Human cytomegalovirus induces cellular tyrosine kinase signaling and promotes glioma cell invasiveness

Given our previous findings that human cytomegalovirus (HCMV) nucleic acids and proteins are expressed in human malignant glioma in vivo, we investigated cellular signaling events associated with HCMV infection of human glioma and astroglial cells. HCMV infection caused rapid activation of the phosphatidylinositol-3 kinase (PI-3K) effector AKT kinase in human astro-glial and fibroblast cells, and induced tyrosine phosphorylation of phospholipase Cγ (PLCγ). Co-immunoprecipitation experiments revealed association of the p85 regulatory subunit of PI-3K with a high-molecular weight protein phosphorylated on tyrosine, following short-term exposure to HCMV. In contrast to a previous report, we were unable to confirm the identity of this high-molecular weight protein as being the epidermal growth factor receptor (EGFR). Stimulation of glioma and fibroblast cell lines over-expressing EGFR with HCMV (whole virus) or soluble glycoprotein B did not induce tyrosine phosphorylation of the receptor, as did the genuine ligand, EGF. Furthermore, we found that expression levels of the human ErbB1-4 receptors were not rate-limiting for HCMV infection. Dispensability of EGFR function during early HCMV infection was substantiated by demonstration of viral immediate early gene expression in cells lacking the EGFR gene, indicating that HCMV may promote oncogenic signaling pathways independently of EGFR activation. Among non-receptor cellular kinases, HCMV infection induced phosphorylation of focal adhesion kinase (FAK) Tyr397, which is indispensable for integrin-mediated cell migration and invasion. HCMV-induced FAK activation was paralleled by increased extracellular matrix-dependent migration of human malignant glioma but not normal astro-glial cells, suggesting that HCMV can selectively augment glioma cell invasiveness. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Erythropoietin signaling promotes invasiveness of human head and neck squamous cell carcinoma

Erythropoietin (Epo) is used for managing anemia in cancer patients. However, recent studies have raised concerns for this practice. We investigated the expression and function of Epo and the erythropoietin receptor (EpoR) in tumor biopsies and cell lines from human head and neck cancer. Epo responsiveness of the cell lines was assessed by Epoetin-alpha-induced tyrosine phosphorylation of the Janus kinase 2 (JAK2) protein kinase. Transmigration assays across Matrigel-coated filters were used to examine the effects of Epoetin-alpha on cell invasiveness. In 32 biopsies, we observed a significant association between disease progression and expression of Epo and its receptor, EpoR. Expression was highest in malignant cells, particularly within hypoxic and infiltrating tumor regions. Although both Epo and EpoR were expressed in human head and neck carcinoma cell lines, only EpoR was upregulated by hypoxia. Epoetin-alpha treatment induced prominent JAK2 phosphorylation and enhanced cell invasion. Inhibition of JAK2 phosphorylation reduced both basal and Epo-induced invasiveness. Our findings support a role for autocrine or paracrine Epo signaling in the malignant progression and local invasiveness of head and neck cancer. This mechanism may also be activated by recombinant Epo therapy and could potentially produce detrimental effects in rhEpo-treated cancer patients.     

Aberrant methylation of EYA4 promotes epithelial‐mesenchymal transition in esophageal squamous cell carcinoma

EYA4, one of the four members of the EYA gene family, is associated with several human cancers. However, its biological functions and molecular mechanisms in the progression of cancer, particularly in esophageal squamous cell carcinoma (ESCC), remain unknown. In the present study, we found that EYA4 was underexpressed and hypermethylated in most of the ESCC cell lines tested (85.7%, 6/7). Treatment with 5‐aza‐dC and/or trichostatin A (TSA) restored EYA4 expression in ESCC cell lines, which indicates that EYA4 expression was epigenetically regulated. Similarly, EYA4 was aberrantly hypermethylated in ESCC tissues (78%, 39/50) and downregulation of EYA4 occurred in approximately 65% of primary ESCC at protein level where it was associated significantly with TNM stage and lymph node metastases. Knockdown of EYA4 in KYSE30 and KYSE70 ESCC cells using small hairpin RNA increased migration and invasive motility in vitro. Conversely, the overexpression of EYA4 in KYSE180 and KYSE450 promoted an epithelial phenotype, which consisted of decreased migration and invasion abilities and a decrease in TGF‐β1‐induced epithelial‐mesenchymal transition. Mechanistically, EYA4 overexpression reduced the phosphorylation of Akt and glycogen synthase kinase (GSK) 3β, which led to the inactivation of slug. In addition, we found that TGF‐β1 decreased EYA4 expression in both a dose‐dependent and a time‐dependent manner in KYSE30 cells, accompanied by an increase in the expression of DNA methyltransferases, especially DNMT3A. In summary, EYA4 is frequently hypermethylated in ESCC and may function as a tumor suppressor gene in the development of ESCC.     

