C-Jun Transcription Factor Oncogenic Activation in Oral Carcinoma.

BackgroundThe tumor suppressor Programmed Cell Death 4 (PDCD4) has been found to be under-expressed in several cancers and associated with disease progression and metastasis. There are no current studies characterizing PDCD4 expression and its clinical relevance in Oral Squamous Cell Carcinoma (OSCC). Since nodal metastasis is a major prognostic factor in OSCC, we focused on determining whether PDCD4 under-expression was associated with patient nodal status and had functional relevance in OSCC invasion. We also examined PDCD4 regulation by microRNA 21 (miR-21) in OSCC.ResultsPDCD4 mRNA expression levels were assessed in 50 OSCCs and 25 normal oral tissues. PDCD4 was under-expressed in 43/50 (86%) OSCCs, with significantly reduced mRNA levels in patients with nodal metastasis (p = 0.0027), and marginally associated with T3-T4 tumor stage (p = 0.054). PDCD4 protein expression was assessed, by immunohistochemistry (IHC), in 28/50 OSCCs and adjacent normal tissues; PDCD4 protein was absent/under-expressed in 25/28 (89%) OSCCs, and marginally associated with nodal metastasis (p = 0.059). A matrigel invasion assay showed that PDCD4 expression suppressed invasion, and siRNA-mediated PDCD4 loss was associated with increased invasive potential of oral carcinoma cells. Furthermore, we showed that miR-21 levels were increased in PDCD4-negative tumors, and that PDCD4 expression may be down-regulated in OSCC by direct binding of miR-21 to the 3'UTR PDCD4 mRNA.ConclusionsOur data show an association between the loss of PDCD4 expression, tumorigenesis and invasion in OSCC, and also identify a mechanism of PDCD4 down-regulation by microRNA-21 in oral carcinoma. PDCD4 association with nodal metastasis and invasion suggests that PDCD4 may be a clinically relevant biomarker with prognostic value in OSCC.

Development of a 4-nitroquinoline-1-oxide model of lymph node metastasis in oral squamous cell carcinoma

Introduction: Oral squamous cell carcinoma (OSCC) is the 3 rd most pervasive malignancy in India. The indicative of oral malignancy is the mechanism of invasion and metastasis, which is facilitated by numerous enzyme systems, chiefly matrix metalloproteinases (MMPs). Among them, MMP-9 performs a major role in the facilitation of these processes by its ability to degrade the basal lamina and extracellular matrix. Oral carcinomas can develop from the progression of premalignant oral conditions. The early tissue changes occurring in oral potentially malignant disorders (OPMDs) lead to cellular alteration with expression of MMP-9, indicating that these MMPs influence the disease process. Materials and Methods: This retrospective cross-sectional study included five study groups comprising histopathologically diagnosed cases of oral lichen planus (10 cases), oral submucous fibrosis (10 cases), oral leukoplakia (10 cases), OSCC (10 cases), and normal oral mucosa (10 cases). Results: After evaluation and comparison of the area and intensity of staining of MMP-9 among the groups, it is significantly appreciated in OSCC (100% positive expression) and OPMD disorders when compared to the mucosa free of lesions. Conclusion: MMP-9 can be considered a reliable marker to assess invasion and prognosis in OSCC. The expression of MMP-9 in OPMDs suggests alteration in the epithelial cells, thereby prompting early management to curb lesion carcinogenesis.

Background: Various environmental factors have been reported to play key role in the development of oral squamous cell carcinoma (OSCC). A lesser known risk factor of oral cancer in India is the uncontrolled use of areca nut chewing. In North-East India, Areca nut, locally called as “Tamol” in Assam, is raw betel nut, lime and betel leaf without tobacco, which are more effective as compared to dried which can be the important contributing factor for OSCC. Objectives: The aim of the study was to detect the association between XRCC1 polymorphisms and increased risk of OSCC in tamol chewers population in Assam, India. Methods: 50 OSCC patients, 50 tamol chewers and 50 controls were enrolled in the study. XRCC1 Arg399Gln polymorphisms were determined by using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Results: There was a significant association for XRCC1 codon 399 (Arg/Gln+Gln/Gln) (p<0.05; OR=1.909, CI= 0.8622- 4.227) with the wild type in cancer sample as compared with control sample. Similarly, the positive association for 399G/G (p<0.05; OR =2.842, CI = 0.919-8.79) genotypes with oral carcinoma and control sample. In case of tamol chewers, the AA genotype was found to be associated with 2-fold (OR- 2.25, CI= 0.709-7.14) increase risk of developing oral cancer while GA+AA genotype was associated with one and half fold (OR-1.62, CI=0.7354- 3.568) risk of developing oral cancer. Conclusions: Based on these results, the XRCC1399G>A genotype could be used as a useful molecular biomarker to predict genetic susceptibility in tamol chewers population and its susceptibility to develop OSCC.

