Raghu Radhakrishnan

HOXA5 , a member of the HOX family, has been characterized as a tumor suppressor in several cancers. However, its regulation and role in oral cancer (OC) remain unclear. HOXA5 expression was assessed by transcriptome analysis and qRT-PCR, while methylation was evaluated using methyl-capture sequencing and bisulfite Sanger sequencing in a clinical cohort comprising OPMD, LN(-) and LN(+) groups and in vitro models. The effects of DNA methylation and retinoic acid were examined using DNMT inhibitors (DNMTi) and/or retinoic acid (RA). Promoter activity and RARB occupancy were assessed by dual-luciferase reporter and ChIP-qPCR assays, respectively. Effects of ectopic HOXA5 expression were evaluated using functional assays, whole-transcriptome profiling, and in-silico analyses. HOXA5 was upregulated and relatively hypomethylated in OPMD and the dysplastic DOK cell line, whereas its expression did not differ between LN(-) or LN(+) tumors and matched normal tissues despite increased promoter methylation. In SCC9 cells, HOXA5 was downregulated and promoter hypermethylated. Weak but significant inverse correlations between expression and promoter methylation were observed. DNMTi and/or RA treatment reversed HOXA5 expression. HOXA5- EPD2 region showed the highest promoter activity and RARB occupancy at the promoter regions was validated. HOXA5 overexpression reduced proliferation, migration, and invasion, while inducing S-phase arrest and apoptosis. Transcriptome and enrichment analyses identified pathways associated with cancer-related phenotypes, while protein interaction and in silico drug sensitivity analyses identified candidate drug-gene associations for further investigation in OC. Our findings support a role for DNA methylation in HOXA5 regulation and suggest tumor-suppressive properties of HOXA5 in OC.

Identifying biomarkers of epithelial‒mesenchymal transition (EMT) and metastasis play a decisive role in the prognosis and clinical management of Oral cancer (OC). Homeobox A9 (HOXA9), an imperative regulator of embryogenesis and endothelial cell proliferation, is aberrantly expressed in several malignancies, including OC. To date, HOXA9-mediated molecular mechanisms and their functional roles in EMT and OC metastasis remain poorly understood. In this study, we observed significant upregulation of HOXA9 in OC samples with lymph node-positive stage and higher histological grade. Notably, we demonstrated that retroviral-mediated knockdown of HOXA9 impairs proliferation, migration, and invasion while promoting apoptosis and cell cycle arrest. A substantial reduction in tumor volume and diminished lung metastasis was observed in nude mice receiving HOXA9-knockdown cells. Transcriptomic analysis of HOXA9-depleted cells revealed the downregulation of multiple pathways, with the most significant being “pathways in cancer”. We further demonstrated that HOXA9 transcriptionally activates VIM to promote EMT and it also facilitates β-catenin nuclear translocation, which subsequently activates downstream target genes driving Wnt/β-catenin cascade. Our study also unveils HOXA9-driven molecular interplay in which HOXA9-dependent transcriptional activation of VIM triggers Wnt/β-catenin cascade in OC. Furthermore, we demonstrated that hypomethylated CpGs at HOXA9 promoter (spanning −4201bp to −3574bp upstream of TSS) in stage III/IV tumors showed a significant inverse correlation between hypomethylation and gene upregulation. Consistently, analysis of the same distal promoter region revealed the enrichment of activating histone modifications, further supporting its transcriptional permissive state. Collectively, our study uncovers a novel mechanism by which epigenetically altered HOXA9 serves as a potential inducer of EMT through regulation of VIM/Wnt-β-catenin/EMT signaling axis. These findings signify HOXA9 as a promising biomarker in OC and targeting HOXA9 could be an effective strategy to improve clinical outcome of patients diagnosed with advanced stages.

