MicroRNA-125b regulates proliferation and radioresistance of oral squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs) are involved in essential biological activities, and have been reported to exhibit differential expression profiles in various cancers. Our previous study demonstrated that intercellular adhesion molecule-2 (ICAM2) inhibition induces radiosensitisation in oral squamous cell carcinoma (OSCC) cells. Thus, we hypothesised that certain miRNAs play crucial roles in radioresistance in OSCC by regulating ICAM2 expression. METHODS: Because predicted target gene analyses revealed that microRNA-125b (miR-125b) potentially regulates ICAM2 mRNA expression, we examined the association between miR-125b and radioresistance. The expression of miR-125b was investigated by real-time quantitative reverse transcriptase-PCR. For a functional analysis, miR-125b was transfected to OSCC-derived cells. RESULTS: A downregulated expression of miR-125b was found in OSCC-derived cell lines and OSCC samples. The miR-125b-transfected cells showed a decreased proliferation rate, enhanced radiosensitivity to X-ray irradiation and diminished ICAM2 mRNA expression. Moreover, miR-125b expression correlated with OSCC tumour staging and survival. CONCLUSION: These findings suggested that the downregulated miR-125b expression was associated with proliferation and radioresistance mechanisms, probably through ICAM2 signalling. Thus, controlling the expression or activity of miR-125b might contribute to suppressing proliferation and overcoming radioresistance in OSCC.

Oncolytic activity of Sindbis virus in human oral squamous carcinoma cells.

BACKGROUND: Sindbis virus (SIN) infection causes no or only mild symptoms (fever, rash, and arthralgia) in humans. However, SIN has a strong cytopathic effect (CPE) on various cancer cells. This study focuses on the oncolytic activity of SIN AR399 on oral cancer cells compared with reovirus, a well-known oncolytic virus that targets cancer cells. METHODS: We analysed the cytotoxicity and growth of SIN in 13 oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, H-1, Sa-3, KON, KOSC-2, OK-92, HO-1-N1, SCC-4, SAT, SKN-3) and normal human oral keratinocytes (NHOKs). RESULTS: Sindbis virus infection induced CPE in 12 OSCC cell lines at a low multiplicity of infection (MOI) of 0.01, but not in the OSCC cell line, HSC-4 or NHOKs. Sindbis viral growth was not observed in NHOKs, whereas high SIN growth was observed in all OSCC cell lines, including HCS-4. The cytotoxicity and growth of SIN was the same as reovirus at an MOI of 20 in 12 OSCC cell lines. The CPE was shown, by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assays, to be apoptotic cell death. Furthermore, quantitative RT-PCR of mRNA in HSC-3 and HSC-4 cells after SIN infection showed that activation of caspases, cytochrome c, and IkappaBalpha was associated with SIN-induced apoptosis. CONCLUSION: As a replication-competent oncolytic virus, SIN may be a useful therapeutic modality for oral cancers.

Aberrant Collagen Cross-linking in Human Oral Squamous Cell Carcinoma.

Tumor progression is a complex process involving extracellular matrix (ECM) remodeling and stiffening. However, the mechanisms that govern these processes and their roles in tumor progression are still poorly understood. In this study, we performed bioinformatics, immunohistochemical, and biochemical analyses to examine if collagen cross-linking is associated with tumor stage and regional lymph node metastasis (RLNM) in oral squamous cell carcinoma (OSCC). We found that the genes encoding key enzymes for cross-linking are frequently overexpressed in oral, head, and neck cancers. Specifically, the enzymes lysyl hydroxylase 2 (LH2) or lysyl oxidase (LOX) and LOX-like 2 (LOXL2) were significantly upregulated in late-stage tumors and associated with poor patient prognosis. The protein levels of these enzymes in the primary human OSCC were also significantly increased in late-stage tumors and markedly elevated in the RLNM-positive tumors. Notably, while overall LOX/LOXL2-catalyzed collagen cross-links were enriched in late-stage and RLNM-positive tumors, LH2-mediated stable cross-links were significantly increased. To our knowledge, this is the first study to investigate the association of collagen cross-linking and expression of key enzymes regulating this process with OSCC stage. The data indicate a critical role for collagen cross-linking in OSCC tumor progression and metastasis, which may provide insights into development of novel therapeutic strategies to prevent OSCC progression.

Inhibition of ICAM2 induces radiosensitization in oral squamous cell carcinoma cells.

