osteoprotegerin and Mouth-Neoplasms

Bone invasion by oral cancer is a common clinical problem, which affects the choice of treatment and predicts a poor prognosis. Unfortunately, the molecular mechanism of this phenomenon has not been fully elucidated. Current studies have revealed that oral cancer cells modulate the formation and function of osteoclasts through the expression of a series of signal molecules. Many signal pathways are involved in this process, of which receptor activator of nuclear factor-κB ligand/receptor activator of nuclear factor-κB/osteoprotegerin signaling pathway attracted much attention. In this review, we introduce recent progress in molecular mechanisms of bone invasion by oral cancer.. 口腔癌易于发生颌骨侵犯，影响患者预后，而这一现象的分子机制尚未完全阐明。目前研究发现，口腔癌细胞通过一系列信号分子的表达直接或者间接影响破骨细胞的形成和功能，有多条信号通路参与其调控，其中核因子κB受体活化因子配体/核因子κB受体活化因子/骨保护素信号通路的调节备受关注。本文就口腔癌颌骨侵犯的分子机制研究进展进行综述。.

Topics: Bone and Bones; Bone Resorption; Humans; Mouth Neoplasms; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

Squamous cell carcinomas (SCCs) of the gingiva frequently invade the mandible or maxilla; this invasion is associated with a worse prognosis. The bone destruction associated with carcinomal invasion is mediated by osteoclasts rather than directly by the carcinoma. Therefore, if the cellular and molecular mechanisms by which oral SCC regulates bone invasion were known, it could inform the development of new therapeutic targets. Recently, dysregulation of the functional equilibrium in the receptor activator of NF-κB ligand (RANKL)/RANK/osteoprotegerin (OPG) triad has been shown to be responsible for osteolysis associated with the development of malignant tumors in bone sites. Furthermore, the administration of OPG or soluble RANK prevents bone metastasis by cancer cells. In this review, we discuss recent findings indicating that bone invasion by oral SCC is mediated via RANKL/RANK and may be successfully prevented by RANKL inhibition.

Topics: Animals; Bone and Bones; Bone Neoplasms; Carcinoma, Squamous Cell; Humans; Mandible; Mice; Molecular Targeted Therapy; Mouth Neoplasms; NF-kappa B; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

The objective of this study was to assess the remodeling-associated gene expression in the mandible of patients diagnosed with oral squamous cell carcinoma (OSCC), investigating the cortical microarchitecture, and their influence on disease-free survival (DFS) and overall survival (OS) rates. A total of twenty-four patients who underwent mandibulectomy for OSCC treatment had two bone fragments harvested from the mandible for gene expression (RANK, RANKL, OPG, and SOST), and microarchitecture analysis, including bone volume, surface, mineral density, degree of anisotropy, and fractal dimension. The prognosis of the patients was assessed. The results revealed that RANK, RANKL, and SOST were predominantly downregulated, while OPG was completely downregulated. Tumors located adjacent to the posterior region of the mandible (p = 0.02), with a bone mineral density below 1.03 g/cm3 HA (p = 0.001), and a bone volume less than 86.47% (p = 0.03) were associated with poor outcomes. In conclusion, bone-remodeling-associated genes exhibited downregulation in the cortex of the mandible in OSCC patients. Additionally, the tumor's location within the mandible, bone volume, and cortical bone mineral density were identified as factors impacting DFS.

Topics: Carcinoma, Squamous Cell; Gene Expression; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Osteoprotegerin; Prognosis; RANK Ligand; Squamous Cell Carcinoma of Head and Neck

