Integrative analysis reveals associations between oral microbiota dysbiosis and host genetic and epigenetic aberrations in oral cavity squamous cell carcinoma.

Dysbiosis of the human oral microbiota has been reported to be associated with oral cavity squamous cell carcinoma (OSCC) while the host-microbiota interactions with respect to the potential impact of pathogenic bacteria on host genomic and epigenomic abnormalities remain poorly studied. In this study, the mucosal bacterial community, host genome-wide transcriptome and DNA CpG methylation were simultaneously profiled in tumors and their adjacent normal tissues of OSCC patients. Significant enrichment in the relative abundance of seven bacteria species (Fusobacterium nucleatum, Treponema medium, Peptostreptococcus stomatis, Gemella morbillorum, Catonella morbi, Peptoanaerobacter yurli and Peptococcus simiae) were observed in OSCC tumor microenvironment. These tumor-enriched bacteria formed 254 positive correlations with 206 up-regulated host genes, mainly involving signaling pathways related to cell adhesion, migration and proliferation. Integrative analysis of bacteria-transcriptome and bacteria-methylation correlations identified at least 20 dysregulated host genes with inverted CpG methylation in their promoter regions associated with enrichment of bacterial pathogens, implying a potential of pathogenic bacteria to regulate gene expression, in part, through epigenetic alterations. An in vitro model further confirmed that Fusobacterium nucleatum might contribute to cellular invasion via crosstalk with E-cadherin/ß-catenin signaling, TNFα/NF-κB pathway and extracellular matrix remodeling by up-regulating SNAI2 gene, a key transcription factor of epithelial-mesenchymal transition (EMT). Our work using multi-omics approaches explored complex host-microbiota interactions and provided important insights into genetic and functional basis in OSCC tumorigenesis, which may serve as a precursor for hypothesis-driven study to better understand the causational relationship of pathogenic bacteria in this deadly cancer.

Notch signaling genes and CD8+ T-cell dynamics: Their contribution to immune-checkpoint inhibitor therapy in oral squamous cell carcinoma: A retrospective study.

BACKGROUND: Aberrant Notch signaling pathway has been related with the tumorigenesis in head and neck region, involving oral cavity. Here, we report the correlation between mutations in the Notch signaling pathway and CD8+ T-cell infiltration via PD-L1, which lead to enhanced antitumor immunity and may target for immune-checkpoint inhibitors (ICIs) therapy. METHODS: This retrospective study analyzed the results of immunohistochemical staining for PD-L1 and CD8+ T-cell infiltration in 10 patients and whole-exome sequencing (WES) was conducted on five of these patients to identify frequently mutated genes. RESULTS: Four of 10 patients were positive for PD-L1 and CD8+ T. By analyzing WES in three of these four patients, we notably identified the mutations of NOTCH1, FBXW7, and noncoding RNA intronic mutation in NOTCH2NLR in two of these three patients. This study may enable better selection of ICI therapy with CD8+ T-cell infiltration via PD-L1 expression for oral squamous cell carcinoma patients with mutations in Notch signaling pathway.

Potential targets of diosgenin for the treatment of oral squamous cell carcinoma and their bioinformatics and transcriptional profiling analyses.

Diosgenin can inhibit the proliferation and cause apoptosis of various tumor cells, and its inhibitory effect on oral squamous cell carcinoma (OSCC) and its mechanism are still unclear. In this study, we predicted the targets of diosgenin for the treatment of OSCC through the database, then performed bioinformatics analysis of the targets, and further verified the effect of diosgenin on the activity of OSCC cell line HSC-3, the transcriptional profile of the targets and the molecular docking of the targets with diosgenin. The results revealed that there were 146 potential targets of diosgenin for OSCC treatment, which involved signaling pathways such as Ras, TNF, PI3K-AKT, HIF, NF-κB, and could regulate cellular activity through apoptosis, autophagy, proliferation and differentiation, inflammatory response, DNA repair, etc. Diosgenin significantly inhibited HSC-3 cell activity. The genes such as AKT1, MET1, SRC1, APP1, CCND1, MYC, PTGS2, AR, NFKB1, BIRC2, MDM2, BCL2L1, MMP2, may be important targets of its action, not only their expression was regulated by diosgenin but also their proteins had a high binding energy with diosgenin. These results suggest that diosgenin may have a therapeutic effect on OSCC through AKT1, MMP2 and other targets and multiple signaling pathways, which is of potential clinical value.

