epidermal-growth-factor and Squamous-Cell-Carcinoma-of-Head-and-Neck

Head and neck squamous cell carcinoma (HNSCC) develops from the mucosal epithelium of the oral cavity, pharynx, and larynx, and is the most common malignancy of the head and neck, the incidence of which continues to rise. The epidermal growth factor receptor is thought to play a key role in the pathogenesis of HNSCC. Inhibition of epidermal growth factor receptor has been identified as an effective target for the treatment of HNSCC. Many phytochemicals have emerged as potential new drugs for the treatment of HNSCC. A systematic search was conducted for research articles published in PubMed, and Medline on relevant aspects. This review provides an overview of the available literature and reports highlighting the in vitro effects of phytochemicals on epidermal growth factor in various HNSCC cell models and in vivo in animal models and emphasizes the importance of epidermal growth factor as a current therapeutic target for HNSCC. Based on our review, we conclude that phytochemicals targeting the epidermal growth factor receptor are potentially effective candidates for the development of new drugs for the treatment of HNSCC. It provides an idea for further development and application of herbal medicines for cancer treatment.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Phytochemicals; Squamous Cell Carcinoma of Head and Neck

Glycosylation change is one of the landmark events of tumor occurrence and development, and tumor cells may be inhibited by regulating the aberrant expression of glycosyltransferases. Currently, fucosyltransferase VI (FUT6), which is involved in the synthesis of α-1, 3 fucosyl bond, has been detected to be closely associated with multiple tumors, but its function and mechanism in head and neck squamous cell carcinoma (HNSCC) still need further research. In this study, FUT6 knockdown and overexpression strategies were used to investigate the effects of FUT6 on cell proliferation, migration, and invasion, as well as the growth and metastasis of HNSCC in a xenografts mouse model. The protein expression levels of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK), Signal Transducer and Activator of Transcription (STAT), protein kinase B (AKT), c-Myc, and epithelial-mesenchymal transition (EMT) markers were determined by western blot analysis. Our research found that the mRNA expression of FUT6 was lower in HNSCC tissues than in normal mucosal epithelial tissues. In Cal-27 and FaDu cells, FUT6 overexpression inhibited cell proliferation, migration and invasion, causing upregulation of ZO-1 and E-cadherin, downregulation of N-cadherin and Vimentin, and finally decreased the phosphorylation levels of EGFR, ERK, STAT, and c-Myc. In HSC-3 cells, knockdown of FUT6 promoted cell proliferation, migration and invasion, downregulating ZO-1 and E-cadherin, upregulating N-cadherin and Vimentin, and increased the phosphorylation levels of EGFR, ERK, STAT, and c-Myc. In the HNSCC xenografts mouse, FUT6 overexpression inhibited tumor growth and metastasis. In summary, FUT6 controls the proliferation, migration, invasion, and EGF-induced EMT of HNSCC by regulating EGFR/ERK/STAT signaling pathway, indicating its potential future therapeutic application for HNSCC.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Fucosyltransferases; Head and Neck Neoplasms; Humans; Mice; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Vimentin

Epithelial-to-mesenchymal transition (EMT) of malignant cells is a driving force of disease progression in human papillomavirus-negative (HPV-negative) head and neck squamous cell carcinomas (HNSCC). Sustained hyper-activation of epidermal growth factor receptor (EGFR) induces an invasion-promoting subtype of EMT (EGFR-EMT) characterized by a gene signature ("'EGFR-EMT_Signature'") comprising 5´-ectonucleotidase CD73. Generally, CD73 promotes immune evasion via adenosine (ADO) formation and associates with EMT and metastases. However, CD73 regulation through EGFR signaling remains under-explored and targeting options are amiss.. CD73 functions in EGFR-mediated tumor cell dissemination were addressed in 2D and 3D cellular models of migration and invasion. The novel antagonizing antibody 22E6 and therapeutic antibody Cetuximab served as inhibitors of CD73 and EGFR, respectively, in combinatorial treatment. Specificity for CD73 and its role as effector or regulator of EGFR-EMT were assessed upon CD73 knock-down and over-expression. CD73 correlation to tumor budding was studied in an in-house primary HNSCC cohort. Expression correlations, and prognostic and predictive values were analyzed using machine learning-based algorithms and Kaplan-Meier survival curves in single cell and bulk RNA sequencing datasets.. CD73/NT5E is induced by the EGF/EGFR-EMT-axis and blocked by Cetuximab and MEK inhibitor. Inhibition of CD73 with the novel antagonizing antibody 22E6 specifically repressed EGFR-dependent migration and invasion of HNSCC cells in 2D. Cetuximab and 22E6 alone reduced local invasion in a 3D-model. Interestingly, combining inefficient low-dose concentrations of Cetuximab and 22E6 revealed highly potent in invasion inhibition, substantially reducing the functional IC. In sum, CD73 is an effector of EGF/EGFR-mediated local invasion and a potential therapeutic target and candidate predictive marker for advanced HPV-negative HNSCC.

Topics: 5'-Nucleotidase; Cetuximab; Epidermal Growth Factor; ErbB Receptors; GPI-Linked Proteins; Head and Neck Neoplasms; Humans; Papillomavirus Infections; Squamous Cell Carcinoma of Head and Neck

Head neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors which ranks the sixth incidence in the world. Although treatments for HNSCC have improved significantly in recent years, its recurrence rate and mortality rate remain high. Myosin genes have been studied in a variety of tumors, however its role in HNSCC has not been elucidated. GSE58911 and GSE30784 gene expression profile analysis were performed to detect significantly dys-regulated myosin genes in HNSCC. The Cancer Genome Atlas (TCGA) HNSCC database was used to verify the dys-regulated myosin genes and study the relationship between these genes and prognosis in HNSCC. The results showed that MYL1, MYL2, MYL3, MYH2, and MYH7 were down-regulated, while MYH10 was up-regulated in patients with HNSCC. Interestingly, MYL1, MYL2, MYH1, MYH2, and MYH7 were shown to be unfavorable prognostic markers in HNSCC. It is also worth noting that MYL1 was a specific unfavorable prognostic biomarker in HNSCC. MYL1, MYL2, MYL3, MYH2, MYH7, and MYH10 promoted CD4 + T cells activation in HNSCC. MYL1 was proved to be down-regulated in HNSCC tissues compared to normal tissues at protein levels. MYL1 overexpression had no effect on proliferation, but significantly promoted migration of Fadu cells. MYL1 increased EGF and EGFR protein expression levels. Moreover, there is a positive correlation between MYL1 expression and Tcm CD8 cells, Tcm CD4 + cells, NK cells, Mast cells, NKT cells, Tfh cells and Treg cells in HNSCC. Overall, MYL1 facilitates tumor metastasis and correlates with tumor immune infiltration in HNSCC and these effects may be associated with the EGF/EGFR pathway.