Anterior gradient protein 2 expression in high grade head and neck squamous cell carcinoma correlated with cancer stem cell and epithelial mesenchymal transition

Anterior gradient protein 2 (AGR2) is a novel biomarker with potential oncogenic role. We sought to investigate the diagnostic and prognostic role of AGR2 on head and neck squamous cell carcinoma (HNSCC) with an emphasis on its correlation of cancer stemloid cells (CSC) and epithelial mesenchymal transition (EMT). We found that AGR2 protein levels were higher in HNSCC than in normal oral mucosa. High levels of AGR2 were associated with the T category, pathological grade and lymph node metastasis of HNSCC. Expression of AGR2 increased in recurring HNSCC after radiotherapy and in post cisplatin-based chemotherapeutic tissues. In HNSCC cell lines, knock-down of AGR2 induced apoptosis, reduced sphere formation, and down-regulated Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog and Oct4. In addition, over-expressed AGR2 in transgenic mice with spontaneous HNSCC was associated with lost function of Tgfbr1 and/or lost function of Pten. In vitro knockdown TGFBR1 in HNSCC cell lines increased AGR2 expression. These results suggest that AGR2 is involved in EMT and self-renewal of CSC and may present a potential therapeutic target (oncotarget) for HNSCC.     

Corticotropin Releasing Factor promotes breast cancer cell motility and invasiveness

Introduction  

Cancer cells secrete bioactive peptides that act in an autocrine or paracrine fashion affecting tumor growth and metastasis. Corticotropin-releasing factor (CRF), a hypothalamic neuropeptide that controls the response to stress, has been detected in breast cancer tissues and cell lines. CRF can affect breast cancer cells in an autocrine or paracrine manner via its production from innervating sympathetic neurons or immune cells.     

C4.4A as a biomarker of head and neck squamous cell carcinoma and correlated with epithelial mesenchymal transition

C4.4A, a member of the Ly6/uPAR family of membrane proteins, has been identified as a metastasis-associated molecule, but little is known about its actual expression and possible function in head and neck squamous cell carcinoma (HNSCC). To explore diagnostic and prognostic roles of C4.4A in HNSCC, we investigated the expression of C4.4A in human HNSCC tissue array which contains 43 HNSCC, 6 epithelial dysplasia and 16 normal oral mucosa. Expression of C4.4A was significantly increased in epithelial dysplasia and HNSCC when compared with normal oral mucosa. Moreover, high C4.4A expression indicated a rather poor prognosis of HNSCC patients. To better understand the function of C4.4A in HNSCC progression, we investigated epithelial to mesenchymal transition (EMT) associated proteins including transforming growth factor (TGF-β1), Slug and CD147 in HNSCC. The expression of TGF-β1, Slug, and CD147 was significantly increased in HNSCC when compared with normal oral mucosa. Meanwhile, the expression of C4.4A was significantly correlated with TGF-β1, Slug, and CD147 in HNSCC tissue array. Furthermore, knockdown of C4.4A decreased the cell invasion and migration in CAL27 cell line and suppressed the EMT with increased E-cadherin and decreased N-cadherin and Slug. Our study demonstrated that C4.4A was a potential marker for prognosis of HNSCC, and C4.4A participated in EMT program in HNSCC progression.     