Lipid droplets-related Perilipin-3: potential immune checkpoint and oncogene in oral squamous cell carcinoma.

BACKGROUND: Lipid droplets (LDs) as major lipid storage organelles are recently reported to be innate immune hubs. Perilipin-3 (PLIN3) is indispensable for the formation and accumulation of LDs. Since cancer patients show dysregulated lipid metabolism, we aimed to elaborate the role of LDs-related PLIN3 in oral squamous cell carcinoma (OSCC). METHODS: PLIN3 expression patterns (n = 87), its immune-related landscape (n = 74) and association with B7-H2 (n = 51) were assessed by immunohistochemistry and flow cytometry. Real-time PCR, Western blot, Oil Red O assay, immunofluorescence, migration assay, spheroid-forming assay and flow cytometry were performed for function analysis. RESULTS: Spotted LDs-like PLIN3 staining was dominantly enriched in tumor cells than other cell types. PLIN3high tumor showed high proliferation index with metastasis potential, accompanied with less CD3+CD8+ T cells in peripheral blood and in situ tissue, conferring immunosuppressive microenvironment and shorter postoperative survival. Consistently, PLIN3 knockdown in tumor cells not only reduced LD deposits and tumor migration, but benefited for CD8+ T cells activation in co-culture system with decreased B7-H2. An OSCC subpopulation harbored PLIN3highB7-H2high tumor showed more T cells exhaustion, rendering higher risk of cancer-related death (95% CI 1.285-6.851). CONCLUSIONS: LDs marker PLIN3 may be a novel immunotherapeutic target in OSCC.

Supramolecular nanodrug targeting CDK4/6 overcomes BAG1 mediated cisplatin resistance in oral squamous cell carcinoma.

Chemoresistance to cisplatin remains a significant challenge affecting the prognosis of advanced oral squamous cell carcinoma (OSCC). However, the specific biomarkers and underlying mechanisms responsible for cisplatin resistance remain elusive. Through comprehensive bioinformatic analyses, we identified a potential biomarker, BCL2 associated athanogene-1 (BAG1), showing elevated expression in head and neck squamous cell carcinoma (HNSCC). Since OSCC represents the primary pathological type of HNSCC, we investigated BAG1 expression in human tumor tissues and cisplatin resistant OSCC cell lines, revealing that silencing BAG1 induced apoptosis in cisplatin-resistant cells both in vitro and in vivo. This effect led to impaired cell viability of cisplatin resistant OSCC cells and indicated a positive correlation between BAG1 expression and the G1/S transition during cell proliferation. Based on these insights, the administration of a CDK4/6 inhibitor in combination with cisplatin effectively overcame cisplatin resistance in OSCC through the CDK4/6-BAG1 axis. Additionally, to enable simultaneous drug delivery and enhance synergistic antitumor efficacy, we developed a novel supramolecular nanodrug LEE011-FFERGD/CDDP, which was validated in an OSCC orthotopic mouse model. In summary, our study highlights the potential of a combined administration of CDK4/6 inhibitor and cisplatin as a promising therapeutic regimen for treating advanced or cisplatin resistant OSCC.

PHA-665752's Antigrowth and Proapoptotic Effects on HSC-3 Human Oral Cancer Cells.

