transforming-growth-factor-beta and Mouth-Neoplasms

Oral cancer is a common malignancy worldwide, accounting for 1.9% to 3.5% of all malignant tumors. Transforming growth factor β (TGF-β), as one of the most important cytokines, is found to play complex and crucial roles in oral cancers. It may act in a pro-tumorigenic and tumor-suppressive manner; activities of the former include cell cycle progression inhibition, tumor microenvironment preparation, apoptosis promotion, stimulation of cancer cell invasion and metastasis, and suppression of immune surveillance. However, the triggering mechanisms of these distinct actions remain unclear. This review summarizes the molecular mechanisms of TGF-β signal transduction, focusing on oral squamous cell and salivary adenoid systemic carcinomas as well as keratocystic odontogenic tumors. Both the supporting and contrary evidence of the roles of TGF-β is discussed. Importantly, the TGF-β pathway has been the target of new drugs developed in the past decade, some having demonstrated promising therapeutic effects in clinical trials. Therefore, the achievements of TGF-β pathway-based therapeutics and their challenges are also assessed. The summarization and discussion of the updated knowledge of TGF-β signaling pathways will provide insight into the design of new strategies for oral cancer treatment, leading to an improvement in oral cancer outcomes.

Topics: Carcinogenesis; Cytokines; Humans; Mouth Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

The increasing incidence of resistance to chemotherapeutic agents has become a major issue in the treatment of oral cancer (OC). Epithelial-mesenchymal transition (EMT) has attracted a great deal of attention in recent years with regard to its relation to the mechanism of chemotherapy drug resistance. EMT-activating transcription factors (EMT-ATFs), such as Snail, TWIST, and ZEB, can activate several different molecular pathways, e.g., PI3K/AKT, NF-κB, and TGF-β. In contrast, the activated oncological signal pathways provide reciprocal feedback that affects the expression of EMT-ATFs, resulting in a peritumoral extracellular environment conducive to cancer cell survival and evasion of the immune system, leading to resistance to multiple chemotherapeutic agents. We present an overview of evidence-based chemotherapy for OC treatment based on the National Comprehensive Cancer Network (NCCN) Chemotherapy Order Templates. We focus on the molecular pathways involved in drug resistance related to the EMT and highlight the signal pathways and transcription factors that may be important for EMT-regulated drug resistance. Rapid progress in antitumor regimens, together with the application of powerful techniques such as high-throughput screening and microRNA technology, will facilitate the development of therapeutic strategies to augment chemotherapy.

Topics: Antineoplastic Agents; Cell Line, Tumor; Drug Resistance; Drug Resistance, Neoplasm; Drug Therapy; Epithelial-Mesenchymal Transition; Humans; Mouth Neoplasms; NF-kappa B; Phosphatidylinositol 3-Kinases; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Transforming growth factor β (TGF-β) signaling either promotes or inhibits tumor formation and/or progression of many cancer types including squamous cell carcinoma (SCC). Canonical TGF-β signaling is mediated by a number of downstream proteins including Smad family proteins. Alterations in either TGF-β or Smad signaling can impact cancer. For instance, defects in TGF-β type I and type II receptors (TGF-βRI and TGF-βRII) and in Smad2/3/4 could promote tumor development. Conversely, increased TGF-β1 and activated TGF-βRI and Smad3 have all been shown to have tumor-promoting effects in experimental systems of human and mouse SCCs. Among TGF-β/Smad signaling, only TGF-βRII or Smad4 deletion in mouse epithelium causes spontaneous SCC in the mouse model, highlighting the critical roles of TGF-βRII and Smad4 in tumor suppression. Herein, we review the dual roles of the TGF-β/Smad signaling pathway and related mechanisms in SCC, highlighting the potential benefits and challenges of TGF-β/Smad-targeted therapies.

Topics: Animals; Carcinoma, Squamous Cell; Esophageal Neoplasms; Humans; Models, Biological; Mouth Neoplasms; Signal Transduction; Skin Neoplasms; Smad Proteins; Transforming Growth Factor beta

Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer.

Topics: Animals; Hedgehog Proteins; Humans; MicroRNAs; Molecular Targeted Therapy; Mouth Neoplasms; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta; Wnt Proteins

Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-β superfamily, regulate many cellular activities including cell migration, differentiation, adhesion, proliferation and apoptosis. Use of recombinant human bone morphogenic protein?2 (rhBMP?2) in oral and maxillofacial surgery has seen a tremendous increase. Due to its role in many cellular pathways, the influence of this protein on carcinogenesis in different organs has been intensively studied over the past decade. BMPs also have been detected to have a role in the development and progression of many tumors, particularly disease-specific bone metastasis. In oral squamous cell carcinoma - the tumor type accounting for more than 90% of head and neck malignancies- aberrations of both BMP expression and associated signaling pathways have a certain relation with the development and progression of the disease by regulating a range of biological functions in the altered cells. In the current review, we discuss the influence of BMPs -especially rhBMP-2- in the development and progression of oral squamous cell carcinoma.

Topics: Bone Morphogenetic Protein 2; Carcinogenesis; Carcinoma, Squamous Cell; Disease Progression; Humans; Mouth Neoplasms; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta

Integrins are a family of heterodimeric cell surface receptors, which are expressed on most cells where they mediate cell-cell and cell-extracellular matrix (ECM) interactions. The alphavbeta6 integrin is epithelial-specific and binds to the ECM proteins fibronectin, vitronectin and tenascin, and also to the latency associated peptide of TGF-beta. Unlike most epithelial integrins, alphavbeta6 is not expressed constitutively by healthy oral epithelia, but is up-regulated during tissue remodelling, including that accompanying wound healing and carcinogenesis. Although, the data at present have been generated principally from in vitro studies, there is increasing evidence to suggest that alphavbeta6 may promote carcinoma progression: alphavbeta6 has been shown to modulate invasion, inhibit apoptosis, regulate protease expression and activate TGF-beta1. This review examines the current literature, and discusses the possible role of alphavbeta6 in wound healing, and in the development and progression of oral squamous cell carcinoma.

Topics: Antigens, Neoplasm; Apoptosis; Carcinoma, Squamous Cell; Cell Communication; Disease Progression; Epithelial Cells; Extracellular Matrix; Humans; Integrins; Mouth Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation; Wound Healing

The role of transforming growth factor-beta (TGF-beta) in epithelial malignancy is complex, but it is becoming clear that, in the early stages of carcinogenesis, the protein acts as a potent tumor suppressor, while later, TGF-beta can function to advance tumor progression. We review the evidence to show that the pro-oncogenic functions of TGF-beta are associated with (1) a partial loss of response to the ligand, (2) defects of components of the TGF-beta signal transduction pathway, (3) over-expression and/or activation of the latent complex, (4) epithelial-mesenchymal transition, and (5) recruitment of signaling pathways which act in concert with TGF-beta to facilitate the metastatic phenotype. These changes are viewed in the context of what is known about the pathogenesis of oral cancer and whether this knowledge can be translated into the development of new therapeutic modalities.

Topics: Animals; Carcinoma; Cell Transformation, Neoplastic; DNA-Binding Proteins; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; Neoplasm Metastasis; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

Oral squamous cell carcinoma (OSCC) is a malignant neoplasm with high mortality and morbidity. The role of circRNA and its molecular mechanism in OSCC remains largely unknown. The study aims to explore the role of a novel circular RNA (circLDLRAD3) in OSCC and its underlying mechanism. PCR and fluorescence in situ hybridization were used to explore the expression features of circLDLRAD3 in OSCC. The effects of circLDLRAD3 on the behaviour of OSCC were investigated using CCK-8, colony formation assay, transwell and animal experiments. Bioinformatics analysis along with dual luciferase reporter assay and RIP assay were used to reveal the interaction between circLDLRAD3, miR-558 and Smad4. It was revealed that circLDLRAD3 exhibited low expression status in OSCC. CircLDLRAD3 inhibits proliferation, migration, and invasion of OSCC cells both in vitro and in vivo. Mechanistically, circLDLRAD3 could bind with miR-558 to positively regulate its target gene Smad4 expression. Rescue experiments further confirmed both miR-558 overexpression and Smad4 knockdown could reverse the influence of circLDLRAD3 on OSCC phenotypes. Moreover, circLDLRAD3 regulate the TGF-β signalling pathways to influence EMT through miR-558/Smad4 axis. Our study found that circLDLRAD3 is downregulated in OSCC and verified its tumour suppressor function and mechanism in OSCC through sponging miR-558 to regulate miR-558/Smad4/TGF-β axis. The characterization of such regulating network uncovers an important mechanism underlying OSCC progression, which could provide promising targets targeted therapy strategies for OSCC in the future.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; In Situ Hybridization, Fluorescence; MicroRNAs; Mouth Neoplasms; RNA, Circular; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta

The acquisition of motility via epithelial-mesenchymal transition (EMT) and osteoclast induction are essential for the invasion and metastasis of oral squamous cell carcinoma (OSCC) to bone. However, the molecule suppressing both EMT and osteoclastogenesis is still unknown. In this study, we found that cellular communication network factor 6 (CCN6) was less produced in a human OSCC cell line, HSC-3 with mesenchymal phenotype, than in HSC-2 cells without it. Notably, CCN6 interacted with bone morphogenetic protein 2 (BMP2) and suppressed the cell migration of HSC-3 cells stimulated by BMP2. Moreover, knockdown of CCN6 in HSC-2 cells led to the promotion of EMT and enhanced the effect of transforming growth factor-β (TGF-β) on the promotion of EMT. Furthermore, CCN6 combined with BMP2 suppressed EMT. These results suggest that CCN6 strongly suppresses EMT in cooperation with BMP2 and TGF-β. Interestingly, CCN6 combined with BMP2 increased the gene expression of receptor activator of nuclear factor-κB ligand (RANKL) in HSC-2 and HSC-3 cells. Additionally, CCN6 interacted with RANKL, and CCN6 combined with RANKL suppressed RANKL-induced osteoclast formation. In metastatic lesions, increasing BMP2 due to the bone destruction led to interference with binding of CCN6 to RANKL, which results in the promotion of bone metastasis of OSCC cells due to continuous osteoclastogenesis. These findings suggest that CCN6 plays dual roles in the suppression of EMT and in the promotion of bone destruction of OSCC in primary and metastatic lesions, respectively, through cooperation with BMP2 and interference with RANKL.

Topics: Bone Morphogenetic Protein 2; Carcinoma, Squamous Cell; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Head and Neck Neoplasms; Humans; Mouth Neoplasms; RANK Ligand; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta

Numerous studies have demonstrated an association between periodontitis and oral squamous cell carcinoma (OSCC), and periodontal pathogens such as

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Mice; Mouth Neoplasms; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta; Treponema denticola; Treponemal Infections

Tumor-associated neutrophils (TANs) are the major cellular component of the tumor microenvironment and have been shown to release of different bioactive molecules such as B-cell activating factor (BAFF). The data on the interactions between OSCC cells and neutrophils are limited and do not explain the actual role of the BAFF in the development of the OSCC. In the present study we examined the direct effect of neutrophils-derived BAFF on the OSCC cell line CAL-27 proliferation and apoptosis. PMNs of OSCC patients and healthy control were isolated from whole blood and separated by magnetic selection with monoclonal anti-human CD16 antibodies. CD-16 - positive neutrophils were incubated in the presence of TGF-β and/or LPS as well as flavonoids (luteolin and quercetin). CAL-27 cells were co-incubated with supernatants of neutrophils. BAFF expression in neutrophils, BAFF-R expression on CAL-27 cells and apoptosis of CAL-27 cells were assessed by flow cytometry. To determine the CAL-27 cells proliferation, the MTT test was used. Expression of select mitochondrial proteins in CAL-27 cells were measured by Western blot. Neutrophils from OSCC patients showed significantly higher expression of BAFF than those from the healthy controls. The results obtained revealed upregulation of the proliferation and downregulation of the apoptosis of the CAL-27 cells in the presence of the supernatants of TGF-β-treated neutrophils. Flavonoids reduced BAFF expression in neutrophils of patients with OSCC and control group. Lower intensity of apoptosis in CAL-27 cells was associated with the increased expression of anti-apoptotic Bcl-2, Mcl-1 and activated form of PI3K kinase (pPI3K) and simultaneously reduced expression of pro-apoptotic Bax protein in the presence of rhBAFF, as well as of supernatants of neutrophils derived from OSCC patients. In conclusion, the data presented confirm the previously suggested role of neutrophil-derived BAFF in OSCC development. The favorable effects of examined flavonoids on tumor-promoting BAFF expression in neutrophils suggest that they might be promising candidates as chemo-preventive agents in the therapy of patients with OSCC.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; B-Cell Activating Factor; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Flavonoids; Humans; Longevity; Mouth Neoplasms; Neutrophils; Transforming Growth Factor beta; Tumor Microenvironment

Although substantial progress has been made in cancer biology and treatment, the prognosis of oral squamous cell carcinoma (OSCC) is still not satisfactory because of local tumor invasion and frequent lymph node metastasis. The tumor microenvironment (TME) is a potential target in which cancer-associated fibroblasts (CAFs) are of great significance due to their interactions with cancer cells. However, the exact mechanism is still unclear. Therefore, we focus on the crosstalk between cancer cells and CAFs and discover that CAFs are the main source of TGF-β1. Transwell assays and western blot analysis further prove that CAFs activate the TGF-β1/Smad pathway to promote OSCC invasion. Through survival analysis, we confirm that CAF overexpression is correlated with poor overall survival in OSCC. To further elucidate the origin and role of CAFs in OSCC, we analyze single-cell RNA sequencing (scRNA-seq) data from 14 OSCC tumor samples and identify four distinct cell types, including CAFs, in the TME, indicating high intratumoral heterogeneity. Then, two subtypes of CAFs, namely, myofibroblasts (mCAFs) and inflammatory CAFs (iCAFs), are further distinguished. Based on the differentially upregulated genes of mCAFs and iCAFs, GO enrichment analysis reveals their different roles in OSCC progression. Furthermore, the gene expression pattern is dynamically altered across pseudotime, potentially taking part in the transformation from epithelial to mCAFs or iCAFs through the epithelial to mesenchymal transition.

Topics: Cancer-Associated Fibroblasts; Carcinoma, Squamous Cell; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Fibroblasts; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Single-Cell Analysis; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Microenvironment

Transforming growth factor β (TGF-β) increases epithelial cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). TGF-β also inhibits cell proliferation by inducing G1 phase cell-cycle arrest. However, the correlation between these tumor-promoting and -suppressing effects remains unclear. Here, we show that TGF-β confers higher motility and metastatic ability to oral cancer cells in G1 phase. Mechanistically, keratin-associated protein 2-3 (KRTAP2-3) is a regulator of these dual effects of TGF-β, and its expression is correlated with tumor progression in patients with head and neck cancer and migratory and metastatic potentials of oral cancer cells. Furthermore, single-cell RNA sequencing reveals that TGF-β generates two populations of mesenchymal cancer cells with differential cell-cycle status through two distinctive EMT pathways mediated by Slug/HMGA2 and KRTAP2-3. Thus, TGF-β-induced KRTAP2-3 orchestrates cancer cell proliferation and migration by inducing EMT, suggesting motile cancer cells arrested in G1 phase as a target to suppress metastasis.

Topics: Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Keratins; Mouth Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Head and Neck Neoplasms; Humans; Macrophages; Mouth Neoplasms; Phenotype; Receptor, Transforming Growth Factor-beta Type I; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta; Tumor Microenvironment

The immune responses play significant roles in the onset, progression, and outcome of oral squamous cell carcinoma (OSCC). CD4. In a carcinogen-induced mouse OSCC model, interleukin-23 receptor (IL-23R) expression on Tregs and Treg function were determined by flow cytometry. IL-23R overexpression in Tregs was achieved by lentiviral infection, followed by evaluation of the expression of Forkhead box P3 (Foxp3), T-bet, retineic-acid-receptor-related orphan nuclear receptor gamma t, and cytokines by flow cytometry. Adoptive transfer assays were applied to analyze the function of IL-23R. IL-23R. This study unveils Treg heterogeneity, thus deepening the understanding of Treg biology and tumor immunity in OSCC.

Topics: Animals; Carcinoma, Squamous Cell; Forkhead Transcription Factors; Head and Neck Neoplasms; Interleukin-10; Interleukin-23; Mice; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Reserpine is a natural indole alkaloid isolated from Rauwolfia serpentina and has potent antioxidant, antimicrobial, and anti-mutagenic properties. Accordingly, this study aimed to investigate the effect of reserpine on DNA repair, cell proliferation, invasion and apoptosis in 7,12-dimethylbenz[a]anthracene(DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Transforming growth factor-β (TGF-β) was found to induce Smad2, 3 and 4 phosphorylation triggering Smad3/Snail mediated DNA repair proteins and Smad2/4 nuclear translocation. In contrast, reserpine inhibits TGF-β dependent Smad2/3/4 phosphorylation, thereby blockage Smad3/Snail activation and Smad2/4 nuclear translocation. Interruption of these oncogenic signaling pathways leads to downregulating ERCC1, XPF, Ku70, DNA-PKcs, PCNA, cyclin D1, HIF-1α, IL-6, Mcl-1 and stimulates Bax, cytochrome C, Apaf-1, caspase-9, caspase-3 and PARP protein expressions. This study provides therapeutic potential of reserpine in inhibiting DNA repair, cell proliferation, and invasion while simultaneously inducing apoptosis via modulation TGF-β signals.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apoptosis; Carcinogenesis; Cell Proliferation; Cricetinae; DNA Repair; Down-Regulation; Male; Molecular Docking Simulation; Mouth Mucosa; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; Reserpine; Signal Transduction; Transforming Growth Factor beta

The role of neutrophils in cancer has been the subject of intense research in recent years. One major theme that has emerged is that not all neutrophils are equal in the field of cancer. However, it remains unclear what induces the protumorigenic or antitumorigenic phenotype predominate in tumor. Therefore, this study aimed to investigate what factors induce which of these two phenotypes of neutrophil predominate in OSCC and to explore the role of neutrophil polarization on tumor.. Immunofluorescence and immunohistochemistry staining were used to observe neutrophil infiltration and the expression of TGF-β1 and IL-17A in OSCC tissues. Recombinant human TGF-β1 and IL-17A were used to modulate neutrophil polarization. OSCC cell (SCC9 and SAS cell lines) migration, proliferation, invasion, stemness, and EMT were analyzed after treatment with conditioned medium from TGF-β1/IL-17A-activated neutrophils. The levels of neutrophil-associated markers in OSCC tissues and peripheral blood were examined by immunofluorescence staining and quantitative PCR.. Our data showed neutrophil infiltration and elevated expression of TGF-β1 and IL-17A in OSCC tissues. The cooperative effect of TGF-β1 and IL-17A promoted neutrophils to take on a protumor phenotype in vitro. TGF-β1/IL-17A-activated neutrophils remarkably induced cell migration, proliferation, invasion, stemness, and EMT in OSCC cells. Additionally, OSCC patients showed increased expression of MMP9 and decreased expression of CCL3 in circulating neutrophils.. TGF-β1 and IL-17A cooperated to augment the protumor functions of neutrophils, thereby promoting the progression of OSCC cells. In addition, the combination of neutrophil-associated markers may serve as a predictive method to screen for patients with OSCC.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Extracellular Matrix Proteins; Head and Neck Neoplasms; Humans; Interleukin-17; Mouth Neoplasms; Neutrophils; Phenotype; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta; Transforming Growth Factor beta1

Oral squamous cell carcinoma (OSCC) frequently invades nearby bone and bone involvement determines the prognosis of patients. Growth factors, stored in the bone matrix and released during bone destruction, are known as key components in the bone-tumor interaction. However, the coordination of growth factor signals and the precise mechanism of bone destruction in oral cancer are still unclear. In the study, we investigated the differential cytokine expression profile of oral cancer cells by TGF-β treatment and the function of altered expression of cytokines on the osteoclast differentiation. We established TGFBR2-knockdown cells using small hairpin RNA. TGF-β was treated to both TGFBR2 expressing and knockdown cells and the culture supernatants were analyzed using a cytokine array kit. We found that the TGF-β inhibited IGFBP3 level and enhanced MMP9 level. We confirmed this regulation of IGFBP3 and MMP9 by TGF-β using ELISA and zymography, respectively. IGFBP3 is known as to modulate the bioavailability of IGF1, which is abundant in the bone microenvironment and regulates osteoclast differentiation. Therefore, we further analyzed the function of IGFBP3 on osteoclastogenesis. Although IGFBP3 increased the viability of murine bone marrow macrophages, the osteoclast differentiation of these cells was blocked by IGFBP3 in a dose-dependent manner. These results revealed a novel pathway for the regulation of osteoclastogenesis by oral cancer cells, which may be a new therapeutic target for osteolysis induced by oral cancer infiltrating into the bone.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Gene Knockdown Techniques; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Macrophages; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred ICR; Mouth Neoplasms; Osteoclasts; Osteogenesis; Osteoprotegerin; RANK Ligand; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta

Patients with oral squamous cell carcinoma (OSCC) bone invasion are surgically treated with bone resection, which results in severe physical and psychological damage. Here, we investigated the potential of fractalkine (CX3CL1), which is regulated by transforming growth factor (TGF-β), as a novel biomarker for correct prediction and early detection of OSCC-associated bone invasion. TGF-β knockdown and treatment with a TGF-β-neutralizing antibody decreased the level of fractalkine in the culture media of HSC-2 and YD10B OSCC cells. Treatment with a fractalkine-neutralizing antibody reduced TGF-β-stimulated invasion by HSC-2 and YD10B cells. Fractalkine treatment increased the viability, invasion, and uPA secretion of both OSCC cell lines. Furthermore, OSCC cell bone invasion was assessed following subcutaneous inoculation of wild-type or TGF-β knockdown OSCC cells in mouse calvaria. TGF-β knockdown prevented erosive bone invasion, reduced the number of osteoclasts at the tumor-bone interface, and downregulated fractalkine expression in mouse tumor tissues. Our results indicate that the production of fractalkine is stimulated by TGF-β and mediates TGF-β-induced cell invasion in several OSCC cell lines showing an erosive pattern of bone invasion. Fractalkine may be a useful predictive marker and therapeutic target for OSCC-induced bone destruction.

