Normal Human Oral Keratinocytes Research Articles

BCAT1 promotes cell proliferation, migration, and invasion via the PI3K-Akt signaling pathway in oral squamous cell carcinoma.

To analyze the expression, biological function of branched chain amino-acid transaminase 1 (BCAT1) in oral squamous cell carcinoma (OSCC). Real-time PCR and immunohistochemistry were used to analyze the expression of BCAT1 protein in OSCC and normal oral tissues. Based on the clinicopathological information of patients, the relationship between the expression of BCAT1 protein and other clinicopathological factors was analyzed. Real-time PCR and western blot assays were used to analyze the expression of BCAT1 gene and protein in normal human oral keratinocytes (HOK) and human OSCC cells, respectively. After BCAT1 overexpression or knockdown, the proliferation, cell cycle, migration, and invasion of human OSCC cells were analyzed by CCK8, flow cytometry, wound healing, and transwell invasion assays, respectively. After adding the BCAT1 inhibitor EGR240 to OSCC cells, the changes in cell proliferation, migration, and invasion ability in OSCC cells were analyzed. Based on the TCGA database, the involved signal pathway in BCAT1-related and BCAT1-binding genes was obtained for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, verified by western blot assays. After inhibiting PI3K, the effect of BCAT1 on the expression of the downstream phosphorylated protein of the PI3K-Akt signaling pathway was analyzed by western blot assays. The relationship between the expression of BCAT1 and EMT-related protein of OSCC cells was also analyzed. The expression of BCAT1 gene and protein were upregulated in OSCC tissue, which positively correlated with the pathological grade of patients with OSCC. Compared with normal oral keratinocytes, BCAT1 gene and protein were upregulated in OSCC cells. BCAT1 overexpression promoted the proliferation, migration, and invasion of OSCC cells. BCAT1 knockdown or inhibition could reduce the proliferation, migration, and invasion abilities of OSCC cells. The results of bioinformatics analysis and Western bolt showed that BCAT1 could regulate the activation of PI3K-Akt signaling pathway, and promote epithelial-mesenchymal transition (EMT) of OSCC cells. BCAT1 could promote the proliferation, migration, and invasion of OSCC cells via PI3K-Akt signaling pathway, which is a potential therapeutic target for OSCC.

Angiopoietin-like 4 is upregulated by amphiregulin and activates cell proliferation and migration through p38 kinase in head and neck squamous cell carcinoma.

Angiopoietin-like 4 is a molecular hallmark that correlates with the growth and metastasis of head and neck squamous cell carcinoma, one of the most prevalent cancers worldwide. However, the molecular mechanisms by which angiopoietin-like 4 promotes head and neck squamous cell carcinoma tumorigenesis are unclear. Using well-characterized cell lines of head and neck squamous cell carcinoma development, including human normal oral keratinocytes, dysplastic oral keratinocytes, oral leukoplakia-derived oral keratinocytes, and head and neck squamous cell carcinoma cell lines, HN13, HN6, HN4, HN12, and CAL27, we investigated the signaling pathways upstream and downstream of angiopoietin-like 4-induced head and neck squamous cell carcinoma tumorigenesis. We found that both epidermal growth factor receptor ligands, epithelial growth factor, and amphiregulin led to angiopoietin-like 4 upregulation in normal oral keratinocytes and dysplastic oral keratinocytes and cooperated with the activation of hypoxia-inducible factor-1 in this effect. Interestingly, amphiregulin and angiopoietin-like 4 were increased in dysplastic oral keratinocytes and head and neck squamous cell carcinoma cell lines, and amphiregulin-induced activation of cell proliferation was dependent on angiopoietin-like 4. Although both p38 mitogen-activated protein kinases (p38 MAPK) and protein kinase B (AKT) were activated by angiopoietin-like 4, only pharmacological inhibition of p38 MAPK was sufficient to prevent head and neck squamous cell carcinoma cell proliferation and migration. We further observed that angiopoietin-like 4 promoted the secretion of interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin-1 alpha (IL-1α), vascular endothelial growth factor, platelet-derived growth factor (PDGF), and tumour necrosis factor alpha (TNF-α), cytokines and chemokines previously implicated in head and neck squamous cell carcinoma pathogenesis. Our results demonstrate that angiopoietin-like 4 is a downstream effector of amphiregulin and promotes head and neck squamous cell carcinoma development both through direct activation of p38 kinase as well as paracrine mechanisms.

