Truncated P-cadherin is produced in oral squamous cell carcinoma

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

Cyclooxygenase-2 is involved in S100A2-mediated tumor suppression in squamous cell carcinoma

S100A2 is considered a putative tumor suppressor due to its loss or down-regulation in several cancer types. However, no mechanism has been described for the tumor suppressor role of S100A2. In this study, ectopic expression of S100A2 in the human malignant squamous cell carcinoma cell line KB resulted in a significant inhibition of proliferation, migration, and invasion. Moreover, S100A2 significantly reduced the number of colonies (>or=0.5 mm) formed in semisolid agar and decreased tumor growth and burden in nude mice. cDNA microarray analysis was used to compare mRNA expression profiles of vector- and S100A2-expressing isogenic cells. Among the genes deregulated by S100A2, the expression of cyclooxygenase-2 (COX-2) mRNA was significantly suppressed by S100A2 (2.4-fold). Western blot analysis confirmed that S100A2 reduced the expression of COX-2 protein in stably and transiently transfected KB and RPMI-2650 cells. COX-2 is frequently overexpressed in various types of cancer and plays an important role in tumor progression. Partial restoration of COX-2 expression attenuated the antitumor effect of S100A2 both in vitro and in vivo. Although the interplay between S100A2 and COX-2 remains to be clarified, these findings first showed a potent antitumor role of S100A2 in squamous cell carcinoma partly via reduced expression of COX-2.     

Podocalyxin is crucial for the growth of oral squamous cell carcinoma cell line HSC-2

Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies and therapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal, breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and poor outcomes of oral cancers. PODXL overexpression was strongly detected using our previously established anti-PODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG_2a-type, 47-mG_2a. In previous studies, we also generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the function of PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to be lower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2 cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth was observed for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The influence of PODXL on tumor growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed to be significantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together, indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo.     

Heterogeneity of cancer-associated fibroblasts in head and neck squamous cell carcinoma

Cancer-associated fibroblasts (CAFs) consist of heterogeneous cellular populations that contribute critical roles in head and neck squamous cell carcinoma (HNSCC). A series of computer-aided analyses were performed to determine various aspects of CAFs in HNSCC, including their cellular heterogeneity, prognostic value, relationship with immune suppression and immunotherapeutic response, intercellular communication, and metabolic activity. The prognostic significance of CKS2+ CAFs was verified using immunohistochemistry. Our findings revealed that fibroblasts group demonstrated prognostic significance, with the CKS2+ subset of inflammatory CAFs (iCAFs) exhibiting a significant correlation with unfavorable prognosis and being localized in close proximity to cancer cells. Patients with a high infiltration of CKS2+ CAFs had a poor overall survival rate. There is a negative correlation between CKS2+ iCAFs and cytotoxic CD8+ T cells and natural killer (NK) cells, while a positive correlation was found with exhausted CD8+ T cells. Additionally, patients in Cluster 3, characterized by a high proportion of CKS2+ iCAFs, and patients in Cluster 2, characterized by a high proportion of CKS2- iCAFs and CENPF-/MYLPF- myofibroblastic CAFs (myCAFs), did not exhibit significant immunotherapeutic responses. Moreover, close interactions was confirmed to exist between cancer cells and CKS2+ iCAFs/ CENPF+ myCAFs. Furthermore, CKS2+ iCAFs demonstrated the highest level of metabolic activity. In summary, our study enhances the understanding of the heterogeneity of CAFs and provided insights into improving the efficacy of immunotherapies and prognostic accuracy for HNSCC patients.     

Role of EZH2 in oral squamous cell carcinoma carcinogenesis

Oral squamous cell carcinoma (OSCC) is a common human malignancy with high incidence rate and poor prognosis. Although the polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a crucial role in cell proliferation and differentiation during the occurrence and development progress of several kinds of malignant tumors, the impact of EZH2 on the development and progression of OSCC is unclear. In this study, we demonstrate that EZH2 is overexpressed in OSCC cells and clinical tissue. With in vitro RNAi analysis, we generated stable EZH2 knocking down cell lines from two OSCC cell lines, with two sh-RNAs targeting to EZH2, respectively. We found that knocking down of EZH2 could decrease the proliferation ability and induce apoptosis of OSCC cells. Moreover, we demonstrated that of EZH2 inhibition decreased the migration and metastasis of OSCC cells. In conclusion, the results of the current study demonstrated an association between EZH2 expression and OSCC cell development. We recommend that EZH2 acts as an oncogene and plays an important role in OSCC carcinogenesis.     