Pituitary tumor-transforming 1 increases cell motility and promotes lymph node metastasis in esophageal squamous cell carcinoma

Human pituitary tumor-transforming 1 (PTTG1)/securin is a putative oncoprotein that is overexpressed in various tumor types. However, the involvement of PTTG1 in gastrointestinal cancer development and progression remains unclear. In this study, we investigated the clinical significance and biological effects of PTTG1 in esophageal squamous cell carcinoma (ESCC). Immunohistochemical studies performed on 113 primary ESCC specimens revealed a high prevalence of PTTG1 overexpression (60.2%), which was significantly associated with lymph node metastasis (regional, P = 0.042; distant, P = 0.005), advanced tumor stage (P = 0.028), and poorer overall survival (P = 0.017, log-rank test; P = 0.044, Cox proportional hazard model). Eleven ESCC cell lines expressed PTTG1 protein at levels 2.4 to 6.6 times higher than those in normal esophageal epithelial cells (HEEpiC). PTTG1 protein expression was confined to the nucleus in HEEpiC cells but present in both the cytoplasm and nucleus in ESCC cells. Two small interfering RNAs (siRNA) inhibited PTTG1 mRNA and protein expression in three ESCC cell lines by 77% to 97%. In addition, PTTG1 down-regulation by these siRNAs significantly reduced cell motility in all three ESCC cell lines (P < 0.01) in vitro, as well as popliteal lymph node metastases of ESCC cells in nude mice (P = 0.020). Global gene expression profiling suggested that several members of the Ras and Rho gene families, including RRAS, RHOG, ARHGAP1, and ARHGADIA, represented potential downstream genes in the PTTG1 pathway. Taken together, these findings suggest that PTTG1 overexpression promotes cell motility and lymph node metastasis in ESCC patients, leading to poorer survival. Thus, PTTG1 constitutes a potential biomarker and therapeutic target in ESCCs with lymph node metastases.     

Metadherin regulates metastasis of squamous cell carcinoma of the head and neck via AKT signalling pathway-mediated epithelial–mesenchymal transition

Our recent study suggested that metadherin (MTDH) is overexpressed in laryngeal squamous cell carcinoma. Here, we further investigated its role in promoting metastasis of squamous cell carcinoma of the head and neck (SCCHN). An immunohistochemistry analysis demonstrated that MTDH is elevated and positively correlated with metastasis in 189 primary SCCHN tissues. In vitro experiments demonstrated that MTDH overexpression enhanced the migratory and invasive ability of SCCHN cells. Moreover, MTDH induced epithelial–mesenchymal transition (EMT) by both regulating morphological changes and mediating the expression of the biomolecular makers E-cadherin and vimentin. In addition, MTDH mediated AKT activation, and all of the above effects were nearly completely blocked by the inhibition of AKT. Our results suggested that MTDH might promote the metastasis of SCCHN through AKT signalling pathway mediated-EMT.     

Caveolin-1-negative head and neck squamous cell carcinoma primary tumors display increased epithelial to mesenchymal transition and prometastatic properties

Distant metastases arise in 20-30% of patients with squamous cell carcinoma of the head and neck (HNSCC) in the 2 years following treatment. Therapeutic options are limited and the outcome of the patients is poor. The identification of predictive biomarkers of patient at risk for distant metastasis and therapies are urgently needed. We previously identified a clinical subgroup, called “R1” characterized by high propensity for rapid distant metastasis. Here, we showed that “R1” patients do not or at very low level express caveolin-1 (Cav1). Low or no expression of Cav1 is of bad prognosis. Disappearance of Cav1 enables cells to undergo epithelial-mesenchymal transition (EMT). EMT is associated with enhanced migration and invasion. Our study uncovered a new target, α₅β₁ integrin. Targeting α₅β₁ integrins might not only prevent metastasis of HNSCC but also delay the development of the primary tumor by reducing tumor cell viability. Cav1 detection might be taken into consideration in the future in the clinic not only to identify patients at high risk of metastasis but also to select patient who might benefit from an anti-integrin therapy.     