Topics: Animals; Biomarkers; Carcinoma, Squamous Cell; Cell Line, Tumor; Chemokine CX3CL1; Head and Neck Neoplasms; Humans; Mice; Mouth Neoplasms; Neoplasm Invasiveness; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta; Transforming Growth Factor beta1

Epithelial-mesenchymal transition (EMT) has been contributed to increase migration and invasion of cancer cells. However, the correlate of Naa10p and IKKα with EMT in oral squamous cell carcinoma (OSCC) is not yet fully understood. In our present study, we found N-α-acetyltransferase 10 protein (Naa10p) and IκB kinase α (IKKα) were abnormally abundant in oral squamous cell carcinoma (OSCC). Bioinformatic results indicate that the expression of Naa10p and IKKα is correlated with TGF-β1/Smad and EMT-related molecules. The Transwell migration, invasion, qRT-PCR and Western blot assay indicated that Naa10p repressed OSCC cell migration, invasion and EMT, whereas IKKα promoted TGF-β1-mediated OSCC cell migration, invasion and EMT. Mechanistically, Naa10p inhibited IKKα activation of Smad3 through the interaction with IKKα directly in OSCC cells after TGF-β1 stimulation. Notably, knockdown of Naa10p reversed the IKKα-induced change in the migration, invasion and EMT-related molecules in OSCC cells after TGF-β1 stimulation. These findings suggest that Naa10p interacted with IKKα mediates EMT in OSCC cells through TGF-β1/Smad, a novel pathway for preventing OSCC.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; I-kappa B Kinase; Male; Mouth Neoplasms; N-Terminal Acetyltransferase A; N-Terminal Acetyltransferase E; Protein Binding; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Oral Potentially Malignant Disorders (OPMDs) including Oral Lichen Planus (OLP) are associated with risk of transformation to oral squamous cell carcinoma (OSCC). Available data show that innate immune cells involving polymorphonuclear neutrophils (PMNs) with their ability to neutrophil extracellular traps (NETs) formation are likely to be directly involved in development of cancer. Examination of NETs generation by TGF-β - induced neutrophils of OLP patients showed increased amounts of traps with MPO, H3Cit and cfDNA, known to be released with NETs. The presence of excessive amounts of NETs components may lead to numerous adverse consequences associated with potential transformation to OSCC. Bacterial-related infection may enhance the NETs formation and lead to consequences resulting from the excessive number of individual elements of these networks. It is likely that regulating NETs release by the flavonoids presented herein may be beneficial not only for inhibiting OLP development, but also in reducing risk of transformation to OSCC.

Topics: Cell Transformation, Neoplastic; Extracellular Traps; Female; Humans; Lichen Planus, Oral; Male; Middle Aged; Mouth Neoplasms; Neutrophils; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta

Oral squamous cell carcinoma (OSCC) is associated with high morbidity and ranks sixth among malignancies worldwide. Increasing evidence suggests that microRNAs (miRNAs or miRs) play a critical role in regulating cancer stem cells (CSCs), which drive the proliferation and spread of OSCC. Therefore, based on the alteration of aberrantly expressed miR-495 and homeobox C6 (HOXC6) by Gene Expression Omnibus (GEO) analysis, we subsequently explore the potential effect of miR-495 on the progression of CSCs in OSCC.. After the isolation of CSCs from the clinical tissue samples of OSCC patients, the expression of miR-495 and HOXC6 was determined, followed by the validation of the relationship between miR-495 and HOXC6. Subsequently, gain- and loss-function approach was performed to detect the role of miR-495 and HOXC6 in cell proliferation, migration, invasion, cell cycle entry, apoptosis, and epithelial-mesenchymal transition (EMT) of CSCs in OSCC, as well as the tumor growth in vivo.. HOXC6 was highly expressed while miR-495 was poorly expressed in OSCC. HOXC6 was verified to be a target gene of miR-495, and miR-495 could inhibit the activation of the TGF-β signaling pathway. CSCs with miR-495 overexpression or HOXC6 silencing exhibited reversed EMT process; reduced abilities of proliferation, migration, and invasion; and promoted cell apoptosis in vitro. Moreover, inhibited tumor growth was observed in vivo after injection with miR-495 agomir or sh-HOXC6. In contrast, the downregulation of miR-495 showed an induced role in the progression of OSCC.. These findings suggest that miR-495 may suppress HOXC6 to inhibit EMT, proliferation, migration, and invasion while promoting apoptosis of CSCs in OSCC by inhibiting the TGF-β signaling pathway.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Head and Neck Neoplasms; Homeodomain Proteins; Humans; MicroRNAs; Mouth Neoplasms; Neoplastic Stem Cells; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer-associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF-β, which induces endothelial-to-mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor-infiltrating inflammatory cells secrete various cytokines, including TNF-α. However, the role of TNF-α in TGF-β-induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF-α on TGF-β-induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF-β and TNF-α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF-β and TNF-α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF-β type I receptor, TGF-β2, activin A, and integrin αv, suggesting that TNF-α enhanced TGF-β-induced EndMT by augmenting TGF-β family signals. Furthermore, oral squamous cell carcinoma-derived cells underwent epithelial-to-mesenchymal transition (EMT) in response to humoral factors produced by TGF-β and TNF-α-cultured ECs. This EndMT-driven EMT was blocked by inhibiting the action of TGF-βs. Collectively, our findings suggest that TNF-α enhances TGF-β-dependent EndMT, which contributes to tumor progression.

Topics: Biomarkers; Cancer-Associated Fibroblasts; Carcinoma, Squamous Cell; Cell Line; Cells, Cultured; Endothelial Cells; Epithelial-Mesenchymal Transition; Humans; Inflammation Mediators; Mouth Neoplasms; NF-kappa B; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Necrosis Factor-alpha

Bone mesenchymal stem cells (BMSCs) play critical roles in tumour microenvironment. However, molecular mechanisms of how BMSCs to be recruited and effect subsequent tumour progression are poorly understood in oral squamous cell carcinoma (OSCC).. The distribution of CXCL8 was detected by immunohistochemical staining in OSCC tissues. The chemotaxis of conditioned media from different epithelial cells to BMSCs was examined by trans-well assay. Real-time quantitative PCR (qPCR) and ELISA were used to detect the expression of related cytokines and chemokine receptors. The migration of BMSCs was observed in BALB/c nude mice. The roles of BMSCs in proliferation, migration and invasion of OSCC were detected by CCK-8, flow cytometry and trans-well assay. Epithelial-mesenchymal transition (EMT)-related markers were analysed by qPCR and Western blot in vitro, and growth was evaluated in BALB/c nude mice using subcutaneously implanted OSCC in nude mouse model in vivo.. Using OSCC, we show CXCL8, secreted by OSCC, binds to exclusively CXCR2 in BMSCs to facilitate migration of BMSCs to OSCC. TGF-β secreted by BMSCs subsequently induces EMT of OSCC to promote their proliferation, migration and infiltration. We also showed that the Ras/Raf/Erk axis plays a critical role in tumour progression.. Our results provide the molecular basis for BMSC recruitment into tumours, and how this process leads to tumour progression and leads us to develop a novel OSCC treatment target.

Topics: Animals; Cadherins; Carcinoma, Squamous Cell; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Interleukin-8; Male; Mesenchymal Stem Cells; Mice, Nude; Mouth Neoplasms; Receptors, Interleukin-8B; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Tumor progression is governed by various growth factors and cytokines in the tumor microenvironment (TME). Among these, transforming growth factor-β (TGF-β) is secreted by various cell types residing in the TME and promotes tumor progression by inducing the epithelial-to-mesenchymal transition (EMT) of cancer cells and tumor angiogenesis. TGF-β comprises three isoforms, TGF-β1, -β2, and -β3, and transduces intracellular signals via TGF-β type I receptor (TβRI) and TGF-β type II receptor (TβRII). For the purpose of designing ligand traps that reduce oncogenic signaling in the TME, chimeric proteins comprising the ligand-interacting ectodomains of receptors fused with the Fc portion of immunoglobulin are often used. For example, chimeric soluble TβRII (TβRII-Fc) has been developed as an effective therapeutic strategy for targeting TGF-β ligands, but several lines of evidence indicate that TβRII-Fc more effectively traps TGF-β1 and TGF-β3 than TGF-β2, whose expression is elevated in multiple cancer types. In the present study, we developed a chimeric TGF-β receptor containing both TβRI and TβRII (TβRI-TβRII-Fc) and found that TβRI-TβRII-Fc trapped all TGF-β isoforms, leading to inhibition of both the TGF-β signal and TGF-β-induced EMT of oral cancer cells, whereas TβRII-Fc failed to trap TGF-β2. Furthermore, we found that TβRI-TβRII-Fc suppresses tumor growth and angiogenesis more effectively than TβRII-Fc in a subcutaneous xenograft model of oral cancer cells with high TGF-β expression. These results suggest that TβRI-TβRII-Fc may be a promising tool for targeting all TGF-β isoforms in the TME.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; HEK293 Cells; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neovascularization, Pathologic; Protein Isoforms; Receptors, Fc; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Transforming Growth Factor beta; Tumor Microenvironment

The aim of this study was to explore the mechanisms by which oral cancer acquires resistance to gemcitabine.. Oral squamous cell carcinoma (OSCC) cells were treated with gemcitabine upon infection or with a lentivirus harboring short hairpin RNA (shRNA) targeted to transforming growth factor-β (TGF-β). Then, Western blot, ELISA, migration assay, MTT assay, and animal experiments were used to explore the mechanism of resistance to gemcitabine treatment.. After the treatment of non-transfected cells with gemcitabine, NF-κB and AKT activities were increased, which may have induced the OSCC resistance to gemcitabine. Then, we found that TGF-β downregulation effectively reduced NF-κB and AKT phosphorylation levels after the administration of gemcitabine and increased the OSCC sensitivity to gemcitabine, resulting in cell death and the blunting of OSCC resistance to gemcitabine. The EMT was also reduced by TGF-β downregulation combined with gemcitabine treatment.. Cellular levels of TGF-β constitute an important factor in gemcitabine resistance and TGF-β silencing might represent a novel and potent strategy for overcoming OSCC resistance to gemcitabine.

Topics: Animals; Cell Line, Tumor; Deoxycytidine; Down-Regulation; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gemcitabine; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mice; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

The human Obg-like ATPase 1 (OLA1) protein has been reported to play an important role in cancer cell proliferation. The molecular mechanism underlying OLA1 regulated oral metastasis is still unknown. We investigated in this study the regulatory role of OLA1 playing in oral squamous cell metastasis.. A series of in vitro assays were performed in the cells with RNAi-mediated knockdown or overexpression to expound the regulatory function of OLA1 in oral cancer. We found that the endogenous level of OLA1 in a highly metastatic oral squamous cell line was significantly lower than that in low metastatic oral cells as well as in normal oral cells. Escalated expression of OLA1 resulted in a reduced ability of metastasis in highly metastatic cells, and enhanced its sensitivity to the paclitaxel treatment. Further analysis of the EMT markers showed that Snail, Slug, N-cadherin were up-expressed significantly. Meanwhile, E-cadherin was significantly down-regulated in the oral cancer cells with OLA1-knocked down, suggesting that OLA1 inactivated EMT process. Furthermore, we found that OLA1 suppressed oral squamous cell metastasis by suppressing the activity of a TGFβ/SMAD2/EMT pathway.. Our data suggests that OLA1 may be developed as a potential target for the treatment of oral cancer metastasis.

Topics: Adenosine Triphosphatases; Cadherins; Carcinoma; Cell Line, Tumor; Down-Regulation; GTP-Binding Proteins; Humans; Mouth Neoplasms; Neoplasm Metastasis; Paclitaxel; Signal Transduction; Smad2 Protein; Snail Family Transcription Factors; Transforming Growth Factor beta; Up-Regulation

Oral squamous cell carcinoma (OSCC) is a prevalent cancer that develops in the head and neck area and has high annual mortality despite optimal treatment. microRNA-218 (miR-218) is a tumour inhibiting non-coding RNA that has been reported to suppress the cell proliferation and invasion in various cancers. Thus, our study aims to determine the mechanism underlying the inhibitory role of miR-218 in OSCC. We conducted a bioinformatics analysis to screen differentially expressed genes in OSCC and their potential upstream miRNAs. After collection of surgical OSCC tissues, we detected GREM1 expression by immunohistochemistry, RT-qPCR and Western blot analysis, and miR-218 expression by RT-qPCR. The target relationship between miR-218 and GREM1 was assessed by dual-luciferase reporter gene assay. After loss- and gain-of-function experiments, OSCC cell proliferation, migration and invasion were determined by MTT assay, scratch test and Transwell assay, respectively. Expression of TGF-β1, Smad4, p21, E-cadherin, Vimentin and Snail was measured by RT-qPCR and Western blot analysis. Finally, effects of miR-218 and GREM1 on tumour formation and liver metastasis were evaluated in xenograft tumour-bearing nude mice. GREM1 was up-regulated, and miR-218 was down-regulated in OSCC tissues, and GREM1 was confirmed to be the target gene of miR-218. Furthermore, after up-regulating miR-218 or silencing GREM1, OSCC cell proliferation, migration and invasion were reduced. In addition, expression of TGF-β signalling pathway-related genes was diminished by overexpressing miR-218 or down-regulating GREM1. Finally, up-regulated miR-218 or down-regulated GREM1 reduced tumour growth and liver metastasis in vivo. Taken together, our findings suggest that the overexpression of miR-218 may inhibit OSCC progression by inactivating the GREM1-dependent TGF-β signalling pathway.

Topics: Adult; Aged; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Computational Biology; Databases, Genetic; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Reporter; Heterografts; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice; MicroRNAs; Middle Aged; Models, Biological; Mouth Neoplasms; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; RNA Interference; Signal Transduction; Transforming Growth Factor beta

FOXO3a has been widely regarded as a tumor suppressor. It also plays a paradoxical role in regulating the cancer stem cells (CSCs), responsible for tumor-initiation, chemo-resistance, and recurrence in various solid tumors, including oral squamous cell carcinoma (OSCC). This study aims to uncover the role of FOXO3a and its importance for a non-canonical pathway of TGFβ in regulating the OSCC stemness.. We identified FOXO3a expression in OSCC tissues and cell lines using immunohistochemistry and western blot. The correlation between FOXO3a and stemness was evaluated. Stable cell lines with differential expression of FOXO3a were constructed using lentiviruses. The effects of FOXO3a on stem-cell like properties in OSCC was further evaluated in vitro and in vivo. We also explored the effect of TGFβ on FOXO3a with respect to its expression and function.. Our findings suggest that FOXO3a was widely expressed and negatively correlated with the stemness in OSCC. This regulation can be abolished by TGFβ through phosphorylation, nuclear exclusion, and degradation in the non-Smad pathway. We also observed that non-Smad AKT-FOXO3a axis is essential to regulate stemness of CSCs by TGFβ.. TGFβ induces stemness through non-canonical AKT-FOXO3a axis in OSCC. Our study provides a foundation to understand the mechanism of CSCs and a possible therapeutic target to eliminate CSCs.

Topics: Adult; Aged; Animals; Biomarkers; Carcinoma, Squamous Cell; Cell Line, Tumor; Female; Forkhead Box Protein O3; Gene Expression; Humans; Immunohistochemistry; Male; Mice; Middle Aged; Mouth Neoplasms; Mutation; Neoplastic Stem Cells; Phosphorylation; Proteolysis; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta

There is increasing evidence that the expression of CD109, a GPI-anchored cell surface protein is dysregulated in squamous cell carcinoma (SCC). However, the functional role of CD109 in SCC progression is poorly understood. In current study, we demonstrate that CD109 is a critical regulator of epithelial phenotype in SSC cells. CD109 levels inversely correlate with TGF-β signaling, EMT, migration, and invasion in cultured SCC cells. CRISPR/Cas9-mediated knockout CD109 (CD109 KO) in SCC cells represses epithelial traits and promotes the mesenchymal phenotype, as evidenced by elevated expression of mesenchymal proteins and markers of epithelial to mesenchymal transition. Treatment with recombinant CD109 protein causes CD109 KO cells to regain their epithelial traits. CD109 loss results in pronounced alterations of gene expression as detected by microarray analysis and in dysregulation of 15 important signalling pathways as shown by KEGG pathway cluster analysis. Validation using 52 human oral SCC tumor samples show that CD109 levels inversely correlate with tumor grade and the activation state of one such pathway, the TGF-β signaling pathway. Taken together, our findings highlight a novel role for CD109 as a gatekeeper of the epithelial phenotype by regulating TGF-β pathway in SCC cells.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; CRISPR-Cas Systems; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Male; Middle Aged; Mouth Neoplasms; Neoplasm Grading; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Signal Transduction; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) exerts its versatile function (oncogenic or tumor suppressive role) during the carcinogenesis in tumor microenvironment-dependent manner. Considering the tumor heterogeneity, spatial and temporal distribution of TGF-β in oral squamous cell carcinoma (OSCC) remained to be elucidated.. Formalin-fixed, paraffin-embedded sections derived from 73 patients with OSCC were immunostained, revealing expression patterns of TGF-β, both at the regions of tumor center (TC) and invasive tumor front (ITF).. The TGF-β levels on tumor cells, fibroblast-like cells (FLCs), and tumor-infiltrating lymphocytes (TILs) were comparable and showed to be cell-type-independent manner. Although TC regions harbored less positive staining of TGF-β than ITF in tumor cells (TGF-β. Tumor cell-derived TGF-β at TC regions, but not at ITF, could be a promising predictor for disease recurrence and poor prognosis of patients with OSCC.

Topics: Carcinoma, Squamous Cell; Female; Humans; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Prognosis; Survival Analysis; Transforming Growth Factor beta; Tumor Microenvironment

To investigate the role of the long non-coding ribonucleic acid (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) in the proliferation and apoptosis of the oral squamous cell carcinoma (OSCC) cells by regulating the transforming growth factor-beta (TGF-β)/Smad pathway.. Human OSCC cells were cultured, and then transfected with small interfering (si)-ANRIL to inhibit the lncRNA ANRIL and ANRIL-OE to overexpress the lncRNA ANRIL. Next, the flow cytometry was carried out to detect the apoptosis rate, the proliferation was determined via methyl thiazolyl tetrazolium (MTT) assay, and the changes in the protein level were detected through Western blotting (WB).. The lncRNA ANRIL was highly expressed in the tissues and serum of patients. The proliferation ability of the cells transfected with si-ANRIL was significantly reduced, while that of the cells transfected with ANRIL-OE was overtly increased. The apoptosis rate was (9.21±5.22)%, (22.3±1.34)%, and (13.21±6.22)% in lncRNA ANRIL-OE group, si-ANRIL group and control group, respectively. The protein expression level of the apoptotic protein active caspase-3 was lowered after the treatment with ANRIL-OE, and the key molecules of the TGF-β/Smad pathway were notably down-regulated after inhibiting ANRIL with si-ANRIL.. The lncRNA ANRIL regulates the TGF-β/Smad signaling pathway to promote the proliferation and suppress the apoptosis of OSCC cells.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; RNA, Long Noncoding; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Up-Regulation

Oral squamous cell carcinoma (OSCC) has a very poor prognosis because of its highly invasive nature, and the 5-year survival rate has not changed appreciably for the past 30 years. Although cylindromatosis (CYLD), a deubiquitinating enzyme, is thought to be a potent tumour suppressor, its biological and clinical significance in OSCC is largely unknown. This study aimed to clarify the roles of CYLD in OSCC progression. Our immunohistochemical analyses revealed significantly reduced CYLD expression in invasive areas in OSCC tissues, whereas CYLD expression was conserved in normal epithelium and carcinoma in situ. Furthermore, downregulation of CYLD by siRNA led to the acquisition of mesenchymal features and increased migratory and invasive properties in OSCC cells and HaCaT keratinocytes. It is interesting that CYLD knockdown promoted transforming growth factor-β (TGF-β) signalling by inducing stabilization of TGF-β receptor I (ALK5) in a cell autonomous fashion. In addition, the response to exogenous TGF-β stimulation was enhanced by CYLD downregulation. The invasive phenotypes induced by CYLD knockdown were completely blocked by an ALK5 inhibitor. In addition, lower expression of CYLD was significantly associated with the clinical features of deep invasion and poor overall survival, and also with increased phosphorylation of Smad3, which is an indicator of activation of TGF-β signalling in invasive OSCC. These findings suggest that downregulation of CYLD promotes invasion with mesenchymal transition via ALK5 stabilization in OSCC cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Aged; Cell Line, Tumor; Cell Movement; Deubiquitinating Enzyme CYLD; Down-Regulation; Enzyme Stability; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Mouth Neoplasms; Neoplasm Invasiveness; Phosphorylation; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Smad3 Protein; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta

Topics: Biomechanical Phenomena; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Epithelial-Mesenchymal Transition; Extracellular Matrix; Fibrosis; Humans; Matrix Metalloproteinases; Mechanotransduction, Cellular; Mouth Neoplasms; Oral Submucous Fibrosis; Signal Transduction; Transforming Growth Factor beta

Snail, also called Snai1, is a key regulator of EMT. Snail plays crucial roles in cancer progression, including resistance to anti-tumor drugs and invasion by various cancer cells. Slug, also known as Snai2, is also involved in the aggravation of certain tumors. In this study, we examined the roles of Slug in human oral squamous cell carcinoma (OSCC) cells. Slug is highly expressed in these cells, and Slug siRNA effectively represses anti-tumor drug resistance and invasive properties. In addition, transforming growth factor (TGF)-β upregulates the expression of Snail and Slug and promotes resistance to anti-tumor drugs in OSCC cells. Surprisingly, Slug siRNA appears to upregulate Snail expression considerably in OSCC cells. Snail siRNA also appears to upregulate Slug expression. Thus, either Slug or Snail siRNA alone partially mitigates malignant phenotypes in the presence of TGF-β, whereas both Slug and Snail siRNAs together dramatically suppress them. Therefore, Slug and Snail in tandem, but not alone, are potential therapeutic targets for nucleic acid medicines to treat oral cancer.

