Oral Squamous Cell Carcinoma Primary Tumors Research Articles

Single-cell RNA sequencing of OSCC primary tumors and lymph nodes reveals distinct origin and phenotype of fibroblasts

Desmoplasia in fibroblasts within metastatic lymph nodes (MLNs) serves as an indicator of extranodal extension (ENE), which led mortality in oral squamous cell carcinoma (OSCC). However, systematic studies on fibroblasts in MLNs are lacking. Therefore, this study characterized the differences in phenotype, function, and origin of fibroblasts between primary tumors (PTs) and lymph nodes (LNs) in OSCC.We generated single-cell maps of PTs and paired MLNs and draining LNs from three OSCC patients. The transcriptomic atlas, pseudotime analysis, intercellular communication networks and enrichment analysis of the single cells were characterized. Phenotype and function heterogeneity of fibroblast cells between PTs and MLNs were further verified in vitro.Among 44,052 fibroblasts, we identified two distinct subpopulations of cancer-associated myofibroblastic cells (mCAFs): RGS4+ mCAF1 and COMP + mCAF2. Notably, they exhibited distinct distributions, with mCAF1 predominantly localized in the PTs and mCAF2 in the MLNs. Moreover, pseudotime analysis revealed their distinct origins: mCAF1 originated from inherent normal myofibroblastic cells in the PT, whereas mCAF2 originated from fibroblastic reticular cells in the LNs. Further functional experiments using primary fibroblasts revealed that, compared to mCAF1, mCAF2 in MLNs exhibited weaker crosstalk with immune cells but enhanced extracellular matrix activity, which is closely linked to ENE formation in OSCC. Additionally, we identified two fibroblast subgroups in a transforming state, indicating a potential epithelial–mesenchymal transition.Our research offers profound insights into the heterogeneity of fibroblasts between the PT and MLN in OSCC, serving as an essential resource for future drug discovery endeavors.

Potential of uPAR, αvβ6 Integrin, and Tissue Factor as Targets for Molecular Imaging of Oral Squamous Cell Carcinoma: Evaluation of Nine Targets in Primary Tumors and Metastases by Immunohistochemistry.

No clinically approved tumor-specific imaging agents for head and neck cancer are currently available. The identification of biomarkers with a high and homogenous expression in tumor tissue and minimal expression in normal tissue is essential for the development of new molecular imaging targets in head and neck cancer. We investigated the expression of nine imaging targets in both primary tumor and matched metastatic tissue of 41 patients with oral squamous cell carcinoma (OSCC) to assess their potential as targets for molecular imaging. The intensity, proportion, and homogeneity in the tumor and the reaction in neighboring non-cancerous tissue was scored. The intensity and proportion were multiplied to obtain a total immunohistochemical (IHC) score ranging from 0-12. The mean intensity in the tumor tissue and normal epithelium were compared. The expression rate was high for the urokinase-type plasminogen activator receptor (uPAR) (97%), integrin αvβ6 (97%), and tissue factor (86%) with a median total immunostaining score (interquartile range) for primary tumors of 6 (6-9), 12 (12-12), and 6 (2.5-7.5), respectively. For the uPAR and tissue factor, the mean staining intensity score was significantly higher in tumors compared to normal epithelium. The uPAR, integrin αvβ6, and tissue factor are promising imaging targets for OSCC primary tumors, lymph node metastases, and recurrences.

Gene microarray analysis revealed a potential crucial gene RACK1 in oral squamous cell carcinoma (OSCC)

ABSTRACTOral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide, which appears as a consequence of multiple molecular genetic events in various chromosomes and genes. In order to unveil the possible mechanisms underlying OSCC tumorigenesis, the OSCC-related gene expression variance and the gene interaction network should be further investigated. Herein, we conducted the NimbleGen Human Gene Expression Microarray to analyze expression heterogeneity between OSCC primary tumor tissue and its adjacent normal tissue from two patients. A total number of 7872 out of 32,448 detected genes are differentially expressed in OSCC. Gene ontology (GO) analysis demonstrated that these differentially expressed transcripts were critical in a series of metabolic processes, cancer-related signal pathways, and biological regulations. KEGG signaling pathway enrichment suggested a number of pathways (metabolic process and immune response) which are frequently enrolled during cancer progression. 15 most differential regulated genes between OSCC tumor and non-tumor were confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the interaction network analysis of these confirmed genes by STRING database showed the two subunits of RACK1 had direct interaction with 14 differential proteins. This bioinformatics research lends support about the critical role of RACK1 which functions as a key node protein driving OSCC development.