Topics: Biomarkers; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Neoplasms, Second Primary; Prognosis; Squamous Cell Carcinoma of Head and Neck

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the role of RNA-binding proteins (RBPs) in EGFR-associated metastasis of HNSCC remains unclear. In this study, we reveal that RBPs, specifically nucleolin (NCL) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), correlated with the mesenchymal phenotype of HNSCC. The depletion of RBPs significantly attenuated EGF-induced HNSCC metastasis. Intriguingly, the EGF-induced EMT markers, such as fibronectin, were regulated by RBPs through the ERK and NF-κB pathway, followed by the enhancement of mRNA stability of fibronectin through the 5' untranslated region (5'-UTR) of the gene. The upregulation of fibronectin triggered the integrin signaling activation to enhance tumor cells' attachment to endothelial cells and increase endothelial permeability. In addition, the concurrence of EGFR and RBPs or EGFR and fibronectin was associated with overall survival and disease-free survival of HNSCC. The in vivo study showed that depletion of NCL, hnRNPA2B1, and fibronectin significantly inhibited EGF-promoted extravasation of tumor cells into lung tissues. The depletion of fibronectin or treatment with integrin inhibitors dramatically attenuated EGF-induced HNSCC metastatic nodules in the lung. Our data suggest that the RBPs/fibronectin axis is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.

Topics: 5' Untranslated Regions; Endothelial Cells; Epidermal Growth Factor; ErbB Receptors; Fibronectins; Head and Neck Neoplasms; Humans; Integrins; Squamous Cell Carcinoma of Head and Neck

Various cell types secrete exosomes into their surrounding extracellular space, which consequently affect the function and activity of recipient cells. Numerous studies have showed that tumor cell-derived exosomes play important roles in tumor growth and progression. Although a variety of endocytic pathways are reportedly involved in the cellular uptake of exosomes, detailed mechanisms remain unknown. The present study demonstrated that treatment with recombinant epidermal growth factor (EGF) time- and dose-dependently promoted cellular uptake of oral squamous cell carcinoma (OSCC) cell-derived exosomes into OSCC cells themselves. Conversely, EGF receptor (EGFR) knockdown and treatment with EGFR inhibitors, including erlotinib and cetuximab, abrogated OSCC cell uptake of exosomes. The macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked the effects of active EGF/EGFR signaling on uptake of OSCC cell-derived exosomes. These EGFR inhibitors also suppressed OSCC cell-derived exosome-induced proliferation, migration, invasion, stemness, and chemoresistance of OSCC cells. Taken together, the data presented herein suggest that EGFR inhibitors might inhibit the malignant potential of OSCC cells through direct inhibition of not only EGFR downstream signaling pathway but also cellular uptake of OSCC cell-derived exosomes through macropinocytosis.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Exosomes; Humans; Mouth Neoplasms; Neoplastic Stem Cells; Pinocytosis; Protein Kinase Inhibitors; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Head and neck squamose cell carcinoma (HNSCC) is an aggressive group of tumors that are generally heterogeneous. Despite treatment advances, disease-free survival has not significantly improved. Therefore, it is of great importance to understand the molecular etiology of HNSCC and genetic alterations in the signal pathways in order to develop new therapeutic approaches. In this study, firstly we used a cytokine array to analyze the secretomes of HNSCC patients and healthy controls. In the next step, the results from the cytokine sequence were validated by qRT-PCR and western blot, including genes in the associated signaling pathway. In array analysis, the levels of EGF, IGF-1, IGFBP-1, and PDGFBB were significantly higher in patients than in the controls. The results of qRT-PCR analyses showed that expression levels of

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cytokines; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor I; Placenta Growth Factor; Receptor, Platelet-Derived Growth Factor beta; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

The expression of programmed death ligand-1 (PD-L1) is controlled by complex mechanisms. The elucidation of the molecular mechanisms of PD-L1 expression is important for the exploration of new insights into PD-1 blockade therapy. Detailed mechanisms of the in situ expression of PD-L1 in tissues of oral squamous cell carcinomas (OSCCs) have not yet been clarified. We examined the mechanisms of PD-L1 expression focusing on the phosphorylation of downstream molecules of epidermal growth factor (EGF) and interferon gamma (IFN-γ) signaling in vitro and in vivo by immunoblotting and multi-fluorescence immunohistochemistry (MF-IHC), respectively. The in vitro experiments demonstrated that PD-L1 expression in OSCC cell lines is upregulated by EGF via the EGF receptor (EGFR)/PI3K/AKT pathway, the EGFR/STAT1 pathway, and the EGFR/MEK/ERK pathway, and by IFN-γ via the JAK2/STAT1 pathway. MF-IHC demonstrated that STAT1 and EGFR phosphorylation was frequently shown in PD-L1-positive cases and STAT1 phosphorylation was correlated with lymphocyte infiltration and EGFR phosphorylation. Moreover, the phosphorylation pattern of the related molecules in PD-L1-positive cells differed among the cases investigated. These findings indicate that PD-L1 expression mechanisms differ depending on the tissue environment and suggest that the examination of the tissue environment and molecular alterations of cancer cells affecting PD-L1 expression make it necessary for each patient to choose the appropriate combination drugs for PD-1 blockade cancer treatment.

Topics: B7-H1 Antigen; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Interferon-gamma; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Programmed Cell Death 1 Receptor; Squamous Cell Carcinoma of Head and Neck

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck

Epidermal growth factor receptor (EGFR) is often overexpressed in head and neck squamous cell carcinoma (HNSCC) and represents a top candidate for targeted HNSCC therapy. However, the clinical effectiveness of current Food and Drug Administration (FDA)-approved drugs targeting EGFR is moderate, and the overall survival rate for HNSCC patients remains low. Therefore, more effective treatments are urgently needed. In this study, we generated a novel diphtheria toxin-based bivalent human epidermal growth factor fusion toxin (bi-EGF-IT) to treat EGFR-expressing HNSCC. Bi-EGF-IT was tested for in vitro binding affinity, cytotoxicity, and specificity using 14 human EGFR-expressing HNSCC cell lines and three human EGFR-negative cancer cell lines. Bi-EGF-IT had increased binding affinity for EGFR-expressing HNSCC compared with the monovalent version (mono-EGF-IT), and both versions specifically depleted EGFR-positive HNSCC, but not EGFR-negative cell lines, in vitro. Bi-EGF-IT exhibited a comparable potency to that of the FDA-approved EGFR inhibitor, erlotinib, for inhibiting HNSCC tumor growth in vivo using both subcutaneous and orthotopic HNSCC xenograft mouse models. When tested in an experimental metastasis model, survival was significantly longer in the bi-EGF-IT treatment group than the erlotinib treatment group, with a significantly reduced number of metastases compared with mono-EGF-IT. In addition, in vivo off-target toxicities were significantly reduced in the bi-EGF-IT treatment group compared with the mono-EGF-IT group. These results demonstrate that bi-EGF-IT is more effective and markedly less toxic at inhibiting primary HNSCC tumor growth and metastasis than mono-EGF-IT and erlotinib. Thus, the novel bi-EGF-IT is a promising drug candidate for further development.