Expression levels of Akt in nimesulide-treated squamous carcinoma cell lines of the head and neck

Nimesulide, a selective cyclooxygenase-2 (COX-2) inhibitor, is known to induce apoptosis in various cell lines. So far, the molecular targets that account for this effect are unknown. Akt and extracellular-signal regulated kinase (ERK) are both important signaling molecules, which promote survival in different types of cancer, including head and neck cancer. The aim of our study was to describe the effect of treatment with nimesulide and ERK inhibitor PD 98059 on the expression patterns of Akt within the head and neck cancer cell lines SCC 9 and SCC 25. SCC 9 and SCC 25 cells were cultured in RPMI and treated with nimesulide and PD 98059. Protein expression levels were semi-quantified by Western blot analysis. Apoptosis was examined using the M30 antibody that binds to a caspase cleaved cytoskeletal protein for detection of early apoptosis in immunofluorescence studies. In both cell lines, nimesulide treatment induced apoptosis and resulted in a decrease of Akt expression. ERK inhibition had no effect on Akt expression, and combined application of both agents had the same effect as treatment with nimesulide alone. In conclusion, nimesulide induces apoptosis and promotes down-regulation of Akt-expression in squamous cell carcinoma cells, independently of ERK activity.     

MicroRNA-138 suppresses invasion and promotes apoptosis in head and neck squamous cell carcinoma cell lines

Metastasis is a critical event in the progression of head and neck squamous cell carcinoma (HNSCC). To identify microRNAs associated with HNSCC metastasis, six paired HNSCC cell lines with different metastatic potential were examined. Using microarrays, a panel of differentially expressed microRNAs was identified, including reduction of miR-138 in highly metastatic cells. Ectopic transfection of miR-138 suppressed cell invasion and led to cell cycle arrest and apoptosis. Knockdown of miR-138 enhanced cell invasion and suppressed apoptosis. Thus, our results suggested miR-138 acts as a tumor suppresser and may serve as a therapeutic target for HNSCC patients at risk of metastasis.     

Expression of MTA1 promotes motility and invasiveness of PANC-1 pancreatic carcinoma cells

The human metastasis-associated protein 1 (MTA1) is a constituent of the nucleosome-remodelling and -deacetylation complex. Its expression has been correlated with the invasion and metastasis of epithelial neoplasms. To address the functional consequences of MTA1 expression in pancreatic carcinoma cells, we have established PANC-1 pancreatic carcinoma cells that stably express MTA1 as an enhanced green fluorescent fusion protein (EGFP-MTA1). Here, we demonstrate that heterologous expression of EGFP-MTA1 markedly enhanced the cellular motility and the invasive penetration of epithelial barriers by the cells. Expression of EGFP-MTA1 had no effect on substrate-independent growth, but reduced substrate-dependent cell proliferation. In addition, the organisation of the cytokeratin filament system and the localisation of the actin cytoskeleton-associated protein IQGAP1 were distinctly altered in EGFP-MTA1-expressing cells. These results indicate that enhanced expression of MTA1 promotes the acquisition of an invasive, metastatic phenotype, and thus enhances the malignancy of pancreatic adenocarcinoma cells by modulation of the cytoskeleton.     

Inhibitor of DNA binding-1 induces mesenchymal features and promotes invasiveness in thyroid tumour cells

Characterisation of molecular mechanisms that control tumour invasion is a crucial step for the identification of molecular markers to apply in cancer diagnosis and treatment.In this work, we have investigated the role of Id1 in thyroid tumours.We demonstrate that Id1 participates to tumour progression by powering the invasion capacity of cancer cells. We prove that the overexpression of Id1 in thyroid tumour cells profoundly alters cell morphology and growth, increasing migration and invasion properties of the cells. Analysis in human thyroid tumours reveals that Id1 is expressed in invading cells and its expression is associated with an increased metastatic potential of non-anaplastic tumours. The gene expression study supports these observations demonstrating that Id1 modulates a number of genes known to control invasion, aggressiveness and pharmacological resistance in different type of human tumours. Finally, we demonstrate that the pro-invasive effect of Id1 is accompanied by the acquisition of mesenchymal features in thyroid tumour cells. This suggests that the trans-differentiation towards a more immature condition is the mechanism through which Id1 promotes thyroid tumour metastatic spreading.This study identifies Id1 as part of the pro-metastatic programme of thyroid cancer and suggests its possible utilisation as a prognostic marker.