Toll-like receptor 2 activation implicated in oral squamous cell carcinoma development

Recent studies have revealed that Toll-like receptors (TLRs) are highly expressed and activated in many types of cancer. Physiologically, TLR2 recognizes bacteria and other microorganisms in the oral cavity; however, the role of TLR2 in oral squamous cell carcinoma (OSCC) is unclear. In this study, we demonstrated that TLR2 is highly expressed in OSCC in comparison with adjacent non-malignant tissue. TLR2 was also expressed in OSCC-derived cell lines, and its expression was activated by ligands derived from bacteria and mycoplasma. Furthermore, to elucidate the mechanism of OSCC progression via TLR2 signal transduction, we focused on microRNAs (miRNAs) that are induced by TLR2 activation. Interestingly, ligand activation of TLR2 induced the expression of miR-146a and we found that downregulation of caspase recruitment domain–containing protein 10 (CARD10) mRNA in OSCC-derived cell lines. Moreover, knockdown of CARD10 induced resistance to cisplatin-induced apoptosis in OSCC cells. These findings suggest that the activation of TLR2 by bacterial components can enhance the progression of OSCC and may be implicated in acquired resistance to cisplatin-induced apoptosis through regulation of the miR-146a pathway.     

SATB2 overexpression promotes oral squamous cell carcinoma progression by up-regulating NOX4

While atypical expression of special AT-rich sequence-binding protein 2 (SATB2) has been approved associated with tumor progression, metastasis and unfavourable prognosis in various carcinomas. However, in oral squamous cell carcinoma (OSCC), both the expressive state and associated functions of SATB2's are still undefined. Here we show that, in clinical samples from a retrospective cohort of 58 OSCC patients, high expression of SATB2 is associated with poor prognosis of OSCC patients. In this study, we investigated SATB2 is highly expressed in OSCC tissues and cell lines, which can promote OSCC cells' proliferation, migration, invasion and tumor growth. According to sequencing results based on previous literature, we identified NOX4 is a bona fide downstream target of SATB2, when it was knockdown, OSCC's proliferation can be partially suppressed. Furthermore, NOX4 knockdown inhibits tumorigenicity, which can be rescued partially by ectopic expression of SATB2 in HNSCC cell line, and vice versa. Collectively, our findings not only indicate overexpression of SATB2 triggers the proliferative, migratory and invasive mechanisms which are important in the malignant phenotype of OSCC, but also identify NOX4 as the downstream gene for SATB2. These findings indicate that SATB2 may play a key role in OSCC tumorigenicity and may be a future target for the development of new therapeutic regimens.     

Cancer stem cell model in oral squamous cell carcinoma

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

Crosstalk between oral squamous cell carcinoma cells and cancer-associated fibroblasts via the TGF-β/SOX9 axis in cancer progression

Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial–mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-β1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-β1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-β/SOX9 axis.     

IGF2BP2 maybe a novel prognostic biomarker in oral squamous cell carcinoma

Aim: The main of the present study was to investigate the role of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) in oral squamous cell carcinoma (OSCC) with the overarching of providing new biomarkers or potential therapeutic targets for OSCC.Methods: We combined datasets downloaded from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and samples collected from the clinic to evaluate the expression of IGF2BP2 in OSCC. IGF2BP2 survival analysis was respectively performed based on TCGA, GEO, and clinical samples. Correlations between IGF2BP2 expression and clinicopathological parameters were then analyzed, and signaling pathways associated with IGF2BP2 expression were identified using gene set enrichment analysis (GSEA 4.1.0). Moreover, an IGF2BP2 co-expressed gene network was constructed, followed by gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on IGF2BP2 co-expressed genes. Finally, TIMER and CIBERSORT were used to analyze the correlations among IGF2BP2, IGF2BP2-coexpressed genes, and tumor-infiltrating immune cells (TICs).Results: IGF2BP2 was highly expressed in OSCC and significantly correlated with overall survival of OSCC patients (P<0.01). High IGF2BP2 expression correlated with poor overall survival. The GSEA results showed that cell apoptosis-, tumor-, and immune-related pathways were significantly enriched in samples with high IGF2BP2 expression. Furthermore, GO and KEGG enrichment analyses results of IGF2BP2 co-expressed genes indicated that these genes are mainly associated with immunity/inflammation and tumorigenesis. In addition, IGF2BP2 and its co-expressed genes are associated with TICs (P<0.01).Conclusion: IGF2BP2 may be a potential prognostic biomarker in OSCC and correlates with immune infiltrates.     

