Cancer-associated fibroblasts induce matrix metalloproteinase-mediated cetuximab resistance in head and neck squamous cell carcinoma cells

A growing body of evidence suggests that components of the tumor microenvironment, including cancer-associated fibroblasts (CAF), may modulate the treatment sensitivity of tumor cells. Here, we investigated the possible influence of CAFs on the sensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines to cetuximab, an antagonistic epidermal growth factor receptor (EGFR) antibody. Cetuximab treatment caused a reduction in the proliferation rate of HNSCC cell lines, whereas the growth of HNSCC-derived CAF cultures was unaffected. When tumor cells were cocultured with CAFs in a transwell system, the cetuximab-induced growth inhibition was reduced, and a complete protection from growth inhibition was observed in one of the tumor cell lines investigated. Media that had been conditioned by CAFs offered protection from cetuximab treatment in a concentration-dependent manner, suggesting that the resistance to treatment was mediated by CAF-derived soluble factors. The coculture of HNSCC cell lines with CAFs resulted in an elevated expression of matrix metalloproteinase-1 (MMP-1) in both the tumor cells and CAFs. Moreover, the CAF-induced resistance was partly abolished by the presence of an MMP inhibitor. However, CAFs treated with siRNA targeting MMP-1 still protected tumor cells from cetuximab treatment, suggesting that several MMPs may cooperate to facilitate resistance or that the protective effect is mediated by another member of the MMP family. These results identify a novel CAF-dependent modulation of cetuximab sensitivity and suggest that inhibiting MMPs may improve the effects of EGFR-targeted therapy. Mol Cancer Res; 10(9); 1158-68. ?2012 AACR.     

Cyr61 increases matrix metalloproteinase-3 expression and cell motility in human oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) has a striking tendency to migrate and metastasize. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effects of Cyr61 on human OSCC cells are largely unknown. In this study, we found that Cyr61 increased the migration and the expression of matrix metalloproteinases-3 (MMP)-3 in human OSCC cells. αvβ5 or α6β1 monoclonal antibody (mAb), focal adhesion kinase (FAK) inhibitor, and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the Cyr61-induced increase of the migration and MMP-3 up-regulation of OSCC cells. Cyr61 stimulation increased the phosphorylation of FAK, MEK, and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors suppressed the cell migration and MMP-3 expression enhanced by Cyr61. Moreover, Cyr61 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-3 promoter. Taken together, our results indicate that Cyr61 enhances the migration of OSCC cells by increasing MMP-3 expression through the αvβ3 or α6β1 integrin receptor, FAK, MEK, ERK, and NF-κB signal transduction pathway.     

CX3CL1 induces cell migration and invasion through ICAM-1 expression in oral squamous cell carcinoma cells

Human oral squamous cell carcinoma (OSCC) has been associated with a relatively low survival rate over the years and is characterized by a poor prognosis. C-X3-C motif chemokine ligand 1 (CX3CL1) has been involved in advanced migratory cells. Overexpressed CX3CL1 promotes several cellular responses related to cancer metastasis, including cell movement, migration and invasion in tumour cells. However, CX3CL1 controls the migration ability, and its molecular mechanism in OSCC remains unknown. The present study confirmed that CX3CL1 increased cell movement, migration and invasion. The CX3CL1-induced cell motility is upregulated through intercellular adhesion molecule-1 (ICAM-1) expression in OSCC cells. These effects were significantly suppressed when OSCC cells were pre-treated with CX3CR1 monoclonal antibody (mAb) and small-interfering RNA (siRNA). The CX3CL1-CX3CR1 axis activates promoted PLCβ/PKCα/c-Src phosphorylation. Furthermore, CX3CL1 enhanced activator protein-1 (AP-1) activity. The CX3CR1 mAb and PLCβ, PKCα, c-Src inhibitors reduced CX3CL1-induced c-Jun phosphorylation, c-Jun translocation into the nucleus and c-Jun binding to the ICAM-1 promoter. The present results reveal that CX3CL1 induces the migration of OSCC cells by promoting ICAM-1 expression through the CX3CR1 and the PLCβ/PKCα/c-Src signal pathway, suggesting that CX3CL1-CX3CR1-mediated signalling is correlated with tumour motility and appealed to be a precursor for prognosis in human OSCC.     

