Combination treatment with ferroptosis and autophagy inducers significantly inhibit the proliferation and migration of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC), a malignancy originating from mucosal epithelial cells. Currently, triggering apoptotic cell death with anticancer drugs is the main way to inhibit OSCC cells. However, the capability to trigger apoptosis in tumors is constrained by the intrinsic resistance of tumor cells to apoptosis, hampering its effectiveness. Thus, utilizing alternative modes of non-apoptotic cell death offers new therapeutic possibilities, such as using a drug combination strategy to simultaneously induce ferroptosis and autophagy has the potential to improve OSCC therapy. In this study, we found the ferroptosis inducer RSL3 has certain inhibitory effects on the proliferation and migration of OSCC cells. Interestingly, our studies showed that RSL3 is also associated with autophagy activation. Based on this finding, we tried to combine RSL3 with the autophagy inducer LYN-1604 to improve the therapeutic effect. The results demonstrated that simultaneous regulation of autophagy and ferroptosis significantly reduced the proliferation and migration of OSCC cells. Taken together, we demonstrated the therapeutic potential of RSL3 in OSCC cells and proposed that simultaneous activation of autophagy and ferroptosis have synergistic effects, which would provide valuable clues for further exploration of targeted therapy for OSCC.

Relationship of biglycan and decorin expression with clinicopathological features and prognosis in patients with oral squamous cell carcinoma.

OBJECTIVE: This study focused on investigating relation between biglycan (BGN) and decorin (DCN) expression and prognostic outcome for oral squamous cell carcinoma (OSCC) cases. MATERIAL AND METHODS: BGN and DCN mRNA and protein expression was detected by qRT-PCR and Western-blotting (WB) assays from 31 OSCC samples as well as healthy samples. This work harvested 101 paraffin-embedded OSCC together with 30 healthy samples, and conducted immunohistochemical (IHC) staining for assessing pathological changes. Association of DCN with BGN within OSCC was explored by Spearman's analysis. Survival rate was explored by Kaplan-Meier (KM) approach. Multivariate analysis was conducted by Cox regression. RESULTS: WB and qRT-PCR results showed BGN up-regulation (p < 0.001, p < 0.0001) whereas DCN down-regulation (p < 0.0001, p < 0.0001) with fresh OSCC tissues; the expression of BGN and DCN associated with the OSCC histopathological grade. IHC results suggested elevated BGN level (p < 0.0001) whereas DCN down-regulation (p < 0.0001) with paraffin embedded OSCC tissues. The expression of BGN and DCN associated with histopathologic grades and tumor stage of OSCC. The result of Spearman's analysis demonstrated significant association between the expression of BGN and DCN in OSCC. Survival analysis revealed that patients with higher BGN/lower DCN level showed poor overall survival (OS) as well as tumor-specific survival (TSS). Multivariate analysis proved that BGN and DCN independently predicted the prognosis of OS and TSS. CONCLUSION: BGN and DCN expression levels can be adopted for predicting OSCC prognostic outcome.

Evaluation of CK19 and VEGF gene expression, ki67 antigen, p53 protein, C-erb-B2, and their relationship in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the ten most common malignant tumors globally. This study aimed to evaluate the expression changes of Cytokeratin 19 (CK19), vascular endothelial growth factor (VEGF), P53, ki67, and c-ert-B2 in OSCC patients. For this purpose, 30 patients were selected as the case group and 30 healthy individuals as the control group. The expression of CK19 and VEGF genes in their blood serum was measured. Also, the expression of ki67, P53, and c-ert-B2 markers in squamous cell carcinoma was evaluated using immunohistochemistry. T-test was used to analyze the data. The results showed that the presence of CK19 marker in people with OSCC was positive in 17 out of 30 patients and VEGF marker in 23 out of 30 patients. The mean of ki67 positive, P53 positive, and Cerb-B2 positive cells were 399.4, 221.4, and 26.8, respectively. The correlation test between the indices showed a statistical correlation between the incidence of ki67 and P53 (r = 91.5% and p = 0.02). While statistical correlation was not seen between the incidence of ki67 and Cerb-B2 index (r = -1.7% and p = 0.97) and P53 and C-erb-B2 index (r = -13% and p = 0.8) (p <0.05). In general, the expression of VEGF and CK19 genes is higher in patients with OSCC than in healthy individuals. Therefore, examining the expression level of these two biomarkers in the blood of OSCC patients can be considered as a diagnostic screening method in the early stages of the disease. The immunohistochemical study of squamous cell carcinoma can also be used as a diagnostic screening test in the early stages of the disease.