c-Met is a tyrosine-kinase receptor, and its aberrant activation plays critical roles in tumorigenesis, invasion, and metastatic spread in many human tumors. PHA-665752 (PHA) is an inhibitor of c-Met and has antitumor effects on many hematological malignancies and solid cancers. However, the activation and expression of c-Met and its role and the antitumor effect of PHA on human oral squamous cell carcinoma (OSCC) cells remain unclear. Here, we investigated the activation and expression of c-Met and the effects of PHA on the growth of a highly tumorigenic HSC-3 human OSCC cell line with high c-Met phosphorylation and expression. Of note, c-Met was highly expressed and phosphorylated on Y1234/1235 in HSC-3 cells, and PHA treatment significantly suppressed the growth and induced apoptosis of these cells. Moreover, PHA that inhibited the phosphorylation (activation) of c-Met further caused the reduced phosphorylation and expression levels of Src, protein kinase B (PKB), mammalian target of rapamycin (mTtor), and myeloid cell leukemia-1 (Mcl-1) in HSC-3 cells. In addition, the antiangiogenic property of PHA in HSC-3 cells was shown, as evidenced by the drug's suppressive effect on the expression of hypoxia-inducible factor-1α (HIF-1α), a critical tumor angiogenic transcription factor. Importantly, genetic ablation of c-Met caused the reduced growth of HSC-3 cells and decreased Src phosphorylation and HIF-1α expression. Together, these results demonstrate that c-Met is highly activated in HSC-3 human oral cancer cells, and PHA exhibits strong antigrowth, proapoptotic, and antiangiogenic effects on these cells, which are mediated through regulation of the phosphorylation and expression of multiple targets, including c-Met, Src, PKB, mTOR, Mcl-1, and HIF-1α.

Mass spectrometry-based proteomics profiling of dogs with and without oral diseases: a systematic review.

BACKGROUND: Understanding the distinct proteomics profiles in dogs' oral biofluids enhances diagnostic and therapeutic insights for canine oral diseases, fostering cross-species translational research in dentistry and medicine. This study aimed to conduct a systematic review to investigate the similarities and differences between the oral biofluids' proteomics profile of dogs with and without oral diseases. METHODS: PubMed, Web of Science, and Scopus were searched with no restrictions on publication language or year to address the following focused question: "What is the proteome signature of healthy versus diseased (oral) dogs' biofluids?" Gene Ontology enrichment and the Kyoto Encyclopedia of Genes and Genomes pathway analyses of the most abundant proteins were performed. Moreover, protein-protein interaction analysis was conducted. The risk of bias (RoB) among the included studies was assessed using the Joanna Briggs Institute (JBI) Critical Appraisal Checklist for Studies Reporting Prevalence Data. RESULTS: In healthy dogs, the proteomic analysis identified 5,451 proteins, with 137 being the most abundant, predominantly associated with 'innate immune response'. Dogs with oral diseases displayed 6,470 proteins, with distinct associations: 'defense response to bacterium' (periodontal diseases), 'negative regulation of transcription' (dental calculus), and 'positive regulation of transcription' (oral tumors). Clustering revealed significant protein clusters in each case, emphasizing the diverse molecular profiles in health and oral diseases. Only six studies were provided to the JBI tool, as they encompassed case-control evaluations that compared healthy dogs to dogs with oral disease(s). All included studies were found to have low RoB (high quality). CONCLUSION: Significant differences in the proteomics profiles of oral biofluids between dogs with and without oral diseases were found. The synergy of animal proteomics and bioinformatics offers a promising avenue for cross-species research, despite persistent challenges in result validation.

Differential Expression of MicroRNA MiR-145 and MiR-155 Downstream Targets in Oral Cancers Exhibiting Limited Chemotherapy Resistance.

New evidence has suggested that non-coding microRNAs play a significant role in mediating and modulating chemotherapy resistance, particularly among oral cancers. One recent study found that the upregulation of miR-145 and the downregulation of miR-155 strongly correlated with a limited chemotherapy resistance to Cisplatin, 5-Fluorouracil, and Paclitaxel, although the mechanism(s) responsible for these observations remain unidentified. Using commercially available cell lines of oral squamous cell carcinoma, RNA was isolated, converted into cDNA, and subsequently screened for the expression of downstream targets of miR-145 and miR-155 using qPCR. These results demonstrated the upregulation of miR-21, miR-125, miR-133, miR-365, miR-720, and miR-1246, as well as the downregulation of miR-140, miR-152, miR-218, miR-221, and miR-224. This screening also confirmed the differential expression and regulation of mir-145 and miR-155 among the cell lines with limited chemotherapy resistance (SCC15). In addition, several downstream targets of these specific microRNAs were upregulated by all oral cancer cell lines, such as MBTD1 and FSCN1, or downregulated in all cell lines, such as CLCN3, FLI-1, MRTFB, DAB, SRGAP1, and ABHD17C. However, three miR-145 downstream targets were identified in the least chemotherapy-resistant cells, exhibiting the differential upregulation of KCNA4 and SRGAP2, as well as the downregulation of FAM135A, with this expression pattern not detected in any of the other oral cancer cell lines. These data strongly support that the differential regulation of these three downstream targets may be related to the chemosensitivity of this oral cancer cell line. The potential involvement of these targets must be further investigated to determine how and whether mechanisms of these cellular pathways may be involved in the observed lack of chemotherapy resistance. These data may be important to design targets or treatments to reduce chemotherapy resistance and improve patient treatment outcomes.