Background: Conventional therapies for oral submucous fibrosis (OSF) are largely conservative and have limited ability to modify the underlying fibrotic process or prevent disease progression. This limitation highlights the need for targeted molecular therapies. MicroRNAs (miRNAs) have recently emerged as promising anti‐fibrotic regulators capable of modulating key profibrotic signalling pathways. Objective: To systematically map and synthesise available evidence on antifibrotic miRNA‐based therapeutic interventions in OSF, including their mechanisms of action, delivery strategies, molecular targets and treatment outcomes. Methods: This scoping review followed the PRISMA‐ScR guidelines and employed the Arksey and O’Malley framework. A comprehensive search was performed in various databases such as PubMed, Scopus, Web of Science, Embase, Cochrane Library and Google Scholar to collect pertinent data. Studies considered eligible comprised of experimental, in vitro, in vivo, and quasi‐experimental research assessing anti‐fibrotic miRNAs in OSF models. Results: Of the 329 records identified, 10 studies fulfilled the inclusion criteria. Most employed in vitro fibrotic oral fibroblast models, with two incorporating in vivo animal models. Several miRNAs with antifibrotic properties were identified, including miR‐29b, miR‐760‐3p, miR‐145 inhibitor, miR‐424 inhibitor, miR‐503, miR‐30b‐5p, miR‐181a‐5p, miR‐29c, miR‐375 and miR‐204, which attenuate fibrotic responses by targeting genes and signalling pathways involved in fibrosis, including TGF‐β/Smad, RAS/RAF/MEK/ERK, and epithelial–mesenchymal transition (EMT). Conclusion: Anti‐fibrotic miRNA‐based interventions demonstrate promising antifibrotic activity in preclinical models of OSF by modulating the profibrotic pathways and reducing fibrosis. Advanced delivery platforms have helped to overcome challenges associated with miRNA delivery; however, evidence remains largely limited to in vitro and in vivo studies and does not establish reversal of established fibrosis or clinical efficacy in patients. Future research should prioritise standardisation of experimental models and well‐designed clinical trials for better understanding of therapeutic relevance and translational potential of miRNA in OSF.

Background: Histopathological risk assessment models in oral squamous cell carcinoma (OSCC) have had varying degrees of success in stratifying patients, with models developed on retrospective cohorts being able to predict recurrence and metastasis. In spite of mounting evidence on their impact on survival outcomes, the incorporation of histopathological features, including perineural invasion (PNI) and the worst pattern of invasion, in staging and risk stratification is still lacking. Thus, our study aimed to assess and characterise histological prognostic indicators in OSCC, elucidate their significance in relation to patient prognosis, and develop a risk assessment model by correlating these indicators with survival parameters and risk stratification. Methods: A retrospective chart audit was performed, and histologic parameters were staged according to the American Joint Committee on Cancer’s 8th Edition Cancer Staging Manual and objectively scored based on the College of American Pathologists’ (CAP) Protocol 2018. Pearson’s chi‐square test and Kaplan–Meier survival analysis were carried out to identify the significant correlations between specific histopathological indicators and patient prognosis. Results: Higher grades of pattern of invasion were significantly associated with greater PNI and depth of invasion ( p < 0.05). Depth of invasion was also significantly associated with extranodal extension (ENE) ( p < 0.05). Survival analysis showed a clear trend with higher grades of the worst pattern of invasion and depth of invasion, as well as PNI, as possible independent predictors of poorer survival outcomes. Conclusion: The development of a risk stratification model based on these indicators provides a valuable tool for enhancing patient outcomes. Incorporation of detailed histopathological analysis into routine clinical decision‐making improves survival rates and treatment efficacy in OSCC patients.

Background: The trigeminal sensory system releases numerous neuropeptides like neurokinin A (NKA), substance P (SP), and calcitonin gene-regulated protein (CGRP), which play crucial functions in orofacial pain. Pulpitis increases the production of these neuropeptides in pulp and peripheral body fluids like saliva and the GCF. Pain perception is the most investigated and well-researched role of SP. Moreover, SP was the first neuropeptide to be recognized in dental pulp. Methods: Searches were performed in PubMed, Scopus, EBSCOhost, Epistemonikos, grey literature databases, and other available databases until July 2024. Three independent reviewers performed a grey literature search on Google to attain maximum coverage beyond peer review, and articles were resolved with agreement among the reviewers. The selection criteria were in vivo studies that evaluated SP levels in GCF at the site of tooth pain and a healthy tooth. Meta-analysis was performed using standardized mean differences by the method of Cohen using the random-effects inverse variance model with the DerSimonian-Laird estimator for tau². Results: Six articles were included in the review according to the selection criteria. Five studies concluded that there is a significant elevation of levels of SP in GCF in a painful tooth when compared to a healthy tooth. These five studies were incorporated in the meta-analysis. However, one study concluded similar levels of SP in GCF in painful and healthy teeth. Conclusions: The results indicate that SP concentrations in GCF could rise due to dental pain and inflammation. These findings must be interpreted conservatively since several possible confounding factors, such as differences in sampling methods, pain determination criteria, patient age, systemic health status, drug use, and periodontal status, could affect SP levels. Due to the paucity of studies and heterogeneity of methods, more high-quality research must be conducted to replicate these associations and define the clinical usefulness of SP as a pain biomarker for dentistry.