Topics: Antineoplastic Agents; Biomarkers; Cell Line, Tumor; Cytokines; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; Snail Family Transcription Factors; Transforming Growth Factor beta

Topics: Cadherins; Carcinoma, Squamous Cell; cdc42 GTP-Binding Protein; Cell Line; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Focal Adhesion Kinase 1; Humans; Mebendazole; Mouth Neoplasms; rhoA GTP-Binding Protein; Transforming Growth Factor beta

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Recently, BMP7 has been demonstrated to be produced by salivary glands and contribute to embryonic branching in mice. The BMP7 in saliva is thought to be delivered to the oral cavity and is expected to contact with stratified squamous epithelial cells which line the surface of oral mucosa. In this study, we attempted to investigate the effects of BMP7 on oral epithelial cells.. The expression of BMP receptors was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). OSCCs were stimulated with human recombinant BMP7 (hrBMP7) and the phosphorylation status of Smad1/5/8 was examined by western blotting. For microarray analysis, Ca9-22 cells were stimulated with 100 ng/mL of hrBMP7 and total RNA was extracted and subjected to real-time PCR. The 5'-untranslated region (5'-UTR) of IL-17 F gene was cloned to pGL4-basic vector and used for luciferase assay. Ca9-22 cells were pre-incubated with DM3189, a specific inhibitor of Smad1/5/8, for inhibition assay.. All isoforms of type I and type II BMP receptors were expressed in both Ca9-22 and HSC3 cells and BMP7 stimulation resulted in the phosphorylation of Smad1/5/8 in both cell lines. The microarray analysis revealed the induction of interleukin-17 F (IL-17 F), netrin G2 (NTNG2) and hyaluronan synthase 1 (HAS1). Luciferase assay using the 5'-UTR of the IL-17 F gene revealed transcriptional regulation. Induced IL-17 F production was further confirmed at the protein level by ELISA. Smad1/5/8 inhibitor pretreatment decreased IL-17 F expression levels in the cells.

Topics: Bone Morphogenetic Protein 7; Carcinoma, Squamous Cell; Epithelial Cells; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Hyaluronan Synthases; Interleukin-17; Mouth Neoplasms; Netrins; Phosphorylation; Recombinant Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) and its receptors are considered as a novel target in cancer chemotherapy. Gramine, an indole alkaloid, possesses various pharmacological properties including antiproliferative and anticancer. However, the anti-angiogenic property remains unexplored.. The present study was designed to evaluate the anti-angiogenic and apoptosis induction properties of gramine through inhibiting TGF-β on DMBA induced oral squamous cell carcinoma (OSCC) in the hamster buccal pouch (HBP).. The effects of gramine on TGF-β signalling in DMBA induced carcinogenic events such as angiogenesis and apoptosis were analysed by studying the mRNA expression using RT-PCR, protein expression by western blot and histopathological analysis using haematoxylin and eosin (H & E) staining.. Gramine significantly inhibited phosphorylation and nuclear translocation of Smad2 and Smad4 by blocking activity of the TGFβ-RII, RI and activation of inhibitory Smad7. Gramine inhibited angiogenic markers such as MMP-2, MMP-9, HIF-1α, VEGF, and VEGF-R2 as well as increased TIMP-2 expression. Furthermore, gramine induced apoptosis in DMBA induced tumour bearing animals by up regulating the pro apoptotic proteins Bax, cytochrome C, apaf-1, caspase-9 caspase-3 and PARP.. In this study, we clearly demonstrated that gramine treatment diminishes angiogenesis and induces apoptosis in hamster buccal pouch (HBP) carcinogenesis by modulating TGF-β signals.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Alkaloids; Angiogenesis Inhibitors; Animals; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspase 9; Cheek; Cricetinae; Indole Alkaloids; Male; Mesocricetus; Mouth Neoplasms; Neovascularization, Pathologic; Signal Transduction; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta

Topics: Adult; B7-H1 Antigen; Carcinoma, Squamous Cell; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class I; HLA-E Antigens; HLA-G Antigens; Humans; Immunologic Factors; Interleukin-10; Leukoplakia, Oral; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Precancerous Conditions; Transforming Growth Factor beta

Numerous studies implicate miR-146a as pleiotropic regulator of carcinogenesis; however, its roles in carcinogenesis are not fully understood. A clue from expression analyses of miR-146a-5p in all 13 oral squamous cell carcinoma (OSCC) cell lines examined and in OSCC tissues, whole blood and whole saliva of OSCC patients in vivo revealed that miR‑146a-5p expression was highly upregulated. Particularly, we widened the view of its upregulation in saliva, implicating that high miR-146a-5p expression is not only correlated closely to the development of human oral cancer, but also to a possible candidate as a diagnostic marker of OSCC. Indeed, further examination showed that exogenous miR-146a-5p expression showed pleiotropic effects on cell proliferation and apoptosis which were partially based on the contextual responses of activation of JNK, downstream of TRAF6 that was targeted by miR-146a-5p in normal human keratinocytes and OSCC cell lines. TRAF6 suppression by a TRAF6-specific siRNA resulted in contradictory consequences on cellular processes in normal and OSCC cells. Notably, TRAF6 downregulation by both miR-146a-5p and TRAF6-specific siRNA deactivated JNK in SCC-9, but not in normal human keratinocytes. In support of the proliferation-promoting effect of miR-146a-5p, silencing of endogenous miR-146a-5p significantly reduced proliferation of SCC-9. Together, these results suggest that miR-146a-5p affects proliferation and apoptosis in a cellular context-dependent manner and selectively disarms the TRAF6-mediated branch of the TGF-β signaling in OSCC cell lines by sparing Smad4 involvement.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Genetic Variation; Humans; Intracellular Signaling Peptides and Proteins; MicroRNAs; Mouth Neoplasms; RNA, Small Interfering; Smad4 Protein; TNF Receptor-Associated Factor 6; Transforming Growth Factor beta

To investigate the expression of Smad4, Smad7 and Caveolin-1 in the process of carcinogenesis of oral mucosa in Wistar rats, and to understand the changes of TGF-β/Smad signaling pathway and Caveolin-1 in oral cancer.. Palatal mucosal carcinogenesis specimens of Wistar rats were obtained from School of Stomatology, Zhengzhou University, which included 5 samples of normal mucosa, 10 samples of simple hyperplasia mucosa, 6 samples of mild mucosal dysplasia, 7 samples of moderate mucosal dysplasia, 13 samples of mucosa severe mucosal dysplasia, and 28 samples of oral cancer tissue. The expression of Smad4, Smad7 and Caveolin-1 was detected by immunohistochemistry. SPSS15.0 software package was used for statistical analysis.. The expression of Smad4 decreased in normal and hyperplastic epithelia, dysplasticepithelia and oral cancer gradually, the difference of the expression among the three groups was significant (P<0.05). The expression of Smad7 and Caveolin-1 increased in normal and hyperplastic epithelia, dysplasticepithelia and oral cancer gradually, respectively; the difference of the expression among the 3 groups was significant (P<0.05). Spearman correlation analysis showed that Smad4 was negatively correlated with Smad7, Smad4 was negatively correlated with caveolin-1, Smad7 was positively correlated with Caveolin-1 (P<0.05).. Synergistic effects may exist among Smad4, Smad7 and caveolin-1 in carcinogenesis of oral cancer.

Topics: Animals; Carcinogenesis; Caveolin 1; Mouth Mucosa; Mouth Neoplasms; Rats; Rats, Wistar; Signal Transduction; Smad4 Protein; Smad7 Protein; Transforming Growth Factor beta

Our aim is to evaluate the expression of SATB1 in human oral squamous cell carcinomas (OSCC) and its role in the invasiveness and metastasis of OSCC.. A human OSCC tissue microarray was used to evaluate the expression pattern of SATB1. SATB1 mRNA knockdown was performed in human OSCC cell lines SCC25 and Cal27 to assess the function of SATB1 in the invasiveness and metastasis of OSCC.. SATB1 is highly expressed in human OSCC determined by immunohistochemistry, and its nuclear/cytoplasmic ratio of histoscore is significantly correlated with patients' prognosis. Reduced cell motility, invasiveness, expression of epithelial to mesenchymal transition (EMT) markers (N-cadherin and β-catenin), and elevated expression of epithelial markers were observed in SATB1-knockdown cells in in vitro studies. Depletion of SATB1 also restored a cobblestone-like morphology in TGF-β1-treated cells.. These findings suggest SATB1 may play an important role in OSCC invasiveness and metastasis.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Humans; Lymphatic Metastasis; Matrix Attachment Region Binding Proteins; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Staging; Transforming Growth Factor beta

High recurrence and lower survival rates in patients with oral squamous cell carcinoma (OSCC) are associated with its bone invasion. We identified the oncogenic role of RUNX3 during bone invasion by OSCC. Tumor growth and the generation of osteolytic lesions were significantly inhibited in mice that were subcutaneously inoculated with RUNX3-knockdown human OSCC cells. RUNX3 knockdown enhanced TGF-β-induced growth arrest and inhibited OSCC cell migration and invasion in the absence or presence of transforming growth factor-β (TGF-β), a major growth factor abundant in the bone microenvironment. RUNX3 knockdown induced cell cycle arrest at the G1 and G2 phases and promoted G2 arrest by TGF-β in Ca9.22 OSCC cells. RUNX3 knockdown also inhibited both the basal and TGF-β-induced epithelial-to-mesenchymal transition by increasing E-cadherin expression and suppressing the nuclear translocation of β-catenin. In addition, the expression and TGF-β-mediated induction of parathyroid hormone-related protein (PTHrP), one of key osteolytic factors, was blocked in RUNX3-knockdown OSCC cells. Furthermore, treating human osteoblastic cells with conditioned medium derived from RUNX3-knockdown OSCC cells reduced the receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin ratio compared with treatment with conditioned medium from RUNX3-expressing cells. These findings indicate that RUNX3 expression in OSCC cells contributes to their bone invasion and the resulting osteolysis by inducing their malignant behaviors and production of osteolytic factors. RUNX3 alone or in combination with TGF-β and PTHrP may be a useful predictive biomarker and therapeutic target for bone invasion by oral cancer.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Core Binding Factor Alpha 3 Subunit; Epithelial-Mesenchymal Transition; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Kaplan-Meier Estimate; Male; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplasm Invasiveness; Osteoblasts; Osteolysis; Paracrine Communication; Parathyroid Hormone-Related Protein; RANK Ligand; RNA Interference; Signal Transduction; Skull; Squamous Cell Carcinoma of Head and Neck; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Microenvironment

Stromal cells in the tumor microenvironment (TME) closely interact with tumor cells and affect tumor cell behavior in diverse manners. We herein investigated the mechanisms by which cancer-associated fibroblasts (CAFs) affect the functional polarization of tumor-associated macrophages (TAMs) in oral squamous cell carcinoma (OSCC) in vitro and in human cancer samples. The expression of CD68, CD14, CD163, CD200R, CD206, HLA-G, CD80, and CD86 was higher in CD14-positive cells co-cultured with the culture supernatants of CAFs established from OSCC specimens (CAF-educated cells) than in control cells. The gene expression level of ARG1, IL10, and TGFB1 was increased in CAF-educated cells. CAF-educated cells suppressed T cell proliferation more strongly than control cells, and the neutralization of TGF-β IL-10, or arginase I significantly restored T cell proliferation. We then investigated the relationship between the infiltration of CAFs and TAMs using tissue samples obtained from patients with OSCC. The infiltration of CAFs was associated with the numbers of CD68-positive and CD163-positive macrophages. It also correlated with lymphatic invasion, vascular invasion, lymph node involvement, and the TNM stage. The infiltration of CAFs was identified as an independent prognostic factor in OSCC. Our results indicate that CAFs play important roles in shaping the tumor immunosuppressive microenvironment in OSCC by inducing the protumoral phenotype of TAMs. Therapeutic strategies to reverse CAF-mediated immunosuppression need to be considered.

Topics: Adult; Aged; Aged, 80 and over; Arginase; Biomarkers, Tumor; Cancer-Associated Fibroblasts; Carcinoma, Squamous Cell; Cell Proliferation; Culture Media, Conditioned; Female; Head and Neck Neoplasms; Humans; Immune Tolerance; Interleukin-10; Kaplan-Meier Estimate; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Macrophages; Male; Middle Aged; Mouth Neoplasms; Paracrine Communication; Phenotype; Proportional Hazards Models; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; T-Lymphocytes; Time Factors; Transforming Growth Factor beta; Tumor Microenvironment

Natural killer (NK) cells are important immune effector cells against tumors especially in the absence or reducing MHC class I antigen. Downregulation of CD16 receptor is accompanied by decreasing NK cell-killing activity. It has also been shown that some of tumor cells can evade from immune system through producing transforming growth factor beta (TGF-β) and affect prognosis. The objective of this study was to evaluate the prognostic significance of CD57(+) and CD16(+) cells and TGF-β expression in samples of oral squamous cell carcinoma (OSCC).. CD57, CD16, and TGF-β expressions were examined immunohistochemically in 57 cases of OSCC. The relationship between markers' expression and clinicopathologic data using bivariate and multivariate analysis was assessed.. Multivariate analysis revealed that CD57 expression [HR 17.34 (95% CI 3.815-78.830); P < 0.001] and mode of invasion [HR 0.362 (95% CI 0.138-0.947); P = 0.038] correlated with survival rate, but no relation between CD57 expression and mode of invasion was seen (P = 0.96). Furthermore, no correlation between CD57, CD16, and TGF-β expression was found.. These findings suggest that CD57 expression and mode of invasion are independent prognostic factors of survival in OSCC patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; CD57 Antigens; Down-Regulation; Female; GPI-Linked Proteins; Head and Neck Neoplasms; Humans; Immunohistochemistry; Iran; Killer Cells, Natural; Male; Middle Aged; Mouth Neoplasms; Prognosis; Receptors, IgG; Squamous Cell Carcinoma of Head and Neck; Survival Rate; Transforming Growth Factor beta

Oral submucous fibrosis (OSMF) is a pre-malignant condition that is strongly associated with the areca nut alkaloids, arecoline (ARC) and arecaidine (ARD). The condition is characterised by the presence of senescent fibroblasts in the subepithelial mesenchyme which have the potential to promote malignancy in the neighbouring epithelial cells. We tested the hypothesis that areca nut alkaloids induce senescence in oral fibroblasts and promote the secretion of invasion-promoting transforming growth factor β (TGF-β) and matrix metalloproteinase-2 (MMP-2).. Two oral fibroblast lines were treated for 48h with ARC and ARD. Senescence-associated β-galactosidase (SA-βGal) activity, Ki67 (cycling cells), large 53BP1 foci (irreparable DNA strand breaks) and p16(INK) (4A) (late senescence) were used as markers of cellular senescence and were quantified using indirect immunofluorescence and the ImageJ program. TGF-β and MMP-2 levels were measured using ELISA. Statistical analyses were performed with the two-tailed unpaired t-test where n = 3 and the Wilcoxon-Mann-Whitney test where n = 6.. ARC (100 and 300 μM) and ARD (30 and 100 μM) significantly (P < 0.05) induced fibroblast senescence, as determined by the increased expression of SA-βGal, 53BP1 staining and CDKN2A/p16(INK) (4A) ; there was also a non-significant reduction in Ki67 staining. Treated cells also showed a three- fivefold increase in TGF-β and a small non-significant increase in MMP-2.. Areca nut alkaloids induce senescence in oral fibroblasts and promote increased secretion of TGF-β and perhaps MMP-2 that may create a tissue environment thought to be critical in the progression of OSMF to malignancy.

Topics: Areca; Arecoline; beta-Galactosidase; Cell Cycle; Cell Line; Cellular Senescence; Disease Progression; DNA Damage; Fibroblasts; Humans; Matrix Metalloproteinase 2; Mouth Neoplasms; Oral Submucous Fibrosis; Transforming Growth Factor beta; Tumor Suppressor p53-Binding Protein 1

Oral squamous cell carcinoma (OSCC) has been reported as the most prevalent cancer of the head and neck region, while early diagnosis remains challenging. Here we took a comprehensive bioinformatics study on microarray data of 326 OSCC clinical samples with control of 165 normal tissues. The cell interaction pathways of ECM-receptor interaction and focal adhesion were found to be significantly regulated in OSCC samples. Further analysis of the topological properties and expression consistency identified that three hub genes in the gene interaction network, MMP9, PDIA3 and BGH3, were consistently up-expressed in OSCC samples. When being validated on additional microarray datasets of 41 OSCC samples, the validation rate of over-expressed BGH3, MMP9, and PDIA3 reached 90%, 90% and 84% respectively. At last, immuno-histochemical assays were done to test the protein expression of the three genes on newly collected clinical samples of 35 OSCC, 20 samples of pre-OSCC stage, and 12 normal oral mucosa specimens. Their protein expression levels were also found to progressively increase from normal mucosa to pre-OSCC stage and further to OSCC (ANOVA p = 0.000), suggesting their key roles in OSCC pathogenesis. Based on above solid validation, we propose BGH3, MMP9 and PDIA3 might be further explored as potential biomarkers to aid OSCC diagnosis.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Cluster Analysis; Databases, Genetic; Extracellular Matrix Proteins; Gene Expression Profiling; Humans; Immunohistochemistry; Matrix Metalloproteinase 9; Mouth Mucosa; Mouth Neoplasms; Protein Disulfide-Isomerases; Protein Interaction Maps; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Cancer stem cells (CSCs) drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC), we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44(high)EpCAM(low/-) CD24(+) cell surface marker profile. Treatment with TGFβ and retinoic acid (RA) enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER) stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; CD24 Antigen; Cell Line; Cell Line, Tumor; Drug Resistance, Neoplasm; Epithelial Cell Adhesion Molecule; Epithelial-Mesenchymal Transition; Female; Humans; Hyaluronan Receptors; Male; Mice; Mice, Inbred NOD; Mice, SCID; Mouth Neoplasms; Neoplastic Stem Cells; Phenotype; Thapsigargin; Transforming Growth Factor beta; Tretinoin

MicroRNAs (miRNAs) are involved in the tumourigenesis of various cancers by regulating their downstream targets. To identify the changes of miRNAs in oral squamous cell carcinoma (OSCC), we investigated the expression profiles of miRNAs in 40 pairs of OSCC specimens and their matched non-tumour epithelial tissues. Our data revealed higher miR-455-5p expression in the tumour tissues than in the normal tissues; the expression was also higher in oral cancer cell lines than in normal keratinocyte cell lines. MiR-455-5p knockdown reduced both the anchorage-independent growth and the proliferative ability of oral cancer cells, and these factors increased in miR-455-5p-overexpressing cells. Furthermore, by analysing the array data of patients with cancer and cell lines, we identified ubiquitin-conjugating enzyme E2B (UBE2B) as a target of miR-455-5p, and further validated this using 3'-untranslated region luciferase reporter assays and western blot analysis. We also demonstrated that UBE2B suppression rescued the impaired growth ability of miR-455-5p-knockdown cells. Furthermore, we observed that miR-455-5p expression was regulated, at least in part, by the transforming growth factor-β (TGF-β) pathway through the binding of SMAD3 to specific promoter regions. Notably, miR-455-5p expression was associated with the nodal status, stage, and overall survival in our patients, suggesting that miR-455-5p is a potential marker for predicting the prognosis of patients with oral cancer. In conclusion, we reveal that miR-455-5p expression is regulated by the TGF-β-dependent pathway, which subsequently leads to UBE2B down-regulation and contributes to oral cancer tumourigenesis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Animals; Carcinogenesis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Mouth Neoplasms; Neoplasm Staging; Prognosis; Signal Transduction; Smad Proteins; Survival Rate; Transforming Growth Factor beta; Ubiquitin-Conjugating Enzymes; Up-Regulation

Aberrant activation of oncogenic signaling pathways plays a pivotal role in tumor initiation and progression. The purpose of the present study was to investigate the chemopreventive and therapeutic efficacy of blueberry in the hamster buccal pouch (HBP) carcinogenesis model based on its ability to target TGF-β, PI3K/Akt, MAPK and NF-κB signaling and its impact on invasion and angiogenesis. Squamous cell carcinomas were induced in the HBP by 7,12-dimethylbenz[a]anthracene (DMBA). The effect of blueberry on the oncogenic signaling pathways and downstream events was analyzed by quantitative real-time PCR and immunoblotting. Experiments with the ECV304 cell line were performed to explore the mechanism by which blueberry regulates angiogenesis. Blueberry supplementation inhibited the development and progression of HBP carcinomas by abrogating TGF-β and PI3K/Akt pathways. Although blueberry failed to influence MAPK, it suppressed NF-κB activation by preventing nuclear translocation of NF-κB p65. Blueberry also modulated the expression of the oncomiR miR-21 and the tumor suppressor let-7. Collectively, these changes induced a shift to an anti-invasive and anti-angiogenic phenotype as evidenced by downregulating matrix metalloproteinases and vascular endothelial growth factor. Blueberry also inhibited angiogenesis in ECV304 cells by suppressing migration and tube formation. The results of the present study suggest that targeting oncogenic signaling pathways that influence acquisition of cancer hallmarks is an effective strategy for chemointervention. Identification of modulatory effects on phosphorylation, intracellular localization of oncogenic transcription factors and microRNAs unraveled by the present study as key mechanisms of action of blueberry is critical from a therapeutic perspective.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Active Transport, Cell Nucleus; Animals; Benz(a)Anthracenes; Blueberry Plants; Carcinogens; Cell Line, Transformed; Dietary Supplements; Freeze Drying; Fruit; Gene Expression Regulation, Neoplastic; Humans; Male; Mesocricetus; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms, Squamous Cell; Neovascularization, Pathologic; Random Allocation; Signal Transduction; Transcription Factor RelA; Transforming Growth Factor beta

Recombinant human bone morphogenetic protein2 (rhBMP2 ), a member of the TGF? family, has been used widely in recent years to regenerate defects of the maxillary and mandible bones. Such defects are sometimes caused by resection of oral squamous cell carcinoma (OSCC) yet the biologic effects of rhBMP2 on these carcinomas are not fully clear. The objective of this study was to determine histologically whether rhBMP2 produces adverse effects on angiogenesis during induction of OSCC, a biologic process critical for tumor formation in an experimental model in the buccal pouch of golden Syrian hamsters. Buccal cavities were exposed to painting with 0.5% DMBA in liquid paraffin three times a week for 14 weeks, then biopsies were taken. Division was into 2 groups: a study group of 10 hamsters receiving 0.25?g/ml of rhBMP2 in the 3rd and 6th weeks; and a control group of 10 hamsters which did not receive any additional treatment. VEGF expression and microvessel density were measured but no differences were noted between the two groups. According to this study, rhBMP2 does not stimulate angiogenesis during induction of OCSSs.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Bone Morphogenetic Protein 2; Carcinoma, Squamous Cell; Cheek; Cricetinae; Humans; Male; Mesocricetus; Microvessels; Morphogenesis; Mouth Neoplasms; Neovascularization, Pathologic; Recombinant Proteins; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Topics: Activating Transcription Factor 2; Areca; Cell Line, Transformed; Female; Fibrosis; Humans; Male; MAP Kinase Kinase 4; Mouth Mucosa; Mouth Neoplasms; Nuts; Oncogene Protein p65(gag-jun); Plant Extracts; Precancerous Conditions; Transforming Growth Factor beta

Oral Squamous Cell Carcinoma (OSCC) is the sixth most common cancer worldwide. OSCC invasion into the lymph nodes and mandible correlates with increased rates of recurrence and lower overall survival. Tumors that infiltrate mandibular bone proliferate rapidly and induce bone destruction. While survival rates have increased 12% over the last 20 years, this improvement is attributed to general advances in prevention, earlier detection, and updated treatments. Additionally, despite decades of research, the molecular mechanisms of OSCC invasion into the mandible are not well understood. Parathyroid Hormone-related Protein (PTHrP), has been shown to be essential for mandibular invasion in OSCC animal models, and our previous studies demonstrate that the transcription factor Gli2 increases PTHrP expression in tumor metastasis to bone. In OSCC, we investigated regulators of Gli2, including Hedgehog, TGFβ, and Wnt signaling to elucidate how PTHrP expression is controlled. Here we show that canonical Hedgehog and TGFβ signaling cooperate to increase PTHrP expression and mandibular invasion in a Gli2-dependent manner. Additionally, in an orthotopic model of mandibular invasion, inhibition of Gli2 using shRNA resulted in a significant decrease of both PTHrP expression and bony invasion. Collectively, our findings demonstrate that multiple signaling pathways converge on Gli2 to mediate PTHrP expression and bony invasion, highlighting Gli2 as a therapeutic target to prevent bony invasion in OSCC.

