Oral Squamous Cell Carcinoma Cell Lines Research Articles

NCAPD2 promotes the malignant progression of oral squamous cell carcinoma via the Wnt/β-catenin pathway

ABSTRACT Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer, with a poor prognosis, yet the underlying mechanism needs further exploration. Non-SMC condensin I complex subunit D2 (NCAPD2) is a widely expressed protein in OSCC, but its role in tumor development is unclear. This study aimed to explore NCAPD2 expression and its biological function in OSCC. NCAPD2 expression in OSCC cell lines and tissue specimens was analyzed using quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Cancer cell growth was evaluated using cell proliferation, 5-Ethynyl-2’-deoxyuridine (EdU) staining, and colony formation assays. Cell migration was evaluated using wound healing and Transwell assays. Apoptosis was detected using flow cytometry. The influence of NCAPD2 on tumor growth in vivo was evaluated in a mouse xenograft model. NCAPD2 expression was significantly higher in OSCC than that in normal oral tissue. In vitro, the knockdown of NCAPD2 inhibited OSCC cell proliferation and promoted apoptosis. NCAPD2 depletion also significantly inhibited the migration of OSCC cells. Moreover, NCAPD2 overexpression induced inverse effects on OSCC cell phenotypes. In vivo, we demonstrated that downregulating NCAPD2 could inhibit the tumorigenicity of OSCC cells. Mechanically, OSCC regulation by NCAPD2 involved the Wnt/β-catenin signaling pathway. These results suggest NCAPD2 as a novel oncogene with an important role in OSCC development and a candidate therapeutic target for OSCC.

Restoring BARX2 in OSCC reverses partial EMT and suppresses metastasis through miR-186-5p/miR-378a-3p-dependent SERPINE2 inhibition.

Tumor cells undergoing partial epithelial-mesenchymal transition (pEMT) are pivotal in local invasion and lymphatic metastasis of oral squamous cell carcinoma (OSCC), yet the mechanisms behind pEMT reversal remain poorly understood. In this study, the loss of BARX2 expression was revealed during the process of oral epithelial carcinogenesis and identified to activate the pEMT program, facilitate metastasis, and be associated with poor prognosis. Restoring BARX2 expression in OSCC cell lines effectively reversed tumor pEMT, evident in E/N-Cadherin switching, reduced cell invasion, proliferation, and stemness, and inhibited murine lung metastasis. BARX2 re-expression negatively correlated with several pEMT markers, notably SERPINE2, which was enriched in the invasive OSCC front, enhancing stemness and promoting metastasis, particularly in cervical lymph nodes. Furthermore, rescuing SERPINE2 impaired the inhibitory effect of BARX2 on the pEMT programs and reconstructed ECM through re-expression of MMP1. Mechanistically, we identified that BARX2 inhibited SERPINE2 through activating miR-186-5p and miR-378a-3p. These miRNAs, upregulated by BARX2, post-transcriptionally degraded SERPINE2 mRNA via targeting specific sequences. Blocking miR-186-5p and miR-378a-3p effectively abolished the negative regulatory effect of BARX2 on SERPINE2. Overall, our findings highlight BARX2 as a partial EMT-reverser in OSCC, providing fresh therapeutic prospects for restoring BARX2 signaling to inhibit invasion and metastasis.

Crosstalk between cancer cells and macrophages promotes OSCC cell migration and invasion through a CXCL1/EGF positive feedback loop.

C-X-C motif chemokine ligand 1 (CXCL1) and epithelial growth factor (EGF) are highly secreted by oral squamous cell carcinoma (OSCC) cells and tumor-associated macrophages, respectively. Recent studies have shown that there is intricate "cross-talk" between OSCC cells and macrophages. However, the underlying mechanisms are still poorly elucidated. The expression of CXCL1 was detected by immunohistochemistry in OSCC clinical samples. CXCL1 levels were evaluatedby RT‒PCR and ELISA in an OSCC cell line and a normal epithelial cell line. The expression of EGF was determined by RT‒PCR and ELISA. The effect of EGF on the proliferation of OSCC cells was evaluated by CCK-8 and colony formation assays. The effect of EGF on the migration and invasion ability and epithelial-mesenchymal transition (EMT) of OSCC cells was determined by wound healing, Transwell, RT‒PCR, Western blot and immunofluorescence assays. The polarization of macrophages was evaluated by RT‒PCR and flow cytometry. Western blotting was used to study the molecular mechanism in OSCC. The expression of C-X-C motif chemokine ligand 1 (CXCL1) was higher in the OSCC cell line (Cal27) than in immortalized human keratinocytes (Hacat cells). CXCL1 derived from Cal27 cells upregulates the expression of epithelial growth factor (EGF) in macrophages. Paracrine stimulation mediated by EGF further facilitates the epithelial-mesenchymal transition (EMT) of Cal27 cells and initiates the upregulation of CXCL1 in a positive feedback-manner. Mechanistically, EGF signaling-induced OSCC cell invasion and migration can be ascribed to the activation of NF-κB signaling mediated by the epithelial growth factor receptor (EGFR), as determined by western blotting. OSCC cell-derived CXCL1 can stimulate the M2 polarization of macrophages and the secretion of EGF. Moreover, EGF significantly activates NF-κB signaling and promotes the migration and invasion of OSCC cells in a paracrine manner. A positive feedback loop between OSCC cells and macrophages was formed, contributing to the promotion of OSCC progression.