Cancer stem cell model in oral squamous cell carcinoma

The concept of "field cancerization" describes the presence of histological abnormal tissue surrounding oral squamous cell carcinoma (OSCC). Molecular model of multistep carcinogenesis indicates that an accumulation of genetic alterations forms the basis for the OSCC progression with genetic heterogeneity. Furthermore, we reviewed cancer stem cell (CSC) model, which suggests functional heterogeneity in the tumor mass and current supporting evidence in OSCC. According to CSC model, prevention from carcinogen exposure and eliminating the particular CSCs instead of targeting tumor mass could help obtain a more long-lasting therapeutic effect.     

Tumor cell and carcinoma-associated fibroblast interaction regulates matrix metalloproteinases and their inhibitors in oral squamous cell carcinoma

Co-culture of periodontal ligament (PDL) fibroblasts and SCC-25 oral squamous carcinoma cells (OSCC), results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs). Paracrin circuits between CAFs and OSCC cells were hypothesized to regulate the gene expression of matrix remodeling enzymes in their co-culture, which was performed for 7days, followed by analysis of the mRNA/protein expression and activity of metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and other relevant genes. Interleukin1-β, transforming growth factor-β1, fibronectin and αvβ6 integrin have shown to be involved in the regulation of the MMP and TIMP gene expression in co-culture of CAFs and tumor cells. In addition, these cells also cooperated in activation of MMP pro-enzymes. It is particularly interesting that the fibroblast-produced inactive MMP-2 has been activated by the tumor-cell-produced membrane-type 1 matrix metalloproteinase (MT1-MMP). The crosstalk between cancer- and the surrounding fibroblast stromal-cells is essential for the fine tuning of cancer cells invasivity.     

Oct4 downregulation-induced inflammation increases the migration and invasion rate of oral squamous cell carcinoma

Inflammatory changes are involved in tumor cell proliferation,migration,and invasion.Tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) play important...     

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

Angiotensin II (AngII) and its type receptor (AT1-R) play important roles in the development of cardiac hypertrophy. Low level of high density lipoprotein (HDL) is also an independent risk factor for cardiac hypertrophy. We therefore investigated in the present study whether HDL inhibits cardiac hypertrophy relatively to inhibition of AngII and AT1-R in both in vitro and in vivo experiments. Stimulation of cultured cardiomyocytes of neonatal rats with AngII for 24 h and infusion of AngII in mice for 2 weeks resulted in marked cardiac hypertrophic responses including increased protein synthesis, enlarged sizes of cardiomyocytes and hearts, upregulated phosphorylation levels of protein kinases and reprogrammed expression of specific genes, all of which were significantly attenuated by the treatment with HDL. Furthermore, AngII-treatment induced upregulation of AT-R expression either in cultured cardiomyocytes or in hearts of mice and HDL significantly suppressed the upregulation of AT1-R. Our results suggest that HDL may abrogate AngII-induced cardiac hypertrophy through downregulation of AT1-R expression.     

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133⁺ cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.     

Fucoxanthin induced apoptotic cell death in oral squamous carcinoma (KB) cells

Fucoxanthin (Fx) is an active compound commonly found in the many types of seaweed with numerous biological activities. The main goal of this investigation is to explore the effect of Fx against the cell proliferation, apoptotic induction and oxidative stress in the oral squamous (KB) cell line. Cytotoxicity of Fx was determined by MTT assay. The intracellular ROS production, mitochondrial membrane potential (MMP) and apoptosis induction in KB cells were examined through DCFH-DA, Rhodamine-123 and DAPI, and dual staining techniques. Effect of Fx on the antioxidant enzymes and lipid peroxidation in the KB cells was studied through the standard procedures. Fx treated KB cells showed morphological changes and reduced cell survival, which is exhibited by the cytotoxic activity of 50 µM/ml (IC50) Fx against the KB cells. The Fx treatment considerably induced the apoptotosis cells (EB/AO) and decreased the MMP (Rh-123) in KB cells. Further, it was pointed out that there was an increased lipid peroxidation (LPO) with decreased antioxidants (CAT, SOD and GSH). These results concluded that Fx has the cytotoxic effect against KB cells and has the potential to induce the apoptosis via increased oxidative stress. Hence, the Fx can be a promising agent for the treatment of oral cancer and it may lead to the development of cancer therapeutics.     