We recently identified genes and molecular pathways related to radioresistance of oral squamous cell carcinoma (OSCC) using Affymetrix GeneChip. The current study focused on the association between one of the target genes, intercellular adhesion molecule 2 (ICAM2), and resistance to X-ray irradiation in OSCC cells, and evaluated the antitumor efficacy of combining ICAM2 small interfering RNA (siRNA) and X-ray irradiation. Downregulation of ICAM2 expression by siRNA enhanced radiosensitivity of OSCC cells with the increased apoptotic phenotype via phosphorylation (ser473) of AKT and activation of caspase-3. Moreover, overexpression of ICAM2 induced greater OSCC cell resistance to the X-ray irradiation with the radioresistance phenotype. These results suggested that ICAM2 silencing is closely related to sensitivity of OSCC cells to radiotherapy, and that ICAM2 may be an effective radiotherapeutic target for this disease.

Monophasic epithelial synovial sarcoma arising in the temporomandibular joint.

Synovial sarcoma is a mesenchymal spindle-cell tumour that occurs infrequently in the head and neck. It originates from unknown stem cells differentiating into mesenchymal and/or epithelial structures. Most synovial sarcomas are biphasic in character, consisting of epithelial and spindle-cell elements. Here is reported a case of monophasic epithelial synovial sarcoma arising in the temporomandibular joint. The tumour was of a predominantly epithelial pattern, although a minute area of sarcomatous cells was found. The primary mode of treatment was wide en-bloc excision. Two years after surgery, the patient died of hepatocellular carcinoma, but there was no evidence of synovial sarcoma recurrence.

Localization of a novel tumor suppressor gene associated with human oral cancer on chromosome 4q25.

Recent cytogenetic and molecular studies with highly polymorphic microsatellite markers have implicated allele loss involving chromosome 4 in several human cancers, which suggests the presence of multiple tumor suppressor gene (TSG) loci. However, there has been no detailed analysis of loss of heterozygosity (LOH) on chromosome 4 in oral squamous cell carcinoma (OSCC). To determine the location of a putative TSG associated with OSCC on chromosome 4, polymerase chain reaction (PCR) analysis of microsatellite polymorphisms corresponding to 17 loci was performed to screen 32 patients with OSCC. LOH was observed in the majority of the tumors (75%) in at least one of the loci. The loci on the long arm exhibited a significantly higher frequency of deletions (66%) than those of the short arm (25%). Among the loci tested, frequent LOH was centered at D4S1573 on 4q25, which represents a region of about 4 centimorgans (cM). However, no commonly deleted regions were found on the short arm of the chromosome. We detected microsatellite instability (MI) in 31% of the cases. MI was also observed more frequently on the long arm (28%) than the short arm (6%). Thus, our data indicate that alterations of chromosome 4 regions, especially the long arm, are associated with OSCC tumorigenesis and that the 4q25 region may harbor at least one putative TSG.

Normal human cementum-derived cells: isolation, clonal expansion, and in vitro and in vivo characterization.

Cultures of primary human cementum-derived cells (HCDCs) were established from healthy premolar teeth extracted for orthodontic reasons. Cementum was manually dissected, fragmented, and digested twice with collagenase. Following a thorough wash to remove liberated cells, the remaining cementum fragments were plated in Dulbecco's modified Eagle's medium/F12 medium containing 10% fetal bovine serum. Discrete colonies that contained cells exhibiting fibroblast-like morphology were visible after 14-21 days of culture. When the colonies became sufficiently large, cells from individual colonies were isolated and subcultured. Cementum-derived cells exhibited low levels or no alkaline phosphatase activity and mineralized in vitro to a lesser degree than human periodontal ligament (PDL) cells and human bone marrow stromal cell (BMSC) cultures. To study differentiation capacities of HCDCs, cells were attached to hydroxyapatite/tricalcium phosphate ceramic and transplanted subcutaneously into immunodeficient mice. The transplants were harvested 3, 6, and 8 weeks after transplantation and evaluated histologically. In human BMSC transplants, new bone tissue was formed with a prominent osteoblastic layer and osteocytes embedded in mineralized bone matrix. No osseous tissue was formed by PDL cells. Of six single colony-derived strains of HCDCs tested, three formed a bone-like tissue that featured osteocyte/cementocyte-like cells embedded within a mineralized matrix and which was lined with a layer of cells, although they were somewhat more elongated than osteoblasts. These results show that cells from normal human cementum can be isolated and expanded in vitro. Furthermore, these cells are capable of differentiating and forming mineralized tissue when transplanted into immunodeficient mice.

Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro.

The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen alpha chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of alpha chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.

Cementum-forming cells are phenotypically distinct from bone-forming cells.

Normal human cementum-derived cells (HCDCs), expanded in vitro, formed mineralized matrix when attached to a ceramic carrier and transplanted subcutaneously into immunodeficient mice. The mineralized matrix elaborated by transplanted HCDC exhibited several features identical to cementum in situ and was significantly different from bone deposited by similarly transplanted human bone marrow stromal cells (BMSCs). No bone marrow formation and very few or no tartrate-resistant acid phosphatase (TRAP)-positive cells (osteoclasts and osteoclastic precursors) were found in HCDC transplants. In contrast, in BMSC transplants both hematopoiesis and TRAP-positive cells were routinely observed. Furthermore, compared with BMSC-derived matrix, HCDC-derived matrix was less cellular, numerous empty lacunae were present, and fewer cells were found on the cementum matrix/ceramic carrier interface. The organization of collagen fibers in HCDC-derived matrix, as visualized by using the Picrosirus red staining method, was similar to cementum, with typical unorganized bundles of collagen fibers. In contrast, bone matrix elaborated by transplanted BMSC had lamellar structure, identical to mature bone in situ. Finally, cementocytes embedded in the cementum-like matrix were immunopositive for fibromodulin and lumican, whereas osteocytes within the bonelike matrix were negative. This pattern is consistent with the cementum and bone in situ, respectively. These results indicate that human cementum cells are phenotypically distinct from bone cells and provide further validation of the combined in vitro/in vivo model of human cementogenesis recently developed in our laboratory.

Differential expression of decorin and biglycan genes during mouse tooth development.

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.

Low levels of NPM gene expression in UV-sensitive human cell lines.

Nucleophosmin (NPM) is a major nuclear matrix protein associated with neoplastic growth in various cell types. We recently suggested that expression of the NPM gene is involved in an increased resistance to UV irradiation in human cells against the cell-killing effects of UV (mainly 254nm wavelength far-ultraviolet ray) [Y. Higuchi, K. Kita, H. Nakanishi, X-L. Wang, S. Sugaya, H. Tanzawa, H. Yamamori, K. Sugita, A. Yamaura, N. Suzuki, Biochem. Biophys. Res. Commun. 248 (1998) 597-602]. In the present study, expression levels of the NPM gene were examined in human cell lines with a high sensitivity to UV cell-killing. Cockayne syndrome patient-derived cell lines, CSAI and CSBI, and the Xeroderma pigmentosum patient-derived cell line, XP2OS(SV), XP13KY, XP3KA, XP6BE(SV), XP101OS and XP3BR(SV), have been investigated for their NPM mRNA expression with Northern blotting analysis. All of these UV-sensitive cells demonstrated lower expression levels compared with those of normal fibroblast cells, FF, or an UV-resistant cell line, UH(r)-10; quite a lower level of expression in XP205(SV) cells after UV irradiation in contrast to a distinguishable increase in the expression in UV(r)- cells. These results confirmed an intimate correlation between degree of UV sensitivity and expression levels of the NPM gene in human cells.

Allelic imbalance on the long arm of chromosome 5 in oral squamous cell carcinoma.

To obtain a more detailed estimate of chromosome 5 loci involvement in oral squamous cell carcinoma (SCC), thirty-two oral SCCs were examined for loss of heterozygosity (LOH) on the long arm of chromosome 5 (5q) by PCR-LOH assay using thirteen microsatellite markers. LOH was observed in 16 (51.6%) of 31 informative cases. The incidence or the number of regions showing LOH was significantly higher in moderately and poorly differentiated tumors than in the well differentiated ones. Among the loci tested, D5S178 exhibited LOH in 11 (39.3%) of 28 cases, suggesting this to be the site, at 5q, of a novel candidate tumor suppressor gene. These data indicate that the incidence of LOH at chromosome 5q is high and is associated with oral tumor differentiation.

Mutational state of p16/cdkn2 and vhl genes in squamous-cell carcinoma of the oral cavity.

