Overexpression of MMP13

Matrix metalloproteinase 13 (MMP13) plays a central role in the MMP activation cascade that enables degradation of the extracellular matrix and basement membranes, and it is identified as a potential driver in oral carcinogenesis. Therefore, this study aims to determine the copy number, mRNA, and protein expression of MMP13 in oral squamous cell carcinoma (OSCC) and to associate these expressions with clinicopathological parameters. Copy number, mRNA, and protein expression analysis of MMP13 were determined using real-time quantitative PCR and immunohistochemistry methods in OSCC samples. The correlations between MMP13 expressions and clinicopathological parameters were evaluated, and the significance of MMP13 as a prognostic factor was determined. Despite discrepancies between gene amplification and mRNA and protein overexpression rates, OSCC cases showed high amplification of MMP13 and overexpression of MMP13 at both mRNA and protein levels. High level of MMP13 protein expression showed a significant correlation with lymph node metastasis (P=0.011) and tumor staging (P=0.002). Multivariate Cox regression model analysis revealed that high level of mRNA and protein expression of MMP13 were significantly associated with poor prognosis (P<0.050). Taken together, these observations indicate that the MMP13 protein overexpression could be considered as a prognostic marker of OSCC

Chromosomal alterations and gene pathways of tongue and cheek squamous cell carcinoma / Vincent Chong Vui King.

Introduction: Tongue and cheek squamous cell carcinoma (SCC) have different behaviours. In order to understand these behaviours, there is a need to look into the chromosomal alterations and gene pathways that maybe associated with oral cancer at these sites. Therefore, the objective of this study is to determine the chromosomal aberrations and gene pathways involved in tongue and cheek SCC using high resolution array based comparative genomic hybridization (aCGH). Methodology: A genome wide screening with array CGH (SurePrint G3 CGH 1x1M microarray) was performed using gDNA from 20 snap frozen fresh tissues consisting of 12 tongue and 8 cheek SCC (samples from the Malaysian Oral Cancer Database and Tumour Bank System [MOCDTBS] coordinated by OCRCC-UM). Cytosure Software was used to detect the chromosomal aberrations and candidate genes related to the selected regions. Pathway analysis was done using MetaCoreTM software for selected genes. Results: The mean number of chromosomal aberrations per tumour for tongue SCC (22.75±26.58) was higher than cheek SCC (8.63±11.89). The most common amplified regions in tongue SCC were 8q24.22 (33.33%), 8q24.3 (33.33%), 11q13.2 (33.33%), 12q13.13 (33.33%), 14q32.33 (33.33%) and for cheek SCC the most common amplified region was 22q12.3 (25%). For the deleted regions, the most common for tongue SCC were 2q21.1 (16.67%), 6q21 (16.67%) and for cheek SCC were 2q22.1 (25%), 7q35 (25%), 19q13.33 (25%). The most significant pathway involved in tongue SCC was cell adhesion extracellular matrix (ECM) remodelling pathway, while for cheek SCC; it was cadherin-mediated cell adhesion pathway. Conclusion: This study showed that the sites of oral cancer origin have a great influence over the variations in chromosomal aberrations and gene pathways. Nevertheless, the identified chromosomal aberrations genes and their interactive pathways revealed from the present research are worth for further investigations on oral carcinogenesis. (Acknowledgment: Grant of UMRG085/09HTM and PS017/2010A

MMP13 and ISG15 are potential driver genes in oral squamous cell carcinoma / Vincent Chong Vui King