CD271 mRNA/hnRNPA2B1 complex promotes proliferation and stemness in oral and head and neck squamous cell carcinoma.

RNAs, such as noncoding RNA, microRNA, and recently mRNA, have been recognized as signal transduction molecules. CD271, also known as nerve growth factor receptor, has a critical role in cancer, although the precise mechanism is still unclear. Here, we show that CD271 mRNA, but not CD271 protein, facilitates spheroid cell proliferation. We established CD271-/- cells lacking both mRNA and protein of CD271, as well as CD271 protein knockout cells lacking only CD271 protein, from hypopharyngeal and oral squamous cell carcinoma lines. Sphere formation was reduced in CD271-/- cells but not in CD271 protein knockout cells. Mutated CD271 mRNA, which is not translated to a protein, promoted sphere formation. CD271 mRNA bound to hnRNPA2B1 protein at the 3'-UTR region, and the inhibition of this interaction reduced sphere formation. In surgical specimens, the CD271 mRNA/protein expression ratio was higher in the cancerous area than in the noncancerous area. These data suggest CD271 mRNA has dual functions, encompassing protein-coding and noncoding roles, with its noncoding RNA function being predominant in oral and head and neck squamous cell carcinoma.

WNT3 promotes chemoresistance to 5-Fluorouracil in oral squamous cell carcinoma via activating the canonical β-catenin pathway

Background5-Fluorouracil (5FU) is a primary chemotherapeutic agent used to treat oral squamous cell carcinoma (OSCC). However, the development of drug resistance has significantly limited its clinical application. Therefore, there is an urgent need to determine the mechanisms underlying drug resistance and identify effective targets. In recent years, the Wingless and Int-1 (WNT) signaling pathway has been increasingly studied in cancer drug resistance; however, the role of WNT3, a ligand of the canonical WNT signaling pathway, in OSCC 5FU-resistance is not clear. This study delved into this potential connection.Methods5FU-resistant cell lines were established by gradually elevating the drug concentration in the culture medium. Differential gene expressions between parental and resistant cells underwent RNA sequencing analysis, which was then substantiated via Real-time quantitative PCR (RT-qPCR) and western blot tests. The influence of the WNT signaling on OSCC chemoresistance was ascertained through WNT3 knockdown or overexpression. The WNT inhibitor methyl 3-benzoate (MSAB) was probed for its capacity to boost 5FU efficacy.ResultsIn this study, the WNT/β-catenin signaling pathway was notably activated in 5FU-resistant OSCC cell lines, which was confirmed through transcriptome sequencing analysis, RT-qPCR, and western blot verification. Additionally, the key ligand responsible for pathway activation, WNT3, was identified. By knocking down WNT3 in resistant cells or overexpressing WNT3 in parental cells, we found that WNT3 promoted 5FU-resistance in OSCC. In addition, the WNT inhibitor MSAB reversed 5FU-resistance in OSCC cells.ConclusionsThese data underscored the activation of the WNT/β-catenin signaling pathway in resistant cells and identified the promoting effect of WNT3 upregulation on 5FU-resistance in oral squamous carcinoma. This may provide a new therapeutic strategy for reversing 5FU-resistance in OSCC cells.

