Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin: suppression of Notch1, stabilization of beta-catenin and pre-adipocyte factor 1

Pasteurella multocida toxin (PMT) is a potent mitogen and a specific activator of Gq-dependent signalling pathways. PMT impairs osteoblast differentiation and causes bone loss and fat reduction in vivo. We examined the effect of PMT on cell signalling pathways involved in 3T3-L1 adipocyte differentiation. We demonstrate that PMT treatment before or together with differentiation induction factors inhibits adipogenesis and prevents upregulation of important adipocyte markers - peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha). Moreover, PMT completely downregulates PPARgamma and C/EBPalpha expression in mature adipocytes. Differentiation of pre-adipocytes into adipocytes requires the suppression of pre-adipocyte factor 1 (Pref1) and Wnt signalling, along with the degradation of beta-catenin. PMT prevents downregulation of Pref1 and beta-catenin under differentiation-inducing conditions. In addition, PMT treatment downregulates expression of Notch1, a protein responsible for cell fate decision and implicated in regulation of adipogenesis in 3T3-L1 cells. PMT action on adipogenesis was not reversed by cyclosporin A, an inhibitor of Galphaq-PLC-calcium-dependent calcineurin activation. Our results reveal new pathways involved in PMT action on cellular physiology and differentiation. Our study further demonstrates that the effect of PMT on Pref1/PPARgamma/C/EBPalpha expression and adipogenesis does not occur just through activation of the Galphaq-calcium-calcineurin pathway, but involves Wnt/beta-catenin and Notch1 signalling pathways, two signalling pathways strongly linked to cancer predisposition, neurological and immunological dysfunctions, and fat and bone development.     

Inter-cellular adhesion disruption and the RAS/RAF and beta-catenin signalling in lung cancer progression

Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and their loss has been correlated with poor prognosis in human cancer. Mutational activation of protein kinases and loss of cell adhesion occur together in human lung adenocarcinoma but how these two pathways interconnect is only poorly understood. Mouse models of human lung adenocarcinoma with oncogene expression targeted to subtypes of lung epithelial cells led to formation of adenomas or adenocarcinomas that lacked metastatic potential. Conditional genetic abrogation of epithelial tumour cell adhesion in mice with benign lung tumours induced by oncogenic RAF kinase has been demonstrated to induce intratumourous vascularization (angiogenic switch), progression to invasive adenocarcinoma and micrometastasis. Importantly, breaking cell adhesion in benign oncogene-driven lung tumour cells activated β-catenin signalling and induced the expression of several genes that are normally expressed in intestine rather than the lung. I will discuss potential routes to nuclear β-catenin signalling in cancer and how nuclear β-catenin may epigenetically alter the plasticity of tumour cells during malignant progression.     

Global gene expression profiles of canine macrophages and canine mammary cancer cells grown as a co-culture in vitro

Background

Solid tumours comprise various cells, including cancer cells, resident stromal cells, migratory haemopoietic cells and other. These cells regulate tumour growth and metastasis. Macrophages constitute probably the most important element of all interactions within the tumour microenvironment. However, the molecular mechanism, that guides tumour environment, still remains unknown. Exploring the underlying molecular mechanisms that orchestrate these phenomena has been the aim of our study. A co-culture of canine mammary cancer cells and macrophages was established and maintained for 72 hrs. Having sorted the cells, gene expression in cancer cells and macrophages, using DNA microarrays, was examined. The results were confirmed using real-time qPCR and confocal microscopy. Moreover, their ability for migration and invasion has been assessed.

Results

Microarray analysis showed that the up-regulated genes in the cancer cell lines are involved in 15 highly over-manifested pathways. The pathways that drew our diligent attention included: the inflammation pathway mediated by chemokine and cytokine, the Toll receptor signalling pathway and the B cell activation. The up-regulated genes in the macrophages were involved in only 18 significantly over-manifested pathways: the angiogenesis, the p53 pathway feedback loops2 and the Wnt signalling pathway. The microarray analysis revealed that co-culturing of cancer cells with macrophages initiated the myeloid-specific antigen expression in cancer cells, as well as cytokine/chemokine genes expression. This finding was confirmed at mRNA and protein level. Moreover, we showed that macrophages increase cancer migration and invasion.

Conclusions

The presence of macrophages in the cancer environment induces acquisition of the macrophage phenotype (specific antigens and chemokines/cytokines expression) in cancer cells. We presumed that cancer cells also acquire other myeloid features, such as: capabilities of cell rolling, spreading, migration and matrix invasion (what has also been confirmed by our results). It may, perhaps, be the result of myeloid-cancer cell hybrid formation, or cancer cells mimicking macrophages phenotype, owing to various proteins secreted by macrophages.     

