High expression of Talin-1 is associated with poor prognosis in patients with nasopharyngeal carcinoma.

BACKGROUND: Talin-1 is a cytoskeletal protein that plays an important role in tumourgenesis, migration and metastasis in several malignant tumors. The aim of this study was to evaluate the expression and prognostic value of Talin-1 in nasopharyngeal carcinoma (NPC). METHODS: Talin-1 mRNA and protein expression were examined in NPC cell lines and clinical nasopharyngeal tissues by quantitative RT-PCR, agarose gel electrophoresis and western blotting. The expression of Talin-1 was analyzed by immunohistochemical staining in 233 paraffin-embedded NPC specimens with clinical follow-up data and cox regression analysis was used to identify independent prognostic factors. The functional role of Talin-1 in NPC cell lines was evaluated by small interfering RNA-mediated depletion of the protein followed by the wound healing and transwell invasion assays. RESULTS: The expression of Talin-1 was significantly upregulated in most NPC cell lines and clinical tissues at both the mRNA and protein levels. High expression of Talin-1 was significantly associated with distant metastasis (P = 0.001) and patient death (P = 0.001). In addition, high expression of Talin-1 was associated with significantly poorer overall survival (OS: HR, 2.15; 95% CI, 1.28-3.63; P = 0.003) and poorer distant metastasis-free survival (DMFS: HR, 2.39; 95% CI, 1.38-4.15; P = 0.001). Cox regression analysis indicated that high expression of Talin-1 and TNM stage were independent prognostic indicators (both P < 0.05). Stratified analysis demonstrated that high expression of Talin-1 was associated with significantly poorer survival in patients with advanced stage disease (stage III-IV, HR, 1.91; 95% CI, 1.09-3.35; P = 0.02 for OS and HR, 2.22; 95% CI, 1.24-3.99; P = 0.006 for DMFS). Furthermore, the depletion of Talin-1 suppressed the migratory and invasive ability of NPC cells in vitro. CONCLUSIONS: Our data demonstrate that high expression of Talin-1 is associated with significantly poorer OS and poorer DMFS in NPC and depletion of Talin-1 expression inhibited NPC cell migration and invasion. Talin-1 may serve as novel prognostic biomarker in NPC.

Overexpression of CIP2A is an independent prognostic indicator in nasopharyngeal carcinoma and its depletion suppresses cell proliferation and tumor growth.

BACKGROUND: Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that acts as a prognostic marker for several human malignancies. In this study, we investigated the clinical significance of CIP2A and its function in nasopharyngeal carcinoma (NPC). METHODS: Quantitative RT-PCR, western blot, and immunohistochemistry analyses were used to quantify CIP2A expression in NPC cell lines and clinical samples. Kaplan-Meier curves were used to estimate the association between CIP2A expression and patient survival. The functional role of CIP2A in NPC cell lines was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation and xenograft growth. RESULTS: CIP2A levels were upregulated in NPC cell lines and clinical samples at both the mRNA and protein levels (P < 0.01). Patients with high CIP2A expression had poorer overall survival (HR, 1.98; 95% CI, 1.16-3.34; P = 0.01) and poorer disease-free survival (HR, 1.68; 95% CI, 1.07-2.62; P = 0.02) rates than patients with low CIP2A expression. In addition, CIP2A expression status was an independent prognostic indicator for NPC patients. The depletion of CIP2A expression inhibited c-Myc protein expression in NPC cell lines, suppressed cell viability, colony formation, and anchorage-independent growth in vitro, and inhibited xenograft tumor growth in vivo. CONCLUSIONS: Our data demonstrate that high CIP2A expression in patients was associated with poor survival in NPC, and depletion of CIP2A expression inhibited NPC cell proliferation and tumor growth. Thus, these results warrant further investigation of CIP2A as a novel therapeutic target for the treatment of NPC.

DNA-methylome-derived epigenetic fingerprint as an immunophenotype indicator of durable clinical immunotherapeutic benefits in head and neck squamous cell carcinoma.