Extracellular vesicles from Aggregatibacter actinomycetemcomitans exhibit potential antitumorigenic effects in oral cancer: a comparative in vitro study

Aggregatibacter actinomycetemcomitans is an opportunistic Gram-negative periodontopathogen strongly associated with periodontitis and infective endocarditis. Recent evidence suggests that periodontopathogens can influence the initiation and progression of oral squamous cell carcinoma (OSCC). Herein we aimed to investigate the effect of A. actinomycetemcomitans-derived extracellular vesicles (EVs) on OSCC cell behavior compared with EVs from periodontopathogens known to associate with carcinogenesis. EVs were isolated from: A. actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; Porphyromonas gingivalis; Fusobacterium nucleatum; and Parvimonas micra. The effect of EVs on primary and metastatic OSCC cells was assessed using cell proliferation, apoptosis, migration, invasion, and tubulogenesis assays. A. actinomycetemcomitans-derived EVs reduced the metastatic cancer cell proliferation, invasion, tubulogenesis, and increased apoptosis, mostly in CDT- and LPS O-antigen-dependent manner. EVs from F. nucleatum impaired the metastatic cancer cell proliferation and induced the apoptosis rates in all OSCC cell lines. EVs enhanced cancer cell migration regardless of bacterial species. In sum, this is the first study demonstrating the influence of A. actinomycetemcomitans-derived EVs on oral cancer in comparison with other periodontopathogens. Our findings revealed a potential antitumorigenic effect of these EVs on metastatic OSCC cells, which warrants further in vivo investigations.

The involvement of epidermal growth factor receptor/protein kinase Bsignaling in the tumor intrinsic PD-L1-induced malignant potential of oral squamous cell carcinoma.

Various antigen-presenting cells and tumor cells-expressing PD-L1 inhibits antitumor immune responses in the tumor microenvironment. Recently, numerous studies have shown that tumor cell intrinsic PD-L1 also plays important roles in tumor growth and progression. On the other hand, oral squamous cell carcinoma (OSCC) cells overexpress epidermal growth factor receptor (EGFR) and EGFR signal pathway exacerbates tumor progression. Therefore, this study assessed whether tumor-intrinsic PD-L1 facilitates malignant potential of OSCC cells through regulation of EGFR signaling. Two OSCC cell lines, SAS and HSC-3, were transfected with PD-L1 and EGFR-specific small interfering RNA (siRNA). Influences of PD-L1 knockdown on malignant potentials of OSCC cells were examined by Cell Counting kit-8 assay, transwell assay, sphere formation assay, flow cytometry, and Western blot. Effects of PD-L1 and EGFR knockdown on each expression were examined by quantitative real-time PCR (qRT-PCR), Western blot, and flow cytometry. Transfection of an PD-L1-siRNA into OSCC cells decreased the abilities of proliferation, stemness, and mobility of these cells significantly. PD-L1 knockdown also decreased EGFR expression through the promotion of proteasome- and lysosome-mediated degradation and following activation of the EGFR/protekin kinase B (AKT)signal pathway. Meanwhile, EGFR knockdown did not influence PD-L1 expression in SAS and HSC-3 cells, but treatment with a recombinant human EGF induced its expression. Treatment with erlotinib and cetuximab suppressed rhEGF-induced PD-L1 expression and localization in the cellular membrane of both OSCC cells. OSCC cells-expressing PD-L1 induced by EGF stimulation may promote malignancy intrinsically via the activation of the EGFR/AKT signaling cascade.

Marine-derived antimicrobial peptide piscidin-1 triggers extrinsic and intrinsic apoptosis in oral squamous cell carcinoma through reactive oxygen species production and inhibits angiogenesis

Cancer of the head and neck encompasses a wide range of cancers, including oral and oropharyngeal cancers. Oral cancer is often diagnosed at advanced stages and has a dismal prognosis. Piscidin-1, a marine antimicrobial peptide (AMP) containing approximately 22 amino acids, also exhibits significant anticancer properties. We investigated the possible anti-oral cancer effects of piscidin-1 and clarified the mechanisms underlying these effects. We treated the oral squamous cell carcinoma cell lines OC2 and SCC4 with piscidin-1. Cell viability and the expression of different hallmark apoptotic molecules, including reactive oxygen species (ROS), were tested using the appropriate MTT assay, flow cytometry and western blotting assays, and human umbilical vein endothelial cell (HUVEC) wound healing, migration, and tube formation (angiogenesis) assays. Piscidin-1 increases cleaved caspase 3 levels to induce apoptosis. Piscidin-1 also increases ROS levels and intensifies oxidative stress in the endoplasmic reticulum and mitochondria, causing mitochondrial dysfunction. Additionally, it decreases the oxygen consumption rates and activity of mitochondrial complexes I–V. As expected, the antioxidants MitoTEMPOL and N-acetylcysteine reduce piscidin-1–induced ROS generation and intracellular calcium accumulation. Piscidin-1 also inhibits matrix metalloproteinase (MMP)-2/-9 expression in HUVECs, affecting migration and tube formation angiogenesis. We demonstrated that piscidin-1 can promote apoptosis via both intrinsic and extrinsic apoptotic pathways and findings indicate that piscidin-1 has anti-proliferative and anti-angiogenic properties in oral cancer treatment. Our study on piscidin-1 thus provides a basis for future translational anti-oral cancer drug research and a new theoretical approach for anti-oral cancer clinical research.