Topics: Animals; Bone and Bones; Carcinoma, Squamous Cell; Cell Line, Tumor; Disease Models, Animal; Gene Expression; Hedgehog Proteins; Heterografts; Humans; Mice; Mouth Neoplasms; Neoplasm Invasiveness; Nuclear Proteins; Parathyroid Hormone-Related Protein; Signal Transduction; Transforming Growth Factor beta; Zinc Finger Protein Gli2

To investigate the expression of nuclear factor κappa-B (NF-κB) and transforming growth factor β receptor II (TGF-βRII) and their clinical pathological significance in oral squamous cell carcinoma(OSCC).. The expression of NF-κB and TGF-βRII in 60 OSCC samples, 20 adjacent tissues, 29 metastatic lymph nodes and 10 non-metastatic lymph nodes were detected by immunohistological method. Statistical analysis was performed with SPSS 20.0 software package.. The expression of NF-κB in OSCC and metastatic lymph nodes was significantly higher than that in the adjacent tissues and non-metastatic lymph nodes, respectively (P<0.05). However, the expression of TGF-βRII in OSCC and metastatic lymph nodes was significantly lower than that in the adjacent tissues and non-metastatic lymph nodes, respectively (P<0.05). NF-κB was negatively correlated with TGF-βRII. The expression of NF-κB in patients with lower differentiation, lymphatic metastasis and higher grade were significantly higher than in patients with higher differentiation, lower grade and without lymphatic metastasis (P<0.05), respectively. NF-κB was a predicator of OSCC.. NF-κB is negatively correlated with TGF-βRII.NF-κB and TGF-βRII are associated with the progression of OSCC. NF-κB may promote angiogenesis through down-regulating the expression of TGF-βRII, which promotes progression of OSCC.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Down-Regulation; Humans; Lymph Nodes; Lymphatic Metastasis; Mouth Neoplasms; NF-kappa B; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) plays a significant role in the regulation of the tumor microenvironment. To explore the role of TGF-β in oral cancer-induced bone destruction, we investigated the immunohistochemical localization of TGF-β and phosphorylated Smad2 (p-Smad2) in 12 surgical specimens of oral squamous cell carcinoma (OSCC). These studies revealed TGF-β and p-Smad2 expression in cancer cells in all tested cases. Several fibroblasts located between cancer nests and resorbing bone expressed TGF-β in 10 out of 12 cases and p-Smad2 in 11 out of 12 cases. Some osteoclasts also exhibited p ∼ Smad2 expression. The OSCC cell line, HSC3, and the bone marrow-derived fibroblastic cell line, ST2, synthesized substantial levels of TGF-β. Culture media derived from HSC3 cells could stimulate Tgf-β1 mRNA expression in ST2 cells. Recombinant TGF-β1 could stimulate osteoclast formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264 cells. TGF-β1 could upregulate the expression of p-Smad2 in RAW264 cells, and this action was suppressed by the addition of a neutralizing antibody against TGF-β or by SB431542. Transplantation of HSC3 cells onto the calvarial region of athymic mice caused bone destruction, associated with the expression of TGF-β and p-Smad2 in both cancer cells and stromal cells. The bone destruction was substantially inhibited by the administration of SB431542. The present study demonstrated that TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC-induced bone destruction.

Topics: Animals; Bone Resorption; Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Humans; Mice; Mouth Neoplasms; Osteoclasts; RANK Ligand; Smad2 Protein; Stromal Cells; Transforming Growth Factor beta

Although tumour budding is an adverse prognostic factor for many cancer types, the molecular mechanisms governing this phenomenon are incompletely understood. Therefore, understanding the molecular basis of tumour budding may provide new therapeutic and diagnostic options. We employ digital image analysis to demonstrate that the number of tumour buds in cytokeratin-stained sections correlates with patients having lymph node metastases at diagnosis. The tumour bud count was also a predictor of overall survival, independent of TNM stage. Tumour buds and paired central tumour areas were subsequently collected from oral squamous cell carcinoma (OSCC) specimens, using laser capture microdissection, and examined with RNA sequencing and miRNA-qPCR arrays. Compared with cells from the central parts of the tumours, budding cells exhibited a particular gene expression signature, comprising factors involved in epithelial-mesenchymal transition (EMT) and activated TGFβ signalling. Transcription factors ZEB1 and PRRX1 were up-regulated concomitantly with the decreased expression of mesenchymal-epithelial (MET) transcription factors (eg OVOL1) in addition to Krüppel-like factors and Grainyhead-like factors. Moreover, miR-200 family members were down-regulated in budding tumour cells. We used immunohistochemistry to validate five markers of the EMT/MET process in 199 OSCC tumours, as well as in situ hybridization in 20 OSCC samples. Given the strong relationship between tumour budding and the development of lymph node metastases and an adverse prognosis, therapeutics based on inhibiting the activation of TGFβ signalling may prove useful in the treatment of OSCC.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; Disease-Free Survival; Drug Design; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Head and Neck Neoplasms; Humans; Immunohistochemistry; In Situ Hybridization; Kaplan-Meier Estimate; Laser Capture Microdissection; Lymphatic Metastasis; Male; MicroRNAs; Middle Aged; Molecular Targeted Therapy; Mouth Neoplasms; Oligonucleotide Array Sequence Analysis; Phenotype; Polymerase Chain Reaction; Predictive Value of Tests; Reproducibility of Results; Retrospective Studies; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Time Factors; Transforming Growth Factor beta

The VAV proteins VAV1, VAV2 and VAV3 have been identified as important molecules in tumorigenesis, tumor growth and cell migration. In addition to the full-length isoforms, a much shorter family member, VAV3.1, also known as VAV3 isoform 2, is known to be differentially expressed in a broad variety of tissues. Furthermore, VAV3.1 was shown to be down-regulated in cultured keratinocytes by the growth factors epidermal growth factor (EGF) EGF and transforming growth factor beta (TGFβ) TGFβ which in turn play important roles in the pathogenesis of oral squamous cell carcinoma (OSCC). Herein we showed that VAV3.1 is underexpressed in OSCC tissue samples compared to corresponding normal mucosa. We further demonstrated a trend of distinctive down-regulation of mRNA for VAV3.1 in tissues of locally advanced OSCC that have already metastasized to regional lymph nodes, indicating an increased malignant potential of tumors with low VAV3.1 mRNA expression. Moreover, in other studies a correlation between increased VAV3 expression and cancer progression was shown. In the present study, the analyzed OSCC tissue samples showed no significant change of VAV3 mRNA expression. Taken together, our data support the hypothesis that molecular interactions and signaling cascades of VAV3 can be regulated or directed by the competing molecule VAV3.1. Additionally, discrete and different functions of VAV3.1 in metastasis and tumorigenesis are conceivable.

Topics: Carcinogenesis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; Neoplasm Metastasis; Protein Isoforms; Proto-Oncogene Mas; Proto-Oncogene Proteins c-vav; Signal Transduction; Transforming Growth Factor beta

Natural killer (NK) cells are the key lymphocytes in solid tumors. Its activity is regulated by both germline encoded receptors and cytokine microenvironment. We conducted a case-control study to investigate the activation status of NK cell in oral squamous cell carcinoma (OSCC). NK cell activation was assessed in context of NK cell cytotoxicity and transcript expression of NK cell receptors (NKp46 and KIRs) and NK cell associated cytokines (IL-1β, IL-2, IL-10, IL-12β, IL-15, IL-18, IL-21, IFN-γ, TNF-α and TGF-β). The results revealed possible mechanisms involved in reduced NK cell activation in peripheral circulation: quantitative deficiency of NK cell number and lowered cytotoxicity together with qualitative NK impairments caused by--(1) decreased expression of NK activating receptor NKp46, (2) increased expression of NK suppressive cytokines--IL-10 and TGF-β and (3) induction of FOXP3(+)CTLA4(+) suppressor cells. On the other hand, in the tumor tissue, escape of NK immune surveillance appeared to be modulated by upregulation of TGF-β and IL-10 together with downregulation of NK cell activating cytokines (IL-2, IL-12β, IL-15, IL-18, IL-21 and IFN-γ) and NK receptors (NKp46 and KIRs). In addition, our study supported the earlier contention that TNF-α and IL-1β expression levels may be used as markers of malignant transformation in oral leukoplakia. In conclusion, the study provided an insight into the negative regulation of NK cell in tumor tissue and peripheral blood of OSCC patients, which can be exploited to boost the current NK cell and cytokine based immunotherapy for the treatment of oral cancer.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; CTLA-4 Antigen; Cytokines; Female; Forkhead Transcription Factors; Humans; K562 Cells; Killer Cells, Natural; Lymphocyte Activation; Male; Middle Aged; Mouth Neoplasms; Natural Cytotoxicity Triggering Receptor 1; Receptors, KIR; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Previous studies have demonstrated that senescent cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), unlike non-senescent CAFs from genetically stable carcinomas (GS-OSCC), promoted keratinocyte invasion in vitro in a paracrine manner. The mechanism by which this occurs is unclear.. Previous work to characterise the senescent-associated secretory phenotype (SASP) has used antibody arrays, technology that is limited by the availability of suitable antibodies. To extend this work in an unbiased manner, we used 2D gel electrophoresis and mass spectroscopy for protein identification. Matrix metalloproteinases (MMPs) were investigated by gelatin zymography and western blotting. Neutralising antibodies were used to block key molecules in the functional assays of keratinocyte adhesion and invasion.. Among a variety of proteins that were differentially expressed between CAFs from GU-OSCC and GS-OSCC, MMP-2 was a major constituent of senescent CAF-CM derived from GU-OSCC. The presence of active MMP-2 was confirmed by gelatine zymography. MMP-2 derived from senescent CAF-CM induced keratinocyte dis-cohesion and epithelial invasion into collagen gels in a TGF-β-dependent manner.. Senescent CAFs from GU-OSCC promote a more aggressive oral cancer phenotype by production of active MMP-2, disruption of epithelial adhesion and induction of keratinocyte invasion.

Topics: Carcinoma, Squamous Cell; Cell Adhesion; Cell Movement; Cells, Cultured; Cellular Senescence; Culture Media, Conditioned; Cyclin-Dependent Kinase Inhibitor p16; Electrophoresis, Gel, Two-Dimensional; Fibroblasts; Humans; Keratinocytes; Mass Spectrometry; Matrix Metalloproteinase 2; Mouth Neoplasms; Paracrine Communication; Phenotype; Proteins; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Regulatory T cells (Tregs), a subset of CD4+ T cells plays a pivotal role in regulating the immune system. An increase in Treg numbers enables cancer progression by dampening the immune system and allowing tumor cells to evade immune detection and destruction. An increase in Treg numbers and expression of inhibitory cytokines including TGF-β and IL-10 are mechanisms by which Tregs exert their immune suppressive function. However, the presence of Tregs and inhibitory cytokines in oral cancer patients is still unclear. In this study, the presence of circulating Tregs in 39 oral cancer patients and 24 healthy donors was examined by studying the presence of the CD4+CD25hiCD127low cell population in their peripheral blood mononuclear cells using flow cytometry. Serum levels of TGF-β and IL-10 were measured by ELISA. T cell subsets of OSCC patients were found to differ significantly from healthy donors where a decrease in CD8+ cytotoxic T cells and an increase in Tregs (CD4+CD25hiCD127low) were observed. Further, the ratio of CD8+ T cells/Tregs was also decreased in patients compared to healthy donors. The presence of Tregs was accompanied by a decrease in IL-10 but not TGF-β secretion in OSCC patients when compared to donors; in addition, the analysis also revealed that an increased presence of Tregs was accompanied by better patient survival. Amongst OSCC patients, smokers had significantly higher levels of TGF-β. It is apparent that the immune system is compromised in OSCC patients and the characterization of the Treg subpopulation could form a basis for improving our understanding of the perturbations in the immune system that occur during OSCC tumorigenesis.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Interleukin-7 Receptor alpha Subunit; Leukocytes, Mononuclear; Male; Middle Aged; Mouth Neoplasms; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The epithelial-to-mesenchymal transition (EMT) process results in a loss of cell-cell adhesion, increased cell mobility, and is crucial for enabling the metastasis of cancer cells. Recently, the enzyme SIRT1 has been implicated in a variety of physiological processes; however, its role in regulating oral cancer metastasis and EMT is not fully elucidated. Here, we propose a mechanism by which the enzyme sirtuin1 (SIRT1) regulates the EMT process in oral cancer by deacetylating Smad4 and repressing the effect of TGF-β signaling on matrix metalloproteinase-7 (MMP7).. The roles of SIRT1 in tumor cell migration/invasion and metastasis to the lungs were investigated using the Boyden chamber assay and orthotopic injections, respectively. RNA interference was used to knockdown either SIRT1 or Smad4 expression in oral squamous cell carcinoma (OSCC) cell lines. Immunoblotting, zymographic assays, and co-immunoprecipitation were used to examine the effects of SIRT1 overexpression on MMP7 expression and activity, as well as on SIRT1/ Smad4 interaction.. We found that compared with normal human oral keratinocytes (HOKs), SIRT1 was underexpressed in OSCC cells, and also in oral cancer tissues obtained from 14 of 21 OSCC patients compared with expression in their matched normal tissues. Overexpression of SIRT1 inhibited migration of OSCC cells in vitro, as well as their metastasis to the lung in vivo. Furthermore, up-regulation of SIRT1 in metastatic OSCCs significantly inhibited the migration and invasion abilities of OSCC cells, while concomitantly increasing the expression of E-cadherin, and decreasing the expressions of mesenchymal markers. We also identified Smad4, a TGF-β-activated transcription factor, as a direct target protein for SIRT1. Overexpression of SIRT1 in OSCC cells led to decreased levels of acetylated Smad4, and inhibition of TGF-β-induced signaling. By associating and deacetylating Smad4, SIRT1 enzyme can influence MMP7 expression, MMP enzyme activity, and consequently, cell migration, invasion, and tumor metastasis in OSCCs.. These findings provide a valuable insight into the potential role of the SIRT1 enzyme in regulating cell migration and invasion in oral squamous cell carcinoma. Our findings suggest the SIRT1/Smad4/MMP7 pathway as a target for oral cancer driven by EMT.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Male; Matrix Metalloproteinase 7; Mice; Mice, SCID; Mouth Neoplasms; Neoplasm Metastasis; Sirtuin 1; Smad4 Protein; Transforming Growth Factor beta

The streptococcal antitumor agent OK-432 is commonly used as an immunopotentiator for immunotherapy in various types of malignant tumors including oral cancer. It has been demonstrated that OK-432 elicits an antitumor effect by stimulating immunocompetent cells, thereby inducing multiple cytokines including interferon (IFN)-γ, interleukin (IL)-2 and IL-12. Serum concentrations of IFN-γ in patients with oral cancer were examined 24 h after administration of OK-432. Serum concentrations of IFN-γ in patients with advanced cancer were significantly lower than those in patients with early cancer. These results suggested that some soluble factors produced by cancer cells may inhibit IFN-γ production with OK-432. Thus, in the present study, an in vitro simulation model was established for the immune status of patients with oral cancer by adding conditioned medium (CM) derived from oral cancer cell lines into a culture of peripheral blood mononuclear cells (PBMCs) derived from a healthy volunteer. We investigated whether soluble factors derived from oral cancer cells affected IFN-γ production from PBMCs following stimulation with OK-432. PBMCs stimulated with OK-432 produced a large amount of IFN-γ; however, both IFN-γ production and cytotoxic activity from PBMCs induced by OK-432 were inhibited by the addition of CM in a dose-dependent manner. In order to examine these inhibitory effects against IFN-γ production, the contribution of inhibitory cytokines such as IL-4, IL-6, IL-10, transforming growth factor-β and vascular endothelial growth factor was investigated. However, neutralization of these inhibitory cytokines did not recover IFN-γ production inhibited by CM. These results indicated that unknown molecules may inhibit IFN-γ production from PBMCs following stimulation with OK-432.

Topics: Antineoplastic Agents; Biological Factors; Cell Line, Tumor; Culture Media, Conditioned; Humans; Interferon-gamma; Interleukins; Leukocytes, Mononuclear; Mouth Neoplasms; Picibanil; RNA, Messenger; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Expression of mucosa-associated lymphoid tissue 1 (MALT1) is inactivated in oral carcinoma patients with worse prognosis. However, the role in carcinoma progression is unknown. Unveiling genes under the control of MALT1 is necessary to understand the pathology of carcinomas.. Gene data set differentially transcribed in MALT1-stably expressing and -marginally expressing oral carcinoma cells was profiled by the microarray analysis and subjected to the pathway analysis. Migratory abilities of cells in response to MALT1 were determined by wound-healing assay and time-lapse analysis.. Totally, 2933 genes upregulated or downregulated in MALT1-expressing cells were identified. The subsequent pathway analysis implicated the inhibition of epidermal growth factor and transforming growth factor-β signalling gene expression, and highlighted the involvement in the cellular movement. Wound closure was suppressed by wild-type MALT1 (66.4%) and accelerated by dominant-negative MALT1 (218.6%), and the velocities of cell migration were increased 0.2-fold and 3.0-fold by wild-type and dominant-negative MALT1, respectively.. These observations demonstrate that MALT1 represses genes activating the aggressive phenotype of carcinoma cells, and suggest that MALT1 acts as a tumour suppressor and that the loss of expression stimulates oral carcinoma progression.

Topics: Caspases; Cell Line, Tumor; Cell Movement; Disease Progression; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lymphoid Tissue; Mouth Neoplasms; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; Neoplasm Proteins; NF-kappa B; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Podoplanin, a transmembrane sialomucin-like glycoprotein, is known to express at high frequency in oral squamous cell carcinomas (OSCC) and possess metastasis-promoting activity such as increased invasion and platelet-aggregating activity. However, the regulatory mechanism of podoplanin expression in OSCC remains unknown.. In the present study, we investigated the podoplanin expression in both clinical specimens from total 80 patients (50 OSCC and 30 pharyngeal SCC) and in 4 OSCC cell lines in vitro.. Immunohistochemical analysis of surgically resected specimens of OSCC revealed podoplanin expression in 70% of OSCC cases with localization primarily in the basal layer of squamous cancer nest and the expression was inversely correlated with squamous cell differentiation. In vitro analysis of OSCC cell lines revealed 36 that podoplanin expression was decreased in response to the squamous cell differentiation (Cytokeratin 10 expression as a marker) induced by treatment with histone deacetylase (HDAC) inhibitors such as sodium butyrate and trichostatin. Furthermore, transforming growth factor-β (TGF-β) significantly enhanced podoplanin expression in OSCC cell lines in line with increased phosphorylation of Smad2. A TGF-β type I receptor inhibitor (SB431542) significantly inhibited such induction of podoplanin expression by TGF-β at both the protein and mRNA level. However, in a subset of OSCC cell line, its expression was only weakly dependent on TGF-β and squamous differentiation.. These results suggest that regulation of podoplanin is not simple, but in the majority of OSCC cell lines, its expression is positively and negatively regulated by TGF-β receptor/Smad signaling pathway and epigenetic mechanism leading to squamous differentiation, respectively.