VAV2 orchestrates the interplay between regenerative proliferation and ribogenesis in both keratinocytes and oral squamous cell carcinoma.

VAV2 is an activator of RHO GTPases that promotes and maintains regenerative proliferation-like states in normal keratinocytes and oral squamous cell carcinoma (OSCC) cells. Here, we demonstrate that VAV2 also regulates ribosome biogenesis in those cells, a program associated with poor prognosis of human papilloma virus-negative (HPV-) OSCC patients. Mechanistically, VAV2 regulates this process in a catalysis-dependent manner using a conserved pathway comprising the RAC1 and RHOA GTPases, the PAK and ROCK family kinases, and the c-MYC and YAP/TAZ transcription factors. This pathway directly promotes RNA polymerase I activity and synthesis of 47S pre-rRNA precursors. This process is further consolidated by the upregulation of ribosome biogenesis factors and the acquisition of the YAP/TAZ-dependent undifferentiated cell state. Finally, we show that RNA polymerase I is a therapeutic Achilles' heel for both keratinocytes and OSCC patient-derived cells endowed with high VAV2 catalytic activity. Collectively, these findings highlight the therapeutic potential of modulating VAV2 and the ribosome biogenesis pathways in both preneoplastic and late progression stages of OSCC.

SALL4 promotes cancer stem-like cell phenotype and radioresistance in oral squamous cell carcinomas via methyltransferase-like 3-mediated m6A modification.

Radioresistance imposes a great challenge in reducing tumor recurrence and improving the clinical prognosis of individuals having oral squamous cell carcinoma (OSCC). OSCC harbors a subpopulation of CD44(+) cells that exhibit cancer stem-like cell (CSC) characteristics are involved in malignant tumor phenotype and radioresistance. Nevertheless, the underlying molecular mechanisms in CD44( + )-OSCC remain unclear. The current investigation demonstrated that methyltransferase-like 3 (METTL3) is highly expressed in CD44(+) cells and promotes CSCs phenotype. Using RNA-sequencing analysis, we further showed that Spalt-like transcription factor 4 (SALL4) is involved in the maintenance of CSCs properties. Furthermore, the overexpression of SALL4 in CD44( + )-OSCC cells caused radioresistance in vitro and in vivo. In contrast, silencing SALL4 sensitized OSCC cells to radiation therapy (RT). Mechanistically, we illustrated that SALL4 is a direct downstream transcriptional regulation target of METTL3, the transcription activation of SALL4 promotes the nuclear transport of ß-catenin and the expression of downstream target genes after radiation therapy, there by activates the Wnt/ß-catenin pathway, effectively enhancing the CSCs phenotype and causing radioresistance. Herein, this study indicates that the METTL3/SALL4 axis promotes the CSCs phenotype and resistance to radiation in OSCC via the Wnt/ß-catenin signaling pathway, and provides a potential therapeutic target to eliminate radioresistant OSCC.

Baicalin induces ferroptosis in oral squamous cell carcinoma by suppressing the activity of FTH1.