Comparison of the expression of TGF-β1, E-cadherin, N-cadherin, TP53, RB1CC1 and HIF-1α in oral squamous cell carcinoma and lymph node metastases of humans and mice.

The aim of the present study was to prove that a mouse model closely simulates human oral cancer progression by comparing the expression levels of transforming growth factor (TGF)-β1, E-cadherin, N-cadherin, tumor protein (TP)53, RB1 inducible coiled-coil (RB1CC)1 and hypoxia inducible factor (HIF)-1α at different stages of oral squamous cell carcinoma (OSCC) in humans and mice. The expression levels of TGF-β1, E-cadherin, N-cadherin, TP53, RB1CC1, and HIF-1α were detected by immunohistochemical staining in normal oral mucosa, oral mucosa dysplasia, OSCC primary tumor and carcinoma tissues from lymph node metastases. Tissue samples were obtained from human specimens and the Balb/c mouse model of lymphatic metastases oral carcinoma, induced by 4-nitroquinoline-1-oxide in drinking water. The results indicated no significant differences in the expression levels of TGF-β1, E-cadherin, N-cadherin, TP53, RB1CC1 and HIF-1α between humans and mice, at any stage of OSCC examined (P>0.05). The expression of TGF-β1, N-cadherin, TP53 and RB1CC1 increased in different stages of OSCC in both humans and mice. The expression of E-cadherin decreased from normal oral mucosa to OSCC, and increased in lymph node metastases in both human and mouse samples. The expression of HIF-1α increased from normal oral mucosa to OSCC, and decreased in lymph node metastases in both human and mouse samples. Additionally, the expression of p53 was positively correlated with that of RB1CC1 in human and mouse samples (r=0.971, P=0.029; r=0.97, P=0.03). Overall, the similar expression of multiple molecules in both human and mouse carcinoma prove that the mouse model of lymphatic metastases from oral carcinoma established in the present study may closely mimic human oral cancer.

Genetic profile of PTEN gene in Indian oral squamous cell carcinoma primary tumors.

Phosphatase and tensin homolog (PTEN) is the tumor suppressor gene located on chromosome10q23.3. Genetic variations in the PTEN gene have been found in various sporadic tumors. However, petite is known about the genetic profile of the PTEN gene in oral squamous cell carcinoma (OSCC), which is the eighth most common neoplasm worldwide and leading cancer in India. The purpose of the present study was to determine the frequency of genetic variations in the tyrosine phosphatase domain of the PTEN gene in an Indian OSCC subset. We analyzed tyrosine phosphatase domain encoded by exon 5 of the PTEN gene in 59 OSCC primary tumors using PCR - direct genomic sequencing. We observed one somatic deletion mutation, IVS4-30delT in three OSCC patients; two of them were at an advanced stage of carcinoma. Moreover, we identified one SNP rs 35560700(C>T), in five OSCC patients with the late stage of oral carcinoma. We identified 5% somatic mutational frequency in the intronic region of the tyrosine phosphatase domain of the PTEN gene; however, mutations were found absent in the coding region. Therefore, PTEN gene mutation is not a frequent event in the pathogenesis of OSCC in the targeted Indian cohort.