Two new tumor-suppressor genes, the cyclin dependent kinase 4 inhibitor gene (p16/CDKN2) and the von Hippel-Lindau disease gene (VHL), have been cloned and mapped on chromosomes 9p and 3p respectively, where putative tumor-suppressor genes of the oral squamous-cell carcinoma (SCC) may be present. In order to elucidate whether abnormalities of these genes could contribute to the tumorigenesis of oral SCC, genomic DNAs from 62 tissue samples of tumors (32 primary SCCs and 30 pre-cancerous lesions) and from 7 oral SCC cell lines were examined by polymerase chain reaction-single strand conformation polymorphism analysis and direct DNA sequencing. Four of 7 (57%) cell lines contained nonsense mutations or missense mutations in the p16/CDKN2 gene and 2 of 32 (6%) primary oral SCCs had nonsense mutations. Particularly, 3 of 4 nonsense mutations detected in the present study were found in codon 80 (CGA-->TGA; Arg-->Stop), suggesting that codon 80 was a mutational hot spot of the p16/CDKN2 gene. No VHL gene mutation was found in any subject. These results suggest that mutation of the VHL gene is not a common factor in the development of human oral SCC. In contrast, the p16/CDKN2 gene may be correlated with the progression of a subtype of this cancer.

Reduced expression of E-cadherin in oral squamous cell carcinoma: relationship with DNA methylation of 5' CpG island.

E-cadherin, a Ca2+-dependent cell-cell adhesion molecule, is involved in the maintenance of the epithelial phenotype. The reduction of its expression is considered to be important in the invasive and metastatic potential of carcinomas. A series of 52 primary oral squamous-cell carcinomas (SCCs) were studied immunohistochemically for E-cadherin expression in microwave-treated paraffin-embedded sections using a monoclonal antibody (HECD-1). Significant correlation was found between reduced E-cadherin expression and tumor dedifferentiation, grade of invasiveness, and lymph node metastasis. The possibility of E-cadherin gene mutation was also investigated by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), but none was found. In addition, to clarify the CpG methylation state around E-cadherin gene promoter in oral SCC, we examined DNA samples by PCR assay after restriction digestion with methylation-sensitive enzyme HpaII. CpG methylation was found in 9 (17%) of 52 primary oral SCCs, but not in the corresponding normal tissues. In particular, 8 of the 9 methylated cases showed reduced expression of E-cadherin and histologically diffuse invasion type of tumor. These results suggest that reduction of E-cadherin expression is associated with the progression of human oral SCCs, and CpG methylation of E-cadherin gene promoter causes reduction of E-cadherin expression in the tumor, resulting in acquisition of the invasive phenotype.

Rare mutations of the growth suppressor genes involved in negative regulation of the cell cycle.

The growth suppressing activity of the retinoblastoma susceptibility gene product, pRb, is down regulated by cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) whose activity is negatively regulated by CDK inhibitors of the p16 family. We have previously reported point mutations of the p16/CDKN2 gene in 4 (57%) of 7 oral squamous cell carcinoma (SCC) cell lines. In the current study, we examined the mutational status of CDK inhibitors, including 3 genes of the p16 family (p16, p15 and p18), in 50 human oral SCCs, and also additional results concerning their loss of heterozygosity in the regions of the p16, p15 and p18 genes. Our results demonstrated that 2 of 50 (4%) primary oral SCCs had nonsense mutations of the p16 gene, and 2 of 50 (4%) showed frameshift mutations of the p18 gene. However, we detected no mutation of the p15 gene in any of the 50 oral SCCs. In addition, no evidence of hypermethylation of the p16 gene was found in our series. To better understand the extent of alterations affecting chromosomes 9p21 (location of the p15/p16 genes) and 1p32 (location of the p18 gene), loss of heterozysity (LOH) on these locations was examined. LOH was detected in 16 of 34 (47%) informative samples that had no detectable mutation of the p15/p16 genes on 9p21, but we found no LOH at 1p32. These results strongly suggest that a putative tumor suppressor gene for oral SCC may be present on chromosome 9p21-22, while the p16, p15 and p18 genes play a minor role in the oncogenesis of this cancer.

Expression of an inhibitor of apoptosis, survivin, in oral carcinogenesis.

A novel inhibitor of apoptosis, survivin, plays a role in oncogenesis. To determine the potential involvement of survivin in oral carcinogenesis, we investigated the distribution of survivin protein expression in oral squamous cell carcinomas (OSCCs) and oral pre-malignant lesions. The mRNA expression level and methylation status of the gene also were evaluated in OSCCs and OSCC-derived cell lines. In immunohistochemistry, 58% of tumors and 37% of pre-malignant lesions examined were positive for survivin, while no immunoreaction was observed in corresponding normal tissues. The reverse-transcription/polymerase chain-reaction revealed similar changes in survivin gene expression levels. Furthermore, of the 9 normal oral tissues with no survivin gene expression, 4 showed methylation of the gene, while no methylation was detected in the corresponding tumorous tissues. The results suggest that survivin plays an important role during oral carcinogenesis, and that the gene expression may be regulated by an epigenetic mechanism.