Glutaredoxin 3 promotes migration and invasion via the Notch signalling pathway in oral squamous cell carcinoma

Substantial evidence indicates that the alteration of the cellular redox status is a critical factor involved in cell growth and death and results in tumourigenesis. Cancer cells have an efficient antioxidant system to counteract the increased generation of ROS. However, whether this ability to survive high levels of ROS has an important role in the growth and metastasis of tumours is not well understood. Glutaredoxin 3 (GLRX3), also known as TXNL2, Grx3 and PICOT, maintains a low level of ROS, thus contributing to the survival and metastasis of several types of cancer. However, little is known about the role of GLRX3 and the underlying mechanisms that suppress oral squamous cell carcinoma (OSCC) progression. Here, by using immunohistochemical staining, we demonstrated that GLRX3 was overexpressed in human OSCC, and enhanced GLRX3 expression correlated with metastasis and with decreased overall patient survival. Knockdown of GLRX3 in human OSCC cell lines reduced Notch activity by reversing the epithelial–mesenchymal transition (EMT), resulting in the inhibition of in vitro migration and invasion. Importantly, knockdown of GLRX3 triggered the generation of ROS. Furthermore, N-acetyl cysteine (NAC), an ROS scavenger, enhanced the effects of GLRX3 knockdown on Notch-dependent EMT. Collectively, these findings suggested the vital roles of GLRX3 in OSCC progression through its relationship with EMT progression, and these data also suggest that a strategy of blocking ROS to enhance the activity of GLRX3 knockdown warrants further attention in the treatment of OSCC.     

Microbial diversity in saliva of oral squamous cell carcinoma

In the oral cavity, chronic inflammation has been observed at various stages of oral squamous cell carcinomas (OSCC). Such inflammation could result from persistent mucosal or epithelial cell colonization by microorganisms. There is increasing evidence of the involvement of oral bacteria in inflammation, warranting further studies on the association of bacteria with the progression of OSCC. The objective of this study was to evaluate the diversity and relative abundance of bacteria in the saliva of subjects with OSCC. Using 454 parallel DNA sequencing, ～58,000 PCR amplicons that span the V4-V5 hypervariable region of rRNAs from five subjects were sequenced. Members of eight phyla (divisions) of bacteria were detected. The majority of classified sequences belonged to the phyla Firmicutes (45%) and Bacteroidetes (25%). Further, 52 different genera containing approximately 860 (16.51%) known species were identified and 1077 (67%) sequences belonging to various uncultured bacteria or unclassified groups. The species diversity estimates obtained with abundance-based coverage estimators and Chao1 were greater than published analyses of other microbial profiles from the oral cavity. Fifteen unique phylotypes were present in all three OSCC subjects.     

CCN5 inhibits proliferation and promotes apoptosis of oral squamous cell carcinoma cells

Oral squamous cell carcinoma (OSCC) is a common cancer with poor prognosis and high mortality. The role of CCN5 has attracted a great focus on the regulation of cancer progression. However, the biological function and mechanism of CCN5 in OSCC are still not well elucidated. The current study was designed to determine the effects of CCN5 on OSCC cell proliferation and apoptosis using two OSCC cell lines. Further, LY294002, a PI3K/AKT antagonist, was employed to explore the mechanism underlying the effects of CCN5 in the regulation of OSCC. The results showed that overexpression of CCN5 in TSCCa cells significantly reduced viable cell number, arrested cell cycle, and suppressed cell‐cycle regulators (cyclin D1, cyclin E, and CDK2). CCN5 overexpression increased the apoptotic ratio and Hoechst‐positive cell number, and altered the apoptotic‐related proteins (caspase‐3/9, Bax, and Bcl‐2). However, CCN5 silencing induced opposite effects on cell proliferation and apoptosis in Tca‐8113 cells. In addition, we observed that CCN5 knockdown increased the expression levels of PI3K (p85α and p110α) and phosphorylated AKT at serine 473 (p‐AKT Ser473) in Tca‐8113 cells. Inhibiting PI3K/AKT signaling with LY294002 rescued the apoptotic process in CCN5‐silenced OSCC cells. Finally, xenograft analysis showed that CCN5 represses tumorigenesis of OSCC cells. These findings together suggest that CCN5 functions as a tumor suppressor for OSCC cell development through inactivation of PI3K/AKT signaling pathway, providing a potential candidate for OSCC therapy.     