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

The aim of the study was to evaluate the mandible cortical bone changes in patients with oral squamous cell carcinoma (OSCC).. Twenty patients who underwent some mandibular bone removal as part of the treatment of OSCC had bone samples collected in two parts: in the proximity of the tumor (BPT) and in the surgical margin (BEP). Cortical microarchitecture was analyzed trough micro-computed tomography, together with texture analysis, followed by microcrack evaluation in histological sections and gene expression of RANK, RANKL, OPG, and sclerostin by quantitative polymerase chain reaction.. Bone surface was higher in BPT (0.005 ± 0.002 vs 0.004 ± 0.002, p = 0.01) compared with BEP. In BPT, the subset of patients without bone invasion presented higher anisotropy (0.83 ± 0.07) compared with the ones with bone invasion (0.70 ± 0.14) (p = 0.04). RANK, RANKL, OPG, and sclerostin were found to be downregulated in the majority of cases in both parts. There were significant correlations between the parameters of microarchitecture and gene expression analysis (p < 0.001 to p < 0.05), most of them related with OPG levels.. The cortex in the mandible in the proximity of the tumor reveals more bone surface than the bone in the surgical margin, and the tumor invasion causes a decrease in anisotropy. RANK, RANKL, OPG, and sclerostin are downregulated in mandible, in both parts analyzed. Correlation tests revealed the association between cortical thickness, bone surface, anisotropy, porosity, bone mineral density, and OPG levels.. The mandible cortical bone microarchitecture changes in the proximity of the squamous cell carcinoma lesion.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Biomarkers, Tumor; Bone Morphogenetic Proteins; Carcinoma, Squamous Cell; Down-Regulation; Female; Genetic Markers; Humans; Male; Mandible; Margins of Excision; Middle Aged; Mouth Neoplasms; Osteoprotegerin; Prospective Studies; RANK Ligand; Real-Time Polymerase Chain Reaction; Receptor Activator of Nuclear Factor-kappa B; X-Ray Microtomography

Oral squamous cell carcinoma (OSCC) is the most common malignancy among oral cancers and shows potent activity for local bone invasion. Receptor activator of nuclear factor κB (RANK) ligand (RANKL) is critical for bone-resorbing osteoclast formation. We previously demonstrated that OSCC tumor cells express high levels of RANKL. In this study, confocal microscopy demonstrated RANKL specific receptor, RANK expression in OSCC tumor cell lines (SCC1, SCC12, and SCC14a). We also confirmed the expression of RANK and RANKL in primary human OSCC tumor specimens. However, regulatory mechanisms of RANKL expression and a functional role in OSCC tumor progression are unclear. Interestingly, we identified that RANKL expression is autoregulated in OSCC tumor cells. The RANKL specific inhibitor osteoprotegerin (OPG) treatment to OSCC cells inhibits autoregulation of RANKL expression. Further, we showed conditioned media from RANKL CRISPR-Cas9 knockout OSCC cells significantly decreased osteoclast formation and bone resorption activity. In addition, RANKL increases OSCC tumor cell proliferation. RANKL treatment to OSCC cells demonstrated a dose-dependent increase in RANK intracellular adaptor protein, TRAF6 expression, and activation of IKK and IκB signaling molecules. We further identified that transcription factor NFATc2 mediates autoregulation of RANKL expression in OSCC cells. Thus, our results implicate RANKL autoregulation as a novel mechanism that facilitates OSCC tumor cell growth and osteoclast differentiation/bone destruction.

Topics: Animals; Bone and Bones; Bone Resorption; Carcinoma, Squamous Cell; Cell Line, Tumor; CRISPR-Cas Systems; Homeostasis; Humans; Mice; Mice, Inbred C57BL; Mouth Neoplasms; NFATC Transcription Factors; Osteoclasts; Osteoprotegerin; RANK Ligand; Signal Transduction; TNF Receptor-Associated Factor 6

The molecular mechanisms involved in the invasion of bone by oral squamous cell carcinomas (OSCC) are poorly understood, and little is known about the role of cancer-associated fibroblasts (CAF), the presence of which confers a poor prognosis.. Clinicopathological data from 277 OSCC cases involving bone resections were reviewed, and 32 cases thoroughly analysed histologically. Immunohistochemistry was used to examine αSMA, RANKL and OPG. Western blotting and qPCR were used to assess myofibroblast (CAF-like) differentiation, RANKL and OPG expression in vitro, and RANKL secretion was analysed by ELISA. Osteoclastogenesis was examined using TRAP staining, multinucleation and pit forming assays.. Fibrous stroma intervened between tumour and bone in the majority of cases, with no direct contact between cancer cells and bone. RANKL and OPG, two proteins key to regulating bone resorption, were expressed in tumour cells as well as fibrous stroma adjacent to bone and αSMA-positive myofibroblastic CAF were consistently seen infiltrating into bone ahead of tumour cells. Human primary osteoblasts cultured with conditioned media from human OSCC-derived cells and human primary CAF showed a significant increase in RANKL and a decline in OPG mRNA expression. RANKL secretion was significantly increased in primary oral fibroblasts induced to differentiate into a CAF-like phenotype by transforming growth factor-β1 (TGF-β1) treatment and in primary CAF. Indirect co-culture of murine macrophages with conditioned media from CAF (experimentally derived and isolated from OSCCs) resulted in a marked increase in osteoclastogenesis (in excess of that provoked by cancer cells) determined by tartrate-resistant acid phosphatase activity, multinucleation and resorption pit formation.. This study is the first to describe a functional role for CAFs in bone invasion and turnover, identifying a novel potential therapeutic target and diagnostic indicator in this difficult to treat bone invasive malignancy.