Simple SummaryThe incidence of oropharyngeal squamous cell carcinomas (OPSCCs) has increased in the last decades, and this seems to be correlated to the infectious epidemiological trend of human papillomavirus (HPV). The prevalence of HPV-positive OPSCCs is approximately 70%, with involvement mainly of the tonsillar area. On the role of HPV in oral squamous cell carcinoma (OSCC), very few studies have investigated the prevalence of HPV in strictly site-codified OSCC, excluding the base of the tongue as distinct oropharyngeal entity. As a result, an inappropriate estimation of HPV infection in OSCC has been observed. We investigated HPV status, using a combination of detection methods, in a sample of 40 subjects with OSCC coded by the latest site classifications. Moreover, we performed a critical review of the studies with the same outcomes. The main finding of our investigation was a low frequency of HPV-positive OSCC, suggesting no significant HPV role in strictly oral carcinogenesis.The aim of this study was to evaluate HPV status in oral squamous cell carcinoma (OSCC), as coded by the latest classifications and applying a combination of detection methods used in clinical practice. Forty-two patients with suspect OSCC were consecutively recruited. Patients underwent an incisional biopsy for histological OSCC diagnosis and HPV identification by PCR DNA and p16 IHC. All lesions were coded by the latest ICD-0-3.2 site/histology classifications, as proposed for OSCC by the National Cancer Institute Surveillance, Epidemiology and End Results Programs. Moreover, a comparative analysis review, critically evaluated by the same site-coded systems and HPV detection methods, was performed. In 40 confirmed cases of OSCC, the frequency of HPV infection was 10% (4/40). Among positive patients, two cases were PCR DNA/p16 IHC positive (high-risk HPV 51, high-risk HPV 67), two cases were PCR DNA positive/p16 IHC negative (high-risk HPV 31 + 68, high-risk HPV 66). Applying the latest site coding systems for OSCC, the frequency of HPV infection in this study and in similar, reviewed investigations was low (from 3.3% to 12.5%). These results suggested no significant HPV role in oral carcinogenesis, particularly where an updated site-coded classification of OSCCs (categorically excluding the base of the tongue) had been performed.

Activation of Toll-like receptor 3 induces apoptosis of oral squamous carcinoma cells in vitro and in vivo

466. Downregulation of ATF2 Desensitizes Oral Squamous Cell Carcinoma to Apoptosis

To study the expression of Ki-67 and the changes of MVD and apoptosis in benign lymphoadenosis of oral mucosa (BLOM). The expression of Ki-67, CD34 and apoptosis were evaluated by SP immunohistochemical staining in 15 BLOM, 9 BLOM with dysplasia, 15 oral squamous cell carcinoma (OSCC). The expression of Ki-67 in BOLM with dysplasia and OSCC was significantly higher than that of BLOM without dysplasia and normal oral mucosa (P < 0.05). The MVD in all BLOM and OSCC was significantly higher than that in normal mucosa (P < 0.05). Apoptosis in BLOM was higher than in normal mucosa and OSCC (P < 0.05). The expression of Ki-67 and MVD in BLOM with dysplasia were between normal oral mucosa and oral carcinoma. The occurrence of apoptosis in BLOM was significantly higher than in normal oral mucosa. The results suggest that BLOM had the potentiality of malignant transformation.

Lin28, a key factor for cellular reprogramming and generation of induced pluripotent stem cell (iPSC), makes a critical contribution to tumorigenicity by suppressing Let-7. However, it is unclear whether Lin28 is involved in regulating cancer stem-like cells (CSC), including in oral squamous carcinoma cells (OSCC). In this study, we demonstrate a correlation between high levels of Lin28B, Oct4, and Sox2, and a high percentage of CD44(+)ALDH1(+) CSC in OSCC. Ectopic Lin28B expression in CD44(-)ALDH1(-)/OSCC cells was sufficient to enhance Oct4/Sox2 expression and CSC properties, whereas Let7 co-overexpression effectively reversed these phenomena. We identified ARID3B and HMGA2 as downstream effectors of Lin28B/Let7 signaling in regulating endogenous Oct4 and Sox2 expression. Let7 targeted the 3' untranslated region of ARID3B and HMGA2 and suppressed their expression, whereas ARID3B and HMGA2 increased the transcription of Oct4 and Sox2, respectively, through promoter binding. Chromatin immunoprecipitation assays revealed a direct association between ARID3B and a specific ARID3B-binding sequence in the Oct4 promoter. Notably, by modulating Oct4/Sox2 expression, the Lin28B-Let7 pathway not only regulated stemness properties in OSCC but also determined the efficiency by which normal human oral keratinocytes could be reprogrammed to iPSC. Clinically, a Lin28B(high)-Let7(low) expression pattern was highly correlated with high levels of ARID3B, HMGA2, OCT4, and SOX2 expression in OSCC specimens. Taken together, our results show how Lin28B/Let7 regulates key cancer stem-like properties in oral squamous cancers.