Truncated P-cadherin is produced in oral squamous cell carcinoma

Cadherins belong to a family of homophilic cell-cell adhesion proteins that are responsible for the establishment of a precise cell architecture and tissue integrity. Moreover, experimental data suggest that loss of intercellular adhesion is inversely correlated with cellular differentiation. Furthermore, dedifferentiation is closely linked to tumor progression. Recently, we have shown that a secreted 50 kDa N-terminal fragment of P-cadherin plays a role in the progression of malignant melanoma. In this study, we have detected both the full-length and the truncated versions of P-cadherin in cell lysates of differentiated head and neck oral squamous cell carcinoma cell lines, whereas in cell lysates of dedifferentiated cell lines, we detected only the truncated 50 kDa version of P-cadherin. Treatment of the cell lines with a recombinantly expressed biotinylated, soluble 50 kDa form of the N-terminal part of P-cadherin revealed a major effect on cell aggregation and migration of oral squamous cell carcinoma cells. However, the 50 kDa N-terminal fragment of P-cadherin did not show any influence on cell proliferation in 2D and 3D cell culture. These results suggest that generation of truncated P-cadherin during the progression of oral squamous carcinoma attenuates tissue integrity, facilitates cellular separation, and leads to the acquisition of a more migratory phenotype.     

Salivary analytes in patients with oral squamous cell carcinoma

Literature data indicates that measurement of certain salivary constituents might serve as a useful diagnostic/prognostic tool in the patients with oral squamous cell carcinoma (OSCC). In 24 patients with OSCC (60 +/- 2.5 yrs) and in 24 controls (24 +/- 3.7 yrs) we have determined levels of salivary magnesium, calcium, copper, chloride, phosphate, potassium, sodium, total proteins and amylase. Sodium, potassium and chloride were determined by indirect potentiometry whereas copper, magnesium and phosphate were determined by atomic absorption spectrophotometry. Total proteins were determined by pyrogalol colorimetric method. Amylase levels were determined by continued colorimetric method. Statistical analysis was performed by use of chi2 test and Spearman's correlation test. The results of this study indicate that the concentrations of sodium and chloride were significantly elevated in patients with OSCC when compared to the controls. However, level of total protein was significantly decreased when compared to the healthy controls. Furthermore, there was a negative correlation between alcohol consumption and total protein concentration in patients with oral carcinoma. We might conclude that in patients with OSCC increased salivary sodium and chloride might reflect their overall dehydration status due to alcohol consumption rather than consequence of OSCC itself.     

A mouse model for oral squamous cell carcinoma

Despite recent advances, the prognosis of oral squamous cell carcinoma is still poor. Therapeutic options such as radiotherapy, chemotherapy, surgery and the novel treatment option gene therapy are being investigated in animal models. Diverse models have been studied to induce oral squamous cell carcinomas. The carcinogenic 4-nitroquinoline-1-oxide (4NQO) model has proven to be successful although until now it is unknown at what time point the established tumor is a representative squamous cell carcinoma and has a suitable volume for scientific treatment. For this end we applied 4NQO 3 times a week during 16 weeks and measured the volume of tumor tissue each week until the end of the experiment at 40 weeks. Concurrent histopathology at different time points up to the end of the experiment revealed that all mice bearing oral tumors were diagnosed with squamous cell carcinoma. Immunohistochemistry with markers cyclin D1 and E-cadherin revealed that the generated mouse oral tumors showed strong similarities with the described immunopathology in human oral tumors. The 4NQO model is a suitable alternative for preclinical gene therapy experiments with primary oral tumors. Future survey of therapeutic options in the carcinogenic 4NQO model should be conducted around 40 weeks after the start of the treatment.     