Topics: Actins; Bone and Bones; Bone Neoplasms; Cancer-Associated Fibroblasts; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Humans; Mitochondrial Proteins; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; Osteogenesis; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Ribosomal Proteins; RNA, Messenger; Transforming Growth Factor beta1

We aimed to determine the prognostic significance of receptor activator of nuclear factor kappa-B ligand (RANKL), RANK and osteoprotegerin (OPG) in patients with oral squamous cell carcinoma (OSCC).. The protein expression of RANKL, RANK and OPG was assessed by immunohistochemistry on pretreatment biopsies of 93 patients with locally advanced OSCC who received preoperative chemoradiotherapy (CRT). The primary endpoint was cancer-specific survival. Secondary endpoints were correlation of biomarkers with bone invasion and pathological tumor response. Kaplan-Meier curves and Cox regression models were used for survival analyses.. A significantly higher OPG expression was demonstrated in patients with malignant bone invasion and non-responders to CRT as compared to patients without bone invasion and responders (p=0.032 and p=0.033, respectively). Multivariate analysis revealed that higher OPG expression was independently associated with shorter cancer-specific survival (p=0.04). The expression status of RANKL and RANK was not significantly related to clinicopathological characteristics and had no impact on survival of OSCC patients.. Upregulation of OPG expression is associated with bone invasion, poor pathological tumor regression to neoadjuvant CRT, and worse long-term cancer-specific survival in patients with locally advanced OSCC. Our results indicate that OPG may be a novel prognostic biomarker in oral cancer.

Topics: Biomarkers, Tumor; Bone Neoplasms; Carcinoma, Squamous Cell; Female; Humans; Male; Mouth Neoplasms; Osteoprotegerin; Prognosis; RANK Ligand; Survival Analysis

The molecular mechanisms underlying bone destruction by invading oral cancer are not well understood. Using IHC, we demonstrated that receptor activator of nuclear factor-κB ligand (RANKL)-positive fibroblasts and cancer cells were located at sites of bone invasion in human oral cancers. HSC3 and HO-1-N-1, human oral cancer cell lines, expressed RANKL and stimulated Rankl expression in the UAMS-32 murine osteoblastic cell line. We discriminated the roles of RANKL synthesized by stromal cells and cancer cells in cancer-associated bone resorption by using species-specific RANKL antibodies against murine RANKL and human RANKL, respectively. Osteoclastogenesis induced by the conditioned medium of HSC3 and HO-1-N-1 cells in a co-culture of murine bone marrow cells and UAMS-32 cells was inhibited by the addition of antibodies against either mouse or human RANKL. HSC3-induced bone destruction was greatly inhibited by the administration of anti-mouse RANKL antibody in a xenograft model. HO-1-N-1-induced bone destruction was inhibited by the administration of either anti-mouse or anti-human RANKL antibody. Bone destruction induced by the transplantation of human RANKL-overexpressing cells (HSC3-R2) was greatly inhibited by the injection of anti-human RANKL antibody. The present study revealed that RANKL produced by both stromal and cancer cells is involved in oral cancer-induced osteoclastic bone resorption. These results provide important information for understanding the cellular and molecular basis of cancer-associated bone destruction and the mechanism of action underlying RANKL antibody (denosumab) therapy.

Topics: Animals; Antibodies; Bone Marrow Cells; Bone Resorption; Cell Line, Tumor; Coculture Techniques; Gene Expression Regulation, Neoplastic; Humans; Mice; Mouth Neoplasms; Neoplasm Invasiveness; Osteoclasts; Osteogenesis; Osteoprotegerin; RANK Ligand; Stromal Cells; Xenograft Model Antitumor Assays

Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed oral malignancy in humans and cats and frequently invades bone. The objective of this study was to determine if feline OSCC serves as a relevant model of human OSCC in terms of osteolytic behavior and expression of bone resorption agonists. Novel feline OSCC cell lines (SCCF2 and SCCF3) were derived from spontaneous carcinomas. Gene expression and osteolytic behavior were compared to an established feline OSCC cell line (SCCF1) and three human OSCC cell lines (UMSCC-12, A253 and SCC25). Interaction of OSCC with bone and murine pre-osteoblasts (MC3T3) was investigated using in vitro co-culture techniques. In vivo bioluminescent imaging, Faxitron radiography and microscopy were used to measure xenograft growth and bone invasion in nude mice. Human and feline OSCC expressing the highest levels of parathyroid hormone-related protein (PTHrP) were associated with in vitro and in vivo bone resorption and osteoclastogenesis. MC3T3 cells had increased receptor activator of nuclear factor κB ligand (RANKL) expression and reduced osteoprotegerin (OPG) expression in conditioned medium from bone-invasive SCCF2 cells compared to minimally bone invasive SCCF3 cells, which was partially reversed with a neutralizing anti-PTHrP antibody. Human and feline OSCC cells cultured in bone-conditioned medium had increased PTHrP secretion and proliferation. Feline OSCC-induced bone resorption was associated with tumor cell secretion of PTHrP and with increased RANKL:OPG expression ratio in mouse preosteoblasts. Bone-CM increased OSCC proliferation and secretion of PTHrP. The preclinical models of feline OSCC recapitulated the bone-invasive phenotype characteristic of spontaneous OSCC and will be useful to future preclinical and mechanistic studies of bone invasive behavior.

Topics: Animals; Bone Resorption; Carcinoma, Squamous Cell; Cats; Cell Line, Tumor; Disease Models, Animal; Humans; Mice; Mouth Neoplasms; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand

This study investigates whether matrix metalloproteinases (MMPs), specifically MMP-2 and MMP-9, interacting with other molecules important in osteoblast differentiation and osteoclastogenesis, could play important roles in the invasion of bone by oral squamous cell carcinoma (OSCC).. Supernatant (conditioned medium, CM) was collected from OSCC cell lines (SCC15 and SCC25), and from cultured osteoblasts (hFOB cell line and a primary culture, OB), and used for indirect co-culture: OSCC cells were treated with CM from osteoblasts and vice versa. Zymogenic activities of MMP-2 and -9, and protein quantities of all molecules studied, were detected by gelatine zymography and Western blotting, respectively. Real-time polymerase chain reaction (PCR) analysed mRNA of these molecules. Targeted molecules were examined by immunohistochemistry in tissue sections of bone-invasive OSCCs.. Zymogenic activities of both MMPs were increased in OSCC cells following culture with CM from hFOB: Twist1 protein expression was increased while Runx2 did not alter. The RANKL/OPG ratio, zymogen and protein expression of MMP-9 were increased in hFOB cells cultured with CM from OSCC lines, while zymogen expression of MMP-2 was decreased. Real-time PCR showed generally similar changes in expression of these molecules. All targeted molecules were expressed in invading malignant keratinocytes, and all but OPG were expressed in osteoclasts of clinical samples.. Crosstalk between different cell types appears to exist in the invasion of bone by OSCC. Understanding and ultimately interfering with the molecules involved may provide therapeutic approaches to inhibit such bone invasion.

Topics: Biomarkers, Tumor; Bone Neoplasms; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Coculture Techniques; Core Binding Factor Alpha 1 Subunit; Culture Media, Conditioned; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mouth Neoplasms; Neoplasm Invasiveness; Nuclear Proteins; Osteoblasts; Osteoclasts; Osteoprotegerin; RANK Ligand; Real-Time Polymerase Chain Reaction; Receptor Cross-Talk; Twist-Related Protein 1

To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first report showing the role of CXCL2 in cancer-associated bone destruction.

Topics: Animals; Bone Resorption; Carcinoma, Squamous Cell; Cell Line, Tumor; Chemokine CXCL2; Coculture Techniques; Culture Media, Conditioned; Humans; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Osteoclasts; Osteoprotegerin; RANK Ligand

Oral squamous cell carcinomas (OSCCs) are malignant tumors that frequently invade the maxilla and mandibular bone. However, the molecular mechanisms underlying bone invasion by OSCC are unclear. Recent studies showed that receptor activator of nuclear factor κB (RANK) was expressed not only in osteoclast precursors but also in tumor cells. Therefore, we examined whether RANK ligand (RANKL)/RANK signaling regulates bone invasion by OSCC cells in vivo and in vitro. We first injected human OSCC B88 cells into the masseter region of nude mice. Mice were treated for 3 weeks with osteoprotegerin (OPG), the decoy receptor for RANKL. Treatment with OPG decreased bone invasion by B88 cells, reduced the number of osteoclasts and increased B88 cell apoptosis. However, OPG did not affect apoptosis and proliferation in B88 cells in vitro, suggesting that the effects of OPG on apoptosis in B88 cells are restricted in a bone environment. RANK was expressed in the B88 cells and in OSCC cells from patients. RANKL induced NF-κB activation and extracellular signal-regulated kinase phosphorylation in B88 cells and enhanced B88 cell migration in a modified chemotaxis chamber equipped with a gelatin-coated filter. OPG inhibited RANKL-induced NF-κB activation, extracellular signal-regulated kinase phosphorylation and cell migration. Our data clearly indicate that RANKL/RANK inhibition suppresses bone invasion by inhibiting osteoclastogenesis and cancer cell migration and by inducing apoptosis of cancer cells via indirect anticancer action in vivo.