Topics: Animals; Cell Line, Tumor; Diphtheria Toxin; Epidermal Growth Factor; Erlotinib Hydrochloride; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Protein Kinase Inhibitors; Recombinant Fusion Proteins; Squamous Cell Carcinoma of Head and Neck; Xenograft Model Antitumor Assays

Lewis y is expressed in oral squamous cell carcinoma (OSCC) cells and tumors. Previously, we reported that Lewis y was not expressed in invasion areas, and attenuation of proliferation and invasion in OSCC cells was caused by over-expression of Lewis y. However, the roles of Lewis y in the attenuation of malignant properties have not been clarified. In this study, we investigated the roles of Lewis y in OSCC.. The levels of Lewis y on EGFR and the phosphorylation levels of EGFR in OSCC cells were analyzed by immunoprecipitation and western blot. EGFR cross-linking and binding kinetics of EGF were performed.. Upon EGF stimulation, phosphorylation and dimer formation of EGFR were more prominent in Lewis y- cells. EGF binding kinetics showed reduced binding sites in Lewis y+ cells.. Lewis y reduced EGF binding to EGFR, leading to suppression of malignant properties through suppression of EGF signaling.

Topics: Binding Sites; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Shape; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Lewis Blood Group Antigens; Mouth Neoplasms; Neoplasm Invasiveness; Phosphorylation; Protein Binding; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Epidermal growth factor receptor (EGFR) is a pro-tumorigenic receptor tyrosine kinase that facilitates growth for cancer cells that overexpress the receptor. Monoclonal anti-EGFR antibody Cetuximab (CTX) provides significant clinical benefit in patients with head and neck squamous cell carcinoma (HNSCC). Missense mutations in the ectodomain (ECD) of EGFR can be acquired under CTX treatment and mimic the effect of large deletions on spontaneous untethering and activation of the receptor. Little is known about the contribution of EGFR ECD mutations to EGFR activation and CTX resistance in HNSCC. We identified two concurrent non-synonymous missense mutations (G33S and N56K) mapping to domain I in or near the EGF binding pocket of the EGFR ECD in patient-derived HNSCC cells that were selected for CTX resistance through repeated exposure to the agent in an effort to mimic what may occur clinically. Structural modeling predicted that the G33S and N56K mutants would restrict adoption of a fully closed (tethered) and inactive EGFR conformation while not permitting association of EGFR with the EGF ligand or CTX. Binding studies confirmed that the mutant, untethered receptor displayed reduced affinity for both EGF and CTX but demonstrated sustained activation and presence at the cell surface with diminished internalization and sorting for endosomal degradation, leading to persistent downstream AKT signaling. Our results demonstrate that HNSCC cells can select for EGFR ECD mutations under CTX exposure that converge to trap the receptor in an open, ligand-independent, constitutively activated state. These mutants impede the receptor's competence to bind CTX possibly explaining certain cases of CTX treatment-induced or de novo resistance to CTX.

Topics: Antineoplastic Agents, Immunological; Cetuximab; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Ligands; Models, Molecular; Mutation, Missense; Primary Cell Culture; Protein Domains; Proto-Oncogene Proteins c-akt; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Tumor Cells, Cultured

Topics: Angiopoietin-Like Protein 4; Animals; Cell Line, Tumor; Cyclooxygenase 2; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Heterografts; Humans; Male; Mice; Mice, SCID; Neoplasm Invasiveness; Squamous Cell Carcinoma of Head and Neck; Up-Regulation

Laryngeal squamous cell carcinoma (LSCC) is a highly disabling disease to the patient, affecting speech, swallowing and respiratory skills. Smoking and alcohol abuse are principal risk factors linked to this disease. Genetic factors can be involved in carcinogenesis by controlling the cell cycle, cell survival, angiogenesis, and invasiveness. Single nucleotide polymorphisms (SNPs) involving specific genes could modulate the risk of LSCC related to known carcinogens by modifying cellular responses, but not all genetic associations are known. In a case-control study, we assess the associations between cyclooxygenase-2 (COX2), epidermal growth factor (EGF), EGF receptor (EGFR), and tumor suppressor P53 SNPs on the risk of LSCC development in the Chilean population. A total of 85 LSCC patients and 95 healthy volunteers were recruited. SNPs genotype were analyzed from genomic DNA by Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (RFLP) and associations were estimated by odds ratios (ORs) using unconditional logistic regressions. A significant association between COX2 and TP53 SNP and LSCC risk was found, with an OR = 3.27 for COX2 c.-1329A>G (rs689466) SNP, and an OR = 1.94 for TP53 c.215C>G, Pro72Arg (rs1042522) SNP. These findings suggest that COX2 c.-1329A>G and TP53 c.215C>G (Pro72Arg) SNPs may be risk factors for LSCC. Through this research, we identify two low penetrance genetic variants that may be evaluated as novel biomarkers for this disease, in South American Mestizo populations.

Topics: Aged; Alcohol Drinking; Biomarkers, Tumor; Case-Control Studies; Chile; Cigarette Smoking; Cyclooxygenase 2; Epidermal Growth Factor; ErbB Receptors; Female; Genetic Predisposition to Disease; Humans; Laryngeal Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Risk Factors; Squamous Cell Carcinoma of Head and Neck; Tumor Suppressor Protein p53

p120-catenin (p120) serves as a stabilizer of the calcium-dependent cadherin-catenin complex and loss of p120 expression has been observed in several types of human cancers. The p120-dependent E-cadherin-β-catenin complex has been shown to mediate calcium-induced keratinocyte differentiation via inducing activation of plasma membrane phospholipase C-γ1 (PLC-γ1). On the other hand, PLC-γ1 has been shown to interact with phosphatidylinositol 3-kinase enhancer in the nucleus and plays a critical role in epidermal growth factor-induced proliferation of oral squamous cell carcinoma (OSCC) cells. To determine whether p120 suppresses OSCC proliferation and tumor growth via inhibiting PLC-γ1, we examined effects of p120 knockdown or p120 and PLC-γ1 double knockdown on proliferation of cultured OSCC cells and tumor growth in xenograft OSCC in mice. The results showed that knockdown of p120 reduced levels of PLC-γ1 in the plasma membrane and increased levels of PLC-γ1 and its signaling in the nucleus in OSCC cells and OSCC cell proliferation as well as xenograft OSCC tumor growth. However, double knockdown of p120 and PLC-γ1 or knockdown of PLC-γ1 alone did not have any effect. Immunohistochemical analysis of OSCC tissue from patients showed a lower expression level of p120 and a higher expression level of PLC-γ1 compared with that of adjacent noncancerous tissue. These data indicate that p120 suppresses OSCC cell proliferation and tumor growth by inhibiting signaling mediated by nuclear PLC-γ1.

Topics: Calcium, Dietary; Carcinoma, Squamous Cell; Catenins; Cell Differentiation; Cell Proliferation; Epidermal Growth Factor; Head and Neck Neoplasms; Mouth Neoplasms; Phospholipase C gamma; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

The aim of this study was to evaluate the expression of Akt, PTEN, Mdm2 and p53 proteins in three different head and neck squamous cell carcinoma (HNSCC) cell lines (HN6, HN19 and HN30), all of them treated with epidermal growth factor (EGF) and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90 protein.. Immunofluorescence and western blot were performed in order to analyze the location and quantification, respectively, of proteins under the action 17-AAG and EGF.. Treatment with EGF resulted in increased levels of Akt, PTEN and p53 in all cell lineages. The expression of Mdm2 was constant in HN30 and HN6 lineages, while in HN19 showed slightly decreased expression. Under the action 17-AAG, in HN6 and HN19, the expression of PTEN and p53 proteins was suppressed, while Akt and Mdm2 expression was reduced. Finally, in the HN30 cell lineage were absolute absence of expression of Akt, Mdm2 and p53 and decreased expression of PTEN.. These data allow us to speculate on the particular utility of 17-AAG for HNSCC treatment through the inhibition of Akt protein expression, especially in the cases that retain the expression of PTEN protein.