Anti-tumor effect of carrimycin on oral squamous cell carcinoma cells in vitro and in vivo

Purpose: Carrimycin is a newly synthesized macrolide antibiotic with good antibacterial effect. Exploratory experiments found its function in regulating cell physiology, proliferation and immunity, suggesting its potential anti-tumor capacity. The aim of this study is to investigate the anti-tumor effect of carrimycin against human oral squamous cell carcinoma cells in vitro and in vivo.Methods: Human oral squamous cell carcinoma cells (HN30/HN6/Cal27/HB96 cell lines) were treated with gradient concentration of carrimycin. Cell proliferation, colony formation and migration ability were analyzed. Cell cycle and apoptosis were assessed by flow cytometry. The effect of carrimycin on OSCC in vivo was investigated in tumor xenograft models. Immunohistochemistry, western blot assay and TUNEL assays of tissue samples from xenografts were performed. The key proteins in PI3K/AKT/mTOR pathway and MAPK pathway were examined by western blot.Results: As the concentration of carrimycin increased, the proliferation, colony formation and migration ability of OSCC cells were inhibited. After treating with carrimycin, cell cycle was arrested in G0/G1 phase and cell apoptosis was promoted. The tumor growth of xenografts was significantly suppressed. Furthermore, the expression of p-PI3K, p-AKT, p-mTOR, p-S6K, p-4EBP1, p-ERK and p-p38 were down-regulated in vitro and in vivo.Conclusions: Carrimycin can inhibit the biological activities of OSCC cells in vitro and in vivo, and regulate the PI3K/AKT/mTOR and MAPK pathways.     

MicroRNA-25 promotes cell migration and invasion in esophageal squamous cell carcinoma

MicroRNAs (miRNAs) as a species of small non coding single stranded RNA of about 21-25 nucleotides have important roles in the development of different cancers. In present study, we found that the expression of miR-25 was up-regulated in 60 esophageal squamous cell carcinoma (ESCC) tissues compared with matched adjacent non-cancer tissues. Moreover, we demonstrated that the up-regulation of miR-25 was significantly correlated with the status of lymph node metastasis and TNM (Tumor, Node and Metastasis) stage. Furthermore, over-expression of miR-25 markedly promoted migration and invasion of ESCC cells. On the contrary, down-regulation of miR-25 inhibited the migration and invasion of cells. E-cadherin(CDH1) is a very important tumor metastasis suppressor. We further identified that miR-25 directly targeted CDH1 3'-untranslated region (3'UTR) and repressed the expression of CDH1. These results, for the first time, demonstrate that miR-25 promotes ESCC cell migration and invasion by suppressing CDH1 expression.     

Interferon-gamma downregulates Hsp27 expression and suppresses the negative regulation of cell death in oral squamous cell carcinoma lines

Interferon-gamma (IFN-gamma) induced cell death in five oral squamous cell carcinoma (SCC) lines. Cell death was specific to IFN-gamma treatment and did not occur with either IFN-alpha or TNF-alpha. IFN-gamma did not induce typical apoptotic phenotype in cells, such as morphological changes and DNA ladder formation. Caspase-3 was partially activated by IFN-gamma. Protein levels of molecular chaperones were examined in cells treated with IFN-gamma. Among these, levels of heat shock protein 27 (Hsp27) were specifically reduced upon IFN-gamma treatment of oral SCC cells. Recombinant clones overexpressing Hsp27 were more resistant to IFN-gamma-induced cell death than parent cells. Conversely, cells expressing a dominant-negative mutant of Hsp27, in which three serine residues (15, 78 and 82) were replaced by glycine, were hypersensitive to the effects of IFN-gamma and exhibited a typical apoptotic phenotype. Pretreatment of cells with IFN-gamma enhanced apoptotic cell death induced by cisplatin. Our data suggest that IFN-gamma suppresses Hsp27 expression in oral SCC cells and blocks the inhibitory effects of this molecular chaperone on apoptotic cell death. Moreover, IFN-gamma initiates the transition of oral SCC cells to the proapoptotic and/or aborted apoptotic state. Hsp27 plays a crucial role in the inhibition of apoptosis of oral SCC cells. Our findings highlight the importance of employing IFN-gamma in combination with certain anticancer drugs as treatments for oral cancer. We suggest that Hsp27 plays a significant role in the IFN-gamma-induced sensitization of oral SCC cells to anticancer drugs.