NUDT1: A potential independent predictor for the prognosis of patients with oral squamous cell carcinoma.

OBJECTIVE: The current study was aimed to investigate the association of nudix hydrolase 1 (NUDT1) levels with the prognosis of oral squamous cell carcinoma (OSCC) patients. MATERIAL AND METHODS: Western immunoblotting and qRT-PCR were used to detect the protein and mRNA levels of NUDT1 in 31 cases of OSCC and normal tissues. The paraffin-embedded 62 cases of OSCC and 18 normal tissues were collected, and the pathological alterations were assessed by immunohistochemistry. The prognosis of all patients was followed up. Kaplan-Meier method was used to analyze the survival rate, and Cox regression was used for multivariate analysis. RESULTS: Both the protein and gene levels of NUDT1 were statistically increased (P = .0007 and P < .0001) in the OSCC tissue and had a significant association with the histopathologic grades of OSCC (P < .0001 and P = .0223). Immunohistochemistry detection of NUDT1 in 62 human OSCC tissues and 18 normal control tissues showed that NUDT1 expression was significantly increased in OSCC tissue and showed a strong association with histopathologic grades (P < .0001) and tumor stage (P = .005). Patients with high NUDT1 expression exhibited poorer overall survival rate (OS) and tumor-specific survival rate (TSS) than those with low NUDT1 expression (P < .0001 and P = .0008), and NUDT1 was independent prognostic factors for OS and TSS (P < .0001 and P < .001). CONCLUSION: The expression level of NUDT1 might be used to predict the prognosis of OSCC patients.

CDH11 inhibits proliferation and invasion in head and neck cancer.

BACKGROUND: In this study, we use a bioinformatics-based strategy to nominate a tumor suppressor gene cadherin-11 (CDH11) and investigate its role in growth and invasion in head and neck squamous cell carcinoma (HNSCC). METHODS: Using the Oncomine™ database to compare HNSCC and normal specimens, CDH11 was nominated as having a role in HNSCC. CDH11 expression in HNSCC was evaluated by immunohistochemistry on a tissue microarray (TMA) and immunoblotting and immunofluorescence of cell lines. The functional impact of CDH11 on proliferation and invasion was evaluated after siRNA-mediated knockdown. RESULTS: In silico analysis suggested that CDH11 is overexpressed in HNSCC compared to normal specimens. HNSCC TMA exhibited a small but significant increase in intensity and proportion of CDH11. By immunoblot analysis, CDH11 was higher in 4/7 HNSCC cell lines compared to normal keratinocytes; CDH11 was highly upregulated in UM-SCC-47 and UM-SCC-74A and detectable in UM-SCC-14A and UM-SCC-29 cell lines. Downregulation of CDH11 in both UM-SCC-29 and UM-SCC-47 using two different siRNAs enhanced proliferation and invasion. CONCLUSION: CDH11 inhibits cell proliferation and invasion of HNSCC. This suggests that CDH11 functions as a tumor suppressor gene in head and neck cancer. Our findings emphasize the importance of verifying in silico findings with functional studies.

