The interplay of EMT and stemness driving malignant transformation of Oral Submucous Fibrosis.

Background: Oral submucous fibrosis (OSF) is a persistent oral mucosal condition that carries an elevated risk of undergoing malignant transformation. Our objective was to elucidate the involvement of epithelial-to-mesenchymal transition (EMT) in OSF and its progression to malignancy by studying a panel of EMT markers, thereby understanding the molecular mechanisms. Methods: An immunohistochemical analysis was done to detect the presence of E-cadherin, N-cadherin, pan-cytokeratin (PanCK), vimentin, α-SMA (alpha-smooth muscle actin), and CD44 in a total of 100 tissue samples. These samples comprised 40 cases of OSF, 20 cases of oral squamous cell carcinoma associated with OSF (OSFSCC), and 40 cases of oral squamous cell carcinoma (OSCC). A whole transcriptomic analysis was performed on a group of seven matched samples encompassing NOM, OSF, OSFSCC, and OSCC. Results: We observed significantly decreased expression of E-cadherin and PanCK, while N-cadherin, vimentin, α-SMA, and CD44 showed significantly higher expression in OSFSCC and OSCC as compared to OSF, both at protein and RNA levels. CD44 expression was noticeably higher in OSFSCC (p < 0.001) than in OSCC. Conclusion: Downregulation of epithelial markers with concomitant upregulation of mesenchymal and stem cell markers suggests the potential role of EMT and stemness in accelerating the pathogenesis and malignant transformation of OSF. The high levels of CD44 expression seen in OSFSCC indicate a high propensity for aggressiveness and acquisition of stem-like characteristics by the cells undergoing EMT.

Exploring the regulatory interactions between mutated genes and homeobox genes in the head and neck cancer progression.

OBJECTIVE: Understanding the regulatory role of homeobox (HOX) and mutated genes in the progression of head and neck cancers is essential, although their interaction remains elusive. This study aims to decipher the critical regulation of mutation driven effects on homeobox genes to enhance our understanding of head and neck cancer progression. METHODS: Genomic mutation data from The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma were analyzed using VarScan2 for somatic variant detection. Mutational clustering, driver mutation identification, and cancer signaling pathway analysis were performed using the OncodriveCLUST method. Harmonizome datasets were retrieved to identify critical cancer driver genes affecting HOX genes. The effects of HPV infection on HOX and mutated genes were assessed using the oncoviral database. Altered pathway activity due to the effects of cancer drivers on HOX genes was analyzed with Gene Set Cancer Analysis. Functional enrichment analysis of gene ontology biological processes and molecular functions was conducted using the ClusterProfiler R package. RESULTS: Significant alterations in HOX genes were observed in head and neck cancer cohorts with mutated TP53, FAT1, and CDKN2A. HOX genes were identified as functionally downstream targets of TP53, signifying transcriptionally mediated regulation. The interaction between HOX genes and mutated TP53, FAT1, and CDKN2A dysregulated the epithelial-to-mesenchymal transition, cell cycle, and apoptosis pathways in head and neck cancer progression. CONCLUSION: The interplay between cancer driver genes and HOX genes is pivotal in regulating the oncogenic processes underlying the pathogenesis of head and neck squamous cell carcinoma.

The regulatory role of HOX interacting lncRNA in oral cancer-An in silico analysis.