Topics: Benzoquinones; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Lineage; Epidermal Growth Factor; Head and Neck Neoplasms; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; PTEN Phosphohydrolase; Squamous Cell Carcinoma of Head and Neck; Tumor Suppressor Protein p53

Tumor microenvironment provides a specialized niche in which a population of stem-like cells is enriched and contributes to cancer progression. Moreover, cancer stem cell (CSC) phenotype has been associated with epithelial-mesenchymal transition (EMT). Here we investigated the effect of tumor microenvironment on the phenotypic characteristics of head and neck cancer cells and expression of CSC markers using a three-dimensional (3D), spheroid, culture system of CAL33 cell line from human tongue squamous cell carcinoma. CAL33 cells derived from 2D monolayer cultures were grown in spheroid cultures containing serum-free medium (epidermal growth factor [EGF], fibroblast growth factor [FGF], and insulin). Adherent CAL33 cells from spheroids or standard control cultures were grown in the presence/absence of serum in combination with hypoxia/normoxia. Markers of EMT, CSC, and hypoxia were analyzed either by Western blotting, immunofluorescence, or reverse transcription quantitative PCR. Spheroid cultures showed hypoxic microenvironment (high carbonic anhydrase IX [CAIX] expression), mesenchymal-like characteristics (reduced E-cadherin and increased vimentin and N-cadherin expression, presence of larger colonies comprised of larger, spread cells with lower density), and increased expression of the CSC marker glioma-associated oncogene homolog 1 (Gli1). These effects were recapitulated in serum-free adherent CAL33 cells maintained for prolonged periods in hypoxia (1% O2) but, in contrast, were completely abolished by the presence of serum. Overall, we found that a combination of hypoxia, EGF and FGF was essential to induce the EMT in adherent CAL33 cell cultures. The addition of serum rapidly reverts the EMT of cells, affects CSC phenotype and, thus, prevents the detection of such cells in tumor cell lines.

Topics: Carbonic Anhydrase IX; Cell Adhesion; Cell Hypoxia; Cell Line, Tumor; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Squamous Cell Carcinoma of Head and Neck; Tongue Neoplasms; Tumor Microenvironment; Zinc Finger Protein GLI1

Cancer cells have been known to overexpress the epidermal growth factor receptor (EGFR) and hence relevant multiple‑targeted therapies have been developed, with a recent clinical application of the antibody‑mediated inhibition of the EGFR. However, this strategy is not useful in cancer cells with mutations in KRAS; a GTPase downstream of EGFR which constitutively activates the pathway without EGF stimulation. Furthermore, mutations in EGFR also reduce the binding of monoclonal antibodies and thereby render them ineffective. In the present study, we designed a chimeric EGF protein fused to the truncated N‑terminal domain fragment of Pseudomonas aeruginosa exotoxin A (EGF‑ETA), which has ADP‑ribosylation activity and induces apoptosis. The EGF‑ETA protein was expressed in E. coli as a His‑tagged fusion. Our results showed that EGF‑ETA significantly inhibited the proliferation of EGFR‑positive A431 epidermoid carcinoma (IC50 27 ng/ml) and HN5 head and neck squamous cell carcinoma (IC50 36 ng/ml) cells. However, its effect on cancer cells with little or no EGFR expression was limited (A549‑IC50 1,000 ng/ml; MCF‑7‑IC50 >10,000 ng/ml). Compared to cetuximab, EGF‑ETA was highly potent in its killing capacity of HN5 cancer cells at 1,000 ng/ml, while cetuximab had little effect at 1,000 ng/ml. Furthermore, EGF‑ETA was just as potent in HCT116 (KRAS G13D) and SW480 (KRAS G12V) colon cancer cell lines harbouring KRAS hyperactivating mutations when compared to KRAS wild‑type HT29 colon cancer cells. Finally, co‑incubation of EGF‑ETA with an anti‑EGF antibody abrogated its effect on the EGFR‑positive A431 cells. Our results show that the chimeric EGF‑ETA toxin is extremely effective against EGFR‑positive cancers and raises the potential to further develop this chimera for use in targeting EGFR‑positive tumours resistant to monoclonal antibodies.

Topics: ADP Ribose Transferases; Antibodies, Anti-Idiotypic; Apoptosis; Bacterial Toxins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Humans; Ligands; Proto-Oncogene Proteins p21(ras); Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Squamous Cell Carcinoma of Head and Neck; Virulence Factors

Overexpression and increased signaling from the epidermal growth factor receptor (EGFR) often changes oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed the roles of OSCC-derived extracellular vesicles (EVs), including exosomes in the trafficking of cetuximab and in epithelial-mesenchymal transition (EMT) of epithelial cells. OSCC cells abundantly expressed EGFR, which was secreted from cells with OSCC-EVs upon EGF stimulations. The OSCC-EGFR-EVs were then able to enter into and transform epithelial cells leading to increased mesenchymal traits with increased vimentin and spindle-like shapes. EGF priming of OSCC cells further increased this EMT-initiating effect of the OSCC-EVs. The internalization and pro-EMT effects of the OSCC-EVs were largely blocked by cetuximab. Thus, OSCC-derived EVs transform normal epithelial cells into a mesenchymal phenotype and anti-EGFR therapeutic antibody cetuximab inhibits such a carcinogenic effect of the OSCC-EVs.

Topics: Cell Line, Tumor; Cell Transformation, Neoplastic; Cetuximab; Epidermal Growth Factor; Epithelial Cells; Epithelial-Mesenchymal Transition; ErbB Receptors; Exosomes; Humans; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide. The majority of HNSCCs overexpress Epidermal Growth Factor Receptor (EGFR), an essential receptor tyrosine kinase (RTK) that promotes HNSCC growth and metastasis. Therefore, EGFR has been used as an important therapeutic target to treat HNSCC. Inhibition of EGFR stimulates autophagy in cancer cells. However, the role of autophagy in EGFR inhibitor-induced cancer suppression is still in a debate. Here, we reveal that the first- and the second-generation EGFR tyrosine kinase inhibitors (TKIs) differentially affect HNSCC autophagy. The second-generation EGFR TKIs have much stronger effects on autophagy than the first-generation TKIs. The second-generation EGFR TKIs not only promote autophagy initiation signaling but also block autophagic flux by disturbing the lysosomes function, indicating a novel mechanism by which EGFR TKIs modulate cancer cell autophagy. Blocking the initiation of autophagy does not affect the second-generation EGFR TKI-induced HNSCC growth suppression. This suggests that the anti-growth effect of the second-generation EGFR TKIs on HNSCC is not dependent on autophagy.