Tra2β exerts tumor-promoting effects via GSK3/β-catenin signaling in oral squamous cell carcinoma.

The splicing factor transformer-2 homolog beta (Tra2β) plays a pivotal role in various cancers. Nonetheless, its role in oral squamous cell carcinoma (OSCC) has not been comprehensively explored. This study sought to discern the influence of Tra2β on OSCC and its underlying mechanisms. We assessed Tra2β expression in OSCC utilizing immunohistochemistry, qRT-PCR, and western blotting techniques. siRNA transfection was used to silence Tra2β. Whole transcriptome RNA sequencing (RNA-seq) analysis was carried out to reveal the alternative splicing (AS) events. KEGG pathway analysis enriched the related pathways. Colony formation, transwell, wound healing, and Annexin V-FITC/PI were employed to appraise the consequences of Tra2β silencing on OSCC. Tra2β was highly expressed in both OSCC tissues and cell lines. Knockdown of Tra2β-regulated AS events with skipped exon (SE) accounts for the highest proportion. Meanwhile, downregulation of Tra2β reduced cell proliferation, migration, and invasion, however increasing cell apoptosis. Moreover, Wnt signaling pathway involved in the function of Tra2β knockdown which was demonstrated directly by a discernible reduction in the expression of GSK3/β-catenin signaling axis. These findings suggest that knockdown of Tra2β may exert anti-tumor effects through the GSK3/β-catenin signaling pathway in OSCC.

Effect of Methanol and Ethyl Acetate Leaf Extracts of Osbeckia octandra L. (Heen Bovitiya) on the Apoptosis and Migration of Human Oral Squamous Cell Carcinoma Cell Lines

Effect of Methanol and Ethyl Acetate Leaf Extracts of Osbeckia octandra L. (Heen Bovitiya) on the Apoptosis and Migration of Human Oral Squamous Cell Carcinoma Cell Lines

Possible Role of miR-375-3p in Cervical Lymph Node Metastasis of Oral Squamous Cell Carcinoma.

No clinically useful predictors of latent cervical lymph node metastasis (LNM) in early oral squamous cell carcinoma (OSCC) are available. In this study, we focused on the microRNAs (miRNAs) involved in the expression of numerous genes and explored those associated with latent cervical LNM in early OSCC (eOSCC). First, microarray and RT-PCR analyses revealed a significant downregulation of miR-375-3p expression in primary eOSCC tissues with latent cervical LNM. Next, we examined the effects of miR-375-3p mimics on the growth and migration of four human OSCC cell lines that do not express miR-375-3p. The overexpression of miR-375-3p significantly suppressed the cell proliferation and migration of human OSCC cells in vitro. Furthermore, miR-375-3p mimics markedly inhibited the subcutaneously xenografted human OSCC tumors. Finally, we found the genes involved in the PI3K-AKT pathway and cell migration as target gene candidates of miR-375-3p in human OSCC cells. These findings suggest that miR-375-3p functions as a tumor suppressive-miRNA in OSCC and may serve as a potential biomarker for the prediction of latent cervical LNM in eOSCC and a useful therapeutic target to suppress OSCC progression.

Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatase Acts as a Tumor Suppressor in Oral Squamous Cell Carcinoma.

The pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) family has been found to have both tumor-suppressor and oncogenic properties across various types and locations of cancer. Given that PHLPP has not been previously studied in oral squamous cell carcinoma (SCC), we conducted an assessment of the expression of both its isoforms in oral SCC tissues and cell lines and compared these findings to their corresponding normal counterparts. In addition, we assessed the relationship between PHLPP and clinicopathological factors and patient survival. Quantitative real-time polymerase chain reaction was used to detect the mRNA levels of PHLPP1 and PHLPP2 in cancerous and normal cell lines in addition to 124 oral SCC and noncancerous adjacent epithelia (N = 62, each). Correlations between their expression rate and clinicopathological parameters were further evaluated in 57 patients. Data were statistically analyzed with t test and paired t test, analysis of variance, Mann-Whitney U, and Cox Regression tests (P < 0.05). We found significantly lower levels of both PHLPP isoforms in oral SCC tissues compared with noncancerous epithelia (P < 0.001, for both). However, in the cell lines, this difference was significant only for PHLPP1 (P = 0.027). The correlation between the two isoforms was significant only in cancerous tissues (P < 0.001). None of the clinicopathologic factors showed significant associations with either of the isoforms and there was no correlation with survival. We showed for the first time that PHLPP1 and PHLPP2 act as tumor suppressors in oral SCC at the mRNA level. The regulation of their mRNA appears to be different between normal and cancerous tissues.