KCTD11 inhibits progression of lung cancer by binding to β-catenin to regulate the activity of the Wnt and Hippo pathways

KCTD11 has been reported to be a potential tumour suppressor in several tumour types. However, the expression of KCTD11 and its role has not been reported in human non-small cell lung cancer (NSCLC). Whether its potential molecular mechanism is related to its BTB domain is also unknown. The expression of KCTD11 in 139 NSCLC tissue samples was detected by immunohistochemistry, and its correlation with clinicopathological factors was analysed. The effect of KCTD11 on the biological behaviour of lung cancer cells was verified in vitro and in vivo. Its effect on the epithelial-mesenchymal transition(EMT)process and the Wnt/β-catenin and Hippo/YAP pathways were observed by Western blot, dual-luciferase assay, RT-qPCR, immunofluorescence and immunoprecipitation. KCTD11 is under-expressed in lung cancer tissues and cells and was negatively correlated with the degree of differentiation, tumour-node-metastasis (TNM) stage and lymph node metastasis. Low KCTD11 expression was associated with poor prognosis. KCTD11 overexpression inhibited the proliferation and migration of lung cancer cells. Further studies indicated that KCTD11 inhibited the Wnt pathway, activated the Hippo pathway and inhibited EMT processes by inhibiting the nuclear translocation of β-catenin and YAP. KCTD11 lost its stimulatory effect on the Hippo pathway after knock down of β-catenin. These findings confirm that KCTD11 inhibits β-catenin and YAP nuclear translocation as well as the malignant phenotype of lung cancer cells by interacting with β-catenin. This provides an important experimental basis for the interaction between KCTD11, β-catenin and YAP, further revealing the link between the Wnt and Hippo pathways.     

EZH2 is a potential prognostic predictor of glioma

The enhancer of zeste homologue 2 (EZH2) is a histone H3 lysine 27 methyltransferase that promotes tumorigenesis in a variety of human malignancies by altering the expression of tumour suppressor genes. To evaluate the prognostic value of EZH2 in glioma, we analysed gene expression data and corresponding clinicopathological information from the Chinese Glioma Genome Atlas, the Cancer Genome Atlas and GTEx. Increased expression of EZH2 was significantly associated with clinicopathologic characteristics and overall survival as evaluated by univariate and multivariate Cox regression. Gene Set Enrichment Analysis revealed an association of EZH2 expression with the cell cycle, DNA replication, mismatch repair, p53 signalling and pyrimidine metabolism. We constructed a nomogram for prognosis prediction with EZH2, clinicopathologic variables and significantly correlated genes. EZH2 was demonstrated to be significantly associated with several immune checkpoints and tumour-infiltrating lymphocytes. Furthermore, the ESTIMATE and Timer Database scores indicated correlation of EZH2 expression with a more immunosuppressive microenvironment for glioblastoma than for low grade glioma. Overall, our study demonstrates that expression of EZH2 is a potential prognostic molecular marker of poor survival in glioma and identifies signalling pathways and immune checkpoints regulated by EHZ2, suggesting a direction for future application of immune therapy in glioma.     

Retracted: CRNDE promotes cell tongue squamous cell carcinoma cell growth and invasion through suppressing miR-384

Tongue squamous cell carcinoma (TSCC) is the most common type of oral cancer and is an aggressive head and neck malignancy. Increasing studies have demonstrated that long noncoding RNAs (lncRNAs) play important roles in diverse biological cell processes, such as cell development, fate decisions, cell differentiation, cell migration, and invasion. In our study, we showed that long noncoding RNA colorectal neoplasia differentially expressed (CRNDE) expression was upregulated in TSCC cell lines and tissues. Overexpression of CRNDE increased the TSCC cell proliferation, cell cycle, and cell invasion. Moreover, ectopic expression of CRNDE inhibited the miR-384 expression in the SCC1 cell and increased the Kirsten Ras (KRAS), cell division cycle 42, and insulin receptor substrate 1 expression, which were the direct target genes of miR-384. We demonstrated that the miR-384 expression was downregulated in the TSCC samples compared with the paired adjacent nontumor samples. The expression of CRNDE was negatively correlated with the expression of miR-384 in the TSCC samples. Overexpression of miR-384 suppressed TSCC cell proliferation, cell cycle, and invasion. Furthermore, we demonstrated that CRNDE promoted TSCC cell proliferation and invasion through inhibiting miR-384 expression. These results suggested that CRNDE acts as an oncogene in the development of TSCC, which partially occurs through inhibiting miR-384 expression.     