BACKGROUND: This study investigated the role of the ferroptosis-related gene FTH1 in oral squamous cell carcinoma (OSCC) and evaluated the therapeutic potential of baicalin in OSCC cell treatment. METHODS: A prognostic model was established by bioinformatic analysis, consisting of 12 ferroptosis related genes (FRGs), and FTH1 was selected as the most significantly up-regulated FRGs. The clinical correlation of FTH1 in OSCC samples was evaluated by both immunohistochemical and bioinformatic characterizations. The effects of FTH1 on migration, invasion, epithelial-mesenchymal transition (EMT) and proliferation were determined by wound healing assays, transwell assays, western blotting and 5'-ethynl 2'-deoxyuridine proliferation assays, respectively. The effects of FTH1 on ferroptosis were tested via ferroptosis markers and Mito Tracker staining. In addition, the therapeutic effects of baicalin on OSCC cells were confirmed using EMT, migration, invasion, proliferation and ferroptosis assays. RESULTS: The 12 FRGs were predictive of the prognosis for OSCC patients, and FTH1 expression was identified as significantly up-regulated in OSCC samples, which was highly associated with survival, immune cell infiltration and drug sensitivity. Moreover, knocking down FTH1 inhibited cell proliferation, EMT and invasive phenotypes, but induced ferroptosis in OSCC cells (Cal27 and SCC25). Furthermore, baicalin directly suppressed expression of FTH1 in OSCC cells, and effectively promoted ferroptosis and inhibited the proliferation as well as EMT by directly targeting FTH1. CONCLUSIONS: This study has demonstrated that FTH1 is a therapeutic target for OSCC treatment, and has provided evidence that baicalin offers a promising alternative for OSCC treatment.

Exploring miR-155-5p and miR-1246 as Diagnostic and Prognostic Markers in Oral Squamous cell carcinoma.

INTRODUCTION: Oral Squamous Cell Carcinoma (OSCC) is one of the leading cancers worldwide, significantly impacting developing nations. This study aimed to explore the diagnostic and prognostic potential of miR-155-5p and miR-1246 in OSCC in the Indian population, as their comparative roles in this context remain unexplored. MATERIAL AND METHODS: The present cross-sectional study comprised 50 histopathologically confirmed OSCC cases, with adjacent normal mucosa as controls. MiRNA expression was assessed via qRT-PCR and correlated with clinicopathological factors. MiRwalk and miRTargetlink were used for miRNA:mRNA interaction prediction, and gprofiler was employed to analyze validated targets for functional insights. RESULTS: The expression analysis showed a significant upregulation of miR-155-5p and miR-1246 in OSCC tissues compared to adjacent controls. Receiver operating curve analysis revealed that miR-1246 exhibited excellent diagnostic accuracy (AUC = 0.94) compared to miR-155-5p (AUC = 0.69). Higher miRNA levels were associated with age and extracapsular extension while overexpression of miR-1246 was correlated significantly with increased tumor size, tumor grade, TNM staging, and depth of invasion. The analysis for target prediction unveiled a set of validated targets, among which were WNT5A, TP53INP1, STAT3, CTNNB1, PRKAR1A, and NFIB. CONCLUSION: miR-155-5p and miR-1246 may be used as potential prognostic biomarkers in OSCC, with miR-1246 demonstrating superior diagnostic accuracy.

Differential effect of cancer-associated fibroblast-derived extracellular vesicles on cisplatin resistance in oral squamous cell carcinoma via miR-876-3p.

Rationale: Platinum-based chemotherapy is commonly used for treating solid tumors, but drug resistance often limits its effectiveness. Cancer-associated fibroblast (CAF)-derived extracellular vesicle (EV), which carry various miRNAs, have been implicated in chemotherapy resistance. However, the molecular mechanism through which CAFs modulate cisplatin resistance in oral squamous cell carcinoma (OSCC) is not well understood. We employed two distinct primary CAF types with differential impacts on cancer progression: CAF-P, representing a more aggressive cancer-promoting category, and CAF-D, characterized by properties that moderately delay cancer progression. Consequently, we sought to investigate whether the two CAF types differentially affect cisplatin sensitivity and the underlying molecular mechanism. Methods: The secretion profile was examined by utilizing an antibody microarray with conditioned medium obtained from the co-culture of OSCC cells and two types of primary CAFs. The effect of CAF-dependent factors on cisplatin resistance was investigated by utilizing conditioned media (CM) and extracellular vesicle (EVs) derived from CAFs. The impacts of candidate genes were confirmed using gain- and loss-of-function analyses in spheroids and organoids, and a mouse xenograft. Lastly, we compared the expression pattern of the candidate genes in tissues from OSCC patients exhibiting different responses to cisplatin. Results: When OSCC cells were cultured with conditioned media (CM) from the two different CAF groups, cisplatin resistance increased only under CAF-P CM. OSCC cells specifically expressed insulin-like growth factor binding protein 3 (IGFBP3) after co-culture with CAF-D. Meanwhile, IGFBP3-knockdown OSCC cells acquired cisplatin resistance in CAF-D CM. IGFBP3 expression was promoted by GATA-binding protein 1 (GATA1), a transcription factor targeted by miR-876-3p, which was enriched only in CAF-P-derived EV. Treatment with CAF-P EV carrying miR-876-3p antagomir decreased cisplatin resistance compared to control miRNA-carrying CAF-P EV. On comparing the staining intensity between cisplatin-sensitive and -insensitive tissues from OSCC patients, there was a positive correlation between IGFBP3 and GATA1 expression and cisplatin sensitivity in OSCC tissues from patients. Conclusion: These results provide insights for overcoming cisplatin resistance, especially concerning EVs within the tumor microenvironment. Furthermore, it is anticipated that the expression levels of GATA1 and miR-876-3p, along with IGFBP3, could aid in the prediction of cisplatin resistance.