Abstract LB329: Interleukin6 as a Potential Prognostic Marker and Therapeutic Target in Oral Squamous Cell Carcinoma

Abstract This study investigates the role of interleukin (IL)-6 in oral squamous cell carcinoma (OSCC), evaluating its potential as a prognostic marker and therapeutic target. This study involved 95 untreated OSCC patients, with pre-surgical serum IL-6 levels measured by Electro chemiluminescence immunoassay (ECLIA). Patients were categorized into high (78 patients) and low (17 patients) IL-6 groups based on a cutoff value of 8 pg/mL.A strong correlation was observed between higher IL-6 levels and lower overall survival (P&amp;lt;0.0001) and disease-free survival (P=0.0283). Serum IL-6 levels also showed a strong positive correlation with certain blood test markers such as neutrophil and lymphocyte fractions and C-reactive protein (CRP), and a negative correlation with albumin levels. These findings suggest a link between systemic inflammation, malnutrition, and the Inflammation-based prognostic score (IBPS).Further analysis through microarray assessed the gene expression in primary tumor tissues, revealing 513 genes with more than a two-fold change in the high IL-6 group compared to controls. Gene Ontology analysis indicated increased metabolic activity in the high IL-6 group and suppressed inflammation and immune-related molecules in the low IL-6 group.Immunohistochemical staining for IL-6 in tumor tissues showed its presence mainly in the stroma, particularly in cancer-associated fibroblasts (CAFs) identified by vimentin and α-SMA positivity. Both OSCC cell lines and CAFs established from OSCC tissues demonstrated high IL-6 secretion, especially in cell lines derived from lymph node metastases. Furthermore, cell lines from primary, lymph node, and lung metastases exhibited high IL-6 secretion, with HRAS mutations also identified. Knockdown of HRAS using siRNA reduced IL-6 secretion.Finally, in vivo experiments using an anti-IL-6 receptor antibody showed that blocking IL-6 signaling reduced the proliferation of OSCC cell lines.The study concludes that IL-6 is a useful prognostic and therapeutic target in OSCC. The wide applicability of IL-6 testing and the clinical use of anti-human IL-6R antibody drugs, combined with aggressive nutritional intervention, suggest a potential paradigm shift in OSCC treatment. Citation Format: Hiroyuki Goda, Tomoko Adachi, Koh-ichi Nakashiro, Nobuyuki Kuribayashi, Daisuke Uchida. Interleukin6 as a Potential Prognostic Marker and Therapeutic Target in Oral Squamous Cell Carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB329.

EHMT2 Suppresses ARRB1 Transcription and Activates the Hedgehog Signaling to Promote Malignant Phenotype and Stem Cell Property in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) represents the primary subtype of head and neck squamous cell carcinoma (HNSCC), characterized by a high morbidity and mortality rate. Although previous studies have established specific correlations between euchromatic histone lysine methyltransferase 2 (EHMT2), a histone lysine methyltransferase, and the malignant phenotype of OSCC cells, its biological functions in OSCC remain largely unknown. This study, grounded in bioinformatics predictions, aims to clarify the influence of EHMT2 on the malignant behavior of OSCC cells and delve into the underlying mechanisms. EHMT2 exhibited high expression in OSCC tissues and demonstrated an association with poor patient outcomes. Artificial EHMT2 silencing in OSCC cells, achieved through lentiviral vector infection, significantly inhibited colony formation, migration, invasion, and cell survival. Regarding the mechanism, EHMT2 was found to bind the promoter of arrestin beta 1 (ARRB1), thereby suppressing its transcription through H3K9me2 modification. ARRB1, in turn, was identified as a negative regulator of the Hedgehog pathway, leading to a reduction in the proteins GLI1 and PTCH1. Cancer stem cells (CSCs) were enriched through repeated sphere formation assays in two OSCC cell lines. EHMT2 was found to activate the Hedgehog pathway, thus promoting sphere formation, migration and invasion, survival, and tumorigenic activity of the OSCC-CSCs. Notably, these effects were counteracted by the additional overexpression of ARRB1. In conclusion, this study provides novel evidence suggesting that EHMT2 plays specific roles in enhancing stem cell properties in OSCC by modulating the ARRB1-Hedgehog signaling cascade.