Type 17 immune response promotes oral epithelial cell proliferation in periodontitis

ObjectivesThis study aims to investigate the effects of type 17 immune response on the proliferation of oral epithelial cells in periodontitis. DesignA time-dependent ligature induced periodontitis mouse model was utilized to explore gingival hyperplasia and the infiltration of interleukin 17A (IL-17A) positive cells. Immunohistochemistry and flow cytometry were employed to determine the localization and expression of IL-17A in the ligature induced periodontitis model. A pre-existing single-cell RNA sequencing dataset, comparing individuals affected by periodontitis with healthy counterparts, was reanalyzed to evaluate IL-17A expression levels. We examined proliferation markers, including proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription (STAT3), Yes-associated protein (YAP), and c-JUN, in the gingival and tongue epithelium of the periodontitis model. An anti-IL-17A agent was administered daily to observe proliferative changes in the oral mucosa within the periodontitis model. Cell number quantification, immunofluorescence, and western blot analyses were performed to assess the proliferative responses of human normal oral keratinocytes to IL-17A treatment in vitro. ResultsThe ligature induced periodontitis model exhibited a marked infiltration of IL-17A-positive cells, alongside significant increase in thickness of the gingival and tongue epithelium. IL-17A triggers the proliferation of human normal oral keratinocytes, accompanied by upregulation of PCNA, STAT3, YAP, and c-JUN. The administration of an anti-IL-17A agent attenuated the proliferation in oral mucosa. ConclusionsThese findings indicate that type 17 immune response, in response to periodontitis, facilitates the proliferation of oral epithelial cells, thus highlighting its crucial role in maintaining the oral epithelial barrier.

The association between the expression level of nuclear paraspeckle assembly transcript 1 and the survival rate of head and neck cancer patients after treatment

Background/purposeThe long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) exhibits diverse and complicated functions in cancer progression. Despite reports suggesting both tumor-suppressive and oncogenic effects in various cancers, its specific role in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study aimed to investigate the association between NEAT1 expression levels and survival outcomes in HNSCC patients. Materials and methodsPaired tissue samples of tumor and non-cancerous matching tissues (NCMT) from 92 HNSCC patients were collected. NEAT1 expression was analyzed using RT-qPCR. Clinical characteristics, treatment received, and survival rates of the patients were assessed to determine the correlation with NEAT1 expression and explore its association with alcohol, betel quid, and cigarette use. Additionally, we examined the effect of arecoline on NEAT1 expression in normal human oral keratinocytes (NHOK) and fibroblasts (NHOF). ResultsThe study revealed a significant downregulation of NEAT1 expression in oral cancer tissues compared to NCMT. Meanwhile, arecoline increased NEAT1 expression in NHOK and NHOF cells. However, patients with downregulated NEAT1 expression exhibited higher overall survival rates, particularly in those who did not receive chemotherapy or radiotherapy. ConclusionNEAT1 expression levels are associated with survival outcomes in HNSCC patients, with upregulated expression indicating a worse prognosis, suggesting this lncRNA might contribute to cancer aggressiveness, especially in the absence of active treatment. These findings indicate NEAT1 may serve as a potential prognostic biomarker in HNSCC, but further research is required to elucidate its role in cancer progression and its potential as a therapeutic target.

Analysis of differentially expressed genes in dysplastic oral keratinocyte cell line and their role in the development of HNSCC

Dysplasia is the presence of abnormal types of cells in a tissue precipitated by over or diminished expression of certain genes. These cells act as a precursor to cancer. Dysplastic oral keratinocyte (DOK) cell lines have an aneuploid complex karyotype. They provide an opportunity to study the action of specific carcinogens on malignant transformations. This study aimed to identify the differentially expressed genes in dysplastic cells and their possible association with head and neck squamous cell carcinoma (HNSCC). These genes can be developed as diagnostic, prognostic, or therapeutic leads.The list of genes related to oral keratinocyte dysplasia and head and neck cancer was accessed from the GEO (Gene Expression Omnibus) database. Gene expression profiling was done between dysplastic oral keratinocytes and normal human oral keratinocytes. Gene expression and Kaplan Meier survival analysis were performed using the UALCAN database to assess the correlations between dysregulated genes identified in dysplastic keratinocytes and primary tumors of HNSCC. The GEO omnibus dataset identified numerous differentially expressed genes of which the top 10 up and downregulated genes in dysplastic oral keratinocytes were curated for further analysis. The expression profile of these genes was assessed using the HNSCC dataset (TCGA, Firehose Legacy). Among all the genes assessed, only one gene, the OLR1 gene encoding oxidized low-density lipoprotein, was found to be overexpressed in both the groups viz., dysplastic keratinocytes and HNSCC cases with a strong correlation with the survival status of patients. There was significant correlation between the gene expression pattern observed in dysplastic keratinocytes and the primary tumor of the HNSCC group, with an exotic gene that was seldom discussed in association with cancer, viz., OLR1. Exploration into other top-ranking differentially expressed genes in dysplastic cases would aid in identifying the candidate gene associated with both phenotypes.