The proto-oncogene Ets-1 is a transcription factor known to regulate the expression of a number of genes involved in extracellular matrix remodelling. It has been postulated to play a role in angiogenesis, cell migration, and regulation of tumour invasion. In betel and tobacco-related oral tumourigenesis, oral squamous cell carcinomas (SCCs) having a poor prognosis often exhibit extensive local disease with spread into regional lymph nodes. At present, no tumour marker is available to predict the metastatic potential of oral carcinomas. To elucidate the involvement of Ets-1 in human oral tumourigenesis, 60 cases of oral SCC, 30 potentially malignant lesions, and 30 samples of normal oral mucosa were examined by immunohistochemistry and the findings corroborated with immunoblotting. The relationship of Ets-1 expression with the degree of differentiation, lymph node metastasis, and tumour stage was determined in oral SCCs. Ets-1 was not detected in the normal oral epithelium. Among the 60 cases of oral SCC, 35 cases (58 per cent) showed positive staining for the Ets-1 protein in tumour cells. In potentially malignant lesions, 14 of 30 cases (47 per cent) showed Ets-1 immunopositivity. Ets-1 expression correlated significantly with tumour stage ( p=0.01) and lymph node metastasis ( p=0.005). The presence of Ets-1 mRNA in oral SCCs was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). These findings suggest that overexpression of Ets-1 in potentially malignant lesions may be related to angiogenesis and its expression in oral SCCs may be one of the factors involved in tumour progression and lymph node metastasis.

To investigate the effects of salinomycin on the proliferation and apoptosis of oral squamous carcinoma cells and to further understand the mechanisms of these effects. The human oral squamous carcinoma cell line CAL-27 was cultured in different concentrations of salinomycin and cisplatin. After co-culture with 0, 1, 2, 4, 8, 16 and 32 μmol/L salinomycin or 0, 1.25, 2.5, 5, 10, 20, 40 and 80 μmol/L cisplatin for 24 hours and 48 hours, the proliferation of oral squamous carcinoma cells were detected by cell counting kit-8(CCK-8) assay. After being exposed to 0, 2, 4, 8 μmol/L salinomycin and 0, 5, 10, 20 μmol/L cisplatin for 48 hours, the cell cycle of oral squamous carcinoma cells was detected by flow cytometry assay, and Western blot analysis was performed to analyze the expressions of cysteine-containing aspartate-specific proteases-3(Caspase-3), cysteine-containing aspartate-specific proteases-9(Caspase-9), poly ADP-ribose polymerase (PARP), protein kinase B (Akt) and phosphorylated protein kinase B (p-Akt) protein in oral squamous carcinoma cells. Both salinomycin and cisplatin significantly inhibited the proliferation of oral squamous cell carcinoma CAL-27 cells in a time- and dose-dependent manner. However, compared with the first-line chemotherapeutic drug cisplatin, salinomycin showed stronger anti-proliferation activity in oral squamous carcinoma cells than cisp-latin (P < 0.001). After being exposed to 8 μmol/L salinomycin, CAL-27 cells exhibited markedly higher proportion in quiescent/ first gap phases (40.40%±1.99% vs. 64.46%±0.90%, P < 0.05), and had a significantly lower proportion in synthesis phases and second gap / mitosis phases (24.32%±2.30% vs. 18.73%±0.61%, P < 0.05; 35.01%±1.24% vs. 16.54%±1.31%, P < 0.05) compared with the dimethyl sulfoxide control group; moreover cisplatin didn't show cell-cycle specific effect on CAL-27. Western blot proved that salinomycin could up-regulate the expressions of Caspase-3 and Caspase-9 protein in oral squamous cell carcinoma CAL-27 cells (P < 0.05). At the same time, the levels of PARP, Akt and p-Akt protein were down-regulated (P < 0.05). Compared with cisplatin, salinomycin has a better inhibitory effect on the proliferation of oral squamous carcinoma cells and blocks the cell cycle process at the quiescent / first gap phase. At the same time, salinomycin could trigger apoptosis of oral squamous carcinoma cells and the mechanism is associated with the Akt/p-Akt signaling pathway.