Elucidating the Anticancer Mechanisms of Cinnamoyl Sulfonamide Hydroxamate: Insights From DNA Content Analysis and Gene Expression Profiling in Squamous Cell Carcinoma.

Oral cancer is a major public health concern worldwide, with oral squamous cell carcinoma (OSCC) being one of its most common subtypes. Despite advances in diagnosis and management of this disease, there remains a need to develop new therapeutic approaches for better outcomes. This study aimed to investigate the molecular mechanisms through which cinnamoyl sulfonamide hydroxamate derivatives exert their anticancer effects on OSCC. The derivatives were synthesized via multi-step processes and then characterized at the molecular level. Flow cytometry assay for DNA content and cell cycle distribution, anisidine/toluidine double staining for apoptosis detection, as well as reverse transcription polymerase chain reaction (RT-PCR) gene expression analysis, were performed on OSCC cell lines exposed to cinnamoyl sulfonamide hydroxamate derivatives. Flow cytometry unveiled remarkable changes in the distribution of cells throughout the OSCC cell line upon treatment with cinnamoyl sulfonamide hydroxamate derivatives. Consequently, it led to a noticeable decrease in cells at the G0/G1 phase, together with an increase at the S phase, thereby indicating a retardation at various points of the cycle. In addition, apoptotic morphological alterations have been observed by anisidine/toluidine double staining after some treatments with the compounds. RT-PCR analysis showed a marked increase in p21 gene expression levels, further supporting the compounds' ability to induce cell cycle arrest and apoptosis. The research highlighted the potential of cinnamoyl sulfonamide hydroxamate derivatives as candidates for oral cancer, particularly OSCC treatment, shedding light on their operation at the molecular level and paving the way for the development of targeted therapies that could aid in the cure of oral cancer.

IL-1α facilitates GSH synthesis to counteract oxidative stress in oral squamous cell carcinoma under glucose-deprivation

Understanding the intrinsic mechanisms underpinning cancer metabolism and therapeutic resistance is of central importance for effective nutrition-starvation therapies. Here, we report that Interleukin 1A (IL1A) mRNA and IL-1α protein facilitate glutathione (GSH) synthesis to counteract oxidative stress and resistance against nutrition-starvation therapy in oral squamous cell carcinoma (OSCC). The expression of IL1A mRNA was elevated in the case of OSCC associated with unfavorable clinical outcomes. Both IL1A mRNA and IL-1α protein expression were increased under glucose-deprivation in vitro and in vivo. The transcription of IL1A mRNA was regulated in an NRF2-dependent manner in OSCC cell lines under glucose-deprivation. Moreover, the IL-1α conferred resistance to oxidative stress via GSH synthesis in OSCC cell lines. The intratumoral administration of siRNAs against IL1A mRNA markedly reversed GSH production and sensitized OSCC cells to Anlotinib in HN6 xenograft models. Overall, the current study demonstrates novel evidence that the autocrine IL-1α favors endogenous anti-oxidative process and confers therapeutic resistance to nutrition-starvation in OSCCs.

Abstract 1727: Silencing of novel m6A reader PRRC2A as a therapeutic strategy for oral cancer