Topics: Adult; Aged; Aged, 80 and over; Animals; Benzamides; Biomarkers, Tumor; Butyrates; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Dioxoles; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Keratin-10; Lymphatic Metastasis; Male; Membrane Glycoproteins; Mice; Mice, Nude; Middle Aged; Mouth Neoplasms; Neoplasm Invasiveness; Pharyngeal Neoplasms; Signal Transduction; Smad2 Protein; Tongue Neoplasms; Transforming Growth Factor beta

To evaluate and characterize macrophage populations (M1/M2) in the tumor microenvironment of oral cavity squamous cell carcinoma (OCSCC). The relationship between macrophages and clinicopathological factors, such as survival data, lymph node metastasis, tumoral proliferation, and WHO histological grading are also analyzed.. The samples consisted of surgically excised specimens from patients with non-metastatic and metastatic OCSCC and normal oral mucosa (control). Immunohistochemistry, flow cytometry, and qRT-PCR were used to evaluate macrophage populations and the expression of pro- (IL-12, IL-23, and INF-γ) and anti-inflammatory (IL-10 and TGF-β) cytokines. The level required for statistical significance was defined as p<0.05.. The data showed a predominance of M2 phenotype (high percentage of IL-10(+)TGF-β(+)) macrophages in the tumor microenvironment of OCSCC. A higher percentage of macrophages expressing TGF-β was seen in the OCSCC group when compared with healthy individuals. The assessment of mRNA expression also presented a greater expression of anti-inflammatory cytokines TGFβ and IL10 in OCSCC when compared with the control group. The percentage of macrophages, demonstrated by immunohistochemistry, was significantly higher in the metastatic OCSCC group than in the non-metastatic and control groups. The log-rank test also showed that the mean survival time for patients with high levels of macrophages was less (44 months) when compared with patients with a low percentage of such cells (93 months).. A predominance of the M2 phenotype in the tumor microenvironment of OCSCC could contribute to local immunosuppression, via TGF-β production, and consequently greater lymph node involvement and reduced patient survival time.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; CD11 Antigens; Cell Count; Cell Proliferation; Cytokines; Female; Follow-Up Studies; Humans; Immune Tolerance; Inflammation Mediators; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-23; Lymphatic Metastasis; Macrophages; Male; Middle Aged; Mouth Neoplasms; Neoplasm Grading; Neoplasm Invasiveness; Retrospective Studies; Survival Rate; Transforming Growth Factor beta; Tumor Microenvironment

The epithelial-mesenchymal transition (EMT) is a crucial step in epithelial cancer invasion and metastasis. The aims of this study were to investigate and validate unidentified micro RNAs (miRNAs) that regulate EMT and to reveal their clinical relevance in epithelial cancer patients. By applying miRNA array screening in a natural epithelial-mesenchymal phenotype cell line pair and in a transforming growth factor β-induced EMT cell model, we found miR-153 was markedly downregulated in the cells that underwent an EMT. A close association was confirmed between inhibition of miR-153 and the EMT phenotype, as well as the invasive ability of epithelial cancer cells. Ectopic expression of miR-153 in mesenchymal-like cells resulted in an epithelial morphology change with decreased cellular invasive ability. On the contrary, transfection of a miR-153 inhibitor in epithelial-like cells led to a mesenchymal phenotype change. In vivo ectopic expression of miR-153 significantly inhibited tumor cell metastasis formation. Data from the dual-luciferase reporter gene assay showed, for the first time, that SNAI1 and ZEB2 were direct targets of miR-153. Inverse correlations were also observed between miR-153 and SNA1 and ZEB2 levels in oral cancer patients' samples. Furthermore, low expression level of miR-153 was found to be significantly related to metastasis and poor prognosis in oral cancer patients. These data demonstrate that miR-153 is a novel regulator of EMT by targeting SNAI1 and ZEB2 and indicate its potential therapeutic value for reducing cancer metastasis.

Topics: 3' Untranslated Regions; Animals; Cell Line, Tumor; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Homeodomain Proteins; Humans; Kaplan-Meier Estimate; Luciferases, Firefly; Lung Neoplasms; Mice; Mice, Nude; MicroRNAs; Mouth Neoplasms; Neoplasm Transplantation; Neoplasms, Glandular and Epithelial; Repressor Proteins; RNA Interference; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Burden; Zinc Finger E-box Binding Homeobox 2

To establish the relevance of the bone morphogenetic protein (BMP) signaling pathway in human oral squamous cell carcinoma (OSCCA) cell lines and determine if there is a biologic impact of stimulating this pathway with recombinant human (rh) BMP-2.. In vitro laboratory investigations and in vivo analysis using an orthotopic animal model for oral cancer.. Gene expression profiles for BMP-2 and components of the BMP-signaling pathway were determined using reverse transcriptase-polymerase chain reaction. In vivo effects were evaluated using Kaplan-Meier survival analysis and studying histopathologic changes in established tumor xenografts with or without rhBMP-2 pretreatment. A phosphokinase array was used to detect levels of activation in signaling kinases.. The BMP-2 gene was expressed in 90% of the 30 OSCCA cell lines tested. Gene expression of all components of the BMP-signaling pathway was highly conserved. Tumor xenografts established with rhBMP-2-treated cells showed more rapid local growth that resulted in worse animal survival as compared to the control group. These tumors had a more poorly differentiated morphology. Changes in protein kinases suggested interactions of BMP-2 signaling with the Wnt-β-catenin, and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways.. Human OSCCA cell lines frequently express BMP-2 and all necessary components of the BMP-signaling pathway. Exogenous treatment of human OSCCA cell lines with rhBMP-2 prior to engraftment in an orthotopic animal model caused the subsequent tumors to be more locally aggressive with worse survival. Continued caution should be used for considering rhBMP-2 for reconstruction of bone defects in oral cancer patients.

Topics: Animals; Bone Morphogenetic Protein 2; Carcinoma, Squamous Cell; Humans; Mice; Mouth Neoplasms; Neoplasm Transplantation; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

MicroRNAs are short non-coding RNAs that regulate gene expression by RNA interference. Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. miR-146a has been reported to regulate Toll-like receptors and cytokine signaling, which are both crucial for inflammation and oncogenesis. This study identifies that areca nut extract, TNFα and TGFβ up-regulates miR-146a in OSCC cells. The increased expression of miR-146a enhanced the oncogenicity of OSCC cells. In addition, a G to C polymorphism (rs2910164), which is located in the pre-miR-146a and has been associated with functional alterations in miR-146a, was significantly more prevalent among OSCC patients having more advanced nodal involvement. Our analysis also suggested a higher miR-146a expression in OSCC tissues of patients carrying C polymorphism. The present study concluded a higher prevalence of the pre-mir-146a C-variant was associated with OSCC progression in patients with this disease.

Topics: Areca; Carcinoma, Squamous Cell; Case-Control Studies; Humans; Keratinocytes; MicroRNAs; Mouth Neoplasms; NF-kappa B; Nuts; Plant Preparations; Polymorphism, Single Nucleotide; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

A tight seal between the epithelium and the dental implant surface is required to prevent bacterial inflammation and soft tissue recession and therefore to demonstrate a long-term success. Surface hydrophilicity was recently shown to promote osseointegration. The aim of this study was to investigate the influence of surface hydrophilicity in combination with surface topography of Ti implant surfaces on the behavior and activation/differentiation of epithelial cells using a set of in vitro experiments mimicking the implant-soft tissue contact.. Hydrophobic acid-etched (A) and coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and modSLA surfaces were produced. The behavior of an oral squamous cell carcinoma cell line (HSC-2) grown on all surfaces was compared through determination of cell attachment and proliferation/viability (CCK-8 and MTT assay), time-lapse microscopy of fluorescence labeled cells and determination of gene expression by real time polymerase chain reaction.. Within the surfaces with similar wettability cell spreading and cell movements observed by time-lapse microscopy after one day of incubation were most pronounced on smoother (A and modA) surfaces compared to rougher (SLA and modSLA) surfaces. Within the surfaces with similar roughness the hydrophilic surfaces (modA and modSLA) showed more cell spreading and cell activity compared to the hydrophobic surfaces (A and SLA). The relative gene expressions of cytokeratin14, integrin α6, integrin β4, vinculin, transforming growth factor (TGF)-β, TGF-β1, and TGF-β3 were decreased in HSC-2 on all four types of Ti surfaces compared to control surfaces (tissue culture polystyrene; p<0.01) and there was no significant difference of gene expression on the four different implant-surfaces.. We have demonstrated that for proliferation and spreading of HSC-2 cells the smoother and hydrophilic surface is optimal (modA). These results suggest that surface hydrophilicity might positively influence the epithelial seal around dental implants. All tested titanium surfaces downregulate cell attachment, cell proliferation, expression of adhesion promoters, and cytokines involved in wound healing in HSC-2 cells compared to control surfaces.

Topics: Acid Etching, Dental; Carcinoma, Squamous Cell; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Coloring Agents; Dental Etching; Dental Implants; Dental Materials; Epithelial Cells; Gene Expression Regulation; Humans; Hydrophobic and Hydrophilic Interactions; Integrin alpha6; Integrin beta4; Keratin-14; Membrane Proteins; Mouth Mucosa; Mouth Neoplasms; Surface Properties; Tetrazolium Salts; Thiazoles; Titanium; Transforming Growth Factor beta; Vinculin; Wound Healing

This study was performed to determine whether T(H)17 cells are involved in the development and metastasis of head and neck squamous cell carcinomas (HNSCCs).. T(H)17 cells frequencies in 67 HNSCC patients and 21 healthy volunteers were examined by flow cytometric analysis. T(H)17 cell-related cytokines in serum (interleukin (IL) 17, transforming growth factor (TGF) β, and IL-6) were evaluated by using enzyme-linked immunosorbent assay.. It was discovered that the higher frequency of T(H)17 cells was in HNSCCs patients (1.0 ± 0.4%). The cell proportions and related cytokine concentrations were consistent with the tumor TNM stage. The IL-6 concentration showed positive correlation with the frequency of T(H)17 cells (r = 0.661) and IL-17 levels (r = 0.597). The TGF-β concentration showed a positive correlation with IL-17 (r = 0.626) but no relationship with T(H)17 cells (r = 0.431).. The present data suggested that T(H)17 cells may be involved in tumor growth and metastasis of HNSCCs. IL-6 may play an important role in T(H)17 cell differentiation and functions, and TGF-β may be related to IL-17 secretion but not to the differentiation of T(H)17 cells.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Female; Flow Cytometry; Humans; Interleukin-17; Interleukin-6; Lymphatic Metastasis; Lymphocyte Count; Male; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Neoplasm Staging; Th17 Cells; Tongue Neoplasms; Transforming Growth Factor beta

T helper 17 (Th17) and regulatory T cells share plasticity in the expression of interleukin (IL)-17 and forkhead box P3 (FOXP3), but their mutual presence in human diseases is unclear.. IL-17 and FOXP3 were analyzed by immunohistostaining and flow cytometry. The cytokine milieu was analyzed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).. Oral squamous cell carcinoma expresses high levels of IL-1β, IL-6, and transforming growth factor (TGF)-β. A unique subset of FOXP3(+) IL-17-producing CD4(+) T cells was consistently identified in tumor-infiltrating lymphocytes from advanced stages of cancer, but not in the circulation, at a frequency of 0.5% to 5.5 % of total CD4(+) T and positively correlated with the frequency of IL-17(+)FOXP3(-) T cells. The IL-17(+)FOXP3(+) T cells express CCR6 and suppress the proliferation of autologous CD4(+) CD25(-) responder T-cells in vitro.. The prevalence of IL-17-producing FOXP3(+) CD4(+) tumor infiltrating lymphocytes is increased in oral squamous cell carcinoma.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Case-Control Studies; CD4-Positive T-Lymphocytes; Female; Flow Cytometry; Forkhead Transcription Factors; Humans; Immunohistochemistry; Interleukin-17; Interleukin-1beta; Interleukin-6; Lymphocytes, Tumor-Infiltrating; Male; Middle Aged; Mouth Neoplasms; Prevalence; Receptors, CCR6; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Premalignant oral lesions have a high incidence of recurrence and progression to malignant disease and, although studies have shown the contribution of transforming growth factor β (TGF-β) to cancer progression, none have been conducted with premalignant oral lesion cells to determine the impact of TGF-β in stimulating properties that are characteristic of more invasive cells. The present study focused on TGF-β-modulation of paxillin and the serine/threonine protein phosphatase PP-1, and the impact on cellular motility. These studies show that TGF-β stimulates premalignant lesion cell motility and up regulates expression of paxillin, as well as its co-localization with PP-1, while concurrently diminishing the level of paxillin serine phosphorylation. The TGF-β-mediated up regulation of paxillin and co-localization with actin, as well as the TGF-β-stimulated motility of premalignant lesion cells, were all blocked by inhibiting PP-1, indicating their dependence on PP-1 activity. These studies suggest interplay between TGF-β and PP-1 in promoting a more malignant phenotype in premalignant oral lesion cells.

Topics: Actins; Animals; Cell Movement; Focal Adhesions; Humans; Mice; Mice, Inbred C57BL; Mouth Neoplasms; Paxillin; Phosphorylation; Protein Phosphatase 1; Protein Transport; Transforming Growth Factor beta; Up-Regulation

To determine if recombinant human bone morphogenetic protein-2 (rhBMP-2) has biological effects on the invasiveness of human oral squamous cell carcinoma (OSCCA) cell lines.. Laboratory investigation using six human OSCCA cell lines, with three cell lines having baseline gene expression of BMP-2 and three cell lines without baseline gene expression of BMP-2.. The invasiveness of each cell line was measured using a matrigel invasion assay with or without stimulation by rhBMP-2. A tumor metastasis quantitative PCR array was used to establish whether observed findings from the invasion assay correlated to changes in gene expression.. There was a significant increase in tumor cell invasion in response to rhBMP-2 in all BMP-2 positive cell lines but no change in the cell lines that did not express the BMP-2 gene. Quantitative PCR revealed that changes in gene expression were distinctly different based on the baseline gene expression of BMP-2 and favored a more metastatic genotype in the BMP-2-positive cells.. Recombinant human BMP-2 has an adverse biological effect on invasiveness of human OSCCA cell lines in vitro. This adverse effect is dependent on the baseline gene expression of BMP-2. Changes in expression of genes involved with tumor metastasis correlated to the invasion assay findings. These data raise concern for the safe application of rhBMP-2 for reconstruction of bone defects in oral cancer patients.

Topics: Bone Morphogenetic Protein 2; Carcinoma, Squamous Cell; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; Neoplasm Invasiveness; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Transforming Growth Factor beta

Human beta-defensin-2 (hBD-2) is a small cationic peptide originally identified from psoriatic skin lesions as an antimicrobial agent of the innate immune system. The expression of hBD-2 is believed to be induced exclusively in epithelial cells by microbial components and certain proinflammatory cytokines, such as interleukin-1 beta (IL-1 beta). In this study, we report, for the first time, that hBD-2 is expressed in vascular endothelial cells associated with oral squamous cell carcinoma (OSCC) and Kaposi's sarcoma lesions, but not in that of normal stroma. Expression of hBD-2 in vascular endothelial cells was further substantiated by in vitro experiments using cultured human umbilical vein endothelial cells (HUVECs). Transforming growth factor beta1 (TGF beta 1) and IL-1 beta, two well-known tumorigenic inflammatory mediators, induce hBD-2 transcript and peptide expression in HUVECs. However, TGF beta 1 does not stimulate hBD-2 expression in oral epithelial cells. In addition, proinflammatory cytokines and microbial reagents do not induce the expression of hBD-1 and hBD-3 in HUVECs. Since hBD-2 has been shown to modulate migration, proliferation, and tube formation of HUVECs in vitro and participate in immune cell trafficking, its expression in vascular endothelial cells located within malignant lesions may play a role in tumor angiogenesis and cancer metastasis.

Topics: beta-Defensins; Carcinoma, Squamous Cell; Cytoplasm; Endothelial Cells; Endothelium, Vascular; Gene Expression; Humans; Interleukin-1beta; Mouth Neoplasms; Neoplasms; Sarcoma, Kaposi; Transforming Growth Factor beta

Experimental data and limited patient experience suggest that rhBMP-2 can be used to regenerate bone in acquired segmental defects of the mandible. Most of these defects are caused by resection of oral squamous cell carcinoma (OSCC) and the biologic effects of rhBMP-2 on these carcinoma cells are unknown. The objective of this study was to determine whether rhBMP-2 produces adverse effects on proliferation and angiogenesis in OSCC, two biologic processes critical to tumor formation. In vitro studies included treating OSCC cells with rhBMP-2 or an adenoviral vector containing the cDNA for BMP-2. In vivo studies involved co-transplantation of OSCC cells with bone marrow stromal cells genetically modified to over express BMP-2, to mimic a clinically relevant scenario for regenerating bone using cell-based therapy in a wound containing microscopic residual disease. Proliferation, as measured by a MTT assay in vitro and tumor growth in vivo was not affected by treatment with BMP-2. Angiogenesis, measured by secretion of the proangiogenic molecules VEGF and IL-8 in vitro and microvessel density in vivo, was not affected. Exposure of OSCC cells to BMP-2 does not stimulate proliferation or angiogenesis. Further studies are needed before using rhBMP-2 for bone tissue engineering in oral cancer-related defects.

Topics: Adenoviridae; Animals; Bone Marrow Transplantation; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Regeneration; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; DNA, Complementary; Female; Genetic Vectors; Humans; Interleukin-8; Mice; Mice, Mutant Strains; Mice, Nude; Microvessels; Mouth Neoplasms; Neoplasm Transplantation; Neovascularization, Pathologic; Recombinant Proteins; Stromal Cells; Transforming Growth Factor beta; Transplantation, Heterologous; Vascular Endothelial Growth Factor A; von Willebrand Factor

Bone invasion is a critical prognostic factor for patients with oral squamous cell carcinoma (OSCC). We established an orthotropic implantation model using the murine OSCC cell line, SCCVII, showing direct invasion of the mandible by OSCC. Using this model, we examined the molecular mechanisms of bone invasion and the role of parathyroid-related protein (PTHrP). We established PTHrP, stable, knock-down SCCVII cells. Knock-down of PTHrP caused decreased osteoclast formation in vitro relative to expression levels of PTHrP. In vivo models showed dramatic suppression of bone invasion in PTHrP knock-down cells, and the degree of suppression was more pronounced than the level of PTHrP knock-down. We looked at an additive role of transforming growth factor-beta (TGF-beta) in PTHrP-mediated bone invasion. TGF-beta induced mRNA expression of PTHrP, showed no inhibitory effect on SCCVII cell proliferation, and caused epithelial mesenchymal trans-differentiation such as changes in the cells. Sections of resected mandibles from patients with invasive OSCC showed a great number of osteoclasts at bone invasion sites, strong expression of PTHrP, and decreased expression of E-cadherin in the tumour cells. Cancer-derived PTHrP appears to play a critical role in bone invasion by OSCC, mediated by osteoclasts. Moreover, TGF-beta appears to act synergistically to accelerate mandibular bone invasion.

Topics: Animals; Bone Neoplasms; Carcinoma, Squamous Cell; Disease Models, Animal; Gene Knockdown Techniques; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C3H; Mouth Neoplasms; Neoplasm Metastasis; Osteoclasts; Parathyroid Hormone-Related Protein; Reverse Transcriptase Polymerase Chain Reaction; Tomography, X-Ray Computed; Transforming Growth Factor beta

Runt-related transcription factor 3 (RUNX3) is a tumor suppressor of cancer and appears to be an important component of the transforming growth factor-beta (TGF-ss)-induced tumor suppression pathway. Surprisingly, we found that RUNX3 expression level in head and neck squamous cell carcinoma (HNSCC) tissues, which is one of the most common types of human cancer, was higher than that in normal tissues by a previously published microarray dataset in our preliminary study. Therefore, here we examined the oncogenic role of RUNX3 in HNSCC.. Frequent RUNX3 expression and its correlation with malignant behavior were observed in HNSCC. Ectopic RUNX3 overexpression promoted cell growth and inhibited serum starvation-induced apoptosis and chemotherapeutic drug induced apoptosis in HNSCC cells. These findings were confirmed by RUNX3 knockdown. Moreover, RUNX3 overexpression enhanced tumorsphere formation. RUNX3 expression level was well correlated with the methylation status in HNSCC cells. Moreover, RUNX3 expression was low due to the methylation of its promoter in normal oral epithelial cells.. Our findings suggest that i) RUNX3 has an oncogenic role in HNSCC, ii) RUNX3 expression observed in HNSCC may be caused in part by demethylation during cancer development, and iii) RUNX3 expression can be a useful marker for predicting malignant behavior and the effect of chemotherapeutic drugs in HNSCC.

Topics: Apoptosis; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Core Binding Factor Alpha 3 Subunit; DNA Methylation; Epithelial Cells; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Neoplasms; RNA, Small Interfering; Transforming Growth Factor beta

Matrix metalloproteinases (MMPs) and transforming growth factor-beta1 (TGF-beta1) are important in many physiological and pathological processes.. Immunohistochemistry for MMP-2, MMP-9, membrane-type 1 MMP (MT1-MMP, MMP-14), tissue inhibitor of matrix metalloproteinase (TIMP)-2, and TGF-beta were performed on normal mucosa, nonatrophic oral lichen planus, atrophic oral lichen planus, and oral squamous cell carcinomas (OSCC) resulting from lichen planus.. Expression of MMPs progressively increased from normal mucosa to nonatrophic oral lichen planus, atrophic oral lichen planus, and OSCCs. Immunoscores of MMPs in atrophic oral lichen planus was significantly greater than nonatrophic oral lichen planus. Moreover, immunoscore of MMP-9 of OSCCs was significantly greater than both atrophic and nonatrophic lichen planus. Furthermore, expression of TIMP-2 and TGF-beta1 paralleled increases seen with MMPs.. Imbalance between MMPs and TIMPs may be involved in cancerization of oral lichen planus. MMP-2, MT1-MMP, and especially MMP-9 may be useful markers for judging potency of malignant transformation from oral lichen planus.