Evodiamine inhibits EPRS expression to regulate glutamate metabolism and proliferation of oral squamous cell carcinoma cells.

The effects of evodiamine (EVO) on oral squamous cell carcinoma (OSCC) are not yet understood. Based on our earlier findings, we hypothesized that evodiamine may affect OSCC cell proliferation and glutamate metabolism by modulating the expression of EPRS (glutamyl-prolyl-tRNA synthetase 1). From GEPIA, we obtained EPRS expression data in patients with OSCC as well as survival prognosis data. An animal model using Cal27 cells in BALB/c nude mice was established. The expression of EPRS was assessed by immunofluorescence, Western blotting, and quantitative PCR. Glutamate measurements were performed to evaluate the impact of evodiamine on glutamate metabolism of Cal27 and SAS tumor cells. transient transfection techniques were used to knock down and modulate EPRS in these cells. EPRS is expressed at higher levels in OSCC than in normal tissues, and it predicts poor prognosis in patients. In a nude mouse xenograft model, evodiamine inhibited tumor growth and the expression of EPRS. Evodiamine impacted cell proliferation, glutamine metabolism, and EPRS expression on Cal27 and SAS cell lines. In EPRS knockdown cell lines, both cell proliferation and glutamine metabolism are suppressed. EPRS's overexpression partially restores evodiamine's inhibitory effects on cell proliferation and glutamine metabolism. This study provides crucial experimental evidence supporting the potential therapeutic application of evodiamine in treating OSCC. Evodiamine exhibits promising anti-tumor effects by targeting EPRS to regulate glutamate metabolism.

Histologic, immunohistochemical, and in situ hybridization study of myxoid stroma in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (oSCC) is a highly invasive malignant neoplasm in cats. Recently, tumor stroma, known as tumor microenvironments, have been considered to play an essential role in tumor progression. However, their role in feline squamous cell carcinoma (SCC) remains unclear. This study aimed to reveal the cancer microenvironment of feline oSCC and evaluate the pathological mechanisms of progression. We used 19 samples from 17 cats with oSCC, which were examined using light microscopy, immunohistochemistry, and in situ hybridization (RNAscope®). Feline oSCCs had two types of stroma, namely fibrotic and myxoid stromal reaction patterns, which were easily distinguished using hematoxylin-eosin staining. The myxoid stroma was rich in hyaluronic acid, which seems to be produced by neoplastic cells. Furthermore, the presence of myxoid stroma was correlated with histological parameters, including the appearance of cancer-associated fibroblasts and tumor budding. Periostin protein expression was also frequently observed in the stroma of feline oSCC and was significantly more common in the myxoid stromal reaction pattern group than in the fibrotic group. Positive signals for periostin mRNA were detected in stromal cancer-associated fibroblasts. This study indicates that the interaction between neoplastic cells and stromal reaction pattern components, such as hyaluronic acid and periostin, may be involved in tumor malignancy. Therefore, we propose that focus be placed not only on the tumor tissue but also on the characterization of the stroma for analyzing feline oSCC.

Knockdown of RNA-binding protein IMP3 suppresses oral squamous cell carcinoma proliferation by destabilizing E2F5 transcript.