Topics: Autophagy; Carcinoma, Squamous Cell; Cell Proliferation; Dose-Response Relationship, Drug; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Squamous Cell Carcinoma of Head and Neck; Treatment Outcome

Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in such cancers as head and neck squamous cell carcinoma (HNSCC); however, whether the metabolic enzyme, pyruvate dehydrogenase kinase 1 (PDK1), mediates EGF-enhanced HNSCC metastasis remains unclear. Of interest, we found that EGF induced PDK1 expression in HNSCC. Tumor cell transformation induced by EGF was repressed by PDK1 knockdown, and the down-regulation of PDK1 expression or inhibition of its activity significantly blocked EGF-enhanced cell migration and invasion. In addition, depletion of PDK1 impeded EGF-enhanced binding of HNSCC cells to endothelial cells as well as the metastatic seeding of tumor cells in lungs. PDK1 depletion inhibited EGF-induced matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. Furthermore, PDK1 overexpression induced MMP-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. Of interest, depletion of fibronectin inhibited PDK1-enhanced MMP-1-3 and MMP-9 expression as well as Rac1/cdc42 activation and tumor invasion. These results demonstrate that EGF-induced PDK1 expression enhances HNSCC metastasis

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Fibronectins; Head and Neck Neoplasms; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Squamous Cell Carcinoma of Head and Neck; Transcriptional Activation; Up-Regulation

Epidermal growth factor (EGF) is important for cancer cell proliferation, angiogenesis and metastasis in many types of cancer. However, the mechanisms involved in EGF-induced head and neck squamous cell carcinoma (HNSCC) metastasis remain largely unknown. In this study, we reveal that angiopoietin-like 4 (ANGPTL4) plays an important role in the regulation of EGF-induced cancer metastasis. We showed that EGF-induced ANGPTL4 expression promoted anoikis resistance and cancer cell migration and invasion in HNSCC. In addition, depletion of ANGPTL4 inhibited EGF-induced cancer cell invasion. Autocrine production of EGF-induced ANGPTL4 regulated the expression of matrix metalloproteinases (MMPs). The induction of MMP-1 gene expression by ANGPTL4-activated integrin β1 signalling occurred through the AP-1 binding site in the MMP-1 gene promoter. Furthermore, down-regulation of MMP-1 impeded EGF- and recombinant ANGPTL4-enhanced HNSCC cell migration and invasion. Depletion of ANGPTL4 significantly blocked EGF-primed extravasation and metastatic seeding of tumour cells and MMP-1 expression in lungs. However, no effect of ANGPTL4 on tumour growth was observed. These results suggest that EGF-induced expression and autocrine production of ANGPTL4 enhances HNSCC metastasis via the up-regulation of MMP-1 expression. Inhibition of ANGPTL4 expression may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Anoikis; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Genes, jun; Head and Neck Neoplasms; Humans; Integrin beta1; Matrix Metalloproteinase 1; Neoplasm Metastasis; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

"Warburg effect", the enhanced glycolysis or aerobic glycolysis, confers cancer cells the ability to survive and proliferate even under stressed conditions. In this study, we explored the role of epidermal growth factor (EGF) in orchestrating Warburg effect, the epithelial-mesenchymal transition (EMT) process, and the acquisition of cancer stem-like cell properties in human oral squamous cell carcinoma (OSCC) cells. Our results showed that EGF induces EMT process in OSCC cells, which correlates with the acquisition of cancer stem-like properties, including the enrichment of CD44+/CD24- population of cancer cells and an increased expression of CSC-related genes, aldehyde dehydrogenase-1 (ALDH1) and Bmi-1. We also showed that EGF concomitantly enhanced L-lactate production, while blocking glycolysis by 2-deoxy-D-glucose (2-DG) robustly reversed EGF-induced EMT process and CSC-like properties in OSCC cells. Mechanistically, we demonstrated that EGF promoted EMT process and CSC generation through EGFR/PI3K/HIF-1α axis-orchestrated glycolysis. Using an orthotopic tumor model of human OSCC (UM-SCC1) injected in the tongue of BALB/c nude mice, we showed that treatment with 2-DG in vivo significantly inhibited the metastasis of tumor cells to the regional cervical lymph nodes and reduced the expression of ALDH1 and vimentin in both in situ tumors and tumor cell-invaded regional lymph nodes. Taken together, these findings have unveiled a new mechanism that EGF drives OSCC metastasis through induction of EMT process and CSC generation, which is driven by an enhanced glycolytic metabolic program in OSCC cells.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; CD24 Antigen; Cell Line, Tumor; Deoxyglucose; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Glycolysis; Head and Neck Neoplasms; Humans; Hyaluronan Receptors; Hypoxia-Inducible Factor 1, alpha Subunit; Isoenzymes; Lactic Acid; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplastic Stem Cells; Phenotype; Phosphatidylinositol 3-Kinase; Polycomb Repressive Complex 1; Retinal Dehydrogenase; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Time Factors; Xenograft Model Antitumor Assays

Head and neck squamous cell carcinoma is frequently associated with aberrant epidermal growth factor receptor (EGFR) signaling, which contributes to tumor growth. Here, the functional importance of EGFR ligands in relation to proliferation and sensitivity to the EGFR-targeted therapy cetuximab was investigated in three tongue cancer cell lines.. The influence of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumor cell proliferation and cetuximab response was evaluated by the addition of recombinant human (rh) proteins or by siRNA-mediated downregulation of the endogenous ligand production. The expression, activation and cellular distribution of EGFR were assessed by Western blot analysis and immunocytochemistry.. EGF downregulation suppressed the proliferation of all investigated tumor cell lines, whereas the response to an increased level of EGF differed between EGFR-overexpressing and EGFR-non-overexpressing cell lines. Furthermore, tumor cells consistently displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing was associated with an improved cetuximab response. The data regarding AR and EPR were inconclusive.. Our data suggest that the amount of EGF is a determinant of the tumor cell proliferation rate and the response to cetuximab treatment in tongue cancer. Thus, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

Topics: Amphiregulin; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cetuximab; Down-Regulation; Drug Resistance, Neoplasm; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Head and Neck Neoplasms; Humans; Immunohistochemistry; Ligands; Molecular Targeted Therapy; Squamous Cell Carcinoma of Head and Neck; Tongue Neoplasms

Head and neck squamous cell carcinoma (HNSCC) represents the most frequent malignancy in the head and neck region, and the survival rate has not been improved significantly over the past three decades. It has been reported the infiltrated macrophages contribute to the malignant progression of HNSCC. However, the crosstalk between macrophages and cancer cells remains poorly understood. In the present study, we explored interactions between monocytes/macrophages and HNSCC cells by establishing the direct co-culture system, and found that the crosstalk promoted the migration and invasion of cancer cells by enhancing the invadopodia formation through a CCL2/EGF positive feedback loop. Our results demonstrated HNSCC cells educated monocytes into M2-like macrophages by releasing C-C motif chemokine ligand 2 (CCL2, or MCP-1). And the M2-like macrophages secreted epithelial growth factor (EGF), which increased the motility of HNSCC cells by enhancing the invadopodia formation. These subcellular pseudopodia degraded extracellular matrix (ECM), facilitating tumor local invasion and distant metastasis. Moreover, EGF up-regulated CCL2 expression in HNSCC cells, which recruited monocytes and turned them into M2-like macrophages, thus forming a positive feedback paracrine loop. Finally, we reported that curcumin, a powerful natural drug, suppressed the production of EGF and CCL2 in macrophages and cancer cells, respectively, blocking the feedback loop and suppressing the migration and invasion of HNSCC cells. These results shed light on the possibilities and approaches based on targeting the crosstalk between cancer cells and monocytes/macrophages in HNSCC for potential cancer therapy.