Persistent injury to oral mucosa due to habitual quid chewing, resulting in the upregulation of inflammatory cytokines and consequential myofibroblastic persistence, emphasizes the role of epigenetic aberration in the pathogenesis of oral submucous fibrosis (OSF). However, there is a dearth of research on the role epistasis plays in the pathophysiology of OSF. Among the important epistatic interactions in the pathophysiology of OSF are those between Phosphatase and Tensin Homologue (PTEN) and Insulin-Like Growth Factor 1, Transforming Growth Factor-β (TGF-β), Cyclooxygenases, and lipoxygenases (LOX). Additionally, PTEN and Nuclear Factor Kappa B (NF-κB) have an epistatic relationship that is particularly mediated by the p65 subunit of NF-κB. Given the importance of epigenetic modification in the pathogenesis of OSF, the potential use of DNA methyltransferase and Histone deacetylase inhibitors as a therapeutic option holds promise. Another in vivo epigenetic therapeutic option for treating OSF is using stimulatory microRNAs against antifibrotic genes and inhibitory microRNAs against profibrotic genes. This review aims to connect numerous epigenetic and epistatic components to the mechanism of OSF. A better understanding of the disease process may provide OSF management with newer therapeutic options. Graphical Abstract

Background: Solitary fibrous tumors encompass a heterogeneous group of spindle cell neoplasms, ranging from biologically low-risk lesions to, in rare instances, highly aggressive tumors with malignant potential. Dedifferentiation in solitary fibrous tumors is uncommon and typically occurs in the retroperitoneum, with extrapleural involvement being among the least frequently reported. Case Presentation: A 13-year-old male presented with a rapidly enlarging mass in the lower jaw of 20 days duration, involving the submandibular triangle and floor of the mouth. Diagnosis: Histopathological examination of the excisional biopsy revealed spindle-shaped cells arranged in compact fascicles with a haphazard distribution, and areas of hyalinization. Immunohistochemical analysis demonstrated positivity for CD34, STAT6, MyoD1, α-SMA, Bcl-2, and CD99, confirming the diagnosis of extrapleural dedifferentiated solitary fibrous tumor (DSFT). Management: The lesion was surgically excised but recurred, likely due to disease progression. Re-excision was planned, but the child died 10 days before surgery.

Background: The hypothesis that aberrant expression of homeobox (HOX) transcription factors contributes to oral cancer progression is gaining prominence. However, the mechanism of regulation involved in the clustered dysregulation of HOX clusters is not clearly known. Results: Our findings revealed that HOXA and HOXB clusters showed significant locus-specific CpG methylation changes compared with the HOXC and HOXD clusters. The constitutively unmethylated regions identified in the HOXA1, HOXA11, HOXB5, HOXB6, HOXB9, HOXC5, HOXC10 and HOXC11 genes may be associated with open chromatin-mediated gene regulation. The methylation of CpG loci within the intron of HOXB9 may serve as a potential marker for distinguishing patients with premalignant and advanced oral tumors. HOXA5 and HOXC9 showed higher transcription factor-mediated interactions with neighboring HOX genes within and across the clusters. Additionally, HOXB9 and HOXC10 were predicted to directly regulate the G2–M checkpoint and hypoxia pathways. HOXA genes can be post-transcriptionally regulated through an antisense-mediated mechanism involving embedded HOX long noncoding RNAs (lncRNAs). Posterior HOX genes were more highly expressed than anterior HOX genes. The HOXC and HOXD cluster gene expression patterns were similar to those of the embedded lncRNAs. HOXA1, HOXC13 and HOXD10 were significantly correlated with the cancer hallmarks driving oral carcinogenesis. Conclusion: The functional consequence of HOX genes dysregulation was driven by diverse DNA and RNA epigenetic mechanisms affecting the transcriptional and post-transcriptional regulation contributing to the oral cancer progression.