Construction of an abnormal glycosylation risk model and its application in predicting the prognosis of patients with head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck, and the incidence rate is increasing year by year. Protein post-translational modification, recognized as a pivotal and extensive form of protein modification, has been established to possess a profound association with tumor occurrence and progression. This study employed bioinformatics analysis utilizing transcriptome sequencing data, patient survival data, and clinical data from HNSCC to establish predictive markers of genes associated with glycosylation as prognostic risk markers. The R procedure WGCNA was employed to construct a gene co-expression network using the gene expression profile and clinical characteristics of HNSCC samples. Multiple Cox Proportional Hazards Regression Model (Cox regression) and LASSO analysis were conducted to identify the key genes exhibiting the strongest association with prognosis. A risk score, known as the glycosylation-related genes risk score (GLRS), was subsequently formulated utilizing the aforementioned core genes. This scoring system facilitated the classification of samples into high-risk and low-risk categories, thereby enabling the prediction of patient prognosis. The association between GLRS and clinical variables was examined through both univariate and multivariate Cox regression analysis. The validation of six core genes was accomplished using quantitative real-time polymerase chain reaction (qRT-PCR). The findings demonstrated noteworthy variations in risk scores among subgroups, thereby affirming the efficacy of GLRS in prognosticating patient outcomes. Furthermore, a correlation has been observed between the risk-scoring model and immune infiltration. Moreover, significant disparities exist in the expression levels of diverse immune checkpoints, epithelial-mesenchymal transition genes, and angiogenic factors between the high and low-risk groups.

Identification and Validation of a Hypoxia-Immune-Based Prognostic mRNA Signature for Oral Squamous Cell Carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a commonly encountered head and neck malignancy. Increasing evidence shows that there are abnormal immune response and chronic cell hypoxia in the development of OSCC. However, there is a lack of a reliable hypoxia-immune-based gene signature that may serve to accurately prognosticate OSCC. METHODS: The mRNA expression data of OSCC patients were extracted from the TCGA and GEO databases. Hypoxia status was identified using the t-distributed Stochastic Neighbor Embedding (t-SNE) algorithm. Both ESTIMATE and single-sample gene-set enrichment analysis (ssGSEA) were used for further evaluation of immune status. The DEGs in different hypoxia and immune status were determined, and univariate Cox regression was used to identify significantly prognostic genes. A machine learning method, least absolute shrinkage and selection operator (LASSO) Cox regression analysis, allowed us to construct prognostic gene signature to predict the overall survival (OS) of OSCC patients. RESULTS: A total of 773 DEGs were identified between hypoxia high and low groups. According to immune cell infiltration, patients were divided into immune high, medium, and low groups and immune-associated DEGs were identified. A total of 193 overlapped DEGs in both immune and hypoxia status were identified. With the univariate and LASSO Cox regression model, eight signature mRNAs (FAM122C, RNF157, RANBP17, SOWAHA, KIAA1211, RIPPLY2, INSL3, and DNAH1) were selected for further calculation of their respective risk scores. The risk score showed a significant association with age and perineural and lymphovascular invasion. In the GEO validation cohort, a better OS was observed in patients from the low-risk group in comparison with those in the high-risk group. High-risk patients also demonstrated different immune infiltration characteristics from the low-risk group and the low-risk group showed potentially better immunotherapy efficacy in contrast to high-risk ones. CONCLUSION: The hypoxia-immune-based gene signature has prognostic potential in OSCC.

miR-34c-5p mediates the cellular malignant behaviors of oral squamous cell carcinoma through targeted binding of TRIM29.