Oral squamous cell carcinoma (OSCC) is an exceptionally aggressive disease with poor prognosis. A major drawback in diagnosis and treatment of OSCC is the lack of knowledge relating to how genetic instability in oral cancer genomes affects oral tumorigenesis. This current study aimed to determine the copy number alterations (CNAs) in OSCC using array comparative genomic hybridization (array CGH) and to determine the expression of candidate genes. Putative candidate gene was identified and further elucidated to explore its potential role(s) in oral tumorigenesis. Materials and Methods: Genome-wide profiling was performed on 75 OSCCs using array CGH. The copy number alterations (CNAs) associated genes that mapped to the amplified and deleted regions were subjected to pathway and network functional analysis using the Ingenuity Pathway Analysis software. The selected putative amplified genes involved in oncogenic networks were further subjected to gene expression analysis using qPCR. The protein expression of the selected putative amplified genes was determined using immunohistochemistry (IHC) technique in the non-cancer oral mucosa, oral epithelial dysplasia (OED) and OSCC samples. Knockdown of the putative amplified gene was performed using small interfering RNA (siRNA) technology in OSCC cell lines and the roles of the gene in cell proliferation, apoptosis, migration and invasion were evaluated. In this study, the frequent CNAs were observed on multiple genomic regions, including amplifications on chromosome 1p, 3q, 5p, 7p, 8q, 9q, 10p, 11q, and deletions on 3p and 8p. Apart from that, this study also demonstrated the significant association between amplification of chromosome 8q, 11q, 7p and 9p and deletion of 8p with clinico-pathological parameters such as tumour size, lymph node metastasis (LNM) and tumour staging. This study also identified novel candidate genes namely matrix metallopeptidase 13 (MMP13) and interferon stimulated gene 15 (ISG15) that linked between cell death and survival, cellular movement and cellular development oncogenic network. Furthermore, this study also demonstrated over-expression of MMP13 (chromosome 11q22.2) and ISG15 (chromosome 1p36.33) as prognostic markers in OSCC. Silencing of ISG15 in OSCC cell lines decreased the tumour cell proliferation, migration, invasion, induced apoptosis and increased the cisplatin sensitivity in oral tumourigenesis. Conclusion: This current study has identified multiple CNAs including amplifications on chromosome 1p, 3q, 5p, 7p, 8q, 9q, 10p, 11q and deletions on 3p and 8p. This study also showed that amplification of the chromosome 7p, 8q, 9p, 11q and genetic signature (+7p8q9p11q) as well as deletion of chromosome 8p was associated with clinico-pathological parameters and poor survival. Apart from that, through the network analysis, the putative amplified genes namely ISG15 and MMP13 were found to be associated with the top oncogenic network namely cell death and survival, cellular movement, cellular development network signalling. This study also has demonstrated the over-expression of MMP13 and ISG15 were associated with lymph node metastasis, tumour staging and poor prognosis. Through the siRNA knockdown of the ISG15 expression inhibited the tumour cell proliferation, migration, invasion and induced cell death in oral tumorigenesis

Development and Radiation Response Assessment in A Novel Syngeneic Mouse Model of Tongue Cancer: 2D Culture, 3D Organoids and Orthotopic Allografts

Oral squamous cell carcinoma (OSCC) are aggressive cancers that contribute to significant morbidity and mortality in humans. Although numerous human xenograft models of OSCC have been developed, only a few syngeneic models of OSCC exist. Here, we report on a novel murine model of OSCC, RP-MOC1, derived from a tongue tumor in a C57Bl/6 mouse exposed to the carcinogen 4-nitroquinoline-1-oxide. Phenotypic characterization and credentialing (STR profiling, exome sequencing) of RP-MOC1 cells was performed in vitro. Radiosensitivity was evaluated in 2D culture, 3D organoids, and in vivo using orthotopic allografts. RP-MOC1 cells exhibited a stable epithelial phenotype with proliferative, migratory and invasive properties. Exome sequencing identified several mutations commonly found in OSCC patients. The LD50 for RP-MOC1 cells in 2D culture and 3D organoids was found to be 2.4 Gy and 12.6 Gy, respectively. Orthotopic RP-MOC1 tumors were pan-cytokeratin+ and Ki-67+. Magnetic resonance imaging of orthotopic RP-MOC1 tumors established in immunocompetent mice revealed marked growth inhibition following 10 Gy and 15 Gy fractionated radiation regimens. This radiation response was completely abolished in tumors established in immunodeficient mice. This novel syngeneic model of OSCC can serve as a valuable platform for the evaluation of combination strategies to enhance radiation response against this deadly disease

A Comprehensive Review of Natural Products as Therapeutic or Chemopreventive Agents against Head and Neck Squamous Cell Carcinoma Cells Using Preclinical Models

Head and neck squamous cell carcinoma (HNSCC) is a type of cancer that arises from the epithelium lining of the oral cavity, hypopharynx, oropharynx, and larynx. Despite the advancement of current treatments, including surgery, chemotherapy, and radiotherapy, the overall survival rate of patients afflicted with HNSCC remains poor. The reasons for these poor outcomes are due to late diagnoses and patient-acquired resistance to treatment. Natural products have been extensively explored as a safer and more acceptable alternative therapy to the current treatments, with numerous studies displaying their potential against HNSCC. This review highlights preclinical studies in the past 5 years involving natural products against HNSCC and explores the signaling pathways altered by these products. This review also addresses challenges and future directions of natural products as chemotherapeutic and chemoprevention agents against HNSCC

Preclinical Prevention Trial of Calcitriol: Impact of Stage of Intervention and Duration of Treatment on Oral Carcinogenesis