Genetic alterations of the PIK3CA oncogene in human oral squamous cell carcinoma in an Indian population

The phosphatidylinositol 3-kinase (PIK3) genes, which code for heterodimeric lipid kinases, consist of a catalytic subunit, p110α (PIK3CA), which regulates cell proliferation, apoptosis, and metastasis. Recently, a high frequency of somatic mutations was observed in the PIK3CA gene in various cancer types, including oral squamous cell carcinoma (OSCC). This study aimed to determine the frequency of oncogenic hotspot mutations in exons 9 and 20 of the PIK3CA gene and its correlation with the clinical characteristics of OSCC patients in an Indian population. We analyzed exons 9 and 20 of the PIK3CA gene using polymerase chain reaction (PCR) and direct genomic sequencing of 50 OSCC primary tumors. We observed two hotspot oncogenic mutations (E542 K, E545 K) in exon 9 and two synonymous mutations (A994 A, T1025 T) in exon 20. Moreover, we identified two single nucleotide polymorphisms (SNPs), rs114587137 (C>T) and rs17849071 (T>G), in intron 9 of the PIK3CA gene. Both oncogenic hotspot mutations were reported primarily in patients with advanced-stage cancer of the buccal mucosa. We have observed a 4% oncogenic mutation frequency of the PIK3CA gene, which plays a minor role in the development of OSCC in an Indian population.

441 The role of méthylation in metastasis of oral squamous cell carcinoma: understanding the OSCC methylome

Background: Oral Squamous Cell Carcinoma (OSCC) is characterized by an increasing incidence and a 60% 5-year survival. The most important prognostic factor for OSCC is the presence of lymph node (LN) metastases but the detection of LN metastases in the clinic by palpation and imaging is inaccurate resulting in under and overtreatment of patients. To improve LN diagnosis new detection methods are needed. DNA methylation studies can be used to identify novel biomarkers, as epigenetics have been established as an important regulator of metastatic potential. The aim of this study is to identify new DNA methylation markers that predict LN metastasis in OSCC. Materials and Methods: Genome-wide methylation patterns of metastatic (n = 6) and non-metastatic OSCC (n = 6) were assessed using MethylCap- Seq. Subsequently, analysis was performed on the most differentially methylated loci to identify pathways and genomic loci associated with LN metastasis. Additionally, the methylation data were combined with expression data of 222 OSCC patients acquired by an expression microarray with a 696 gene panel associated with N-status in OSCC. Finally, we validated these findings on the OSCC samples (n = 174) in The Cancer Genome Atlas (TCGA). Results: We found that genes on chromosome 7 are the most hypermethylated in metastatic OSCC in comparison to non-metastatic OSCC, more specifically a four million bp loci around the EGFR gene. In total 26 genes were found to be differentially methylated by MethylCap- Seq as well as differentially expressed by microarray. In the OSCC TCGA validation cohort, all 26 genes are significantly differentially expressed between metastatic and non-metastatic OSCC. For these 26 differentially expressed genes 88 probes were found in the TCGA Infinium 450k methylation data that are both significantly differentially methylated between the two groups and correlated with mRNA levels. Finally, six of these probes overlapped with the genomic regions annotated by MethylCap-Seq, representing five genes. Conclusion: We identified chromosomal loci, pathways and gene promoters associated with LN metastasis in OSCC patients. For 10 genes the differential methylation, expression and correlation between methylation and expression was validated on an independent OSCC cohort from the TCGA database. Increased understanding of the metastatic OSCC methylome will contribute to the identification of DNA methylation markers, pathways and potential therapeutic targets in OSCC primary tumors that will improve chances of providing the proper lymph node treatment and may result in improvement of survival and quality of life.

Clinicopathological significance and prognostic value of CD133 expression in oral squamous cell carcinoma

ObjectiveThis study investigated the expression of CD44 and CD133, the two surface markers most commonly used to identify cancer stem cells (CSCs), as predictive markers for clinicopathological progression and lymph node metastasis in oral squamous cell carcinoma (OSCC). MethodsOSCC primary tumor specimens obtained from 29 patients with OSCC who underwent resection as well as the normal oral mucosa samples of 14 healthy volunteers were enrolled in this study. We studied expression of CD133 and CD44 using real-time RT-PCR, immunohistochemistry and western blot analysis in primary OSCC, and investigated the correlation of clinicopathological factors. ResultsAlthough CD133 protein expression was not obviously detected, CD133 mRNA expression in primary OSCC tissues significantly correlated with lymph node metastasis, depth of invasion, extranodal invasion, and clinical stage. ConclusionsCD133 mRNA over-expression in OSCC primary tumors could be a novel independent prognostic marker that correlates with tumor progression and lymph node metastasis.