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 < 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.

Metabolic Patterns of High-Invasive and Low-Invasive Oral Squamous Cell Carcinoma Cells Using Quantitative Metabolomics and 13C-Glucose Tracing.

Most current metabolomics studies of oral squamous cell carcinoma (OSCC) are mainly focused on identifying potential biomarkers for early screening and diagnosis, while few studies have investigated the metabolic profiles promoting metastasis. In this study, we aimed to explore the altered metabolic pathways associated with metastasis of OSCC. Here, we identified four OSCC cell models (CAL27, HN6, HSC-3, SAS) that possess different invasive heterogeneity via the transwell invasion assay and divided them into high-invasive (HN6, SAS) and low-invasive (CAL27, HSC-3) cells. Quantitative analysis and stable isotope tracing using [U-13C6] glucose were performed to detect the altered metabolites in high-invasive OSCC cells, low-invasive OSCC cells and normal human oral keratinocytes (HOK). The metabolic changes in the high-invasive and low-invasive cells included elevated glycolysis, increased fatty acid metabolism and an impaired TCA cycle compared with HOK. Moreover, pathway analysis demonstrated significant differences in fatty acid biosynthesis; arachidonic acid (AA) metabolism; and glycine, serine and threonine metabolism between the high-invasive and low-invasive cells. Furthermore, the high-invasive cells displayed a significant increase in the percentages of 13C-glycine, 13C-palmitate, 13C-stearic acid, 13C-oleic acid, 13C-AA and estimated FADS1/2 activities compared with the low-invasive cells. Overall, this exploratory study suggested that the metabolic differences related to the metastatic phenotypes of OSCC cells were concentrated in glycine metabolism, de novo fatty acid synthesis and polyunsaturated fatty acid (PUFA) metabolism, providing a comprehensive understanding of the metabolic alterations and a basis for studying related molecular mechanisms in metastatic OSCC cells.

Astaxanthin Synergizes with Ionizing Radiation (IR) in Oral Squamous Cell Carcinoma (OSCC).

Astaxanthin (ATX) is known for its antioxidant and anti-inflammation functions yet its role in cancers requires more research. This study is aimed to reveal the potential synergetic effect of ATX with ionizing radiation (IR) in OSCC. Cell survival was measured after human OSCC cells including CAL27 and SCC9, and normal human oral keratinocytes (NHOKs) were treated with different concentrations of ATX for 24h. Colony formation assays were performed after OSCC cells were treated with IR, ATX (20μM), or combined and survival fraction was analyzed. Malondialdehyde (MDA), glutathione (GSH), and intercellular iron levels were measured. Western blot method was used to measure the ferroptosis-related proteins, GPX4, SLC7A11, and ACSL4. In xenograft mice model, we evaluated the tumor volumes, tumor growth, and examined the GPX4/ACSL4 proteins in tumor tissues using Immunohistochemistry (IHC). ATX inhibited viability of OSCC cells but not NHOK. In OSCC cells, ATX further enhanced the cell death induced by IR. In addition, ATX promoted the MDA content, Iron levels but inhibited the GSH regulated by IR in cells. ATX could synergize with IR, further inhibiting GPX4, SLC7A11 and promoting ACSL4 in OSCC cells. In vivo, ATX and IR treatment inhibited OSCC tumor growth and the group with combined treatment showed the most inhibitory effect. GPX4 was inhibited by IR and further inhibited in the combined group while ACSL4 was promoted by IR and enhanced more significantly in the combined group. ATX might synergize with IR treatment in OSCC partly via ferroptosis.

Assessment of Oxidative Stress-Induced Oral Epithelial Toxicity.