Topics: Adult; Age Factors; Biomarkers, Tumor; Biopsy, Needle; Case-Control Studies; Cell Transformation, Neoplastic; Cytokines; Disease Progression; Female; Humans; Immunohistochemistry; Lichen Planus, Oral; Male; Metalloproteases; Middle Aged; Mouth Neoplasms; Precancerous Conditions; Probability; Prognosis; Reference Values; Retrospective Studies; Risk Factors; Sensitivity and Specificity; Severity of Illness Index; Sex Factors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Epithelial-mesenchymal transition (EMT) has been considered essential for metastasis, a multistep process including local invasion, intravasation, extravasation, and proliferation at distant sites. However, controversy remains as to whether EMT truly happens and how important it is to metastasis. We studied the involvement of EMT in individual steps of metastasis and found that p12(CDK2-AP1), a down-stream effector of transforming growth factor beta, induced EMT of hamster cheek pouch carcinoma-1 cells by promoting the expression of Twist2. EMT cells have an increased invasive but decreased metastatic phenotype. When s.c. inoculated, both EMT and non-EMT cells established primary tumors, but only EMT cells invaded into the adjacent tissues and blood vessels; however, neither cells formed lung metastases. When i.v. inoculated, only non-EMT cells established lung metastases. Moreover, s.c. inoculation of a mixture of the two cell types resulted in intravasation of both cell types and formation of lung metastasis from non-EMT cells. Our results allowed us to propose a novel model for the role of EMT in cancer metastasis. We showed that EMT and non-EMT cells cooperate to complete the spontaneous metastasis process. We thus hypothesize that EMT cells are responsible for degrading the surrounding matrix to lead the way of invasion and intravasation. Non-EMT cells then enter the blood stream and reestablish colonies in the secondary sites.

Topics: Animals; Cadherins; Cricetinae; Epithelial Cells; Humans; Keratinocytes; Lung Neoplasms; Mesoderm; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Protein Kinases; Transfection; Transforming Growth Factor beta; Tumor Suppressor Proteins

Several lines of evidence demonstrated that the stroma surrounding the tumors plays an important role in the growth and progression of several neoplasms, including oral squamous cell carcinomas (OSCC). We evaluated the presence of myofibroblasts in OSCC and determined whether their presence is associated with clinicopathological features of the tumors. We also investigated the mutual paracrine effects of tumor cells and myofibroblasts on fibroblast-myofibroblast transdifferentiation and tumor cell proliferation. Immunohistochemical analysis showed the approximately 60% of the OSCCs contained myofibroblasts in the stroma of the tumor. Abundant presence of myofibroblasts significantly correlated with N stage, disease stage, regional recurrence, and proliferative potential of the tumor cells. Using OSCC cell lines and primary oral normal fibroblasts (ONF), we demonstrated that tumor cells induced transdifferentiation of ONFs to myofibroblasts via secretion of transforming growth factor-beta 1 (TGF-beta 1). In turn, myofibroblasts secreted factors that stimulated OSCC cell proliferation, as revealed by measuring BrdU incorporation and Ki67 expression. The results of the study suggest that during tumor invasion OSCC-derived TGF-beta 1 promote fibroblast-myofibroblast transdifferentiation, and that tumor cellular proliferation can be induced by factors released from myofibroblasts, which may favor tumor growth.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cell Proliferation; Cell Transdifferentiation; Cell Transformation, Neoplastic; Female; Fibroblasts; Gene Expression Profiling; Humans; Immunohistochemistry; Male; Middle Aged; Mouth Neoplasms; Paracrine Communication; Phenotype; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured

We describe the intramuscular transformation of a hydroxyapatite/osteogenic protein-1 (HA/OP-1) composite implant, into a vascularised pedicled bone flap useful for reconstruction of a hemi-mandible. Extraskeletal induction of a bone flap for transplantation was achieved without the addition of harvested bone, bone marrow, or stem cells. Five months after apparent clinical success, an MRSA infection of the graft led to its failure. The background to ectopically induced bone flaps is introduced, with our experience in a human case presented. The results from this emerging biotechnology are discussed in the light of limited human clinical experience.

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Substitutes; Bone Transplantation; Carcinoma, Squamous Cell; Durapatite; Graft Survival; Humans; Male; Mandible; Methicillin Resistance; Middle Aged; Mouth Neoplasms; Osteogenesis; Pectoralis Muscles; Plastic Surgery Procedures; Staphylococcal Infections; Surgical Flaps; Surgical Wound Infection; Transforming Growth Factor beta

In an orthotopic murine model of head and neck cancer, combined subcutaneous and intratumoral vaccination with recombinant vaccinia virus expressing interleukin-2 (rvv-IL-2) induced significant tumor regression early on therapy. However, its efficacy was restricted by recurrent tumor growth and loco-regional metastases. In this study, we explored the mechanism of tumor metastasis. We compared the levels of expression of a number of molecules involved in tumor metastasis, which included transforming growth factor-beta1 (TGF-beta1), E-cadherin, matrix metalloproteinases (MMPs): MT1-MMP, MMP-2, MMP-9, their tissue inhibitors (TIMPs): TIMP-1/TIMP-2, and pro-angiogenic factors CD31, VEGF-R2, and iNOS between primary and metastatic tumors by real-time RT-PCR and immunohistochemistry. We detected spontaneous lymph node and tongue metastasis. Metastasis was delayed in rvv-IL-2 treated mice. Cultured tumor cells expressed negligible amount of TGF-beta1. Untreated or metastatic tumors, on the other hand, expressed high levels of TGF-beta1 and secreted TGF-beta1 in the sera of tumor-bearing mice. Levels of TGF-beta1 in the sera suddenly jumped at the time when tumor metastasis started. In the metastatic tumors, levels of MT1-MMP, MMP-2, and MMP-9 were significantly elevated (P < 0.001), while levels of TIMP-1/TIMP-2 and E-cadherin were decreased (P < 0.001) compared to control or primary tumors. Levels of CD31, VEGF-R2, and iNOS were also significantly elevated in the metastatic lesions (P < 0.001). The concurrence of high levels of TGF-beta1 in the sera, expression of proteins involved in metastasis and initiation of metastasis suggested possible role of TGF-beta1 in on setting the metastatic cascade in this model.

Topics: Animals; Cadherins; Cell Line, Tumor; Female; Lymph Nodes; Matrix Metalloproteinases; Mice; Mice, Inbred C3H; Mouth Neoplasms; Neoplasm Metastasis; Nitric Oxide Synthase Type II; Platelet Endothelial Cell Adhesion Molecule-1; Reverse Transcriptase Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinases; Tongue Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vaccination; Vaccinia virus; Vascular Endothelial Growth Factor Receptor-2; Viral Vaccines

Although carcinogenesis of oral squamous cell carcinoma (OSCC) has been studied by many investigators in the past decade, the available evidence about its molecular mechanism is inconclusive. The objective of the present study was to compare expression of Smad4, a signaling molecule of the transforming growth factor beta (TGF-beta) pathway, between OSCC and normal oral mucosa. We assayed expression of Smad4 in OSCC and normal oral mucosa by performing immunohistochemistry using paraffin-embedded tissue samples. We also compared expression of Smad4 protein between OSCC lines and normal oral keratinocytes, using Western blot analysis. Smad4 expression was observed in only 60% of OSCC tissue samples, whereas it was observed in 82% of normal oral mucosa samples. Reduced Smad4 expression was clearly observed in all OSCC lines, compared with normal oral keratinocytes. These findings suggest that aberration of the TGF-beta pathway, as indicated by a reduction or absence of Smad4 expression, promotes carcinogenesis of OSCC.

Topics: Biomarkers, Tumor; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Humans; Immunohistochemistry; Mouth Mucosa; Mouth Neoplasms; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

We examined correlations between TGF-beta1, TbetaR-I and TbetaR-II, p12(CDK2-AP1), p21(WAF1), p27(KIP1), Smad2, and p-Smad2 in 125 cases of human oral squamous cell carcinoma (OSCC) to test the hypothesis that resistance to TGF-beta1-induced growth suppression is due to the disruption of its signaling pathway as a consequence of reduced or lost p12(CDK2-AP1). Immunoreactivity for TbetaR-II decreased in OSCC with increasing disease aggressiveness; however, no differences were observed for TbetaR-I and TGF-beta1. The expression of TbetaR-II significantly correlated with p12(CDK2-AP1) and p27(KIP1) (P < .001 and P < .01, respectively). Furthermore, there was a significant relationship between TbetaR-II expression and p-Smad2 (P < .001). The in vivo correlation of the levels of TbetaR-II, p12(CDK2-AP1), and p27(KIP1) was confirmed in normal and OSCC cell lines. Additionally, in vitro analysis of TGF-beta1-treated cells showed that TGF-beta1 treatment of normal keratinocytes suppressed cell growth with upregulation of p-Smad2, p12(CDK2-AP1), and p21(WAF1) expression, whereas there was no effect on OSCC cell lines. These results provide evidence of a link between a disrupted TGF-beta-Smad signaling pathway and loss of induction of cell cycle-inhibitory proteins, especially p12(CDK2-AP1) in OSCC, which may lead to the resistance of TGF-beta1 growth-inhibitory effect on OSCC.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cell Line, Transformed; Cell Line, Tumor; Cyclin-Dependent Kinase 2; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Mouth Neoplasms; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Tumor Suppressor Proteins

We have shown previously that transforming growth factor-beta (TGF-beta) is a potent tumour suppressor in Smad4-deficient human malignant oral keratinocytes but the mechanism by which this occurs is unknown. In the present study, we show that over-expression of TGF-beta1 causes regression of tumours derived from Smad4-deficient oral keratinocytes transplanted orthotopically to athymic mice. Further, tumour regression is associated with the induction of apoptosis without changes in cell proliferation. In vitro, TGF-beta1 did not induce apoptosis directly in these cells but sensitized cells to cisplatin, but not Fas, -induced cell death. The data suggest that TGF-beta1 induces tumour regression in vivo by Smad4-independent pathways that sensitize keratinocytes to mitochondrial-mediated apoptosis.

Topics: Animals; Apoptosis; Cisplatin; DNA-Binding Proteins; Drug Synergism; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplasm Proteins; Neoplasm Regression, Spontaneous; Neoplasm Transplantation; Recombinant Proteins; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Heterologous; Tumor Cells, Cultured

Angiogenesis and tumor-associated immunosuppression are two of the hallmarks of carcinogenesis. In previous studies we demonstrated in vitro that HNSCC tumor cells attract monocytes via monocyte chemotactic protein-1 (MCP-1) and activate them via transforming growth factor-beta 1(TGF-beta1) to secrete interleukin (IL)-1alpha, which in turn stimulates tumor cells to secrete increased levels of the angiogenic and immunosuppressive vascular endothelial growth factor (VEGF). These findings suggest that interaction between the immune system and VEGF-mediated angiogenesis is important for progression of HNSCC. Recent studies in vitro show that retinoic acid (RA) downregulates the release of MCP-1 and TGF-beta1 by tumor cells. Therefore, we investigated the ability of RA to modulate the ability of tumor cells to recruit and activate monocytes for participation in VEGF-mediated angiogenesis and immunosuppression in vivo.. Mice (ten/group) were injected daily with RA (160 microg/kg) for 3 weeks. After that time mice were sacrificed, and paraffin sections of tumors were obtained and stained for VEGF-A, CD68, and PECAM (CD31) by immunohistochemistry. The lungs, liver, and myocardium were analyzed for macro- and micrometastases. The plasma protein levels of VEGF-A and MCP-1 were determined by ELISA.. In RA-treated mice tumors regressed completely and RA prevented metastases (p=0.00) and macrophage infiltration (p=0.007). Treated mice downregulated VEGF-A (0 pg/ml) and MCP-1 (12 pg/ml) in peripheral blood (p=0.001).. Our findings suggest a new therapeutic possibility: the development of treatment protocols that can block each of the ways in which tumors induce new blood vessel growth and immunosuppression of the host.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Chemokine CCL2; Disease Progression; Down-Regulation; Humans; Immune Tolerance; Interleukin-1; Macrophage Activation; Male; Mice; Mice, Inbred A; Mouth Neoplasms; Neoplasm Metastasis; Neoplasm Transplantation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Vascular Endothelial Growth Factor A

The development of an altered stromal microenvironment is a common feature of many tumours including squamous cell carcinoma (SCC), and there is increasing evidence that these changes in the stroma, which include increased expression of proteases and cytokines, may actually promote tumour progression. A common finding is that stromal fibroblasts become 'activated' myofibroblasts, expressing smooth muscle actin and secreting cytokines, proteases and matrix proteins. We show that myofibroblasts are commonly found in the stroma of oral SCC and are often concentrated at the invasive margin of the tumour. Using oral SCC cells and primary oral fibroblasts, we demonstrate that tumour cells directly induce a myofibroblastic phenotype, and that this transdifferentiation is dependent on SCC-derived TGF-beta1. In turn, myofibroblasts secrete significantly higher levels of hepatocyte growth factor/scatter factor compared with fibroblast controls, and this cytokine promotes SCC invasion through Matrigel, a mixture of basement membrane proteins. This is the first time that this double paracrine mechanism has been demonstrated between squamous carcinoma cells and fibroblasts, and emphasises that cancer invasion can be promoted indirectly by the release of tumour-induced host factors from stroma.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Transformation, Neoplastic; Fibroblasts; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Mouth Neoplasms; Muscle, Smooth; Neoplasm Invasiveness; Phenotype; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured

To study the differential expression of transforming growth factor beta 1 (TGF beta 1) in oral carcinoma and stroma lymphocytes by induction chemotherapy and inquire into the mechanism of TGF beta 1 information transmission.. Forty cases of oral tumor were treated with MTX, CDDP and PYM via subcutaneous implantable drug pump, in situ hybridization method was adopted to detect the expression of TGF beta 1 mRNA.. The positive expression of TGF beta 1 mRNA was enhanced in oral carcinoma (P < 0.05). After the induction chemotherapy via subcutaneous implantable drug pump, not only the expression level of TGF beta 1 in malignant cells of invading front zone was up-regulated (P < 0.05), but the expression level of stroma lymphocytes was higher than before.. These data demonstrated that TGF beta 1 not only has transforming potential, but also enhances the malignant progression of oral carcinoma. It was clear that TGF beta 1 can act as a tumor suppressor and a significant stimulator of T-cell-mediated tumor cytotoxity as well.

Topics: Antineoplastic Combined Chemotherapy Protocols; Bleomycin; Carboplatin; Carcinoma, Squamous Cell; Female; Humans; Infusion Pumps, Implantable; Male; Methotrexate; Middle Aged; Mouth Neoplasms; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

The integrin beta6 has been shown to promote invasion and experimental metastasis by oral squamous cell carcinoma (SCC). In this study, we demonstrate that the expression of beta6 by oral SCC9 cells increased activation of the UPA --> MMP3 --> MMP9 pathway. We also demonstrate that the deposition of fibronectin and tenascin-C matrices by SCC9beta6 cells and peritumor fibroblast cocultures is counter-regulated by the UPA --> MMP3 --> MMP9 pathway. Suppression of individual components of this pathway increased the deposition of fibronectin, but decreased tenascin-C matrix assembly by the cocultures. When the SCC9beta6/PTF cocultures were incubated with TGFbeta1, the deposition of fibronectin and tenascin-C as well as the activation of MMP3 and MMP9 was increased. These results indicate that MMP3, MMP9, and TGFbeta1 are important for the modulation, composition, and maintenance of the ECM in oral SCC.

Topics: Animals; Blotting, Western; Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Coculture Techniques; Culture Media, Conditioned; Enzyme Activation; Extracellular Matrix; Fibroblasts; Fibronectins; Humans; Integrin beta Chains; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Mouth Neoplasms; Tenascin; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.

Topics: Biotinylation; Carcinoma, Squamous Cell; Catalysis; Cell Line, Tumor; Collagen; Down-Regulation; Enzyme Activation; Humans; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Mouth Neoplasms; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Promoter Regions, Genetic; Time Factors; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study examined the behaviour of nine human malignant oral keratinocyte cell lines following orthotopic transplantation to the floor of the mouth of athymic mice. Tumourigenesis, local spread, and metastatic dissemination were correlated with known cellular responses to transforming growth factor-beta 1 (TGF-beta 1). Six of nine cell lines were tumourigenic; four of these cell lines showed local spread which was characterized by vascular and bone invasion. Metastatic spread was uncommon, with only 9% of animals with primary tumours developing metastases and these were almost exclusively found in the regional lymph nodes; there was one pulmonary metastasis and no liver deposits. Tumour cell behaviour did not reflect the clinical stage of the original tumours. Cell lines that were resistant to TGF-beta 1-induced growth inhibition were more likely to form primary tumours, exhibit local spread, and metastasize than cells that were growth-inhibited by the ligand. The data demonstrate that tumourigenicity and tumour behaviour in this orthotopic mouse model varied between cell lines and that the pattern of local invasion and metastasis was similar to that seen in human oral cancer. Furthermore, cell lines that were refractory to the growth inhibitory effects of TGF-beta 1 behaved more aggressively than cells that underwent ligand-induced cell-cycle arrest.

Topics: Adult; Aged; Aged, 80 and over; Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Female; Humans; Keratinocytes; Lymphatic Metastasis; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Mouth Neoplasms; Neoplasm Transplantation; Phenotype; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transplantation, Heterologous

Transforming growth factor beta1 (TGFbeta1) is a negative growth regulator in keratinocytes, and in vitro studies lead to the concept that loss of TGFbeta1 responsiveness is a critical step in epithelial carcinogenesis.. To investigate the prognostic relevance of TGFbeta1 expression in head and neck squamous cell carcinoma (HNSCC).. TGFbeta1 distribution was determined by immunohistochemistry in oral cavity/oropharynx (n = 79), larynx (n = 36) and hypopharynx (n = 25) tumors and in matched normal adjacent mucosa. TGFbeta-type I and II receptors were determined in 20 cases of differentiated oral cavity/hypopharynx tumors. Cases were considered positive if displaying reactivity in >10% of the cells.. TGFbeta1-positive expression was found in 47.2% of larynx, 36.7% of oral cavity/oropharynx and in 24% of the hypopharynx tumors. Reactivity in >60% of the cells was displayed only by 11.4% of HNSCC. All normal controls were positive. TGFbeta1-positive expression did not correlate with clinico pathological parameters. An association with differentiation was verified only in oral cavity/oropharynx tumors (P

Topics: Activin Receptors, Type I; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Hypopharyngeal Neoplasms; Immunohistochemistry; Laryngeal Neoplasms; Male; Middle Aged; Mouth Neoplasms; Oropharyngeal Neoplasms; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Survival Rate; Transforming Growth Factor beta; Transforming Growth Factor beta1

Cytokines produced by tumor cells and tumor-infiltrating lymphocytes (TIL) appear to regulate tumor cell growth and the cytotoxic activity of TIL. The objectives of the present study were to investigate cytokine generation patterns in tumor cells and TIL and to examine the influence of cancer therapy on this cytokine production and the cytotoxic activity of TIL.. We determined the levels of cytokines produced by tumor cells and TIL in vitro and measured the cytotoxic activity of TIL against Daudi cells in patients with oral squamous cell carcinoma (OSC) before and 1 week after the start of concomitant chemo-radio-immunotherapy.. Before the therapy, OSC cells generated higher levels of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) than did oral keratinocytes isolated from the noninflamed gingivae of healthy individuals, but both kinds of cells generated similar levels of interleukin (IL)-1beta and IL-6. Compared with peripheral blood mononuclear cells (PBMC) of the patients, TIL produced higher levels of IL-1beta, IL-6, IL-10, TNF-alpha and TGF-beta, whereas their production of IL-12 and interferon-gamma (IFN-gamma) was only slightly higher than that in PBMC. After 1 week of therapy, the cytokine production by OSC cells had largely decreased, while the production of TNF-alpha, IFN-gamma, TGF-beta and IL-12 by TIL had increased greatly, although other cytokine levels were almost constant during the investigations. The cytotoxic activity of TIL was higher than that of PBMC before the therapy, and this activity was strongly increased by 1 week of therapy.. These results suggest that the cytokine productivities of TIL and tumor cells differ from those of PBMC and normal keratinocytes, respectively, and that chemo-radio-immunotherapy modulates in situ cytokine generation, which is advantageous for inhibition of tumor cell growth and activation of TIL.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Chemotherapy, Adjuvant; Cytokines; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-1; Interleukin-10; Interleukin-12; Interleukin-6; Killer Cells, Lymphokine-Activated; Lymphocytes; Male; Middle Aged; Mouth Neoplasms; Radiotherapy, Adjuvant; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The p300 and closely related cAMP response element binding protein (CREB)-binding protein (CBP) acetyltransferases function as global transcriptional coactivators and play important roles in a broad spectrum of biological processes, including cell proliferation and differentiation. A role of p300/CBP in tumor suppression has been proposed from the fact that these coactivators are targeted by viral oncoproteins and that biallelic mutations of p300 have been identified in carcinomas. Here, we show that transcriptional response to the transforming growth factor beta (TGF-beta), an inhibitor of epithelial cell growth, was severely impaired in human carcinoma cell lines carrying p300 mutations accompanied by inactivation of the second allele, and that wild-type expression restored TGF-beta-dependent transcriptional activity. Furthermore, reintroduction of wild-type p300 suppressed the growth of p300-deficient carcinoma cells, whereas p300 did not inhibit the growth of carcinoma cells examined, which have no detectable alterations in p300 protein and retain the TGF-beta-dependent transcriptional response. In addition, tumor-derived mutants missing the bromodomain or glutamine-rich region, which are respectively important for chromatin interaction and coactivator activities, lost the suppressive activity. In contrast, CBP exhibited no or reduced ability to suppress the growth of p300-deficient carcinoma cells. These results provide experimental evidence to show that p300 acts as a suppressor of tumor cell growth and suggest a distinct role of p300 in suppression of epithelial tumors.