Topics: Carcinoma, Squamous Cell; Cell Communication; Cell Differentiation; Cell Movement; Cell Polarity; Chemokine CCL2; Curcumin; Epidermal Growth Factor; Feedback, Physiological; Head and Neck Neoplasms; Humans; Macrophages; Monocytes; Neoplasm Invasiveness; Squamous Cell Carcinoma of Head and Neck

Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinoma (HNSCC) where it has been shown to promote tumor cell invasion upon phosphorylation. One mechanism by which EGFR promotes tumor progression is by activating signal cascades that lead to loss of E-cadherin, a transmembrane glycoprotein of the cell-cell adherence junctions; however mediators of these signaling cascades are not fully understood. One such mediator, RhoC, is activated upon a number of external stimuli, such as epidermal growth factor (EGF), but its role as a mediator of EGF-stimulated migration and invasion has not been elucidated in HNSCC. In the present study, we investigate the role of RhoC as a mediator of EGF-stimulated migration and invasion in HNSCC. We show that upon EGF stimulation, EGFR and RhoC were strongly activated in HNSCC. This resulted in activation of the phosphatidylinositol 3-Kinase Akt pathway (PI3K-Akt), phosphorylation of GSK-3β at the Ser(9) residue, and subsequent down regulation of E-cadherin cell surface expression resulting in increased tumor cell invasion. Knockdown of RhoC restored E-cadherin expression and inhibited EGF-stimulated migration and invasion. This is the first report in HNSCC demonstrating the role RhoC plays in mediating EGF-stimulated migration and invasion by down-regulating the PI3K-Akt pathway and E-cadherin expression. RhoC may serve as a treatment target for HNSCC.

Topics: Cadherins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Gene Knockdown Techniques; Head and Neck Neoplasms; Humans; Models, Biological; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; rho GTP-Binding Proteins; rhoC GTP-Binding Protein; Signal Transduction; Snail Family Transcription Factors; Squamous Cell Carcinoma of Head and Neck; Transcription Factors

The number of epidermal growth factor receptor (EGFR)-targeting drugs in the development for cancer treatment is continuously increasing. Currently used EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors have specific limitations related to toxicity and development of resistance, and there is a need for alternative treatment strategies to maximize the clinical potential of EGFR as a molecular target. This study describes the design and production of a novel EGFR-targeted fusion protein, rGel/EGF, composed of the recombinant plant toxin gelonin and EGF. rGel/EGF was custom-made for administration by photochemical internalization (PCI), a clinically tested modality for cytosolic release of macromolecular therapeutics. rGel/EGF lacks efficient mechanisms for endosomal escape and is therefore minimally toxic as monotherapy. However, PCI induces selective and efficient cytosolic release of rGel/EGF in EGFR-expressing target cells by light-directed activation of photosensitizers accumulated selectively in tumor tissue. PCI of rGel/EGF was shown to be highly effective against EGFR-expressing cell lines, including head and neck squamous cell carcinoma (HNSCC) cell lines resistant to cetuximab (Erbitux). Apoptosis, necrosis and autophagy were identified as mechanisms of action following PCI of rGel/EGF in vitro. PCI of rGel/EGF was further shown as a highly tumor-specific and potent modality in vivo, with growth inhibitory effects demonstrated on A-431 squamous cell carcinoma (SCC) xenografts and reduction of tumor perfusion and necrosis induction in SCC-026 HNSCC tumors. Considering the small amount of rGel/EGF injected per animal (0.1 mg/kg), the presented in vivo results are highly promising and warrant optimization and production of rGel/EGF for further preclinical evaluation with PCI.

Topics: Animals; Antibodies, Monoclonal, Humanized; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Photochemistry; Recombinant Fusion Proteins; Ribosome Inactivating Proteins, Type 1; Squamous Cell Carcinoma of Head and Neck; Toxins, Biological

Overexpression of the epidermal growth factor (EGF) receptor (EGFR) is associated with enhanced invasion and metastasis in head and neck squamous cell carcinoma (HNSCC). Long Pentraxin PTX3 is involved in immune escape in cancer cells. Here, we identified PTX3 as a promoting factor that mediates EGF-induced HNSCC metastasis. EGF-induced PTX3 transcriptional activation is via the binding of c-Jun to the activator protein (AP)-1 binding site of the PTX3 promoter. PI3K/Akt and NF-κB were essential for the PTX3 activation. EGF-induced PTX3 expression was blocked in c-Jun- and NF-κB-knockdown cells. EGF-mediated PTX3 secretion resulted in the enhancement of cell migration and invasion, and interactions between cancer and endothelial cells. The tail-vein injection animal model revealed that depletion of PTX3 decreased EGF-primed tumor cell metastatic seeding of the lungs. In addition, fibronectin, matrix metalloproteinase-9 (MMP9) and E-cadherin were essential components in EGFR/PTX3-mediated cancer metastasis. In conclusion, PI3K/Akt and NF-κB-dependent regulation of AP-1 mediates PTX3 transcriptional responses to EGF. Autocrine production of EGF-induced PTX3 in turn induces metastatic molecules, activating inflammatory cascades and metastasis.

Topics: Animals; C-Reactive Protein; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Heterografts; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, SCID; Neoplasm Metastasis; NF-kappa B; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Serum Amyloid P-Component; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Transcription Factor AP-1; Transfection

Cisplatin is a cytotoxic chemotherapeutic drug frequently used to treat many solid tumors, including head and neck squamous cell carcinoma (HNSCC). EGF receptor (EGFR) inhibitors have also shown efficacy as alternatives to cisplatin in some situations. However, large clinical trials have shown no added survival benefit from the use of these two drugs in combination. Possible explanations for this include overlapping downstream signaling cascades. Using in vitro studies, we tested the hypothesis that cisplatin and EGFR inhibitors rely on the activation of the tumor suppressor STAT1, characterized by its phosphorylation at serine (S727) or tyrosine (Y701) residues. Cisplatin consistently increased the levels of p-S727-STAT1, and STAT1 siRNA knockdown attenuated cisplatin-induced cell death. EGFR stimulation also activated p-S727-STAT1 and p-Y701-STAT1 in a subset of cell lines, whereas EGFR inhibitors alone decreased levels of p-S727-STAT1 and p-Y701-STAT1 in these cells. Contrary to our hypothesis, EGFR inhibitors added to cisplatin treatment caused variable effects among cell lines, with attenuation of p-S727-STAT1 and enhancement of cisplatin-induced cell death in some cells and minimal effect in other cells. Using HNSCC tumor specimens from a clinical trial of adjuvant cisplatin plus the anti-EGFR antibody panitumumab, higher intratumoral p-S727-STAT1 appeared to correlate with worse survival. Together, these results suggest that cisplatin-induced cell death is associated with STAT1 phosphorylation, and the addition of anti-EGFR therapy to cisplatin has variable effects on STAT1 and cell death in HNSCC.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cell Death; Cell Line, Tumor; Cetuximab; Cisplatin; Epidermal Growth Factor; ErbB Receptors; Gene Knockdown Techniques; Head and Neck Neoplasms; Humans; p38 Mitogen-Activated Protein Kinases; Panitumumab; Phosphorylation; Prognosis; RNA, Small Interfering; Squamous Cell Carcinoma of Head and Neck; STAT1 Transcription Factor