Reactive oxygen species (ROS) are highly reactive molecules generated in living organisms and an excessive production of ROS culminates in oxidative stress and cellular damage. Notably, oxidative stress plays a critical role in the pathogenesis of a number of oral mucosal diseases, including oral mucositis, which remains one of cancer treatments' most common side effects. We have shown previously that oral keratinocytes are remarkably sensitive to oxidative stress, and this may hinder the development and reproducibility of epithelial cell-based models of oral disease. Here, we examined the oxidative stress signatures that parallel oral toxicity by reproducing the initial events taking place during cancer treatment-induced oral mucositis. We used three oral epithelial cell lines (an immortalized normal human oral keratinocyte cell line, OKF6, and malignant oral keratinocytes, H357 and H400), as well as a mouse model of mucositis. The cells were subjected to increasing oxidative stress by incubation with hydrogen peroxide (H2O2) at concentrations of 100 μM up to 1200 μM, for up to 24 h, and ROS production and real-time kinetics of oxidative stress were investigated using fluorescent dye-based probes. Cell viability was assessed using a trypan blue exclusion assay, a fluorescence-based live-dead assay, and a fluorometric cytotoxicity assay (FCA), while morphological changes were analyzed by means of a phase-contrast inverted microscope. Static and dynamic real-time detection of the redox changes in keratinocytes showed a time-dependent increase of ROS production during oxidative stress-induced epithelial injury. The survival rates of oral epithelial cells were significantly affected after exposure to oxidative stress in a dose- and cell line-dependent manner. Values of TC50 of 800 μM, 800 μM, and 400 μM were reported for H400 cells (54.21 ± 9.04, p < 0.01), H357 cells (53.48 ± 4.01, p < 0.01), and OKF6 cells (48.64 ± 3.09, p < 0.01), respectively. Oxidative stress markers (MPO and MDA) were also significantly increased in oral tissues in our dual mouse model of chemotherapy-induced mucositis. In summary, we characterized and validated an oxidative stress model in human oral keratinocytes and identified optimal experimental conditions for the study of oxidative stress-induced oral epithelial toxicity.

Induction of S arrest and apoptosis in human oral cancer cells by Rhinacanthin-C extracted from Rhinacanthus nasutus via modulating Akt and p38 signaling pathways

Ethnopharmacological relevanceThe search for effective herbal medicines for complementary treatments is on the rise due to the high incidence of recurrence and mortality rate in human oral cancer. Rhinacanthus nasutus KURZ., an annual herb found mostly in Southeast Asia including Thailand, has been wildly used as a traditional folk medicine for the treatment of several diseases including cancer. However, the anti-cancer effect of Rhinacanthin-C (Rh–C) as a major naphthoquinone compound found in R. nasutus and the underlying mechanism of its action on human oral cancer cells remain unknown. Aim of the studyTo investigate the anti-cancer mechanism of Rh–C extracted from R. nasutus in human oral cancer cells. Materials and methodsThe anti-proliferative effect of Rh–C on human oral squamous cell carcinoma (HSC4) was determined and compared to normal oral cells (human gingival fibroblasts, HGF, and normal oral keratinocytes, NOK) using the SRB colorimetric method. The molecular mechanism of Rh–C was explored using flow cytometry, colorimetric assay, in vitro human topoisomerase II assay, and Western blotting. ResultsRh–C displayed a time- and concentration-dependent growth inhibition on HSC4 and was much less effective on both tested normal oral cells. Rh–C inhibited Akt phosphorylation whereas over-activated p38 MAPK phosphorylation in HSC4 but not in HGF. Rh–C also inhibited topoisomerase II activity. As a result, the cell cycle was arrested in S-phase as the expression of CDK1/2 and Cyclin A2 was decreased. Eventually, the induction of HSC4 cell apoptosis was mediated by increased caspase 3 activity. ConclusionsRh–C isolated from R. nasutus possesses anti-cancer properties on human oral cancer cells by causing the S arrest and the apoptotic induction via modulating Akt/p38 signaling pathways. The results provide molecular bases for further developing Rh–C as a potential drug candidate or a complementary treatment for oral cancer.