Abstract Background: Oral squamous cell carcinoma (OSCC) poses a substantial global health challenge characterized by limited treatment options and unfavorable prognoses. Recent research has illuminated the pivotal role of N6-methyladenosine (m6A) modification and its regulators in cancer biology. However, the precise involvement of a novel m6A regulator (m6A reader), PRCC2A in OSCC remains inadequately understood. Objective: This study aims to unravel the role of PRCC2A in OSCC and ascertain its potential as both a diagnostic biomarker and a therapeutic target. Methods: We collected 76 OSCC tumor samples and 38 adjacent non-tumor samples from OSCC patients. Comprehensive analyses, including RT-qPCR, western blotting, histopathological examination, and associations with clinicopathological features, were performed to assess PRRC2A expression in cancer tissues. Furthermore, we explored its links with key factors like metastasis and its implications for patient prognosis. In vitro experiments were conducted using OSCC cell lines and human normal oral keratinocytes (NOKs). PRCC2A knockdown in OSCC cell lines was assessed for its effects on cell proliferation, apoptosis, cell cycle, migration, invasion, and sphere-forming abilities. The study also delved into its impact on immune cell infiltration and involvement in biological pathways. Results: Our investigation unveiled significant overexpression of PRCC2A in OSCC tissue samples and cell lines, relative to adjacent non-tumor tissues and NOKs (P &amp;lt; 0.001). These findings were further validated by the Cancer Genome Atlas - Head and Neck Squamous Cell Carcinoma (TCGA-HNSC) dataset. PRCC2A expression displayed significant correlations with nodal metastasis, higher tumor grades, and advanced cancer stages in OSCC samples, suggesting its crucial role in driving cancer progression and aggressiveness. Silencing PRCC2A in vitro led to reduced metastasis, diminished tumor colony formation, and increased apoptosis in oral cancer cell lines. Furthermore, the study uncovered PRCC2A intricate connections with immune regulatory genes, highlighting its potential to influence the tumor immune microenvironment. Conclusion: PRCC2A emerges as a promising candidate for both diagnosis and therapeutic intervention in OSCC. Its pronounced overexpression in oral cancer tissues, coupled with its associations with metastasis and intricate interactions with immune regulatory networks, underscores its significance in the context of oral cancer malignancies. This research offers valuable insights into the multifaceted role of PRCC2A in OSCC and lays the groundwork for future investigations and potential clinical applications. Citation Format: Balachander Kannan, Vijayashree Priyadharsini Jayaseelan, Senthil MuruganM, Paramasivam Arumugam. Silencing of novel m6A reader PRRC2A as a therapeutic strategy for oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1727.

Abstract 705: miR-21-5p induces radioresistance through reinforced DNA damage response in oral squamous cell carcinoma

Abstract Cancers arising from oral cavity and oropharynx are commonly treated with radiotherapy, whereas tumor radioresistance results in poor outcome. The prerequisite for developing more effective interventions for these patients is that the molecular mechanisms leading to a radioresistant phenotype in these tumors are clear to researchers. Unfortunately, the radioresistant mechanisms remain elusive to date. We recently identified that a highly conserved and cancer-related microRNA, i.e. miR-21-5p, plays an important role in radioresistance of oral squamous cell carcinoma (OSCC). We analyzed the RNA-sequencing data of 290 tissue samples from TCGA database, and found the expression of miR-21-5p was significantly upregulated in many OSCC samples (N=271) when comparing to adjacent normal tissues (N=19). The difference was further confirmed on our freshly collected OSCC tissues from patients in operation room. We performed RNA-sequencing analysis in 11 OSCC samples and their adjacent normal tissues. Among the 2106 differentially expressed genes between OSCC and normal control groups, we found that several important DNA repair genes (RAD50 double strand break repair protein gene, RAD50; DNA Polymerase Theta, POLQ; Ataxia Telangiectasia and Rad3-related, ATR) were significantly overexpressed in OSCC samples (P&amp;lt;0.05), while positive correlations between miR-21-5p expression and DNA repair genes RAD51 and XRCC4 were identified in the OSCC cohort. Notably, ATR is an important DNA damage response gene, while XRCC4, RAD51, and POLQ are involved in DNA double strand break (DSB) repair through non-homologous end joining, homologous recombination, and microhomology-mediated end joining, respectively. OSCC cell lines were further recruited to study the relations of miR-21-5p with radiation-induced DSB repair. By analyzing kinetics of 53BP1 foci, a biomarker for detecting DSBs, we found that anti-miR-21-5p transfected cells showed more residual foci, indicating of unfinished repair of DSBs, at 5 hours and 18 hours post irradiation. The deficient DSB repair resulted from anti-miR-21-5p transfection were further confirmed by single cell gel electrophoresis with comet assay. Consistently, the same treatment resulted in fewer clonogenic survivors in colony formation assay, demonstrating increased radiosensitivity of OSCC cells when miR-21-5p was antagonized. This work provided novel insights about the mechanism of miR-21-5p-induced radioresistance in OSCC, which may give rise to novel therapeutic strategy for improved treatment of OSCC patients. Citation Format: Qi Liu, Weitao Hu, Xinghan Li, Yongqiang Deng, Lin Ma. miR-21-5p induces radioresistance through reinforced DNA damage response in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 705.