BACKGROUND: This investigation examined the effects of the microRNA miR-34c-5p on the proliferation, migration, and invasion of oral squamous cell carcinoma (OSCC) and the mechanisms involved. METHODS: The Gene Expression Omnibus (GEO) database was used to filter the chips, and the GEO2R software (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to analyze the microarray data (GSE28100 and GSE45238). Gene set enrichment analysis (GSEA) was used to study the relationship between the expression of miR-34c-5p and the distant metastasis and pathological grade of OSCC. The correlation between TRIM29 (tripartite motif containing 29) expression and the malignant clinical phenotype of OSCC was also examined. The mRNA and protein expression levels of miR-34c-5p and TRIM29 were measured by real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot analysis. The proliferation, migration, invasion and apoptosis of the human oral squamous carcinoma cell lines CAL-27 and Tca8113 was assessed by performing cell-counting kit-8 (CCK-8) assays, colony formation assays, transwell tests, wound scratch tests and flow cytometry. Luciferase reporter assays were used to predict the relationship between miR-34c-5p and TRIM29. A xenograft nude model was established and used to evaluate the effect of miR-34c-5p on tumor growth in female BALB/c mice. RESULTS: The expression of miR-34c-5p was significantly correlated with the proliferation, migration, and metastasis of OSCC. Overexpression of miR-34c-5p promoted the proliferation, migration, and invasion of CAL-27 and Tca8113 cells, and suppressed their apoptosis. Inversely, low expression of miR-34c-5p suppressed the proliferation, migration, and invasion of CAL-27 and Tca8113 cells, and promoted their apoptosis. Overexpression of miR-34c-5p promoted tumor growth in the xenograft nude mice model. The expression of TRIM29 was related to malignant clinical phenotype of OSCC. Overexpression of TRIM29 inhibited the proliferation, migration and invasion of CAL-27 and Tca8113 cell, and induced their apoptosis. TRIM29 knockout had just the opposite effect. Importantly, miR-34c-5p binds to TRIM29 and inhibited TRIM29 expression. CONCLUSIONS: MiR-34c-5p regulates the proliferation, migration, invasion, and apoptosis of OSCC through targeted binding of TRIM29. This may represent a novel therapeutic target for the treatment of patients with OSCC.

Squamous cell carcinoma subverts adjacent histologically normal epithelium to promote lateral invasion.

Recurrent and new tumors, attributed in part to lateral invasion, are frequent in squamous cell carcinomas and lead to poor survival. We identified a mechanism by which cancer subverts adjacent histologically normal epithelium to enable small clusters of cancer cells to burrow undetected under adjacent histologically normal epithelium. We show that suppression of DMBT1 within cancer promotes aggressive invasion and metastasis in vivo and is associated with metastasis in patients. Cancer cells via TGFß1 and TNFα also suppress DMBT1 in adjacent histologically normal epithelium, thereby subverting it to promote invasion of a small population of tumor cells. The sufficiency of DMBT1 in this process is demonstrated by significantly higher satellite tumor nests in Dmbt1-/- compared with wild-type mice. Moreover, in patients, invasion of small tumor nests under adjacent histologically normal epithelium is associated with increased risk for recurrence and shorter disease-free survival. This study demonstrates a crucial role of adjacent histologically normal epithelium in invasion and its important role in the tumor microenvironment and opens new possibilities for therapeutic strategies that reduce tumor recurrence.

Inhibition of autophagy by 3-MA enhances IL-24-induced apoptosis in human oral squamous cell carcinoma cells.