Angiopoietin-like 4 induces head and neck squamous cell carcinoma cell migration through the NRP1/ABL1/PXN pathway

ObjectivesThe molecular mechanisms whereby angiopoietin-like 4 (ANGPTL4), a pluripotent protein implicated in cancer development, contributes to head and neck squamous cell carcinoma (HNSCC) growth and dissemination are unclear. Materials and MethodsWe investigated ANGPTL4 expression in human normal oral keratinocytes (NOKs), dysplastic oral keratinocytes (DOKs), oral leukoplakia cells (LEUK1), and HNSCC cell lines, as well as in tissue biopsies from patients with oral dysplasia, and primary and metastatic HNSCC. We further examined the contribution of ANGPTL4 cancer progression in an HNSCC orthotopic floor-of mouth tumor model and the signaling pathways linking ANGPTL4 to cancer cell migration. ResultsANGPTL4 expression was upregulated in premalignant DOKs and HNSCC cell lines compared to NOKs and was increased in tissue biopsies from patients with oral dysplasia, as well as in primary and metastatic HNSCC. We also observed that downregulation of ANGPTL4 expression inhibited primary and metastatic cancer growth in an HNSCC orthotopic tumor model. Interestingly, ANGPTL4 binding to the neuropilin1 (NRP1) receptor led to phosphorylation of the focal adhesion protein, paxillin (PXN), and tumor cell migration; this was dependent on the tyrosine kinase ABL1. Treatment with the ABL1 inhibitor, dasatinib and small interfering RNA silencing of NRP1 or ABL1 expression blocked PXN phosphorylation and tumor cell migration. ConclusionOur findings suggest an early, sustained, and angiogenesis-independent autocrine role for ANGPTL4 in HNSCC progression and expose ANGPTL4/NRP1/ABL1/PXN as an early molecular marker and vulnerable target for the prevention of HNSCC growth and metastasis.

Family with Sequence Similarity 72 (FAM72) − A prospective biomarker for poor prognosis in patients with oral squamous cell carcinoma

ObjectiveTo study the effect of FAM72 on the prognosis of patients with oral squamous cell carcinoma (OSCC) and to explore the relationship between FAM72 and OSCC. DesignWe used a vast array of databases and analytical vehicles to assess the relation between FAM72 and OSCC, including The Cancer Genome Atlas (TCGA), Metascape, and MethSurv. We made a preliminary verification of OSCC lines and tissues by real time quantitative polymerase chain reaction (RT-qPCR). ResultsFAM72 was higher in OSCC than in normal tissues. Analysis of univariate COX data indicated that elevated expression of FAM72A, FAM72B, and FAM72C in OSCC was related to poor overall survival. Moreover, FAM72B and FAM72C were independent of overall survival in multiple COX regression. FAM72A-D and its coexpressed genes in Metascape were analyzed by Gene Ontology (GO), they were enriched in cellular cycle, mitotic and DNA metabolism. Gene set enrichment analysis (GSEA) demonstrated an enrichment in pathways related to cell metabolism. Additionally, high FAM72 expression related to a worse prognosis in OSCC patients. FAM72A-D linked to the infiltration of tumor immune cell in OSCC patients. We found that methylation levels are likely linked to prognosis in OSCC patients. We used RT-qPCR to ascertain the differential FAM72B and FAM72C expression levels in cancer and paracancerous tissues of OSCC, human normal oral keratinocytes (HOK), and human tongue squamous cell carcinoma (Cal-33). ConclusionOur findings indicate that FAM72B and FAM72C are potential molecular markers of poor prognosis in OSCC and may act as novel targets for OSCC treatment strategies.

Anti-Inflammatory Effects of β-Cryptoxanthin on 5-Fluorouracil-Induced Cytokine Expression in Human Oral Mucosal Keratinocytes

Oral mucositis is a typical adverse effect of chemotherapy, causing oral pain that significantly reduces the patient's quality of life. β-cryptoxanthin (β-cry) is a carotenoid abundant in citrus fruits with antioxidant and anti-inflammatory effects. However, the β-cry effect on oral mucositis remains unclear. We investigated the effects of 5-fluorouracil (5-FU) and β-cry on human normal oral mucosal keratinocytes (hOMK). hOMK was seeded on a culture plate and cultured with 5-FU and β-cry. The cell number, mRNA expression of inflammatory cytokines and matrix metalloproteinases (MMPs), and production of inflammatory cytokines in hOMK were evaluated. Additionally, the cell count and inflammatory cytokine production were analyzed when hOMK was co-stimulated with Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) in addition to 5-FU. The numbers of hOMK significantly reduced with 5-FU stimulation, whereas it increased with β-cry treatment. mRNA expression of interleukin (IL)-6, IL-8, metalloproteinase (MMP)-2, and MMP-9 and protein production of IL-6 and IL-8 in hOMK were augmented on 5-FU stimulation. Simultaneously, β-cry treatment significantly suppressed IL-8 and MMP-9 mRNA expression, and IL-8 production was induced on 5-FU stimulation. Co-stimulation with P. gingivalis LPS and 5-FU enhanced IL-6 and IL-8 production in hOMK. β-cry could enhance cell proliferation and suppress 5-FU-induced expression of inflammatory cytokines and MMP in hOMK. Thus, β-cry can alleviate the symptoms of chemotherapy-induced oral mucositis, and its combination with oral care is effective in managing oral mucositis.