The expression level of RNA-binding proteins (RBPs) is dysregulated in oral squamous cell carcinoma (OSCC) and other types of cancer. Among the RBPs, IMP3 is involved in the progression of OSCC. However, the regulation of mRNA fate by IMP3 in OSCC remains less understood. We analyzed the expression level of IMP3 and E2F5 in OSCC tissues and cell lines by immunohistochemistry, qRT-PCR and Western blot. Subsequently, to further investigate the effect of IMP3 on E2F5 expression, we used siRNAs to silence IMP3 expression in OSCC cell lines SCC-25 and SCC-4. The binding site of E2F5 mRNA and IMP3 was confirmed by RNA immunoprecipitation (RIP). Finally, the function of IMP3 and E2F5 was investigated in viro and in xenograft mouse models. Here we report a positive correlation between IMP3 and E2F5 expression in OSCC, which are involved in cell proliferation and cell cycle. Mechanistically, E2F5 mRNA is bound by IMP3 protein, and silencing it leads to a shortened mRNA half-life and reduced protein expression. Also, knockdown of IMP3 inhibited allograft tumor progression in vivo. These studies reveal the molecular mechanism by which IMP3 regulates E2F5 mRNA stability and identify IMP3/E2F5 as a potential therapeutic target in OSCC.

Spatial transcriptomics reveals that metabolic characteristics define the tumor immunosuppression microenvironment via iCAF transformation in oral squamous cell carcinoma.

Tumor progression is closely related to tumor tissue metabolism and reshaping of the microenvironment. Oral squamous cell carcinoma (OSCC), a representative hypoxic tumor, has a heterogeneous internal metabolic environment. To clarify the relationship between different metabolic regions and the tumor immune microenvironment (TME) in OSCC, Single cell (SC) and spatial transcriptomics (ST) sequencing of OSCC tissues were performed. The proportion of TME in the ST data was obtained through SPOTlight deconvolution using SC and GSE103322 data. The metabolic activity of each spot was calculated using scMetabolism, and k-means clustering was used to classify all spots into hyper-, normal-, or hypometabolic regions. CD4T cell infiltration and TGF-ß expression is higher in the hypermetabolic regions than in the others. Through CellPhoneDB and NicheNet cell-cell communication analysis, it was found that in the hypermetabolic region, fibroblasts can utilize the lactate produced by glycolysis of epithelial cells to transform into inflammatory cancer-associated fibroblasts (iCAFs), and the increased expression of HIF1A in iCAFs promotes the transcriptional expression of CXCL12. The secretion of CXCL12 recruits regulatory T cells (Tregs), leading to Treg infiltration and increased TGF-ß secretion in the microenvironment and promotes the formation of a tumor immunosuppressive microenvironment. This study delineates the coordinate work axis of epithelial cells-iCAFs-Tregs in OSCC using SC, ST and TCGA bulk data, and highlights potential targets for therapy.

Cancer-associated fibroblast-derived extracellular vesicles promote lymph node metastases in oral cavity squamous cell carcinoma by encapsulating ITGB1 and BMI1.

BACKGROUND: Extracellular vesicles (EVs) have been revealed to facilitate the development of oral squamous cavity cell carcinoma (OCSCC), while its supporting role in lymph node metastases is under continuous investigation. This study aimed to examine the function of cancer-associated fibroblasts (CAF)-derived EVs (CAF-EVs) during lymph node metastasis in OCSCC and the mechanisms. METHODS: CAF were isolated from OCSCC tissues of patients, and CAF-EVs were extracted and identified. EdU, colony formation, wound healing, and Transwell assays were performed. The OCSCC cells before and after CAF-EVs treatment were injected into mice to probe the effects of CAF-EVs on tumor growth and lymph node metastasis, respectively. The effect of CAF-EVs treatment on transcriptome changes in OCSCC cells was analyzed. Clinical data of patients with OCSCC were analyzed to determine the prognostic significance of the selected genes. Finally, loss-of-function assays were conducted to corroborate the involvement of polycomb complex protein BMI-1 (BMI1) and integrin beta1 (ITGB1). RESULTS: CAF-EVs promoted the malignant behavior of OCSCC cells and accelerated tumor growth and lymph node metastasis in mice. CAF-EVs significantly increased the expression of BMI1 and ITGB1, and the expression of BMI1 and ITGB1 was negatively correlated with the overall survival and relapse-free survival of OCSCC patients. Knockdown of BMI1 or ITGB1 in OCSCC cells abated the promoting effects of CAF-EVs in vitro and in vivo. CONCLUSION: CAF-EVs elicited the metastasis-promoting properties in OCSCC by elevating BMI1 and ITGB1, suggesting that BMI1 and ITGB1 could be potential biomarkers and therapeutic targets for OCSCC.