OBJECTIVES: We aim to elucidate the interaction of long noncoding RNAs with HOX genes and their regulatory role and potential drug candidates in oral cancer. MATERIALS AND METHODS: The interaction network was constructed using RNA Interactome and the RNA Interactome from the Sequencing Experiments database. The differential expression of HOX genes and HOX interacting lncRNAs was assessed using the TCGA-Head and Neck Squamous Cell Carcinoma oral cancer dataset using DESeq2 R-package. Further, the functional enrichment analysis was performed for the differentially expressed HOX genes and HOX-interacting lncRNAs using Gene Ontology, long noncoding RNA Set Enrichment Analysis, lncRNA ontology annotation extractor and repository (Lantern), and LncRNA Ontology tools. Drug-lncRNA interaction and the effect of drugs on lncRNA expression were assessed from the D-lnc tool. RESULTS: A total of 78 unique interactions were identified between HOX and lncRNAs. Differential expression analysis showed 27 HOX genes and 10 HOX-interacting lncRNAs in oral cancer. HOX genes and HOX-interacting lncRNAs were involved in crucial regulatory processes like cell cycle regulation, cell proliferation and migration, epithelial-mesenchymal transition, angiogenesis, and cell signaling pathways. Cancer hallmark analysis from using long noncoding RNA Set Enrichment Analysis showed the involvement of HOTAIR, HOTTIP MIR503HG, and CDKN2B-AS1 in proliferation, migration, and invasion. Panobinostat was the common drug that influenced the expression of HOTAIR, HOTAIRM1, HOTTIP and CDKN2B-AS1. CONCLUSIONS: Differentially expressed HOX-interacting lncRNAs are involved in various regulatory biological processes and cancer hallmark events in oral cancer. CLINICAL RELEVANCE: The creation of interaction networks may expand the existing knowledge of oral cancer signaling pathways and the discovery of novel targets.

Identification of HOX signatures contributing to oral cancer phenotype.

The role of evolutionarily conserved homeobox-containing HOX genes as transcriptional regulators in the developmental specification of organisms is well known. The contribution of HOX genes involvement in oral cancer phenotype has yet to be fully ascertained. TCGA-HNSC HTSeq-counts and clinical data were retrieved from the GDC portal for oral cavity neoplasms. GEO datasets (GSE72627, GSE30784, GSE37991) were accessed and analyzed using GEO2R. Differential HOX gene expression was profiled using the DESeq2 R package with a log2 fold change cut-off (- 1 and + 1) and Benjamini-Hochberg p-adjusted value at ≤ 0.01. Gene set over-representation analysis and semantic analysis associated with the disease ontology was performed using the ClusterProfiler R package, and pathway over-representation analysis was performed using IMPaLa. HOX protein interaction network was constructed using the Pathfind R package. HOX phenotype associations were performed using Mammalian Phenotype Ontology, Human Phenotype Ontology, PhenGenI associations, Jensen tissues, and OMIM entries. Drug connectivity mapping was carried out with Dr. Insight R package. HOXA2 was upregulated in oral dysplasia but silenced during tumor progression. Loss of HOXB2 expression was consistent in the potentially malignant oral lesions as well as in the primary tumor. HOXA7, HOXA10, HOXB7, HOXC6, HOXC10, HOXD10, and HOXD11 were consistently upregulated from premalignancy to malignancy and were notably associated with risk factors. Overrepresentation analysis suggested HOXA10 was involved in the transcriptional misregulation contributing to the oral cancer phenotype. HOX genes subnetwork analysis showed crucial interactions with cell cycle regulators, growth responsive elements, and proto-oncogenes. Phenotype associations specific to the oral region involving HOX genes provide intrinsic cues to tumor development. The 5' HOX genes were aberrantly upregulated during oral carcinogenesis reflecting their posterior prevalence.

In silico interaction of HOX cluster-embedded microRNAs and long non-coding RNAs in oral cancer.

The essential role HOX-associated non-coding RNAs play in chromatin dynamics and gene regulation has been well documented. The potential roles of these microRNAs and long non-coding RNAs in oral cancer development, with their attendant involvement in various cellular processes including proliferation, invasion, migration, epithelial-mesenchymal transition and metastasis is gaining credence. An interaction network of HOX-embedded non-coding RNAs was constructed to identify the RNA interaction landscape using the arena-Idb platform and visualized using Cytoscape. The miR-10a was shown to interact with HOXA1, miR-10b with HOXD10, miR-196a1 with HOXA5, HOXA7, HOXB8, HOXC8, HOXD8, and miR-196a2 with HOXA5. The lncRNAs, HOTAIR interacted with HOXC11, HOTAIRM1 with HOXA1 and HOXA4, HOTTIP with HOXA13, HOXA-AS2 with HOXA3, HOXA11-AS with HOXA11 and HOXD-AS1 with HOXB8. Changes in the HOX cluster-embedded non-coding RNAs have implications for prognosis and overall disease survival. Our review aims to analyze the functional significance and clinical relevance of non-coding RNAs within the HOX cluster in the context of oral carcinogenesis. Elucidating these interactions between the non-coding RNAs and HOX genes in oral cancer development and progression could pave the way for the identification of reliable biomarkers and potential therapeutic targets.