A Novel Preclinical In Vitro 3D Model of Oral Carcinogenesis for Biomarker Discovery and Drug Testing.

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents.

SCARA5 inhibits oral squamous cell carcinoma via inactivating the STAT3 and PI3K/AKT signaling pathways.

Oral squamous cell carcinoma (OSCC) is a common tumor in the world. Despite the rapid development of medical care, OSCC is also accompanied by high incidence and mortality every year. Therefore, it is still necessary to continuously develop new methods or find new targets to treat OSCC. Previous research showed that scavenger receptor class A member 5 (SCARA5) was one of the potential biomarkers of OSCC, and its expression is significantly low in OSCC. This study aimed to explore the role and related molecular mechanisms of SCARA5 in OSCC. In this study, we found that the SCARA5 expression was lower in CAL-27 and SCC-9 cells than that in human normal oral epithelial keratinocytes. SCARA5 overexpression significantly inhibited cell proliferation and induced apoptosis of CAL-27 and SCC-9 cells. In addition, SCARA5 repressed OSCC cell epithelial-mesenchymal transformation (EMT), evidenced by increased E-cadherin expression and reduced N-cadherin expression. Finally, we found that SCARA5 could suppress STAT3, PI3K, and AKT phosphorylation. Therefore, SCARA5 was related to STAT3 and PI3K/AKT signaling pathways in OSCC. In conclusion, SCARA5 inhibited the proliferation and EMT and induced the apoptosis of OSCC cells through the inhibition of STAT3 and PI3K/AKT signaling pathways, thereby exerting a tumor suppressor effect.

Hsa_circ_0000231 affects the progression of tongue squamous cell carcinoma by activating Wnt/β-catenin signaling pathway

Objective: To explore the action mechanism of hsa_circ_0000231 in the occurrence and development of tongue squamous cell carcinoma (TSCC). Methods: Tissue samples of 60 TSCC patients were examined. The patients, including 32 males and 28 females, aged from 36 to 84 years old, underwent surgery in the Affiliated Hospital of Nantong University and Affiliated Tumor Hospital of Nantong University from December 2014 to December 2017. Saliva samples were obtained from healthy volunteers (5 males and 5 females, aged from 40 to 75 years old) and 10 TSCC patients. The TSCC cell lines (CAL-27, Tca-8113 and HN-4) were used. The expression levels of hsa_circ_0000231 in 60 pairs of freshly matched TSCC and para-carcinoma tissue samples, 10 pairs of saliva samples and 3 TSCC cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR). hsa_circ_0000231 gene interference and lentiviral transfection were constructed, hsa_circ_0000231 in TSCC cell lines CAL-27 and Tca-8113 was knocked down, and the expressions of hsa_circ_0000231 in hsa_circ_0000231 interference group (sh-circ) and no-load lentivirus group (negative control) were tested with qRT-PCR. Cells with the highest knock-down efficiency were selected for CCK-8 test, colony formation assay, transwell invasion assay and scratch assay. The expressions of EMT-related proteins including E-cadherin, snail protein, N-cadherin and vimentin and proteins related to Wnt/β-catenin signaling pathway including β-catenin, C-myc, Bcl-2, MMP-9 and Cyclin D1 were measured by western blot. After TSCC cells in the interference group were co-cultured with Wnt/β-catenin pathway activator LiCl, the expressions of above proteins were re-measured by western blot. TSCC cells in interference group and control group were subcutaneously injected into nude mice to compare the effect of hsa_circ_0000231 knockdown on the growths of the tumors grafted subcutaneously in the nude mice. Statistical analysis software 25.0 was used for data analysis, and t-test or chi-square test was used for comparison between groups. Results: hsa_circ_0000231 was highly expressed in the tissue and saliva samples of TSCC patients and cell lines CAL-27, Tca-8113 and HN-4, but lowly expressed in paired para-carcinoma tissues, saliva samples of healthy people and normal human oral keratinocytes (all P<0.05). Log-rank univariate analysis showed that hsa_circ_0000231 expression level, tumor differentiation degree and T stage were related to the survival of TSCC patients (all P<0.05). Multivariate Cox risk regression model analysis suggested that hsa_circ_0000231 expression level (χ2=5.77,P=0.016) and T stage (χ2=5.27,P=0.029) were independent factors for the poor prognosis of TSCC patients. Western blot showed the expressions of snail protein, N-cadherin and vimentin were down-regulated, but E-cadherin was up-regulated in interference group compared with control group. In interference group, the expressions of β-catenin, C-myc, Bcl-2, MMP-9 and CyclinD1 were down-regulated, which were reversed after TSCC cells were co-cultured with LiCl. The knockdown of hsa_circ_0000231 reduced the proliferation, invasion and metastasis abilities of CAL-27 and Tca-8113 cells, which were reversed after TSCC cells were co-cultured with LiCl. The growth rate and volume of the tumors grafted subcutaneously in interference group using LiCl were greater than those in negative control group. Conclusion: hsa_circ_0000231 is an independent prognostic factor of TSCC. Highly expressed hsa_circ_0000231 can promote the proliferation, invasion and metastasis of TSCC cells.