BACKGROUND: Interleukin-24(IL-24), also referred to as melanoma differentiation-associated gene-7(mda-7), is a unique member of the IL-10 gene family, which displays nearly ubiquitous cancer-specific toxicity. The most notable feature of IL-24 is selectively induced growth suppression and apoptosis in various cancer cells, with no harmful effects toward normal cells. Autophagy is a self-protective mechanism in many kinds of tumor cells that respond to anticancer treatment. It is reported that autophagy inhibition could enhance the effects of many kinds of anticancer treatments, including gene therapy. However, whether IL-24 is effective to treat oral squamous cell carcinomas (OSCC) and if autophagy inhibition could improve the anticancer effect of IL-24 towards OSCC is has not been detected. METHODS: MTT assays were carried out to determine the cell proliferation; Transfection was used to gene transfer; Western Blot was performed to detect the protein level of LC3II, P62, Beclin 1, Cleaved caspase-3, ß-Tubulin and ß-actin; Apoptosis rates and cell cycle alteration were analyzed using flow cytometry; Autophagy induction was confirmed by MDC staining, GFP-LC3 staining and transmission electron microscopy. Amount of IL-24 in the culture medium was quantified by ELISA. Apoptosis in vivo was analyzed by TUNEL assay. HE staining was used to observe the morphology of the samples. RESULTS: In the present study, we proved that IL-24 have a novel anticancer effect towards KB cells and that autophagy inhibition could improve the anticancer effect of IL-24. IL-24 treated cells showed autophagy characteristics and autophagy inhibition by 3-methyladenine (3-MA) significantly enhanced IL-24-induced apoptosis. Similar results were obtained in the KB cells xenograft tumor model. CONCLUSIONS: These results suggest that the combination of autophagy inhibitors and IL-24 based on the AdLTR2EF1α-mediated gene transfer could be a promising way to cure OSCC.

Increased expression of USP22 is associated with disease progression and patient prognosis of salivary duct carcinoma.

OBJECTIVES: Ubiquitin-specific protease 22 (USP22) could exhibit a critical function in pathological processes, including oncogenesis and cell cycle progression. This study examines the protein expression of USP22 in salivary duct carcinoma (SDC) in association with patient survival and other clinicopathologic parameters. MATERIALS AND METHODS: Quantitative RT-PCR and immunohistochemistry (IHC) were used to determine the expression of USP22 protein in 44 SDCs in comparison with 20 non-cancerous salivary tissues. Furthermore, we analyzed the correlation between the expression of the USP22 protein and various clinicopathologic factors including survival status of patients with SDC. RESULTS: The incidence of positive USP22 expression was 63.7% in 44 SDC tissues. The mRNA level of USP22 expression in SDC samples was significantly higher than that in non-cancerous salivary tissues (P < 0.001), which was consistent with the IHC result (P < 0.001). Moreover, statistical analysis showed that positive USP22 expression was positively related to pT classification, pN classification and AJCC stage. Notably, high USP22 expression was significantly associated with shorter overall survival (P = 0.023) and disease-specific survival (P = 0.019). Multivariate Cox regression analysis revealed that USP22 expression level was an independent prognostic factor for both overall survival (P < 0.001) and disease-free survival (P < 0.001). CONCLUSION: Our results indicate that activation of USP22 correlates with SDC progression and therapy failure. Overexpression of USP22 may contribute to the progression of SDC and thus may serve as a new molecular marker to predict the prognosis of SDC patients.

USP22 is useful as a novel molecular marker for predicting disease progression and patient prognosis of oral squamous cell carcinoma.

BACKGROUND AND OBJECTIVE: The significance of ubiquitin-specific protease 22 (USP22) as a potential marker has been growing in the field of oncology. The aim of this study was to investigate the role of USP22 and the association with its potential targets in oral squamous cell carcinoma (OSCC). METHODS: Immunohistochemistry was used to determine the expression of USP22 protein in 319 OSCC patients in comparison with 42 healthy controls. The clinical correlations and prognostic significance of the aberrantly expressed protein was evaluated to identify novel biomarker of OSCC. RESULTS: The incidence of positive USP22 expression was 63.32% in 319 conventional OSCC tissues. The protein expression level of USP22 was concomitantly up-regulated from non-cancerous mucosa to primary carcinoma and from carcinomas to lymph node metastasis (P<0.001). Moreover, statistical analysis showed that positive USP22 expression was positively related to lymph node metastasis, Ki67, Cox-2 and recurrence. Furthermore, it was shown that patients with positive USP22 expression had significantly poorer outcome compared with patients with negative expression of USP22 for patients with positive lymph nodes. Multivariate Cox regression analysis revealed that USP22 expression level was an independent prognostic factor for both overall survival and disease-free survival (P<0.001 and P<0.001, respectively). Cancer cells with reduced USP22 expression exhibited reduced proliferation and colony formation evaluated by MTT and soft agar assays. CONCLUSION: To our knowledge, this is the first study that determines the relationship between USP22 expression and prognosis in OSCC. We found that increased expression of USP22 is associated with poor prognosis in OSCC. USP22 may represent a novel and useful prognostic marker for OSCC.