The anticancer activity of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3 or calcitriol) has been widely reported in preclinical models. However, systematic investigation into the chemopreventive potential of calcitriol against the spectrum of oral carcinogenesis has not been performed. To address this gap in knowledge, we conducted a preclinical prevention trial of calcitriol in the 4-nitroquinoline-1-oxide (4NQO) oral carcinogenesis model. C57BL/6 mice were exposed to the carcinogen 4NQO in drinking water for 16 weeks and randomized to control (4NQO only) or calcitriol arms. Calcitriol (0.1 μg i.p, Monday, Wednesday, and Friday) was administered for (i) 16 weeks concurrently with 4NQO exposure, (ii) 10 weeks post completion of 4NQO exposure, and, (iii) a period of 26 weeks concurrent with and following 4NQO exposure. Longitudinal magnetic resonance imaging (MRI) was performed to monitor disease progression until end point (week 26). Correlative histopathology of tongue sections was performed to determine incidence and multiplicity of oral dysplastic lesions and squamous cell carcinomas (SCC). Vitamin D metabolites and calcium were measured in the serum using liquid chromatography-mass spectrometry (LC–MS/MS) and colorimetric assay, respectively. Renal CYP24A1 (24-hydroxylase) and CYP27B1 (1α-hydroxylase) expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Immunostaining of tongue sections for vitamin D receptor (VDR), CYP24A1, and Ki67 was also performed. Non-invasive MRI enabled longitudinal assessment of lesions in the oral cavity. Calcitriol administered concurrently with 4NQO for 16 weeks significantly (P < .001) decreased the number of premalignant lesions by 57% compared to 4NQO only controls. Mice treated with calcitriol for 26 weeks showed highest renal CYP24A1, lowest serum 1,25(OH)2D3 levels and highest incidence of invasive SCC. Immunohistochemistry revealed increased VDR, CYP24A1 and Ki67 staining in dysplastic epithelia compared to normal epithelium, in all four groups. Collectively, our results show that the effects of calcitriol on oral carcinogenesis are critically influenced by the stage of intervention and duration of exposure and provide the basis for exploring the potential of calcitriol for prevention of OSCC in the clinical setting

Prognostic Abilities of Pre- and Post-Treatment Inflammatory Markers in Oral Squamous Cell Carcinoma: Stepwise Modelling

Background and Objectives: Studies examining the importance of inflammatory markers before treatment as prognosticators of OSCC are available, but information on post-therapy inflammatory markers and their prognostic significance is limited. This study aimed to evaluate the prognostic abilities of pre- and post-treatment inflammatory markers in patients with OSCC. Materials and Methods: In this retrospective analysis, information on 151 OSCC patients&rsquo; socio-demographic, clinico-pathological, recurrence, metastasis, and survival data were gathered from clinical records. A multivariable Cox proportional hazards regression (stepwise model) was conducted to identify the prognostic predictors of OS and DFS. The multivariable models&rsquo; performances were evaluated using Harrell&rsquo;s concordance statistics. Results: For OS, high pre-treatment LMR (HR 3.06, 95%CI 1.56, 5.99), and high post-treatment PLC (HR 3.35, 95%CI 1.71, 6.54) and PLR (HR 5.26, 95%CI 2.62, 10.58) were indicative of a poor prognosis. For DFS, high pre-treatment SII (HR 2.59, 95%CI 1.50, 4.48) and high post-treatment PLC (HR 1.92, 95%CI 1.11, 3.32) and PLR (HR 3.44, 95%CI 1.98, 5.07) were associated with increased mortality. The fitness of the OS and DFS stepwise Cox regression models were proven with a time-dependent AUC of 0.8787 and 0.8502, respectively. Conclusions: High pre-treatment levels of LMR and SII and high post-treatment levels of PLC and PLR are independent predictors of a poor prognosis for patients with OSCC

DNA Methylation Profiling Revealed Promoter Hypermethylation-induced Silencing of p16, DDAH2 and DUSP1 in Primary Oral Squamous Cell Carcinoma

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Co-Expression of TWIST1 and ZEB2 in Oral Squamous Cell Carcinoma Is Associated with Poor Survival

<div><p>Oral squamous cell carcinoma (OSCC) is an aggressive disease accounting for more than 260,000 cancer cases diagnosed and 128,000 deaths worldwide. A large majority of cancer deaths result from cancers that have metastasized beyond the primary tumor. The relationship between genetic changes and clinical outcome can reflect the biological events that promote cancer’s aggressive behavior, and these can serve as molecular markers for improved patient management and survival. To this end, epithelial-mesenchymal transition (EMT) is a major process that promotes tumor invasion and metastasis, making EMT-related proteins attractive diagnostic biomarkers and therapeutic targets. In this study, we used immunohistochemistry to study the expression of a panel of transcription factors (TWIST1, SNAI1/2, ZEB1 and ZEB2) and other genes intimately related to EMT (CDH1 and LAMC2) at the invasive tumor front of OSCC tissues. The association between the expression of these proteins and clinico-pathological parameters were examined with Pearson Chi-square and correlation with survival was analyzed using Kaplan Meier analysis. Our results demonstrate that there was a significant differential expression of CDH1, LAMC2, SNAI1/2 and TWIST1 between OSCC and normal oral mucosa (NOM). Specifically, CDH1 loss was significantly associated with Broder’s grading, while diffused LAMC2 was similarly associated with non-cohesive pattern of invasion. Notably, co-expression of TWIST1 and ZEB2 in OSCC was significantly associated with poorer overall survival, particularly in patients without detectable lymph node metastasis. This study demonstrates that EMT-related proteins are differentially expressed in OSCC and that the co-expression of TWIST1 and ZEB2 could be of clinical value in identifying patients with poor survival for appropriate patient management.</p></div