MicroRNA-382-5p Promotes Oral Squamous Cell Carcinoma Development and Progression by Negatively Regulating PTEN Expression

Oral squamous cell carcinoma (OSCC) local recurrence and distant metastasis remain a poorly understood clinical challenge. The objective of this study was to investigate how dysregulation of miR-382-5p impacts invasion and dissemination of OSCC. Tissue samples were collected from 20 subjects with OSCC. Expression levels of miR-382-5p were determined by quantitative real-time polymerase chain reaction (qRT-PCR), and correlations with clinical characteristics were investigated. qRT-PCR was used to determine the miR-382-5p and peptidyl-prolyl cis/trans isomerase (PTEN) expression in tumor tissues, adjacent normal tissues, normal human oral keratinocyte line, and OSCC line (SCC-9). Cell proliferation, invasion, and migration of knock-in and knock-down miR-382-5p transfectants were assessed using cell counting kit-8 and Transwell assays. PTEN was confirmed to be a downstream target using a TargetScan prediction, dual-luciferase reporter assays, and western blot analysis. Statistical analysis of experimental data was performed with SPSS 22.0 software. We found high expression of miR-382-5p and significant downregulation of PTEN in tumor tissues and SCC-9cells from OSCC patients (P<.05). miR-382-5p expression was lower in early stage (I+II) than in late stage (III+IV), while PTEN exhibited higher expression in early stage (I+II) instead of in late stage (III+IV) (P<.05). In addition, overexpression of miR-382-5p promoted the proliferation, invasion, and migration of OSCC cells. However, the proliferation, invasion, and migration of OSCC cells were inhibited after suppression of miR-382-5p. Finally, PTEN is downregulated by miR-382-5p. MiR-382-5p supports proliferation, invasion, and migration of OSCC cells through the PTEN pathway. Further investigation may improve our understanding of OSCC local recurrence and distant metastasis.

IQGAP1 and RNA Splicing in the Context of Head and Neck via Phosphoproteomics.

IQGAP1 (IQ motif-containing GTPase-activating protein 1) scaffolds several signaling pathways in mammalian cells that are implicated in carcinogenesis, including the RAS and PI3K pathways that involve multiple protein kinases. IQGAP1 has been shown to promote head and neck squamous cell carcinoma (HNSCC); however, the underlying mechanism(s) remains unclear. Here, we report a mass spectrometry-based analysis identifying differences in phosphorylation of cellular proteins in vivo and in vitro in the presence or absence of IQGAP1. By comparing the esophageal phosphoproteome profiles between Iqgap1+/+ and Iqgap1-/- mice, we identified RNA splicing as one of the most altered cellular processes. Serine/arginine-rich splicing factor 6 (SRSF6) was the protein with the most downregulated levels of phosphorylation in Iqgap1-/- tissue. We confirmed that the absence of IQGAP1 reduced SRSF6 phosphorylation both in vivo and in vitro. We then expanded our analysis to human normal oral keratinocytes. Again, we found factors involved in RNA splicing to be highly altered in the phosphoproteome profile upon genetic disruption of IQGAP1. Both the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the Cancer Genome Atlas (TCGA) data sets indicate that phosphorylation of splicing-related proteins is important in HNSCC prognosis. The Biological General Repository for Interaction Datasets (BioGRID) repository also suggested multiple interactions between IQGAP1 and splicing-related proteins. Based on these collective observations, we propose that IQGAP1 regulates the phosphorylation of splicing proteins, which potentially affects their splicing activities and, therefore, contributes to HNSCC. Raw data are available from the MassIVE database with identifier MSV000087770.