Topics: Animals; Blotting, Western; Bromodeoxyuridine; Carcinoma; Cell Division; COS Cells; Epithelial Cells; HeLa Cells; Humans; Luciferases; Models, Genetic; Mouth Neoplasms; Mutation; Nuclear Proteins; Plasmids; Trans-Activators; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

This study examined the role of TGF-beta1 in human keratinocyte malignancy. Two carcinoma-derived human oral keratinocyte cell lines, BICR 31 and H314, were selected on the basis of their known resistance to TGF-beta1-induced G(1) arrest, the presence of wild type TGF-beta cell surface receptors and normal Ras. Smad 4 protein was undetectable in both cell lines, but Smad 2 and Smad 3 were expressed at levels comparable with a fully TGF-beta responsive cell line, and treatment of the cells with TGF-beta1 resulted in the phosphorylation of Smad 2. Treatment with exogenous TGF-beta1 resulted in a failure to induce transcription from an artificial Smad-dependent promoter and a failure to down-regulate c-myc, but resulted in an up-regulation of AP-1 associated genes (Fra-1, JunB and fibronectin). Transient transfection of Smad 4 into BICR 31 restored TGF-beta1-induced growth inhibition and Smad-dependent transcriptional activation. Protracted treatment of cells with exogenous TGF-beta1 resulted in the attenuation of cell growth in vitro. To over-express TGF-beta1, both cell lines were transfected with latent TGF-beta1 cDNA; neutralization studies of conditioned media demonstrated that whilst the majority of the peptide was in the latent form, a small proportion was present as the active peptide. Cells that over-expressed endogenous TGF-beta1 grew more slowly in vitro compared to both the vector-only controls and cells that did not over-express the peptide. Orthotopic transplantation of cells that over-expressed endogenous TGF-beta1 to the floor of the mouth in athymic mice resulted in marked inhibition of primary tumor formation compared to controls. Expression of a dominant-negative TGF-beta type II receptor in cells that over-expressed endogenous TGF-beta1 resulted in enhanced cell growth in vitro and diminished the tumor suppressor effect of the ligand in vivo, indicating that the endogenous TGF-beta1 was acting in an autocrine capacity. The results demonstrate that over-expression of endogenous TGF-beta1 in human malignant oral keratinocytes leads to growth inhibition in vivo and tumor suppression in vitro by mechanisms that are independent of Smad 4 expression and TGF-beta1-induced G(1) arrest.

Topics: Animals; Carcinoma; Cell Division; DNA-Binding Proteins; G1 Phase; Humans; Keratinocytes; Kinetics; Ligands; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Transplantation; RNA, Neoplasm; Skin Neoplasms; Smad4 Protein; Trans-Activators; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tumor Suppressor Proteins

Humoral hypercalcemia of malignancy (HHM), a paraneoplastic syndrome associated with epithelial cancers, including squamous cell carcinoma (SCC), is due to expression and secretion of parathyroid hormone-related protein (PTHrP). Transforming growth factor-beta1 (TGFbeta1), expressed by many tumors, has been demonstrated in vitro to increase the half-life of PTHrP mRNA. In this study, oral squamous carcinoma cells (SCC2/88) had a two-fold increase in PTHrP mRNA stability (from 45 to 90 min) in response to treatment with TGFbeta1. In order to examine the mechanism of TGFbeta1-mediated PTHrP mRNA stability, a cell-free assay of mRNA degradation was utilized in which the degradation of in vitro-transcribed mRNA incubated with cytoplasmic protein extracts from SCC2/88 treated with vehicle or TGFbeta1 was measured. In this assay, full-length PTHrP mRNA was not significantly stabilized in TGFbeta1-treated samples when compared to vehicle treated samples. However, there was a striking (>5-fold) increase in PTHrP mRNA half-life in TGFbeta1-treated samples when PTHrP mRNA lacked the 3'-untranslated region (3'-UTR). In contrast, the degradation of 3'-UTR-truncated PTHrP mRNA using the cell-free assay was not altered in vehicle-treated samples. UV cross-linking of PTHrP mRNA and cytoplasmic proteins from cells treated with either vehicle or TGFbeta1 revealed numerous mRNA-binding proteins. TGFbeta1 treatment resulting in decreased binding of 33, 31, 27, 20 and 18 kDa binding proteins to the terminal coding region. These studies revealed that TGFbeta1-induced PTHrP mRNA stability might be, in part, the result of cis-acting sequences within the coding region of the PTHrP mRNA.

Topics: 3' Untranslated Regions; Animals; Carcinoma, Squamous Cell; Cell-Free System; Cross-Linking Reagents; Dichlororibofuranosylbenzimidazole; DNA Primers; Dogs; Enzyme Inhibitors; Gene Expression; Humans; Mouth Neoplasms; Mutagenesis, Site-Directed; Parathyroid Hormone-Related Protein; Polymerase Chain Reaction; Protein Biosynthesis; Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Ultraviolet Rays

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

Topics: Active Transport, Cell Nucleus; Carcinoma; Cell Cycle Proteins; Cell Division; Cell Line; Cell Movement; Cell Nucleus; Cells, Cultured; Disease Progression; DNA-Binding Proteins; Humans; Keratinocytes; Kinetics; Male; Middle Aged; Mouth Neoplasms; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

The objective of this study was to determine the expression of transforming growth factor beta (TGF-beta) subtypes and their relationship with the mechanisms of squamous cell carcinoma (OSCC) growth.. Totally 40 cases of surgical specimens of OSCC resected between 1998 and 2000 and 20 cases of normal human oral mucosa were investigated. Strepto-adridinibiotin complex (SABC) immunohistochemical staining was used to analyze the expression of TGF-beta protein subtypes and their relations with clinical prognosis of OSCC.. Semi-quantitative analysis revealed that the subtypes 1, 2 and 3 of TGF-beta protein could be found in OSCC cells and normal oral epithelial cells, however the intensity of protein expression was different. Comparing with those in normal oral mucosa epithelial cells, the subtypes 1 and 2 of TGF-beta were over-expressed in OSCC cells. The over-expression of subtypes 1 and 2 of TGF-beta protein were associated with their pathological grades, clinical stages and neck lymph node metastasis (P < 0.05), whilst the subtype 3 protein of TGF-beta was not.. It will be useful to detect the expression of TGF-beta subtypes in OSCC, as the subtypes 1 and 2 of TGF-beta may play an important role in OSCC growth and metastasis.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Female; Humans; Lymphatic Metastasis; Male; Middle Aged; Mouth Neoplasms; Prognosis; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

Our study examined the expression of AP-1 family members in keratinocytes derived from the rat-4NQO model of oral carcinogenesis in which extremes of epithelial differentiation and tumour cell aggressiveness are evident. The constitutive expression of JunB was diminished in the undifferentiated, more aggressive tumour phenotype compared with the well-differentiated, less aggressive keratinocytes, whereas the expression of other AP-1 family members (c-jun, junD, c-fos, fra1, fra2 and fosB) was either very weak or variable. After transfection of the undifferentiated keratinocytes with junB cDNA, clonal populations were isolated that expressed similar levels of JunB protein as the well-differentiated cells. Both untransfected and transfected cell lines were keratin negative and vimentin positive. Increased expression of JunB in the transfected cells resulted in up-regulation of c-Jun and Fra1 and an enhanced AP-1 activity as demonstrated by transcriptional activation of the prototypic AP-1 dependent promoter, MMP-1. JunB transfected cells grew more quickly than vector-only controls and were refractory to the growth inhibitory effects of TGF-beta1. Over-expression of JunB resulted in the elevated expression of the AP-1 dependent proteinase, MMP-9, whereas the expression of the AP-1 independent enzyme, MMP-2, was unaffected. JunB transfected keratinocytes were highly invasive in an in vitro assay of tumour cell invasion compared with vector controls. The results indicate that increased expression of JunB above baseline levels in undifferentiated rat keratinocytes does not alter epithelial differentiation but enhances the malignant phenotype in vitro, possibly by altering the dynamics of the AP-1 complex.

Topics: 3T3 Cells; 4-Nitroquinoline-1-oxide; Animals; Bacterial Proteins; Blotting, Northern; Blotting, Western; Carcinogens; Cell Differentiation; Cell Division; Cell Movement; Cells, Cultured; DNA-Binding Proteins; DNA, Complementary; Fos-Related Antigen-2; Genes, Reporter; Genetic Vectors; Keratinocytes; Keratins; Luciferases; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mouth Neoplasms; Neoplasms, Experimental; Phenotype; Plasmids; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Time Factors; Transcription Factor AP-1; Transcription Factors; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Up-Regulation; Vimentin

This study examined the immunocytochemical expression of the transforming growth factor-beta (TGF-beta) isoforms TGF-beta1, TGF-beta2, and TGF-beta3, together with the TGF-beta cell surface receptors TbetaR-I and TbetaR-II, in patient-matched tissue pairs of normal human oral epithelium, primary squamous cell carcinomas, and metastatic lymph node tumour deposits. There were no significant differences in the intensity of TGF-beta isoform specific staining between the normal oral epithelium, the primary tumours, and the lymph node metastases. By contrast, there was significantly less TbetaR-II in the metastases than in the primary tumour and between the primary tumour and the normal oral epithelium. Similar trends were evident with TbetaR-I, but not at a statistically significant level. This study also examined the structure of TbetaR-I and TbetaR-II in normal human oral keratinocytes in vitro and in 14 human oral carcinoma cell lines with known responses to TGF-beta1. No structural abnormalities of TbetaR-II were present in the normal keratinocytes or in 13 of 14 malignant cell lines; in one line, there were both normal and mutant forms of TbetaR-II, the latter being in the form of a frameshift mutation with the insertion of a single adenine base (bases 709-718, codons 125-128), predicting a truncated receptor having no kinase domain. No defects were present in TbetaR-I. The structures of TbetaR-I and TbetaR-II did not correlate with growth inhibition by TGF-beta1. The data suggest that decreased expression of TGF-beta receptors, rather than structural defects of these genes, may be important in oral epithelial tumour progression. In order to examine the functional significance of a specific decrease in TbetaR-II expression, a dominant-negative TbetaR-II construct (dnTbetaR-II) was transfected into a human oral carcinoma cell line with a normal TGF-beta receptor profile and known to be markedly inhibited by TGF-beta1. In those clones that overexpressed the dnTbetaR-II, growth inhibition and Smad binding activity were decreased, whilst the regulation of Fra-1 and collagenase-1 remained unchanged following treatment with TGF-beta1. The results demonstrate that a decrease in TbetaR-II relative to TbetaR-I leads to selective gene regulation with loss of growth inhibition but continued transcription of AP-1-dependent genes that are involved in the regulation of the extracellular matrix.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Disease Progression; Female; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Male; Middle Aged; Mouth Neoplasms; Mutation; Neoplasm Proteins; Protein Isoforms; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

The aim of the present study was to determine the relationships between bone morphogenetic proteins (BMPs), BMP receptor type IA and carcinogenesis of oral epithelium. A retrospective study was performed on material obtained from oral mucosa, including nine cases of normal mucosa (NB), eight cases of nonspecific chronic inflammation (NCI), seven cases of hyperkeratosis (HK), five cases of squamous cell papilloma (SCP), 29 cases of squamous cell carcinoma (SCC) with various grades of differentiation and 10 cases of epithelium adjacent to carcinoma (EAC). Six cases of NB from hard palate (NHP) were chosen as a control group. The benign groups consisted of NCI, HK and SCP. The antibodies against BMP-2/4, -5, receptor BMPR-IA and purified bovine BMP (bBMP-McAb) were utilised using an immunocytochemical method. The results demonstrated that the immunostaining of BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb was weak and not consistent in normal and benign groups. The immunoreactivity level was independent of the clinical and pathological grading of SCC. All cases of SCC showed positive staining for BMP-2/4, BMP-5, BMPR-IA and bBMP-McAb except for three cases and one case of SCC which negatively stained for BMP-2/4 and BMP-5, respectively. The staining intensity and proportion of the positively stained cells were markedly increased in SCC when compared with that of the normal and benign groups except for EAC. The metastatic carcinoma cells in lymph nodes were strongly and positively stained for BMP-2/4 and BMP-5 when compared with the primary lesions. Our results indicate that there was an overexpression of BMP-2/4, BMP-5, bBMP-McAb and BMPR-IA in the high-risk premalignant and malignant lesions of oral epithelium. Our findings suggest that BMP-2/4 and BMP-5 but not BMPR-IA might be involved in the metastasis of oral carcinoma cells.

Topics: Analysis of Variance; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 5; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Carcinoma, Squamous Cell; Case-Control Studies; Cell Membrane; Cytoplasm; Epithelium; Humans; Immunohistochemistry; Leukoplakia, Oral; Lymphatic Metastasis; Mouth Mucosa; Mouth Neoplasms; Neoplasm Proteins; Palate; Papilloma; Precancerous Conditions; Protein Serine-Threonine Kinases; Receptors, Growth Factor; Retrospective Studies; Statistics, Nonparametric; Transforming Growth Factor beta

The symposium on Virus Infections and Tumors of the Oral Mucosae was organized as a joint meeting of the Arbeitskreis Oralpathologie and Oralmedizin and the Arbeitsgemeinschaft Dermatologische Infektiologie (ADI) der Deutschen Dermatologischen Gesellschaft. The main topics of the meeting were herpes virus infections, human papillomavirus (HPV) infections and human immunodeficiency virus (HIV) infections of the oral mucosae. Clinically both diagnostic, differential diagnostic and therapeutic aspects of the virus-associated diseases were discussed in several presentations. Another important issue was the role of these viruses, particularly of HPVs, in the origin and development of oral cancer. Apparently besides smoking and alcohol other risk factors comprise high risk HPVs, immunodeficiency and possibly also genetic factors. Whether neonatal early infections may predispose children to a specific cancer risk in their future life is still at a level of discussion. Some arguments, however were shown that tonsillar carcinoma, which shows the highest prevalence of the high-risk HPV 16- DNA sequences between all oral cancer, is possibly an epidemiologically and etiologically distinct tumor. It is argued that this tumor is probably less dependent on classical carcinogens than other oral malignant tumors.

Topics: DNA, Viral; Genes, p53; Herpesviridae Infections; HIV Infections; Humans; Leukoplakia, Oral; Lichen Planus, Oral; Mouth Neoplasms; Papilloma; Papillomaviridae; Papillomavirus Infections; Transforming Growth Factor beta; Tumor Virus Infections

This study examined the effect of stable transfection of latent transforming growth factor-beta1 (TGF-beta1) cDNA into a predominantly polygonal, 4 nitroquinoline N-oxide (4NQO)-induced rat oral keratinocyte cell line. Seven polygonal and five spindle clonal populations were isolated that overexpressed TGF-beta1 protein by approximately two- to four-fold compared to vector-only transfected controls. Neutralisation experiments indicated that the majority of protein was in the latent form. There was no change in the proportion of polygonal and spindle cells in vitro after transfection with TGF-beta1 cDNA. Polygonal and spindle cells that overexpressed TGF-beta1 produced similar amounts of protein and grew more slowly in vitro than controls. The parent cell line and all transfected cells were growth inhibited (60-75%) by exogenous TGF-beta1. Orthotopic transplantation of the parent and the vector-only control cell lines resulted in primary tumours in the floor of the mouth in almost 100% (20/21) of athymic mice, with no evidence of bone resorption at the site of the primary tumour and pulmonary metastatic tumour deposits in some 40% (7/20) of these animals. The polygonal and spindle cells that overexpressed TGF-beta1 behaved similarly following orthotopic transplantation. A 96% (23/24) primary tumour take was evident following transplantation of cells that overexpressed TGF-beta1, with a significantly (P<0.02) higher number of animals showing bone resorption at the site of the primary tumour (35%; 8/23) compared to controls. By contrast, there was a significant (P<0.03) decrease in the number of animals with pulmonary metastases (4%; 1/23) following transplantation of TGF-beta1 overexpressing cells compared to controls. Overexpression of TGF-beta1 did not alter tumour cell differentiation in vivo. The results demonstrate that endogenous TGF-beta1 functions as a tumour suppressor in the rat-4NQO model of oral carcinogenesis without altering tumour cell morphology or differentiation but can also act to promote local bone resorption.

Topics: Animals; Bone Resorption; Cell Division; Cell Line, Transformed; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Metastasis; Neoplasm Transplantation; Rats; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Invasive potentials of malignant cancer cells are regulated by cell motility factors. To examine the regulation of motility and invasiveness in oral squamous carcinoma, we investigated autocrine- and/or paracrine-acting cell motility factors, using a newly established human cell line (IF cells) from oral squamous cell carcinoma, which has highly invasive and metastatic characteristics. Conditioned medium derived from IF cells stimulated cell scattering and migration of GB-d1 gallbladder carcinoma cells, indicating that IF cells secreted cell motility factors. Using antibodies, IF-derived cell motility factors proved to be transforming growth factor (TGF)-alpha and TGF-beta1. Antibodies against TGF-alpha and TGF-beta1 inhibited autonomous migration of the IF cells. On the other hand, in vitro invasion of IF cells was strongly enhanced by hepatocyte growth factor (HGF) but only slightly by TGF-alpha and TGF-beta1. The conditioned medium from fibroblasts enhanced in vitro invasion of IF cells, an event abrogated by anti-HGF antibody, but not by antibodies against TGF-alpha and TGF-beta1. Importantly, IF cells secreted a factor inducing HGF production in fibroblasts and the factor was identified as interleukin-1, which means that a mutual interaction exists between tumour cells and fibroblasts, as mediated by the HGF/HGF-inducer loop. These results indicate that IF cells utilize TGF-alpha and TGF-beta1 as autocrine-acting motility factors and HGF as a paracrine-acting motility factor, and that invasiveness of IF cells is particularly stimulated by HGF derived from stromal fibroblasts. Utilization of multiple cell motility/invasion factors that act in distinct pathways may confer highly invasive and metastatic potentials in IF oral squamous carcinoma cells.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Movement; Cells, Cultured; Culture Media, Conditioned; Fibroblasts; Gallbladder Neoplasms; Gingival Neoplasms; Hepatocyte Growth Factor; Humans; Models, Biological; Mouth Neoplasms; Neoplasm Invasiveness; Skin; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

This study examined the metastatic capacity of clonal populations of 4NQO-induced rat malignant oral keratinocytes following orthotopic transplantation to athymic mice. Polygonal and spindle cells formed well-differentiated squamous cell carcinomas (keratin positive and vimentin negative) and undifferentiated spindle cell tumours (keratin negative and vimentin positive), respectively, in almost 100% of animals at the site of inoculation (floor of mouth). Transplantation of 5x 10(6) cells of either cell type at high cell density resulted in approximately 50% of animals forming pulmonary metastases. By contrast, inoculation of 2x 10(6) differentiated polygonal cells resulted in the formation of significantly fewer pulmonary metastases than the undifferentiated spindle cells. A single well-differentiated clone of polygonal cells and 3 of 4 of the undifferentiated spindle cell lines produced comparable levels of TGF-beta1. One undifferentiated spindle cell line expressed significantly more TGF-beta1 and, following transplantation orthotopically, fewer animals formed pulmonary metastases despite the formation of primary tumours in almost all grafted animals, suggesting that TGF-beta1 can act as a tumour suppressor in this cell type. All cell lines produced comparable amounts of TGF-beta2. The clones of polygonal cells were markedly inhibited and the spindle cells were only partially inhibited by exogenous TGF-beta1. Both cell types expressed high-affinity TGF-beta cell surface receptors; the ratio of type I to type II TGF-beta receptors was 1.0:<3.0 in the spindle cells and 1.0:17.9 in the polygonal clone. The results suggest that differentiated rat malignant oral keratinocytes are less aggressive and have a decreased potential to metastasise than their undifferentiated spindle cell counterparts. This may be attributable, in part, to a change in TGF-beta receptor profile leading to the partial loss of response to exogenous TGF-beta1.

Topics: 4-Nitroquinoline-1-oxide; Animals; Carcinoma; Carcinoma, Squamous Cell; Cell Differentiation; Cell Transplantation; Clone Cells; Keratinocytes; Lung Neoplasms; Lymphatic Metastasis; Male; Mice; Mice, Nude; Mouth Neoplasms; Rats; Rats, Sprague-Dawley; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

Induction of differentiation is today a useful strategy in cancer therapy but the clinical practice is insufficient in squamous cell carcinomas. We examined the effect of vesnarinone, a differentiation-inducing agent, on the cell cycle and cellular differentiation in four cell lines established from oral squamous cell carcinomas possessing a wild-type or mutated p53. Vesnarinone dose-dependently inhibited cell growth and induced G1 phase accumulation regardless of p53 gene mutation. The expression of involucrin and transglutaminase was increased by 4 days treatment with 60 microg/ml vesnarinone in all cell lines. Although p21 promoter activity was suppressed by vesnarinone, p21-mRNA was stabilized by the agent and expression of p21-mRNA was maintained for a long time. Corresponding to the prolonged p21-mRNA expression, p21 protein was induced by cell treatment with 60 microg/ml vesnarinone for 12 h and longer. The induced p21 protein bound cyclin E and suppressed cyclin E/Cdk2 kinase activity suppressing the phosphorylation of retinoblastoma (Rb) protein. These results suggest that vesnarinone possesses activity to induce p21 protein by stabilizing its mRNA with induction of differentiation of squamous cell carcinoma cells in a p53-independent manner.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Differentiation; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; G1 Phase; Humans; Mouth Neoplasms; Phosphorylation; Promoter Regions, Genetic; Pyrazines; Quinolines; Retinoblastoma Protein; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We examined the effect of the stable transfection of latent TGF-beta 1 cDNA, under the control of a cytomegalovirus promoter in the expression vector pcDNA3, into a 4NQO-induced clonal rat oral keratinocyte cell line that formed undifferentiated spindle cell tumours following subcutaneous transplantation to athymic mice. Test cells containing latent TGF-beta 1 cDNA produced a 2.3-fold increase in TGF-beta 1 protein compared to pcDNA3 controls as demonstrated by ELISA. Neutralisation experiments indicated that the majority of the protein was in the latent form. Untransfected and transfected (containing either TGF-beta 1 cDNA or pcDNA3) cell lines were keratin negative and vimentin positive. Cells transfected with TGF-beta 1 were inhibited more than pcDNA3 controls when cultured in an anchorage dependent or independent environment. Subcutaneous transplantation of cells overproducing TGF-beta 1 resulted in tumours of significantly smaller volume than vector-only controls. Further, orthotopic transplantation of cells containing TGF-beta 1 cDNA to the floor of the mouth in athymic mice markedly inhibited the development of pulmonary metastases compared to vector-only controls. Both test and control cell lines in athymic mice formed undifferentiated tumours with a complete absence of keratin elaboration. Subcutaneous xenografts were recultured and cells containing the TGF-beta 1 cDNA produced a similar amount of TGF-beta 1 peptide as the cells containing pcDNA3 only. The production of TGF-beta 1 by both of the xenograft-derived cell lines was significantly less than the parent, pre-transplanted cell lines and the untransfected cell line. All of the cell lines were inhibited by exogenous TGF-beta 1. Our results demonstrate that autocrine TGF-beta 1 functions as a tumour suppressor in vitro and in vivo in 4NQO-induced spindle tumour cells that are growth inhibited by the ligand. Furthermore, tumour formation in athymic mice is associated with selection for a cell phenotype with diminished autocrine TGF-beta 1 production.