Previous studies in head and neck squamous cell carcinoma (HNSCC) cell lines have revealed that the Ah receptor (AHR) plays a significant role in mediating the "aggressive" phenotype of these cells, which includes enhanced inflammatory signaling (e.g., IL6) and migratory potential. Here we sought to identify putative novel targets of the AHR associated with enhanced tumor invasiveness. Global gene expression analysis identified a number of genes that are repressed upon treatment of OSC-19 or HN30 cells with an AHR antagonist. Three growth factors were targets of AHR activity; amphiregulin (AREG), epiregulin (EREG), and platelet-derived growth factor A (PDGFA) were repressed by an AHR antagonist and further examined. Quantitative PCR analysis, ELISA, and siRNA-mediated knock down of AHR revealed an attenuation of basal and/or induced levels of expression of these growth factors in two HNSCC lines, following AHR antagonism. In silico analysis revealed that these growth factors possess dioxin-like response elements. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene (BP) also elicited similar responses. In conclusion, this study identified AREG, EREG, and PDGFA as growth factor targets of AHR activity associated with metastatic phenotype of HNSCC cells, suggesting that attenuation of AHR activity may be a therapeutic strategy.

Topics: Azo Compounds; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; Epiregulin; Fibroblast Growth Factor 2; Gene Expression; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Platelet-Derived Growth Factor; Pyrazoles; Receptors, Aryl Hydrocarbon; Squamous Cell Carcinoma of Head and Neck

It has been reported that the epidermal growth factor receptor (EGFR) expression is associated with the extracellular matrix metalloproteinase inducer (EMMPRIN) in some solid tumors; however, the relationship of EMMPRIN with EGFR in head and neck cancers is not fully understood. To determine the relationship between EMMPRIN and EGFR in head and neck squamous cell carcinoma (HNSCC), HNSCC cells were stimulated with epidermal growth factor (EGF), a ligand of EGFR. EMMPRIN expression in HNSCC cells was upregulated by EGF. In addition, EGF stimulation induced HNSCC cell invasion and MMP-9 expression. This increase in invasion and MMP-9 expression was abrogated by downmodulation of EMMPRIN. Furthermore, to determine the effects of combined EMMPRIN and EGFR targeting in HNSCC, HNSCC cells were treated with an EMMPRIN function-blocking antibody and the EGFR inhibitor AG1478. This combined treatment resulted in greater inhibition of HNSCC cell proliferation and migration compared with the individual agents alone. These results suggest that EMMPRIN mediates EGFR-induced tumorigenicity and that combined targeting of EMMPRIN and EGFR may be an efficacious treatment approach.

Topics: Basigin; Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Emerging evidence suggests that endothelial cell-secreted factors contribute to the pathobiology of squamous cell carcinoma (SCC) by enhancing invasive migration and resistance to anoikis. Here, we report that SCC cells within the perivascular niche have undergone epithelial to mesenchymal transition (EMT) in a primary human SCC of a patient that developed distant metastases. Endothelial cell-secreted EGF induced EMT of human SCC cells in vitro and also induced acquisition of a stem-like phenotype. In vivo, tumor xenografts vascularized with EGF-silenced endothelial cells exhibited a smaller fraction of cancer stem-like cells (ALDH(+)CD44(+)) and were less invasive than tumors vascularized with control endothelial cells. Collectively, these results demonstrated that endothelial cell-EGF induces EMT and acquisition of stem-like properties by head and neck tumor cells. On this basis, we suggest that vascular endothelial cells contribute to tumor dissemination by secreting factors that endow carcinoma cells with enhanced motility and stemness.

Topics: Aged; Animals; Carcinoma, Squamous Cell; Cell Communication; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Endothelial Cells; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Head and Neck Neoplasms; Heterografts; Humans; Male; Mice; Mice, SCID; Neoplastic Stem Cells; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to γ-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial-mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics.

Topics: Adult; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Head and Neck Neoplasms; Humans; Insulin-Like Growth Factor I; Kruppel-Like Factor 4; Male; Middle Aged; Neoplastic Stem Cells; Receptor, IGF Type 1; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

The epithelial-mesenchymal transition (EMT) is suggested to be a crucial factor for the development of an invasive and metastatic cell phenotype, which is characterized by down-regulation of epithelial adhesive proteins (e.g. E-cadherin) and induction of mesenchymal proteins (e.g. vimentin). Therefore, there is a great clinical interest to specify this phenotype. Different growth factors induce EMT, such as epithelial growth factor (EGF) and transforming growth factor beta 1 (TGFβ1). The role of EMT in human papilloma virus (HPV)-positive squamous cell carcinoma (SCC) is still not understood. The aim of this study was to investigate the expression pattern in p16-positive and -negative SCC cells of vimentin, β-catenin and E-cadherin after stimulation with growth factors.. We incubated the p16-positive CERV196 and p16-negative HNSCC22B SCC cell lines with EGF and EGF/TGFβ1 (10 ng/ml) and detected E-cadherin, vimentin and β-catenin by immunocytochemistry and enzyme-linked immunosorbent assay after 5, 24 and 96 h.. We found a low expression of vimentin in all studied tumor cell lines. The negative control of HNSCC22B cells showed a higher intrinsic level of membranous E-cadherin and β-catenin. We found statistically significant EGF/TGFβ1-induced expression of vimentin dependent on incubation time in p16-negative HNSCC22B cells. Particularly in the presence of EGF, we detected an increase of β-catenin and vimentin expression in p16-positive SCC tumor cell lines in addition to induced cell scattering and unexpected expression of E-cadherin.. In conclusion, E-cadherin, β-catenin and vimentin expression are important features to characterize EMT-like events. We were able to show incomplete EGF-induced EMT with β-catenin expression in p16-positive SCC. Extended studies are required to investigate the mechanistic role of EMT markers, especially in p16-positive SCC, in order to develop new anti-SCC therapies to block EMT progression.