Abstract 5569: Mechano-chemical control of secretory cues regulating immune tolerance, survival and invasion in oral squamous cell carcinomas

Abstract Tissue fibrosis and extracellular matrix (ECM) stiffening have been shown to promote tumor cell proliferation, metastasis, and chemoresistance in a range of cancers. Stiffness of the ECM can lead to changes in cancer cell behavior, including enhanced proliferation, activation of mechano-sensitive signaling pathways, and potential genomic damage, with implications for cancer progression and immune responses. Recent research suggests stiff matrix may also induce the release of tumor-derived small extracellular vesicles (sEVs) that promote tumor growth. Tumor-derived sEVs carry bioactive cargo, such as micro-RNAs (miRNAs) and DNA fragments, and function as a unique form of intracellular communication promoting cell survival, growth, and metastasis. Understanding these mechano-sensing mechanisms is essential for identifying potential targets in precision medicine for solid tumor treatment. However, the influence of tumor stiffness on secretory cues, and their subsequent role in immunomodulation and/or chemo-resistance has not been extensively studied. This project aims to explore how tumor stiffness can alter cell responses to genotoxic stress and how these responses can perpetuate feedback to the immune system through the release of secretory cues such as sEVs. Here, we outline the isolation and characterization of tumor-derived sEVs obtained from culture media of oral squamous cell carcinoma (OSCC) spheroids, cultured at varying stiffness (0.2kPa-1.5kPa). In this project, we use OSCC lines that are either sensitive or resistant to cisplatin (HSC-3 and HN-5, respectively). To explore the immunomodulatory effects of these tumor-derived sEVs, we first established a consistent supply of CD206+ve macrophages differentiated from human induced pluripotent stem cells (iMacs), following the established protocol of Douthwaite et al (2021). iMacs were then treated with sEVs with their resulting immunophenotype, and activation assessed. These preliminary experiments provide a step towards understanding how the biophysical environment influences tumor cell resistance to therapy and how sEVs can modulate the relationship between cancer cells and macrophages. Citation Format: Bethany R. Bareham, Matthew Dibble, Leila Mouhib, Aitor Bermejo Arteagabeitia, Subhankar Mukhopadhyay, Maddy Parsons. Mechano-chemical control of secretory cues regulating immune tolerance, survival and invasion in oral squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5569.

Abstract 3795: Possible role of miR-375-3p in cervical lymph node metastasis of oral squamous cell carcinoma

Abstract Latent cervical lymph node metastasis (LNM) is the most important prognostic factor in early oral squamous cell carcinoma (OSCC). However, no clinically useful predictors of LNM in early OSCC are available. In this study, we focused on the microRNAs (miRNAs) involved in the expression of numerous genes and explored those associated with latent cervical LNM in OSCC. Microarray analysis of miRNA expression showed that the expression levels of miR-375-3p were significantly reduced in primary OSCC tissues with latent cervical LNM. Subsequently, we quantified the expression of miR-375-3p by RT-qPCR and digital PCR. Similar to the results of miRNA microarray analysis, miR-375-3p was significantly downregulated in primary OSCC tissues with latent cervical LNM. The results of digital PCR showed a more significant difference than those of RT-qPCR. When the reference value for predicting latent cervical LNM was set to less than 57.85 copies based on the receiver operating characteristic curve, the sensitivity, specificity, accuracy, and area under the curve were 80%, 100%, 90%, and 0.942, respectively. Next, we examined the effects of miR-375-3p mimics on the growth and migration of four human OSCC cell lines (SAS-L1, HSC2, HSC3, and Ca9-22) that do not express miR-375-3p. When an miR-375-3p mimic was introduced into these cells, cell proliferation and migration were significantly suppressed. Total RNA was extracted from these cells reverse-transfected with miR-375-3p mimic for 48 hours. Microarray analysis and Ingenuity Pathway Analysis (IPA) miRNA target filter showed that miR-375-3p overexpression reduced the expression of 266 genes, of which 37 genes had the target sequences of miR-375-3p in their 3’-untranslated region. Among these target genes, CEPT1 and TIMM8A knockdown significantly inhibited the migration of all human OSCC cells, similar to miR-375-3p mimics. Furthermore, IPA core analysis revealed a decrease in expression of genes involved in the activation of PI3K-AKT pathways by miR-375-3p overexpression. These results indicated that downregulating miR-375-3p expression could promote cervical LNM by enhancing cell proliferation and migration in OSCC. We also found that CEPT1 and TIMM8A as novel target genes of miR-375-3p regulated the migration of human OSCC cells. The expression levels of miR-375-3p in primary OSCC tissues appear to be a useful biomarker for predicting latent cervical LNM. Citation Format: Masato Saika, Koh-ichi Nakashiro, Norihiko Tokuzen, Hiroyuki Shirai, Daisuke Uchida. Possible role of miR-375-3p in cervical lymph node metastasis of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3795.