-rank was performed to plot the survival curve. JMJD2A was knocked or overexpressed with lentivirus. Cell proliferation and colony formation were performed to assess the effects of JMJD2A on glioma cell growth. Xenograft experiment was performed the evaluate the growth rate of glioma cells in vivo. The signaling pathway was analyzed with western blot and mTOR was inhibited with rapamycin. Results:Quantitative real-time PCR and western blot experiments revealed higher expression of JMJD2A and lower levels of H3K9me3/H3K36me3 in glioma tissues than that in normal brain tissues. We showed that knockdown of JMJD2A expression attenuated the growth and colony formation in three lines of glioma cells (U251, T98G, and U87MG), whereas JMJD2A overexpression resulted in opposing effects. Furthermore, we performed in vivo xenograft experiments and our data demonstrated that JMJD2A knockdown reduced the growth of glioma T98G cells in vivo. Further mechanism study implicated that JMJD2A activated the Akt-mTOR pathway and promoted protein synthesis in glioma cells via promoting phosphoinositide-dependent kinase-1 (PDK1) expression. The activation of the Akt-mTOR pathway was also validated in human glioma tissues. Finally, we showed that inhibition of mTOR with rapamycin blocked the effects of JMJD2A on protein synthesis, cell proliferation and colony formation of glioma cells. Conclusions:These findings demonstrated that JMJD2A regulated glioma growth and implicated that JMJD2A might be a promising target for intervention.
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