CDNA and lincRNA microarrays: identification of expression patterns related to metastatization in oral squamous cell carcinoma (1048.6)

Oral squamous cell carcinoma (OSCC) metastatic cascade still a poorly understood mechanism, in terms of gene expression differences between primary site and metastatic cells at lymph nodes. Using 20 matched samples of OSCC cases, being 10 from primary sites and 10 metastastic lymph nodes we performed lincRNA and cDNA microarrays analysis (Agilent`s Sureprint G3 Human Genome V2.0). Our results showed 617 genes and lincRNAs significantly differentially expressed between metastatic lymph nodes and primary tumors. Integrated pathway analysis showed alteration in canonical pathways such as Ras homolog gene family, member A signaling pathway (RhoA pathway; p=0.002) and tumor necrosis factor receptor 2 signaling pathway (TNFR2 pathway; p=0.009). The most up‐regulated genes included SNORA65 (9,045‐fold), ZMAT1 (4,776‐fold) HNRNPU‐AS1 (4,626‐fold), RNU12 (4,557‐fold), TLE4 (4,555‐fold), and the most down‐regulated included MYBPH (‐67,818‐fold), ACTA1 (‐58,551‐fold), TNNC1 (‐23,119‐fold), F2RL2 (‐17,431‐fold) and MYLPF (‐13,785‐fold). Regarding lincRNAs, MALAT1 was significantly overexpressed in metastatic lymph nodes, with a 4.2‐fold increase. Our study shows the existence of several genes and lincRNAs with different expression patterns between OSCC primary tumor and metastatic lymph nodes, and also points to novel genes, which has not yet been implicated in OSCC metastasis, providing new potential targets in oral cancer research.Grant Funding Source: Supported by FAPESP grants 2011/18606‐7‐0 and 2010/08400‐0

Functional Relevance of Urinary-type Plasminogen Activator Receptor-α3β1 Integrin Association in Proteinase Regulatory Pathways

Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin alpha3beta1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of alpha3beta1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of alpha3beta1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/alpha3beta1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (-1967) site in the uPA promoter blocked the uPAR/integrin-mediated transcriptional activation. Because uPAR is redistributed to clustered alpha3beta1 integrins, the requirement for uPAR/alpha3beta1 interaction in uPA regulation was assessed. Clustering of alpha3beta1 in the presence of a peptide (alpha325) that disrupts uPAR/alpha3beta1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin alpha3beta1 clustering. These results were confirmed using a genetic strategy in which alpha3 null epithelial cells reconstituted with wild type alpha3 integrin, but not a mutant alpha3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/alpha3beta1 binding using peptide alpha325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering ofalpha3beta1 integrin promotes uPAR/alpha3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.

Inactivation of p16INK4a in primary tumors and cell lines of head and neck squamous cell carcinoma.

Inactivation of the p16INK4a gene by mutation and deletion is common in head and neck squamous cell carcinoma (HNSCC). The present study demonstrates that hypermethylation of the 5' CpG islands can serve as an alternative mechanism for the inactivation of the p16INK4a gene in this tumor. We studied 11 HNSCC cell lines and 17 oral squamous cell carcinoma (OSCC) primary tumors for p16INK4a gene status by protein/mRNA and DNA genetic/epigenetic analyses to determine the incidence of its inactivation. Our study indicates that: (1) inactivation of p16 protein is frequent in HNSCC cell lines (6/11, 54.5%) and OSCC primary tumors (15/17, 88.2%), (2) inactivation of p16INK4a protein is commonly associated with the presence of gene alteration such as mutation, homozygous deletion and especially aberrant methylation, and (3) genomic sequencing of bisulfite-modified DNA shows that the carcinoma develops a heterogeneous pattern of hypermethylation.