Topics: beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Neoplasm Proteins; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta1; Vimentin

The epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Despite the high expression of EGFR in HNSCC, EGFR inhibitors have only limited success as monotherapy. The Grb2-associated binder (GAB) family of adaptor proteins acts as docking/scaffolding molecules downstream of tyrosine kinase receptors. We hypothesized that GAB1 may amplify EGFR-induced signaling in HNSCCs and therefore could play a role in the reduced sensitivity of HNSCC to EGFR inhibitors. We used representative human HNSCC cell lines overexpressing wild type EGFR, and expressing GAB1 but not GAB2. We demonstrated that baseline Akt and MAPK signaling were reduced in HNSCC cells in which GAB1 expression was reduced. Furthermore, the maximal EGF-induced activation of the Akt and MAPK pathway was reduced and delayed, and the duration of the EGF-induced activation of these pathways was reduced in cells with GAB1 knock-down. In agreement with this, HNSCC cells in which GAB1 levels were reduced showed an increased sensitivity to the EGFR inhibitor gefitinib. Our work demonstrates that GAB1 plays an important role as part of the mechanism of by which EGFR induces induced activation of the MAPK and AKT pathway. Our results identify GAB1 as an amplifier of the EGFR-initiated signaling, which may also interfere with EGFR degradation. These findings support the emerging notion that reducing GAB1 function may sensitize HNSCC to EGFR inhibitors, hence representing a new therapeutic target HNSCC treatment in combination with EGFR targeting agents.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Gefitinib; GRB2 Adaptor Protein; Head and Neck Neoplasms; Humans; Mitogen-Activated Protein Kinase Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

The migratory ability of tumor cells requires cytoskeletal rearrangement processes. Epidermal growth factor receptor (EGFR)-signaling tightly correlates with tumor progression in head and neck squamous cell carcinomas (HNSCCs), and has previously been implicated in the regulation of cytokeratin (CK) expression. In this study, HNSCC cell lines were treated with EGF, and CK expression levels were monitored by Western blot analysis. Changes in cellular morphology were documented by fluorescence- and atomic force microscopy. Some of the cell lines demonstrated an EGF-dependent modulation of CK expression levels. Interestingly, regression of some CK subtypes or initial up-regulation followed by downregulation at higher EGF-levels could also be observed in the tested cell lines. Overall, the influence of EGF on CK expression levels appeared variable and cell-type-dependent. Real-time cellular analysis of EGF-treated and -untreated HNSCC cell lines demonstrated a rise over time in cellular impedance. In three of the EGF-treated HNSCC cell lines, this rise was markedly higher than in untreated controls, whereas in one of the cell lines the gain of cellular impedance was paradoxically reduced after EGF treatment, which was found to correlate with changes in cellular morphology rather than with relevant changes in cellular viability or proliferation. After treating HNSCC cells with EGF, CK filaments frequently appeared diffusely distributed throughout the cytoplasm, and in some cases were found in a perinuclear localization, the latter being reminiscent to observations by other groups. In summary, the data points to a possible role of EGFR in modulating HNSCC cell morphology.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Shape; Cell Survival; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Keratinocytes; Keratins; Microscopy, Atomic Force; Phenotype; Plakophilins; Squamous Cell Carcinoma of Head and Neck

Mtss1 is located within chromosomal region 8q23-24, which is one of the three most commonly amplified regions in head and neck squamous cell carcinoma (HNSCC). Mtss1 is lost in metastatic cells, but confusingly is commonly overexpressed in primary tumors. Here we address possible reasons why Mtss1 is positively selected for in primary tumors. We find that Mtss1 enhances the localization of the epidermal growth factor (EGF) receptor to the plasma membrane, prolonging EGF signaling and resulting in enhanced proliferation in HNSCC. Depletion of Mtss1 results in decreased EGF receptor levels and decreased phosphorylation of Erk1/2 and Akt. However, when cells are at high density and adherent to each other, analogous to conditions in a solid tumor, Mtss1 does not confer any growth advantage, either in basal conditions or following EGF stimulation. This could indicate why Mtss1 might be lost in metastases, but preserved in early primary tumors. This is supported by an organotypic assay showing that Mtss1-expressing cells display a less proliferative more epithelial-like morphology on top of a collagen matrix. Furthermore, xenograft tumors expressing Mtss1 initially grow more rapidly, but later show less proliferation and more differentiation. Mtss1 positively modulates EGF signaling at low cell densities to promote proliferation and, therefore, may be beneficial for the early stages of primary HNSCC tumor growth. However, at high cell densities, Mtss1 impacts negatively on EGF signaling and this suggests why it inhibits metastasis.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Microfilament Proteins; Neoplasm Proteins; Neoplasm Transplantation; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Predictive strategies for the treatment efficacy of cetuximab are currently not available for head and neck cancer. We investigated the correlation between the expression of epidermal growth factor receptor (EGFR) ligands and EGFR expression, and the growth inhibitory activity of cetuximab in a panel of head and neck squamous cell carcinoma (HNSCC) cell lines.. The growth inhibiting effect of cetuximab was measured for eight HNSCC cell lines and correlated with the autocrine production of five EGFR ligands as measured by ELISA, and the mRNA expression of two ligands, as measured by quantitative RT-PCR. EGFR expression was assessed by western blot analysis.. There was a good correlation between the expression of four of the EGFR ligands (TGF-α, amphiregulin, epiregulin and epigen) and the growth inhibiting effect of cetuximab. TGF-α had the highest predictive potential but had to be combined with epigen for full prediction. EGFR expression also correlated with cetuximab sensitivity but less clearly.. The results indicate that the expression of several EGFR ligands has to be used to predict sensitivity to cetuximab in HNSCC. This has to be further evaluated in clinical samples.

Topics: Amphiregulin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; EGF Family of Proteins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epigen; Epiregulin; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glycoproteins; Head and Neck Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor alpha

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Kaplan-Meier Estimate; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Prognosis; Prospective Studies; Squamous Cell Carcinoma of Head and Neck

Epidermal growth factor receptor (EGFR) overexpression correlates with recurrence and with treatment resistance in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to evaluate the relationship of EGFR gene copy number utilizing FISH and protein expression with automated quantitative analysis (AQUA) and to correlate those with patient outcome.. A tissue microarray composed of 102 HNSCC treated with (chemo)radiation was constructed and analyzed for EGFR copy number by FISH (Vysis; Abbott Laboratories) and EGFR protein expression using AQUA analysis of EGFR staining scored on a scale of 0 to 255. We evaluated associations of EGFR FISH status and AQUA score with clinicopathologic parameters and survival prognosis.. Eleven (17.2%) of 64 tumors with FISH results showed EGFR high polysomy and/or gene amplification (FISH positive). Protein levels assessed by AQUA in FISH-positive cases were significantly higher (P = 0.04) than in FISH-negative cases. Using the continuous AQUA scores for EGFR expression, AQUA and FISH showed significant agreement (Pearson's ρ = 0.353, P = 0.04). Patients with high tumor EGFR protein expression had inferior 5-year overall survival (27.7%) compared with those with low tumor EGFR expression (54%; P = 0.029). There was no significant association between EGFR FISH status and overall survival (P = 0.201). In the multivariate model, high tumor EGFR protein expression status remained an independent prognostic factor for overall survival (P = 0.047).. EGFR protein content correlates with gene copy number if protein content is quantitated and automatically analyzed, as with AQUA. EGFR protein levels assessed by AQUA strongly predict for patient outcome in HNSCC, whereas EGFR FISH status does not provide prognostic information.

Topics: Carcinoma; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Gene Dosage; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Male; Neoplasms, Squamous Cell; Predictive Value of Tests; Prognosis; Proteins; Squamous Cell Carcinoma of Head and Neck; Survival Analysis; Tissue Array Analysis

Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro.. Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis.. In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis.. Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Dose-Response Relationship, Radiation; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Gamma Rays; Head and Neck Neoplasms; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Quinazolines; Signal Transduction; Sirolimus; Squamous Cell Carcinoma of Head and Neck; TOR Serine-Threonine Kinases; Tyrphostins