Topics: Animals; Apoptosis; Blotting, Western; Bone Neoplasms; Carcinoma, Squamous Cell; Cell Movement; Cell Proliferation; Electrophoretic Mobility Shift Assay; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Mandibular Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplasm Invasiveness; NF-kappa B; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

Oral squamous cell carcinoma (SCC) cells frequently invade mandibular bone, and bone invasion is a common clinical problem. Recent studies have revealed that bone resorption by osteoclasts is an important step in the progress of bone invasion by oral SCCs. We previously reported that oral SCC cells induce osteoclastogenesis by suppressing osteoprotegerin (OPG) in host cells. In the present study, we examined the effects of oral SCCs on osteoclast function. Both BHY cells, a human oral SCC cell line, and its conditioned medium (BHY-CM) stimulated osteoclast survival by suppressing Bim, a pro-apoptotic protein, depending on extracellular signal-regulated kinase (ERK) and multinucleation. Adding BHY cells but not BHY-CM induced pit-forming activity by osteoclasts and adding OPG abrogated the activity. Thus, oral SCC cells regulate not only osteoclastogenesis but also its function.

Topics: Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carcinoma, Squamous Cell; Cell Survival; Humans; Immunoblotting; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; Osteoclasts; Osteoprotegerin; Proto-Oncogene Proteins; RANK Ligand; Tumor Cells, Cultured

The mechanism of oral squamous cell carcinoma (SCC) invading jawbone remains controversial. Interactions between receptor activator of NF-kappaB (RANK) and its ligand (RANKL) are required for osteoclastogenesis. The binding of RANK and RANKL induces differentiation of osteoclasts, leading to bony destruction. Osteoprotegerin (OPG), a decoy receptor for RANKL, also binds to RANKL by competing with RANK, and this could protect against osseous destruction.. Immunoexpression of RANKL, RANK, and OPG in 25 cases of human buccal SCCs without bony invasion and 15 cases of gingival SCCs with mandibular bony invasion was investigated. Normal oral mucosa from five individuals without betel-quid chewing or cigarette smoking was used as a control. The scores are designated as percentage of positive staining x intensity of staining for each section.. Strong cytoplasmic staining of RANKL proteins is detected in cancer cells of both buccal and gingival SCCs. The same protein is identified in cytoplasm of osteoclasts for all cases involving bony invasion. Strong cytoplasmic staining of RANKL is confined to basal layer for all normal mucosa. A similar staining pattern is noted for RANK protein in all buccal and gingival SCCs. An absence of staining of RANK protein is noted for all normal tissues. Weak to negative cytoplasmic stained OPG protein is present in all buccal and gingival SCCs, but is absent in all normal tissues.. These findings suggest the potential value of the RANK/RANKL/OPG pathway as biomarkers in human oral SCCs.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Differentiation; Coloring Agents; Cytoplasm; Female; Gingival Neoplasms; Humans; Immunohistochemistry; Male; Mandibular Neoplasms; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Staging; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B

To investigate the osteoclastic activity in oral squamous cell carcinoma (OSCC) invading the jaws and to observe the expression of RANKL and OPG, the two bone resorption related cytokines, in these cases.. Twelve cases of OSCC invading the mandible were studied. After pathological diagnosis, operations were done to remove part of the mandible depending upon the X-ray findings. Fresh soft tissue specimens were frozen-sectioned and other specimens with the bone tissue were fixed and decalcified to make paraffin sections. Tartrate-resistant acid phosphatase(TRAP) staining and immunohistochemical staining were then applied to observe the location of osteoclasts and the expression of RANKL and OPG.. TRAP positive multinuclear cells were detected near the interface between the bone and tumor. RANKL positive cells were commonly seen on the endothelium of blood vessel and basement membrane of the epithelium. But OPG reactivities were not seen in these sections.. The bone destruction caused by OSCC is mediated by osteoclasts but not by cancer cell itself. It appears that the differentiation and activation of osteoclasts were induced by OSCC through cytokines like RANKL.