Expression of Snail is associated with myofibroblast phenotype development in oral squamous cell carcinoma

Snail is a regulator of epithelial–mesenchymal transition (EMT) and considered crucial to carcinoma metastasis, myofibroblast transdifferentiation, and fibroblast activation. To investigate the role of Snail in oral squamous cell carcinoma (OSCC), its immunohistochemical expression was analysed in 129 OSCC samples and correlated to nodal metastasis, histological grade, E-cadherin, and alpha smooth-muscle-actin (αSMA). The results were compared to findings in 23 basal cell carcinomas (BCC). Additionally, the influence of TGFβ1 and EGF on Snail, E-cadherin, vimentin, and αSMA expression was analysed in two OSCC cell lines. As a result, Snail-positive cells were mainly found in the stroma of the OSCC invasive front without statistically significant correlation to histological grade or nodal metastasis. Snail was co-localised to αSMA but not to E-cadherin or cytokeratin and showed a significant correlation to the loss of membranous E-cadherin. All BCCs were Snail negative. In OSCC culture, the growth-factor-mediated EMT-like phenomenon was accompanied by αSMA down-regulation. In summary, Snail expression in OSCC is a stromal phenomenon associated with the myofibroblast phenotype and not related to growth-factor-mediated transdifferentiation of the carcinoma cells themselves. Consequently, Snail immunohistochemistry cannot contribute to the prediction of the metastatic potential. Furthermore, stromal Snail expression is suggested to be the result of mutual paracrine interaction of fibro-/myofibroblasts and dedifferentiated carcinoma cells leading to the generation of a special type of carcinoma-associated fibroblasts. M. Franz and K. Spiegel have contributed equally to the study.     

Nuclear survivin promoted by acetylation is associated with the aggressive phenotype of oral squamous cell carcinoma

Defects in apoptotic pathway contribute to development and progression of oral cancer. Survivin, a member of the inhibitors of apoptosis protein (IAP) family, is increased in many types of cancers. However, it is unclear whether increased survivin is associated with oral squamous cell carcinomas (OSCC), and what mechanisms may involve in. In this study, we examined survivin expression in OSCC compared with normal oral tissues via immunohistochemical staining. The results showed that, not only total survivin is increased in OSCCs, but also the subcellular location of survivin is changed in OSCCs compared with normal oral tissues. In most of normal oral tissues, survivin staining was either negative, or cytoplasmic positive/nuclear negative; whereas in most of OSCC tissues, survivin staining was nuclear positive. Statistic analysis indicates that nuclear survivin, rather than total or cytoplasmic one, correlates with tumor TNM stage and differentiation grade. Consistently, in vitro analysis showed that survivin is in cytoplasm in normal human oral kinotinocyte (HOK) cells; whereas it is in nucleus in OSCC HN6 cells. Importantly, treatment of HOK cells with HDAC inhibitor Trichostatin A (TSA) induces survivin acetylation and promotes its nuclear localization. Moreover, nuclear survivin in OSCC cells was acetylated at K129 in its C-terminal, suggesting that the acetylation is important for nuclear location of survivin. Our study demonstrates that it is nuclear survivin, rather than total or cytoplasmic one, associates with TNM stage and tumor grade of OSCC. Thus, we propose nuclear survivin as a prognostic marker for the progression of OSCC.     

Identification of tumor-associated proteins in oral tongue squamous cell carcinoma by proteomics

Oral tongue carcinoma is an aggressive tumor that particularly affects chronic smokers, drinkers and betel squid chewers. Patients often present symptoms at a late stage, and there is a high recurrence rate after treatment. In this article, we report the first proteomic analysis of oral tongue carcinoma to globally search for tumor related proteins. Apart from helping us to understand the molecular pathogenesis of the carcinoma, these proteins may also have potential clinical applications as biomarkers, enabling the tumor to be identified at an early stage in high risk individuals, treatment response to be predicted, and residual or recurrent carcinoma to be detected sooner after treatment. The protein expression profiles of ten oral tongue squamous cell carcinomas and their matched normal mucosal resection margins were examined by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. A number of tumor-associated proteins including heat shock protein (HSP)60, HSP27, alpha B-crystalline, ATP synthase beta, calgranulin B, myosin, tropomyosin and galectin 1 were consistently found to be significantly altered in their expression levels in tongue carcinoma tissues, compared with their paired normal mucosae. The expression profile portrays a global protein alteration that appears specific to oral tongue cancer. The potential of utilizing these tumor related proteins for screening cancer and monitoring recurrence warrants further investigation.     

Salivary analytes in patients with oral squamous cell carcinoma

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

Microbial diversity in saliva of oral squamous cell carcinoma

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

A mouse model for oral squamous cell carcinoma

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