The anti-apoptotic protein lifeguard is expressed in breast cancer cells and tissues

Lifeguard (LFG) is an anti-apoptotic protein that inhibits Fas-mediated death in tumour cells. However, the molecular function of human LFG in the carcinogenesis of human breast cells is uncertain. We studied the expression and function of endogenous LFG in four breast cancer cell lines (MCF-7, MDA-MB-231, T-47D and HS 578T), a human breast epithelial cell line (HS 578Bst), and in healthy and cancerous breast tissues. Molecular (Western blot and RT-PCR) and immunohistochemical techniques were used to investigate the LFG expression. To investigate the breast cancer cell proliferation in the presence of Fas, we performed fluorescent cell viability assays. The possible association of Fas with LFG was analyzed by immunofluorescence microscopy. In this paper, we provide convincing evidence that LFG is overexpressed in several human breast cancer cell lines. More importantly, we found that the LFG expression correlates with high tumour grades in primary breast tumours. Finally, we demonstrated that Fas sensitivity is reduced in breast cancer cell lines expressing LFG. Our results indicated that LFG is strongly expressed in breast cancer epithelial cells. Moreover, the overexpression of LFG correlated with tumour grade and reduced Fas sensitivity. Our findings support the idea that LFG may have a role in the downregulation of apoptosis in breast cancer cells.     

The NKG2D receptor: sensing stressed cells

The activating killer cell lectin-like receptor NKG2D plays a key role in the natural killer (NK) cell-mediated lysis of tumours and infected cells. Unlike other receptors, the ligands recognised by NKG2D are 'induced-self' ligands on stressed cells. This system requires precise regulation because inappropriate expression of NKG2D ligands might compromise NK cell activation. For therapeutic purposes it is essential to understand the mechanisms that regulate the expression and function of the NKG2D system. This review focuses on the importance of the signalling pathways involved in the regulation of the NKG2D receptor and its ligand expression in arming the immune response against infected or tumour cells and for the identification of new molecular targets and therapeutic strategies.     

The membrane-bound mucins: From cell signalling to transcriptional regulation and expression in epithelial cancers

The membrane-bound mucins belong to an ever-increasing family of O-glycoproteins. Based on their structure and localization at the cell surface they are thought to play important biological roles in cell–cell and cell–matrix interactions, in cell signalling and in modulating biological properties of cancer cells. Among them, MUC1 and MUC4 mucins are best characterized. Their altered expression in cancer (overexpression in the respiratory, gastro-intestinal, urogenital and hepato-biliary tracts) indicates an important role for these membrane-bound mucins in tumour progression, metastasis, cancer cell resistance to chemotherapeutics drugs and as specific markers of epithelial cancer cells. Some mechanisms responsible for MUC1 and MUC4 role in tumour cell properties have been deciphered recently. However, much remains to be done in order to understand the molecular mechanisms and signalling pathways that control the expression of membrane-bound mucins during the different steps of tumour progression toward adenocarcinoma and evaluate their potential as prognostic/diagnostic markers and as therapeutic tools. In this review we focus on the molecular mechanisms and signalling pathways known to control the expression of membrane-bound mucins in cancer. We will discuss the mechanisms of regulation at the promoter level (including genetic and epigenetic modifications) that may be responsible for the mucin altered pattern of expression in epithelial cancers.     

Tumour-released exosomes and their implications in cancer immunity

Tumour cells release vesicular structures, defined as microvesicles or exosomes, carrying a large array of proteins from their originating cell. The expression of antigenic molecules recognized by T cells has originally suggested a role for these organelles as a cell-free antigen source for anticancer vaccines. However, recent evidence shows that tumour exosomes may also exert a broad array of detrimental effects on the immune system, ranging from apoptosis in activated antitumour T cells to impairment of monocyte differentiation into dendritic cells and induction of myeloid suppressive cells. Immunosuppressive exosomes of tumour origin can be found in neoplastic lesions and sera from cancer patients, implying a potential role of this pathway in in vivo tumour progression. Through the expression of molecules involved in angiogenesis promotion, stromal remodelling, delivery of signalling pathways through growth factor/receptor transfer, chemoresistance and genetic intercellular exchange, tumour exosomes could represent a versatile tool for moulding host environment. Hence, their secretion by neoplastic cells may in the future become a novel pathway to target for therapeutic intervention in cancer patients.     

Triggering caspase-independent cell death to combat cancer

Caspase-mediated apoptosis is a major hindrance to tumour growth and metastasis. Accordingly, defects in signalling pathways leading to the activation of caspases are common in tumours. Moreover, many tumour cells can unexpectedly survive the activation of caspases. As a result, caspase-independent cell death programmes are gaining increasing interest among cancer researchers. The heterogeneity of cancer cells with respect to their sensitivity to various death stimuli further emphasizes the need for additional death pathways in the therapeutic control of cell death. An understanding of the molecular control of alternative death pathways is beginning to emerge, being comparable with that of the molecular anatomy of apoptosis at the time of the discovery of caspases less than a decade ago. Here, newly discovered triggers and molecular regulators of alternative cell death programmes are reviewed and their potential in future cancer therapy is discussed.     

Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium

Interactions between tumour cells and the extracellular matrix (ECM) strongly influence tumour development, affecting cell survival, proliferation and migration. Many of these interactions are mediated through a family of cell surface receptors named integrins. Fibronectin and its integrin receptors play important roles in tumour development. The alpha5beta 1 integrin interacts with the central cell adhesive region of fibronectin and requires both the RGD and synergy sites for maximal binding. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases. They are capable of digesting the different components of the ECM and basement membrane. The ECM gives structural support to cells and plays a central role in cell adhesion, differentiation, proliferation and migration. Binding of ECM to integrins modulates expression and activity of the different MMPs. Our experimental findings demonstrate that cultivation of human breast cancer cells, MCF-7, in serum free medium in the presence of fibronectin upregulates the activity of MMP-2 and MMP-9. Blocking of alpha5beta 1 integrin with anti-alpha5 monoclonal antibody inhibits the fibronectin-induced MMP activation response appreciably. This strongly indicates alpha5beta 1 mediated signalling events in activation of MMP-2 and MMP-9. Phosphorylation of FAK and PI-3 kinase and the nuclear translocation of ERK and NF-kappaB upon fibronectin binding demonstrate possible participation of the FAK/PI-3K/ERK signalling pathways in the regulation of MMP-2 activity.     

Bmi1 Drives Stem-Like Properties and is Associated with Migration,Invasion, and Poor Prognosis in Tongue Squamous Cell Carcinoma

Bmi1 (B-cell-specific Moloney murine leukemia virus insertion site 1) had been found to involve in self -renewal of stem cells and tumorigenesis in various malignancies. The purpose of this study is to evaluate the role of Bmi1 in the development of tongue squamous cell carcinoma (TSCC) and its functional effect on the migration and invasion of TSCC. Initially, immunohistochemistry revealed that Bmi1 overexpression was a common event in premalignant dysplasia, primary TSCC, and lymph node metastases and was associated with a poor prognosis. A significant correlation between Bmi1 and SOD2 (manganese superoxide dismutase) expression was observed. Side population (SP) cells were used as cancer stem-like cells and further assessed by sphere and colony formation assays, and the expression of stem cell markers. TSCC cells with higher migration and invasion ability (UM1 cell lines) showed a higher proportion of SP cells and Bmi1 expression than TSCC cells with lower migration and invasion ability (UM2 cell lines). Knockdown of Bmi1 in UM1 or SP cells inhibited migration and invasion and decreased the sphere and colony formation, and the expression of stem cell markers and SOD2. Direct binding of C-myc to the Bmi1 promoter was demonstrated by chromatin immunoprecipitation and luciferase assays. Moreover, C-myc knockdown in SP cells inhibited their migration and invasion and decreased the expression of Bmi1 and SOD2. Our results indicate that the deregulation of Bmi1 expression is a frequent event during the progression of TSCC and may have a prognostic value for patients with this disease. The Bmi1-mediated migration and invasion of TSCC is related to cancer stem-like cells and involves the C-myc-Bmi1-SOD2 pathway.     

Glucose dysregulation promotes oncogenesis in human bladder cancer by regulating autophagy and YAP1/TAZ expression

Glucose dysregulation is strongly correlated with cancer development, and cancer is prevalent in patients with Type 2 diabetes (T2D). We aimed to elucidate the mechanism underlying autophagy in response to glucose dysregulation in human bladder cancer (BC). 220 BC patients were included in this retrospective study. The expression of YAP1, TAZ and AMPK, EMT-associated markers, and autophagy marker proteins was analysed by immunohistochemistry, western blotting, and quantitative real-time PCR (qPCR). Further, T24 and UMUC-3 BC cells were cultured in media with different glucose concentrations, and the expression of YAP1, TAZ, AMPK and EMT-associated markers, and autophagy marker proteins was analysed by western blotting and qPCR. Autophagy was observed by immunofluorescence and electron microscopy. BC cell viability was tested using MTT assays. A xenograft nude mouse model of diabetes was used to evaluate tumour growth, metastasis and survival. A poorer pathologic grade and tumour-node-metastasis stage were observed in patients with BC with comorbid T2D than in others with BC. YAP1 and TAZ were upregulated in BC samples from patients with T2D. Mechanistically, high glucose (HG) promoted BC progression both in vitro and in vivo and inhibited autophagy. Specifically, various autophagy marker proteins and AMPK were negatively regulated under HG conditions and correlated with YAP1 and TAZ expression. These results demonstrate that HG inhibits autophagy and promotes cancer development in BC. YAP1/TAZ/AMPK signalling plays a crucial role in regulating glucose dysregulation during autophagy. Targeting these effectors exhibits therapeutic significance and can serve as prognostic markers in BC patients with T2D.