Sclerostin Alters Tumor Cell Characteristics of Oral Squamous Cell Carcinoma and May Be a Key Player in Local Bone Invasion.

Localized jawbone invasion is a milestone in the progression of oral squamous cell carcinoma (OSCC). The factors that promote this process are not well understood. Sclerostin is known to be involved in bone metabolism and there are preliminary reports of its involvement in bone tumors and bone metastasis. To identify a possible involvement of sclerostin in the bone invasion process of OSCC, sclerostin expression was analyzed in vitro in two different human OSCC tumor cell lines by quantitative real-time polymerase chain reaction (qRT-PCR), and the effect of recombinant human (rh)-sclerostin treatment on tumor cell capabilities was evaluated using proliferation, migration, and invasion assays. Undifferentiated human mesenchymal stem cells (hMSCs) were osteogenically differentiated and co-cultured with OSCC tumor cells to demonstrate potential interactions and migration characteristics. Sclerostin expression was evaluated in clinical cases by immunohistochemistry at the OSCC-jawbone interface in a cohort of 15 patients. Sclerostin expression was detected in both OSCC tumor cell lines in vitro and was also detected at the OSCC-jawbone interface in clinical cases. Tumor cell proliferation rate, migration and invasion ability were increased by rh-sclerostin treatment. The migration rate of tumor cells co-cultured with osteogenically differentiated hMSCs was increased. The results presented are the first data suggesting a possible involvement of sclerostin in the bone invasion process of OSCC, which deserves further investigation and may be a potential approach for drug-based tumor therapy.

Nano formulated Resveratrol inhibits PD-L1 in oral cancer cells by deregulating the association between tumor associated macrophages and cancer associated fibroblasts through IL-6/JAK2/STAT3 signaling axis.

Tumor associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) in the tumor microenvironment secrete several cytokines, which involved in tumor initiation, progression, metastatic outgrowth and angiogenesis. However, the association between TAMs and CAFs in the context of tumor development remain unclear. Here, we studied the relationship between TAMs and CAFs along with the involvement of cytokines in the production of cancer-stem-like-cells (CSCs) in oral cancer cells and explored the potential anticancer effects of Nano-formulated Resveratrol (Res-NP) using an activated macrophage-M1 (AM-M1) and activated fibroblast cells as the model system. IL-6 secretion was found to be enhanced in the conditioned-medium (CM) when AM-M1 cells + CAFs-like cells were cocultured together. CSCs-enriched population was developed after addition of CM of AM-M1 +CAFs in H-357 cells and patient-derived-primary-oral-cancer cells. AM-M1 cells+ CAFs-like cells secreted IL-6 enhanced CSCs growth, proliferation, metastasis, and angiogenesis. IL-6 was found to promote PD-L1 expression in CSCs-enriched cells via JAK2/STAT3 pathway, as evident from the enhanced expression of p-JAK2 and p-STAT3. Nevertheless, Res-NP inhibited CSCs proliferation and reduced the expression of metastatic and angiogenic markers, in ovo blood vascularization, NO production and MMPs expression. Res-NP delinked the association between AM-M1 and CAFs by blocking IL-6 production and also disrupted the potential connection between IL-6 and PD-L1 with considerable decrease in p-JAK2 and p-STAT3 expressions. IL-6 depletion inhibited stemness and angiogenesis in oral CSCs by downregulating PD-L1 via JAK2/STAT3 cascade. Similar observations were also observed in Res-NP treated xenograft mice. Thus, data demonstrate that CSCs growth is dependent on IL-6/PD-L1 axis. Res-NP deregulates the association between AM-M1 and CAFs along-with attenuates carcinogenesis in in vitro, in ovo, ex vivo and in vivo model systems by inhibiting PD-L1 via IL-6/JAK2/STAT3 axis.