Topics: Aged; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Male; Mandible; Middle Aged; Mouth Neoplasms; Neoplasm Invasiveness; Osteoclasts; Osteoprotegerin; RANK Ligand

The invasion of oral squamous cell carcinoma (SCC) cells into the mandibular bone is a common clinical problem. It has been reported that BHY cells, a human oral SCC cell line, are capable of invading mandibular bone of nude mice. These results led us to examine possible mechanisms of osteoclastogenesis induced by BHY cells using in vitro culture systems. When BHY cells were cocultured with mouse bone marrow cells (BMCs), only few osteoclasts were formed, even though BHY cells express the receptor activator of NF-kappaB ligand (RANKL). However, adding BHY cells to a coculture of mouse primary osteoblasts (POBs) and BMCs markedly induced osteoclastogenesis in the absence of osteotropic factors. Furthermore, another oral SCC cell line, HSC-2, which does not express RANKL, also induced osteoclastogenesis in our cocultures. These effects were significantly, but not completely, inhibited by adding osteoprotegerin (OPG). In addition, we also found that TNFalpha released from these cells partially contributes to osteoclastogenesis via a RANKL-independent mechanism. Adding BHY or HSC-2 cells suppressed mouse OPG mRNA expression and protein production by POBs in cocultures of POBs and human oral SCC cells. This finding is consistent with the result that BHY cells and HSC-2 cells did not enhance osteoclastogenesis in cocultures of BMCs and POBs from OPG-deficient mice. Immunohistochemical analysis showed a reduction of OPG expression in osteolytic lesions as compared to normal lesions from oral SCC patients. Therefore, oral SCC-induced suppression of OPG expression in POBs appears critical for osteoclastogenesis, rather than expression of RANKL in SCC cells.

Topics: Animals; Bone Marrow Cells; Carcinoma, Squamous Cell; Carrier Proteins; Cell Communication; Cell Differentiation; Down-Regulation; Gene Expression Regulation, Neoplastic; Glycoproteins; Immunohistochemistry; Membrane Glycoproteins; Mice; Mouth Neoplasms; Neoplasm Invasiveness; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

This study examined the mechanisms of osteoclast-mediated bone invasion in a model of oral squamous cell carcinoma (OSCC). C3H/HeN mice were inoculated with SCC VII cells into the masseter region to establish an animal model of mandibular invasion by OSCC.. The mice were divided into three groups: a control group, given daily s.c. injections of saline; group 1, given 2 microg per mouse per day of the bisphosphonate YM529; and group 2, given 10 microg per mouse per day of YM529. After 3 weeks of treatment, the lesions were studied by micro-computed tomography. After tartrate-resistant acid phosphatase (TRAP) staining, the osteoclasts were easily identified, and the percentages of the area occupied by osteoclasts were calculated by computer for each sample. The tumors were analyzed by RT-PCR to determine the mRNA expression of interleukin-6 (IL-6), parathyroid hormone-related protein (PTHrP), tumor necrosis factor-alpha (TNF-alpha), receptor activator of nuclear factor-kappaB (RANK), RANK ligand (RANKL), and osteoprotegerin.. SCC VII cells rapidly multiplied in the masseter muscle of the mice. Bone invasion was evident only in the control group on micro-computed tomography. On TRAP-stained slices, the percentages of osteoclasts in groups 1 and 2 were significantly lower than that in the control group. The mRNA expressions of IL-6, PTHrP, THF-alpha, and RANK decreased as the concentration of YM529 increased.. We conclude that various cancer-derived cytokines play important roles in the invasion of bone by OSCC. YM529, a third-generation bisphosphonate, can suppress osteoclast-mediated bone invasion by OSCC. The mechanism of this effect might involve inhibition of cytokines such as IL-6, PTHrP, TNF-alpha, and RANK by YM529.

Topics: Animals; Body Weight; Bone Resorption; Carcinoma, Squamous Cell; Carrier Proteins; Cell Line, Tumor; Diphosphonates; Disease Models, Animal; Gene Expression; Glycoproteins; Imaging, Three-Dimensional; Imidazoles; Interleukin-6; Male; Mandible; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Mouth Neoplasms; Neoplasm Invasiveness; Osteoclasts; Osteoprotegerin; Parathyroid Hormone-Related Protein; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tomography, X-Ray Computed; Tumor Necrosis Factor-alpha