Frequent microsatellite instability in oral cancer.

In oder to confirm whether microsatellite instability (MI) contributes to oral carcinogenesis, we studied 30 unrelated patients with oral cancer by PCR-MI assay with 14 microsatellite markers. MI was detected in 60% of 30 primary tumors. Tn particular, 12 of these cases presented at least two loci with MI, which were considered as patients with replication error (RER). Moreover, an additional MI, which was not observed in the primary tumor and normal tissue, was observed in one lymph node metastasis. We found significant correlation between RER and lymph node metastasis. These results suggest that RER is a common event in the oncogenesis of oral cancer and may be correlated with the progression of this disease.

Frequent allelic loss/imbalance on the long arm of chromosome 21 in oral cancer: evidence for three discrete tumor suppressor gene loci.

Frequent allelic imbalances including loss of heterozygosity (LOH) and microsatellite instability (MI) on the long arm of chromosome 21 (21q) have been found in several types of human cancer. This study was designed to identify tumor suppressor locus (or loci) associated with oral squamous cell carcinoma (SCC) on 21q. Among 38 patients with oral SCC tested, 15 (44%) of 34 informative cases showed LOH at one or more loci. Deletion mapping of these 15 tumors revealed three discrete commonly deleted regions on the chromosome arm. A minimal region with frequent LOH was found at the marker D21S236 mapped on 21q11.1. Another region of frequent deletion was identified between markers D21S11 and D21S1436 on 21q21, and a further commonly deleted region was found at D21S1254 on 21q22.1. In addition, we have detected MI on the chromosome arm in our oral SCC samples with significant correlation with tumor stage. Thus, our results strongly suggest that allelic imbalances on 21q may be involved in the development of oral SCC; and that at least three different putative tumor suppressor genes contributing to the pathogenesis of this disease are present on 21q.

Evidence of multi-step oncogenesis of oral squamous cell carcinoma.

We examined biopsy samples from one oral cancer and three precancerous lesions of the tongue of an 81-year old woman by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequence analyses using 18 oligonucleotide primer pairs of adenomatous polyposis coli (APC) gene and 5 primers of p53 gene. Normal tongue epithelium adjacent to lesions was used as a control. The four lesions harbored the common mutation of APC gene that was not detected in the control. At codon 1621 in exon 15 of the APC gene there was a C to G substitution resulting in serine (TCA) to stop codon (TGA). No mutation of p53 gene was detected in any samples of the control and three precancerous lesions of the tongue. On the other hand, an A to G substitution at codon 170 in exon 5 of p53 gene resulting in glutamic acid (ACG) to glycine (GCG) was detected in the DNA of her tongue cancer. These results may suggest that the four lesions have the same origin, and that multi-step oncogenesis had occurred, the APC gene alteration being one of the early events in the process of tumorigenesis and p53 gene alteration involved in the late events.

Allelic loss on the short arm of chromosome 8 in oral squamous cell carcinoma.

Allelic deletions on the short arm of chromosome 8 (8p) are frequent events in many different types of malignant tumors, including head and neck tumors and oropharyngeal cancers. These regions are thought to harbor tumor suppressor genes. However, there has been no detailed analysis of loss of heterozygosity (LOH) on the chromosome arm 8p in oral squamous cell carcinoma (SCC). In order to estimate details of 8p loci involved in oral SCC, 32 patients with oral SCCs were examined for the LOH state 8p by PCR-LOH assay using 14 microsatellite markers. Based on our results a detailed deletion map of 8p showed LOH in at least one of the loci tested in 62.5% (20 of 32) of patients; and microsatellite instability (MI) was observed in 50% (16 of 32). The frequent LOH on this chromosome arm was identified at D8S87 and D8S258, mapped on 8p12 and 8p22, respectively. Fisher's exact test revealed no significant statistical correlation between the incidence of LOH or MI and clinicopathological features. Our observations indicate that the short arm of chromosome 8 may play a role in the pathogenesis of oral SCC; and that the two loci identified in this study may be tumor suppressor gene loci on 8p.