Biological implications of the immune factors in the tumour microenvironment of oral cancer.

OBJECTIVE: The objective of this review is to decipher the biological implications of the immune factors in the tumour microenvironment in oral cancer. The restoration of balance between tumour tolerance and tumour eradication by the host immune cells is critical to provide effective therapeutic strategies. DESIGN: The specific role of the stromal and the immune components in oral cancer was reviewed with a tailored search strategy using relevant keywords. The articles were retrieved from bibliometric databases indexed in PubMed, Scopus, and Embase. An in silico analysis was performed to identify potential drug candidates for immunotherapy, by accessing the Drug-Gene Interactions Database (DGIdb) using the rDGIdb package. RESULTS: There is compelling evidence for the role of the cellular and extracellular components of the tumour microenvironment in inducing immunosuppression and progression of oral cancer. The druggable candidates specifically targeting the immune system are a viable option in the treatment of oral cancer as they can regulate the tumour microenvironment. CONCLUSION: A complex interaction between the tumour and the immunological microenvironment influences the disease outcome in oral cancer. Targeting specific components of the immune system might be relevant, as immunotherapy may become the new standard of care for oral cancer.

RARß Expression in Keratinocytes from Potentially Malignant Oral Lesions: The Functional Consequences of Re-Expression by De-Methylating Agents.

Loss of RARß2 expression by promoter methylation is an early event in oral carcinogenesis. Understanding the mechanisms and consequences of RARß loss may aid in understanding the disappointing results of retinoid chemoprevention trials. This study aimed to describe the effects of all-trans retinoic acid (ATRA) and the de-methylating agent 5-Aza-2' deoxycytidine (5-AZA-CdR) on a panel of immortal potentially malignant oral lesion (PMOL) cell cultures. RARß expression was assessed in PMOL tissues by immunohistochemistry. Cells were treated with ATRA ± 5-AZA-CdR, and the effects on the cell cycle and senescence were assessed. In PMOL tissues, RARß expression was variable, but lower in biopsies which gave rise to immortal cell cultures. Treatment of iPMOL cells with ATRA resulted in little change in RARß expression, but the addition of 5-AZA-CdR resulted in significant increases. The effects on the cell cycle and senescence were variable and may be related to 5-AZA-CdR, as this has wider effects on the cell cycle. Overall, the response of iPMOL cells to ATRA and 5-AZA-CdR treatment was variable and is dependent on several factors, including RARß-promoter methylation. These findings may help to explain the lack of consistent effect of retinoids in PMOLs seen in chemoprevention trials.

In silico analysis of HOX-associated transcription factors as potential regulators of oral cancer.

OBJECTIVE: The objective of this study was identification of the transcription factor binding sites (TFBS) in the promoter of HOX genes and elucidation of the comprehensive interaction of transcription factors (TFs)/genes with HOX. METHODOLOGY: Promoter sequences of HOXA3, HOXA5, HOXA9, HOXA10, HOXA13, HOXB5, HOXC10, HOXC12, and HOXD10 were analyzed to predict the TFBS and their targets using TRANSFAC, TRRUST, and Harmonizome. Functional analysis of the processed data sets was carried out using DAVID and GATHER gene annotation tools. A network of regulatory interactions was constructed using NetworkAnalyst and a comprehensive illustration of the TF-gene network was constructed with HOX as a central hub using the Encyclopedia of DNA Elements chromatin immunoprecipitation sequencing data. Further, the enriched network was constructed to elucidate the roles of these genes in the various pathways. RESULTS: Binding sites for E2F1, HNF3α, SP3, and KLF6 were common to promoter regions of all of the HOX genes. The functional annotation and pathway analysis elucidated the regulatory activity of a distinct set of TF-genes in interaction with HOX. A P value ≤.05 and false discovery rate ≤0.01 were considered statistically significant. CONCLUSION: We have confirmed that the predicted TFBSs in the HOX gene promoters function in transcriptional regulation by modulating target gene activity. TF-gene interactions are crucial to understanding oral carcinogenesis.