TRPM7 via calcineurin/NFAT pathway mediates metastasis and chemotherapeutic resistance in head and neck squamous cell carcinoma.

The exact mechanisms of Head and neck squamous carcinoma (HNSCC) chemoresistance and metastatic transformation remain unclear. In recent decades, members of the transient receptor potential (TRP) channel family have been proposed as potential biomarkers and/or drug targets in cancer treatment. First, in a TCGA cohort of HNSCC, TRPM7 is highly expressed in cancer tissues, especially the expression in invasive cancer tissues is statistically significant (p>0.001). In GEO and TCGA cohort, patients with high expression of TRPM7 and NFATC2 have poor overall survival rates. The expression of TRPM7 and NFATC2 showed a positive correlation. Compared to human normal oral keratinocytes (hNOK), TRPM7 is overexpressed in FaDU, SAS, and TW2.6 cell lines. Similarly, patients with HNSCC exhibited higher TRPM7 expression than non-HNSCC subjects, and this high TRPM7 expression was associated with worse 5-year overall survival. Furthermore, TRPM7 inversely correlated with E-cadherin, but positively correlated with Vimentin, NANOG, and BMI-1 mRNA levels. Consistent with this, we demonstrated the overexpression of TRPM7 in cisplatin-resistant subjects, compared to the cisplatin-sensitive counterparts. Moreover, shRNA-mediated silencing of TRPM7 significantly suppressed the migration, invasion, colony formation, and tumorsphere formation of SAS cells, with associated downregulation of Snail, c-Myc, cyclin D1, SOX2, OCT4, and NANOG proteins expression. Finally, compared with the untreated wild-type SAS cells or cisplatin-treated cells, shTRPM7 alone or in combination with cisplatin significantly inhibited tumorsphere and colony formation. These findings serving as the basis for development of novel therapeutic strategies against metastasis and chemoresistance, while providing new insights into TRPM7 biology and activity in HNSCC.

Circ_0004872 inhibits proliferation, invasion, and glycolysis of oral squamous cell carcinoma by sponged miR-424-5p.

ObjectiveOral squamous cell carcinoma (OSCC) is one of the most common oral malignant tumors. circ_0004872 can inhibit the progression of gastric cancer, but its effect on the growth and metastasis of OSCC is still unclear.MethodsqRT‐PCR was used to detect the expression levels of circ_0004872 and miR‐424‐5p in cancer tissues of OSCC patients and adjacent normal tissues, OSCC cell lines, and human normal oral keratinocytes (HOK). CCK‐8, cell colony formation, flow cytometry, and transwell assay were used to detect cell proliferation rate, viability, apoptosis rate, and invasion ability. Use glucose/lactic acid kit to assay cell glycolysis ability. The dual‐luciferase reporter gene experiment and RIP experiment verified the relationship between circ_0004872 and miR‐424‐5p. The protein levels were examined by Western blot.ResultsThe expression of circ_0004872 was significantly downregulated in OSCC tissues and cells, and the overexpression of circ_0004872 inhibited the proliferation, vitality, invasion, and glycolysis of OSCC cells, and promoted apoptosis. The expression of miR‐424‐5p was greatly upregulated in OSCC tissues and OSCC cells. circ_0004872 can adsorb miR‐424‐5p in OSCC cells, and circ_0004872 can reverse the promoting effect of miR‐424‐5p overexpression on the process of OSCC cells.Conclusioncirc_0004872 suppresses the proliferation, invasion, and glycolysis of OSCC cells by sponged miR‐424‐5p, and promotes apoptosis, which can be used as a potential target for early diagnosis and targeted therapy of OSCC.