Increased expression of CD147 and MMP-9 is correlated with poor prognosis of salivary duct carcinoma.

PURPOSE: The aim of this study was to investigate expression of CD147 and MMP-9 in salivary duct carcinoma (SDC) so as to determine whether these two genes may be correlated with poor prognosis of SDC. METHODS: We examined the significance of the CD147 and MMP-9 expression in SDC (n = 35), non-cancerous salivary tissue (n = 20) in previously untreated patients using immunohistochemical staining. Furthermore, we analyzed the correlation between the expression of these two genes and various clinicopathologic factors including survival status of patients with SDC. RESULTS: Positive stain of CD147 and MMP-9 was seen in all 35 cases of tumor samples. A statistical correlation was observed between CD147 and MMP-9 expression in SDC tissues. The incidences of high expression were 45.71% for CD147 and 51.43% for MMP-9 in 35 SDC tissues, respectively. High expression of CD147 and MMP-9 was significantly correlated with clinical feature and shorter progression-free survival (PFS) (P (CD147) = 0.031; P (MMP-9) = 0.020) and overall survival (OS) (P (CD147) = 0.044; P (MMP-9) = 0.013). CONCLUSION: CD147 and MMP-9 expression is correlated with invasion, metastasis and shorter PFS/OS of SDC. Patients with high expression of CD147 and MMP-9 had poor prognosis than SDC patients with low expression.

Expression patterns of USP22 and potential targets BMI-1, PTEN, p-AKT in non-small-cell lung cancer.

BACKGROUND: Recent researches document that an oncogenic role of USP22 activation may contribute to progression and predict the prognosis. We have reported that USP22 mediates cell survival and proliferation by promoting the expression of BMI-1 and upregulation of activated AKT pathway in colon cancer cells. However, little is known about its mechanisms in non-small-cell lung cancer (NSCLC). Here the authors investigated the significance of activation of USP22 and potential targets BMI-1, PTEN and phospho-AKT (p-AKT) in NSCLC. METHODS: Expression levels of USP22, BMI-1, PTEN and p-AKT in samples from 114 patients with NSCLC were evaluated immunohistochemically using the tissue microarray method. Clinical significance was analyzed by multivariate Cox regression analysis, Kaplan-Meier curves and the log-rank test. RESULTS: Immunohistochemically, USP22, BMI-1, p-AKT and PTEN were positive in 66.66%, 78.07%, 71.92% and 43.85% of NSCLC samples, respectively. Statistical correlation analysis showed USP22 to be significantly correlated with BMI-1 (r=0.315, P=0.001), p-AKT (r=0.271, P=0.003), and PTEN (r=-0.384, P<0.0001). NSCLCs with positive expression of USP22, BMI-1, p-AKT, and negative expression of PTEN were significantly correlated to tumor size (P=0.0240), differentiation (P=0.0457), pT classification (P=0.0077), pN classification (P=0.0064), and AJCC stage (P=0.0363) and poor overall survival (P<0.001). Multivariate Cox proportional hazards model analysis showed that the combined 4 markers was the independent prognostic indicator of overall survival (P<0.001; HR, 5.974; 95% CI, 3.307-10.791). CONCLUSIONS: The simultaneous targeting of USP22, and its downstream signal transduction molecules seem highly informative in stratification of the cancer into subgroups with distinct likelihood of therapy failure, which contribute to make decision process regarding the individualized therapy selection and optimization.