Topics: 4-Nitroquinoline-1-oxide; Animals; Cell Division; Keratinocytes; Mice; Mice, Nude; Mouth Neoplasms; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

This study examined inositol phosphate and cAMP regulation by TGF-beta 1, -beta 2 and -beta 3 in normal and tumour-derived human oral keratinocytes. Previous findings indicated that the cell lines expressed TGF-beta cell surface receptors and had a range of response to exogenous TGF-beta 1, -beta 2 and -beta 3 from being refractory to the ligand to marked inhibition. Basal levels of inositol phosphates broadly reflected the differentiation status of the cells as demonstrated by involucrin expression, but did not correlate with responsiveness to TGF-beta 1, as measured previously by thymidine incorporation. Treatment of cells with bradykinin or serum caused up-regulation of inositol phosphate levels; by contrast, TGF-beta 1, -beta 2 and -beta 3 failed to modulate inositol phosphates. In two tumour-derived cell lines, the TGF-beta isoforms had no effect on cAMP levels, despite a significant increase in cAMP using a potent agonist of adenylate cyclase (forskolin). Furthermore, the cAMP analogue, dibutyryl cAMP, failed to mimic the inhibitory or refractory responses of TGF-beta in these cells lines. The results demonstrate that in normal and tumour-derived human oral keratinocytes, TGF-beta signal transduction is not mediated by inositol phosphates or cAMP.

Topics: Bradykinin; Carcinoma, Squamous Cell; Cells, Cultured; Cyclic AMP; DNA; DNA, Neoplasm; Humans; Inositol Phosphates; Melanocytes; Mouth Mucosa; Mouth Neoplasms; Reference Values; Thymidine; Transforming Growth Factor beta; Tumor Cells, Cultured

Thrombospondin (TSP), a cell matrix protein, and transforming growth factor beta (TGF-beta), a growth regulatory protein, play roles in tumor progression. The purpose of this study was to investigate the effects of TSP and TGF-beta on tumor cell invasion.. Tumor cell invasion assays were performed using a modified Boyden chamber apparatus with collagen-coated membranes. The KB oral carcinoma cell line was studied in serum-free media. Invasion was measured as the summation of the number of cells in five representative low-power fields (x 100) traversing the collagen barrier after a 3-hour incubation period. The effects of antibodies against TSP, TGF-beta and the cysteine-serine-valine-threonine-cysteine-glycine (CSVTCG)-specific TSP receptor were also evaluated.. TSP caused a dose-dependent stimulation of tumor cell invasion. Antibodies against TSP, its CSVTCG-specific receptor, and TGF-beta inhibited TSP-promoted invasion by 50% to 71%.. TSP and its CSVTCG-specific receptor promote KB cell invasion of collagen through the production and/or activation of TGF-beta.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Carcinoma, Squamous Cell; CD36 Antigens; Cell Adhesion Molecules; Cell Count; Collagen; Culture Media, Serum-Free; Diffusion Chambers, Culture; Disease Progression; Dose-Response Relationship, Drug; Humans; Immunoglobulin G; Integrins; Membrane Glycoproteins; Membranes, Artificial; Microscopy, Phase-Contrast; Mouth Neoplasms; Neoplasm Invasiveness; Polycarboxylate Cement; Thrombospondins; Transforming Growth Factor beta; Tumor Cells, Cultured

This study examined the effect of transforming growth factor beta-1 (TGF-beta 1) on c-myc, RB1, junB and p53 expression together with pRb phosphorylation, in carcinoma-derived and normal human oral keratinocytes with a range of inhibitory responses to this ligand. Amplification of c-myc was observed in eight of eight tumour-derived cell lines and resulted in corresponding mRNA expression. The down-regulation of c-myc expression by TGF-beta 1 predominantly reflected growth inhibition by TGF-beta 1, but in two of eight tumour-derived cell lines which were partially responsive to TGF-beta 1 c-myc expression was unaltered by this ligand. While RB1 mRNA levels were unaltered by TGF-beta 1, the ligand caused the accumulation of the underphosphorylated form of the Rb protein in all cells irrespective of TGF-beta 1-induced growth arrest. junB expression was up-regulated by TGF-beta 1 in cells with a range of growth inhibitory responses. All cells contained mutant p53. TGF-beta 1 did not affect p53 mRNA expression in both tumour-derived and normal keratinocytes and there was no alteration in p53 protein levels in keratinocytes expressing stable p53 protein following TGF-beta 1 treatment. The data indicate that TGF-beta-induced growth control can exist independently of the presence of mutant p53 and the control of Rb phosphorylation and c-myc down-regulation. It may be that TGF-beta growth inhibition occurs via multiple mechanisms and that the loss of one pathway during tumour progression does not necessarily result in the abrogation of TGF-beta-induced growth control.

Topics: Carcinoma, Squamous Cell; Cells, Cultured; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, jun; Genes, myc; Genes, p53; Humans; Keratinocytes; Mouth Neoplasms; Phosphorylation; Retinoblastoma Protein; Transforming Growth Factor beta

Serum-free cultures of normal human buccal epithelial cells were transfected with a plasmid containing the SV40 T-antigen (SV40T) gene. Two major lines developed that showed extended lifespans (between 30 and 40 weeks) as compared with the controls (approximately 6 weeks). Continued growth through one or two crises generated several sublines. They expressed the epithelial marker keratin and also exhibited nuclear expression of SV40T. The lines showed abnormal karyotypes with both numerical and structural aberrations and variably responded to agents that normally inhibit growth and/or induce terminal differentiation, i.e. transforming growth factor-beta 1 and fetal bovine serum. One of the lines, termed SVpgC2a, developed into an apparently immortal line, since it had undergone more than 700 population doublings from over 2 years in culture. Further characterization of this line demonstrated its clonal origin, with integration of two copies of SV40T at the same site and the presence of both normal retinoblastoma and wild-type p53 proteins. This line showed high resistance to growth inhibition by transforming growth factor-beta 1 and serum similar to that shown by buccal carcinoma cell line SqCC/Y1. Neither SVpgC2a nor its parental lines were tumorigenic when injected into athymic nude mice, whereas the SqCC/Y1 cells induced tumors. The various lines with extended but finite lifespans, complemented by one immortalized line, which retained non-malignant properties upon extended culture, provide a battery of model systems that will be useful for studying mechanisms of human oral carcinogenesis.

Topics: Animals; Antigens, Polyomavirus Transforming; Biomarkers; Blood; Cattle; Cell Differentiation; Cell Division; Cell Line; Cells, Cultured; Culture Media, Serum-Free; Epithelial Cells; Epithelium; Genes, Viral; Humans; Karyotyping; Keratins; Mice; Mice, Nude; Mouth Mucosa; Mouth Neoplasms; Recombinant Proteins; Retinoblastoma Protein; Simian virus 40; Transfection; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Virus Integration

We previously immortalized normal human oral keratinocytes by transfection with recombinant HPV-16 DNA and subsequently exposed the cells to benzo(a)pyrene for 7 days. The exposure to benzo(a)pyrene modified the immortalized cells: the modified cells (HOK-16B-BaP) proliferated in an ordinary culture medium containing physiological calcium level (1.5 mM), but demonstrated only enhanced proliferation capacity without tumor formation in nude mice and failed to show in vitro anchorage-independency. In this study, we further modified the HOK-16B-BaP cells by subculturing the cells in a medium containing benzo(a)pyrene for 6 months. The cells were further modified with a chronic benzo(a)pyrene exposure and were termed HOK-16B-BaP-T cells (1) demonstrated a malignant phenotype in organotypic 'raft' culture, (2) showed in vitro anchorage-independency, (3) developed tumors in nude mice when injected subcutaneously, (4) contained a significantly higher copy number of intact and integrated HPV-16 DNA; (5) contained higher level of HPV-16 E6/E7 messages and E7 protein, (6) were more resistant to transforming growth factor-beta 1 and (7) secreted higher level of vascular endothelial growth factor with molecular weight of 56 kd than parental HOK-16B-BaP cells. However, the levels of p53 and ras proteins and the levels of p53, c-myc and c-fos transcripts in the HOK-16B-BaP-T cells were not different from those in the HOK-16B-BaP cells. The highly conserved coding regions of the p53, c-Ha-ras1, and c-Ki-ras2 genes of the tumor cells were not mutated. These data indicate that the HPV-immortalized human oral keratinocytes can convert to tumorigenic cells by chronic exposure to benzo(a)pyrene. The tumorigenic conversion seems to be associated with (1) the overexpression of viral oncogenes such as E6 and E7 genes, (2) the higher resistance of cells to transforming growth factor-beta 1 and (3) the high secretion of 56 kd vascular endothelial growth factor from the cells.

Topics: Animals; Base Sequence; Benzo(a)pyrene; Carcinoma, Squamous Cell; Cell Division; Cell Line, Transformed; Cocarcinogenesis; DNA Primers; DNA, Viral; Endothelial Growth Factors; Genes, fos; Genes, myc; Genes, ras; Humans; Lymphokines; Mice; Mice, Nude; Molecular Sequence Data; Mouth Neoplasms; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; RNA, Viral; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

This study examined the expression of TGF-beta cell-surface receptors, the response to exogenous TGF-beta 1 and the autocrine production of TGF-beta in normal and squamous cell carcinoma-derived human oral keratinocytes with variable degrees of cellular differentiation. TGF-beta receptor expression, the response to exogenous ligand and the autocrine production of TGF-beta appeared unrelated to cellular differentiation. Cells expressed variable proportions of type-I, -II and -III TGF-beta receptors. The expression of type-III receptors correlated inversely with the expression of type-I receptors, but there was no relationship between type-II and either type-I or type-III TGF-beta receptors. Normal cells and the majority (7 of 8) of tumour-derived keratinocytes were inhibited by exogenous TGF-beta 1 and the degree of inhibition correlated with the expression of type-I, but not type-II or type-III, TGF-beta receptors. One tumour-derived cell line was refractory to exogenous TGF-beta 1 although it expressed all 3 receptor types. Endogenous TGF-beta was produced by both normal and tumour-derived keratinocytes and correlated inversely to the expression of type-I, but not type-II, TGF-beta receptors. Further, cells that produced more autocrine TGF-beta had a diminished response to exogenous TGF-beta 1. The data indicate a complex interaction between the expression of TGF-beta cell-surface receptors, endogenous ligand production and the cellular response to exogenous TGF-beta 1.

Topics: 3T3 Cells; Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Culture Media, Conditioned; Humans; Keratinocytes; Keratins; Mice; Mouth Mucosa; Mouth Neoplasms; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured; Vimentin

The effects of human recombinant transforming growth factor (TGF)-beta 1 were determined on PTH-related protein (PTHrP) production and messenger RNA (mRNA) expression by a canine squamous carcinoma cell line (SCC 2/88) in vitro. TGF-beta increased PTHrP production in a dose- and time-dependent manner (P < 0.05) as measured by RIA, and the effects of TGF-beta treatment persisted up to 72 h after removal. TGF-beta increased PTHrP production by SCC 2/88 cells until cellular confluence, at which time there was no longer a significant increase compared to control. Actinomycin D inhibited the TGF-beta-mediated increase in PTHrP production, suggesting that TGF-beta acted in part by increasing gene transcription. SCC 2/88 cells also produced active TGF-beta as measured by a [3H]thymidine incorporation assay in mink lung epithelial cells. Exposure of SCC 2/88 cells to a neutralizing anti-TGF-beta monoclonal antibody decreased (up to 50%, P < 0.01) basal PTHrP production. TGF-beta increased PTHrP mRNA expression in a dose- and time-dependent manner as evaluated by northern blot analysis. Postconfluent SCC 2/88 cells expressed little mRNA for PTHrP, and there was only a minimal increase in PTHrP mRNA expression in postconfluent TGF-beta-treated cells. These results indicate that TGF-beta increased PTHrP production and mRNA expression in malignant keratinocytes and suggest that TGF-beta may be an important factor in the pathogenesis of humoral hypercalcemia of malignancy.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Count; Dactinomycin; Dogs; Gene Expression; Humans; Kinetics; Mouth Neoplasms; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

The expression pattern of transforming growth factor-beta 1 (TGF-beta 1) during the stages of complete carcinogenesis in the hamster cheek pouch model was studied. The right cheek pouches of 18 male hamsters were treated with 0.5%, 7,12-dimethylbenz[a]anthracene (DMBA) for 16 wk. TGF-beta 1 was detected immunohistochemically in the resulting samples with two different polyclonal monospecific antibodies that recognize intracellular and extracellular forms of TGF-beta 1. In the normal cheek pouch, extracellular protein stained the corium strongly, but the reaction was not evenly distributed. As treatment progressed, the reaction increased in both area and intensity; the peak was reached at 8 wk. Intracellular TGF-beta 1 expression followed a similar pattern, with a peak at 4 wk of treatment. The results of northern blot analysis were concordant with the immunohistochemical results. Overexpression of TGF-beta 1 was also observed in the malignant tumors, but only the extracellular form of the protein was present; intracellular TGF-beta 1 was not detected in these tumors. The expression of TGF-beta 1 in this carcinogenesis model seems to have two formal stages, the first being an overexpression step as a reaction to the uncontrolled growth and the second being one in which tumors have no internal expression of TGF-beta 1 but in which external protein accumulates in the surrounding stroma. A possible explanation of this paradox may be that TGF-beta 1 has functions other than its growth-repressing activity.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cheek; Cricetinae; Disease Models, Animal; Extracellular Space; Immunohistochemistry; Intracellular Fluid; Male; Mesocricetus; Mouth Neoplasms; Protein Processing, Post-Translational; RNA, Messenger; Skin Neoplasms; Transforming Growth Factor beta

Humoral hypercalcemia of malignancy is a paraneoplastic syndrome associated with a variety of solid neoplasms including squamous cell carcinomas of various sites. Parathyroid hormone-related protein (PTHrP) is a newly recognized hormone that has been implicated as one of the major causative factors in the pathogenesis of this syndrome. A canine oral squamous carcinoma cell line (SCC 2/88) was used to investigate the regulation of production of PTHrP in response to agents that alter keratinocyte differentiation/proliferation in vitro.. SCC 2/88 cells grown in serum-free media were exposed to various factors and PTHrP production was measured by radioimmunoassay. This cell line spontaneously produced substantial amounts of PTHrP (up to 7,000 pg/ml) without the need for a fibroblast-feeder layer. Production of PTHrP decreased at cellular confluence, and with increasing passage number.. Epidermal growth factor, cholera toxin, calcium, 1,25-dihydroxyvitamin D, ionomycin, trans-retinoic acid, transforming growth factor-beta 1 and hydrocortisone stimulated production of PTHrP by SCC 2/88 cells to various degrees. Transforming growth factor-beta 1 was the most potent stimulator of PTHrP production, with a maximal stimulation of 25-fold over control. Monensin decreased PTHrP secretion as early as 6 hours post-treatment and by 48 hours, there was no detectable PTHrP in the conditioned cell culture medium. Calcium, cholera toxin, ionomycin, and transforming growth factor-beta 1 decreased keratinocyte proliferation as measured by cell counts at all doses tested.. The results of this study revealed that SCC 2/88 cells spontaneously produced substantial amounts of PTHrP under baseline conditions and that compounds known to affect keratinocyte differentiation/proliferation up-regulated production of PTHrP. These cells will be valuable to investigate the molecular regulation of PTHrP production by squamous cell carcinomas.

Topics: Animals; Calcitriol; Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; Cholera Toxin; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Hydrocortisone; Ionomycin; Keratinocytes; Kinetics; Mouth Neoplasms; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Radioimmunoassay; Time Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Eosinophilia in tissues and/or circulating blood is known to be associated with a wide variety of malignancies but the role of the eosinophil in neoplastic conditions is not known. Using the cheek pouch of the Syrian hamster as an experimental model for oral carcinogenesis, it has recently been shown that eosinophils at sites of developing oral cancer express the multifunctional cytokine, transforming growth factor alpha (TGF-alpha). This study investigated the time course of eosinophil infiltration, tissue eosinophilia associated with malignant epithelium, and eosinophil-derived TGF-alpha mRNA during the 16-week 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral cancer development process. The results reveal that the occasional eosinophil is normally present in the lamina propria of hamster oral mucosa. With progressive DMBA treatments, there is an increase of eosinophils infiltrating into the lamina propria. By weeks 12-16, the number of eosinophils is significantly higher in DMBA-treated pouches than in control pouches treated with the vehicle mineral oil alone. Analysis of the infiltrating eosinophils into fully developed hamster oral carcinomas reveals that tissue eosinophilia is associated with 78% of the stromal areas associated with malignant epithelium, while only 7% of sites associated with non-tumor oral epithelium (normal, hyperplastic-dysplastic) exhibited eosinophilia. Furthermore, the majority of the eosinophils associated with malignant epithelium were found to contain TGF-alpha mRNA. The number of TGF-alpha mRNAs containing eosinophils associated with malignant oral epithelium is significantly higher than that associated with nonmalignant oral epithelium. Together, these results suggest that eosinophils are recruited to tumor-developing sites, that they predominantly associate with malignant epithelium, and that most tumor-associated eosinophils express the cytokine TGF-alpha.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Transformation, Neoplastic; Cricetinae; Eosinophils; Gene Expression; Male; Mesocricetus; Mouth Neoplasms; Precancerous Conditions; RNA, Messenger; Transforming Growth Factor beta

Topics: 4-Nitroquinoline-1-oxide; Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Keratinocytes; Mice; Mice, Nude; Mouth Neoplasms; Rats; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transplantation, Heterologous

Epithelial cell cultures were obtained following tryptic digestion of normal human buccal mucosa. Primary cultures exhibited markedly higher colony-forming efficiencies and growth rates using fibronectin/collagen-coated, as compared to non-coated culture dishes and a serum-free MCDB 153 medium developed for epidermal epithelial cells than a similar medium previously developed for buccal explant outgrowth cultures. At the preferred conditions, the cells could be transferred at least 5-fold, divided at about one population doubling per day, and commonly underwent 60 population doublings resulting in yields of 10(8) to 10(11) cells per cm2 mucosal specimen. Moreover, these conditions successfully cultivated a buccal carcinoma cell line (SqCC/Y1) for several months. The carcinoma cells were resistant to factors that inhibited growth or induced differentiation of normal cells, i.e., transforming growth factor type beta 1, Ca2+, or serum. Karyotype analyses of SqCC/Y1 cells showed 63 to 83 chromosomes per metaphase and consistent occurrences of monosomy 1, tetrasomy 19 and 20, as well as trisomy 22, and at least 7 marker chromosomes, whereas cells obtained from non-cancerous donors were diploid. It is concluded that the similarly defined culture conditions may now be applied to study characteristics of both normal and tumorous buccal epithelial cells.

Topics: Blood; Calcium; Cell Division; Cells, Cultured; Chromosome Banding; Chromosomes, Human; Culture Media, Serum-Free; Culture Techniques; Epithelial Cells; Epithelium; Humans; Karyotyping; Kinetics; Mouth Mucosa; Mouth Neoplasms; Protein Precursors; Reference Values; Transforming Growth Factor beta

This study examined the characteristics of keratinocytes from 4-nitroquinoline N-oxide-treated rat oral epithelium that formed well differentiated carcinomas or spindle cell tumours when transplanted s.c. to athymic mice. Small polygonal-type cells in early culture passage gave rise to xenografts that were well differentiated carcinomas with keratin positivity and a differential reactivity with two anti-vimentin antibodies (positive Vim Dako, negative Vim 3B4). Progressive culture of the polygonal cells resulted in cells of a more stellate-like morphology which, when transplanted, formed spindle cell tumours that were keratin negative and vimentin positive (Vim Dako and Vim 3B4). The staining pattern of the xenografts was similar to that of the cultured cell derivatives. By contrast to normal oral keratinocytes which were stimulated by epidermal growth factor (EGF), the parental and xenograft-derived cells were markedly less responsive and at times refractory to EGF. Low affinity EGF receptors characterized normal keratinocytes whilst parental and xenograft-derived cells from well differentiated carcinomas and spindle cell tumours expressed diminished numbers of low and high affinity and high affinity EGF receptors respectively. Normal keratinocytes and parental tumour cells were inhibited by transforming growth factor (TGF-beta) whereas xenograft-derived cell lines from both well differentiated carcinomas and spindle cell tumours were refractory to TGF-beta. TGF-beta receptors were predominantly of high affinity although xenograft-derived cells, particularly from spindle cell tumours, expressed increased numbers of receptors which were of lower affinity. The results indicate that spindle cell tumours are a variant of well differentiated carcinomas and express an aberrant pattern of differentiation. Resistance to EGF-induced stimulation and TGF-beta-induced inhibition appears not to be an integral part of the tumour phenotype but the pattern of EGF and TGF-beta receptor expression closely follows the degree of tumour differentiation.

Topics: Animals; Carcinoma, Squamous Cell; Cell Differentiation; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Keratinocytes; Male; Mouth Neoplasms; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Thymidine; Transforming Growth Factor beta; Tritium