Tobacco-related oral squamous cell carcinoma (OSCC) is one of the most common cancers involving Indian males. We assessed the association of IL4 promoter -589 T>C, -33 T>C, and IL6-174 G>C functional genetic polymorphisms with tobacco-related OSCC in Asian Indians. The IL4 and IL6 promoter polymorphisms were assessed in 140 patients with OSCC and 120 normal subjects by PCR-RFLP technique, and significance of the data was determined using chi-square test. The frequency of TC, CC genotype, and C allele at IL4 promoter sites -589 and -33 were higher in patients when compared with controls. Consequently, TC/CC genotypes and C allele at both sites appeared as susceptible. However, IL6-174 G>C single-nucleotide polymorphisms (SNP) appeared to be protective in patients with OSCC. Of eight haplotypes, five were associated with two- to seven-fold increased risk of tobacco-related OSCC. These SNPs further showed heterogeneity among different ethnic population, but their distribution in Asian Indians stand closer to other Asian populations. In this study, IL4-589 CC, -33 CC genotype, and *C allele at both sites appeared to be susceptible, while IL6-174 CC genotype and *C allele appeared to be protective in patients with OSCC; hence, these SNPs may be a potential prognostic markers for tobacco-related OSCC in Asian Indians.

Dendritic cells (DCs) are antigen-presenting cells with a unique ability to cross prime T cells and generate strong antitumor responses. This study evaluates the presence and prognostic significance of DCs as well as functional T cells, which accumulate in the microenvironment in patients with oral squamous cell carcinoma (OSCC). Immunohistochemistry for S-100 positive or p55 positive DCs and for T-cell receptor (TcR)-associated zeta-chain expression in tumor-infiltrating lymphocytes (TILs) was performed in 132 paraffin embedded specimens from patients with primary OSCC. The median clinical follow-up for the patients was 50 months. The numbers of intratumoral DCs or TILs expressing the zeta chain were determined microscopically and compared with clinical and pathohistologic prognostic parameters, including disease stage, T stage or tumor grade, lymph node involvement, as well as disease free survival and overall survival. Immunostaining identified DCs in the epithelial compartment of the tumors (S-100 positive) as well as interdigitating reticular DCs (p55 positive) in peritumoral areas. Based on S-100 staining, intratumoral DC infiltrates were low (<10 DCs per high-power field [HPF]) in 20% of OSCC specimens, intermediate (10-20 DCs per HPF) in 42% of OSCC specimens, and high (>20 DCs per HPF) in 37% of OSCC specimens. The number of S-100 positive DCs was positively and significantly correlated with that of p55 and of TILs with normal zeta-chain expression. A low number of infiltrating S-100 positive DCs was more predictive of poor survival (hazard ratio, 7.95) than lymph node involvement (hazard ratio, 3.36) or late T stage (hazard ratio, 2.92). A significant but weaker association of p55 positive DC infiltration with survival was observed. Low density of DCs and low or absent expression of the zeta chain in TILs correlated with each other and predicted the poorest survival and the greatest risk. The number of DCs infiltrating the tumor is a highly significant prognostic parameter in patients with OSCC. Furthermore, the absence or paucity of DCs is strongly linked to abnormalities in the TcR-associated zeta chain in TILs. The two biomarkers, zeta-chain expression in TILs and the number of S-100+ DCs in the tumor, independently predict overall survival, disease free survival, and time to disease recurrence in patients with OSCC.

To study immunohistochemical expression of the epithelial growth factor receptor (EGFR) in oral carcinomas and the head and neck region to examine possible associations with various features of the tumors and survival of the patients. Sections were made from two tissue arrays composed of 206 oral squamous cell carcinomas and 427 squamous cell carcinomas of the head and neck region, respectively, and examined for EGFR expression and Ki-67 labeling index by means of immunohistochemistry, and for EGFR gene amplification by means of fluorescence in situ hybridization. Correlation between resulting parameters and with clinical features was evaluated using chi-square test and Kaplan-Meyer analysis. A statistically significant association was observed for strong EGFR immunohistochemical (IHC) expression with advanced lymph node involvement (P = 0.02). EGFR immunohistochemical expression did not significantly correlate with patient disease specific (DS) or overall survival (OS). EGFR gene amplification was not correlated with any of the tumor features nor to survival of the patients (DS and OS). Epithelial growth factor receptor IHC expression and gene amplification might be suitable to predict locoregional control in oral squamous cell carcinoma patients but an inappropriate predictor for patients survival.

Previous studies of oral carcinomas have shown that both loss of heterozygosity (LOH) and hypermethylation at chromosome 9q33 to 9q34.2 are frequent. The present study investigates the frequency of Notch1 gene methylation and LOH at 9q34.3 region. Gene promoter hypermethylation of the Notch1 gene was analysed by methylation-specific PCR and LOH was analysed using microsatellite markers. We found LOH at 9q34.3 in three patients and methylation of the Notch1 gene only in two patients with oral carcinoma. Comparing with the alterations at 9q33 to 34.2 regions, LOH at 9q34.3 and methylation of the Notch1 gene was less involved in oral squamous cell carcinomas.