BACKGROUND: Cancer immunotherapy provides durable response and improves survival in a subset of head and neck squamous cell carcinoma (HNSC) patients, which may due to discriminative tumor microenvironment (TME). Epigenetic regulations play critical roles in HNSC tumorigenesis, progression, and activation of functional immune cells. This study aims to identify an epigenetic signature as an immunophenotype indicator of durable clinical immunotherapeutic benefits in HNSC patients. METHODS: Unsupervised consensus clustering approach was applied to distinguish immunophenotypes based on five immune signatures in The Cancer Genome Atlas (TCGA) HNSC cohort. Two immunophenotypes (immune 'Hot' and immune 'Cold') that had different TME features, diverse prognosis, and distinct DNA methylation patterns were recognized. Immunophenotype-related methylated signatures (IPMS) were identified by the least absolute shrinkage and selector operation algorithm. Additionally, the IPMS score by deconvolution algorithm was constructed as an immunophenotype classifier to predict clinical outcomes and immunotherapeutic response. RESULTS: The 'Hot' HNSC immunophenotype had higher immunoactivity and better overall survival (p = 0.00055) compared to the 'Cold' tumors. The immunophenotypes had distinct DNA methylation patterns, which was closely associated with HNSC tumorigenesis and functional immune cell infiltration. 311 immunophenotype-related methylated CpG sites (IRMCs) was identified from TCGA-HNSC dataset. IPMS score model achieved a strong clinical predictive performance for classifying immunophenotypes. The area under the curve value (AUC) of the IPMS score model reached 85.9% and 89.8% in TCGA train and test datasets, respectively, and robustness was verified in five HNSC validation datasets. It was also validated as an immunophenotype classifier for predicting durable clinical benefits (DCB) in lung cancer patients who received anti-PD-1/PD-L1 immunotherapy (p = 0.017) and TCGA-SKCM patients who received distinct immunotherapy (p = 0.033). CONCLUSIONS: This study systematically analyzed DNA methylation patterns in distinct immunophenotypes to identify IPMS with clinical prognostic potential for personalized epigenetic anticancer approaches in HNSC patients. The IPMS score model may serve as a reliable epigenome prognostic tool for clinical immunophenotyping to guide immunotherapeutic strategies in HNSC.

Comprehensive Characterization of Immune Landscape Based on Epithelial-Mesenchymal Transition Signature in OSCC: Implication for Prognosis and Immunotherapy.

Current anatomic TNM stage classification fails to capture the immune heterogeneity of oral squamous cell carcinoma (OSCC). Increasing evidence indicates the strong association between epithelial-mesenchymal transition (EMT) and tumor immune response. In this study, we employed an EMT signature to classify OSCC patients into epithelial- (E-) and mesenchymal- (M-) phenotypes using TCGA and GSE41613 transcriptome data. The ESTIMATE and CIRBERSORT analyses implied that the EMT signature genes originated from the stroma of the bulk tissue. The M-subtype tumors were characterized as "immune-hot" with more immune cell infiltration than the E-subtype ones. The low infiltration of active immune cells, the high infiltration of inactive immune cells, and the high expressions of immune checkpoints demonstrated an immunosuppressive characteristic of the M-subtype tumors. Moreover, we developed and validated a novel prognostic classifier based on the EMT score, the expressions of seven immune checkpoints, and the TNM stages, which could improve the prediction efficiency of the current clinical parameter. Together, our findings provide a better understanding of the tumor immune heterogeneity and may aid guiding immunotherapy in OSCC.

Association between APOBEC3H-Mediated Demethylation and Immune Landscape in Head and Neck Squamous Carcinoma.

Immunotherapy has been demonstrated as a promising strategy in controlling head and neck squamous cell carcinoma (HNSC). The AID/APOBEC family is well characterized as DNA mutator and considered to play critical roles in immune responses in HNSC. However, the expression pattern and deamination-dependent demethylation roles of AID/APOBECs in HNSC are unclear. In this study, the RNA-seq and DNA methylation profiles of HNSC from TCGA database and cell-based experiments were applied to analyze the relationships between AID/APOBEC expression levels, patients' clinical outcomes, methylation alterations, and immune responses. Here, we found that APOBEC3H was abnormally upregulated in HNSC patients. HPV+ patients tended to have higher APOBEC3H levels than HPV- patients. Remarkably, patients with high APOBEC3H levels showed a favorable overall survival. Furthermore, tumors with high APOBEC3H levels exhibited a genome-wide DNA hypomethylation pattern. APOBEC3H was identified to demethylate and upregulate CXCL10 and improve CD8+ T cell tumor infiltration in the tumor microenvironment. Collectively, APOBEC3H plays critical roles in CD8+ T cell immune infiltration and activation in HNSC, which may be a potential biomarker for oncoimmunotherapy in HNSC.

Correction to: YPEL3 suppresses epithelial-mesenchymal transition and metastasis of nasopharyngeal carcinoma cells through the Wnt/ß-catenin signaling pathway.

An amendment to this paper has been published and can be accessed via the original article.

Immune Landscape of the B7 and TNFR Families in Oral Squamous Cell Carcinoma.

OBJECTIVE: To understand the immune molecular landscapes of the two major costimulatory and coinhibitory pathways (B7 and TNFR families) in oral squamous cell carcinoma. METHODS: The B7 family members (CD80, CD86, CD274, ICOSLG, CD276, VTCN1, NCR3LG1, HHLA2 and PDCD1LG2) and TNFR family members (TNFSF4, CD40, CD70, TNFSF9, TNFRSF14 and TNFSF18) were used to analyse the costimulatory and coinhibitory pathway alterations in oral squamous cell carcinoma. The online tools UCSC Xena and cBioPortal were used to derive oral squamous cell carcinoma patients' clinical parameters, mRNA levels, mutations, DNA copy number alterations and methylation levels. The correlations between mRNA levels and methylation levels were determined using Spearman's correlation analysis. A Kaplan-Meier survival analysis was performed to examine the relationships between mRNA expression levels and overall survival. RESULTS: Compared with normal oral epithelial tissues, approximately 23.1% of patients showed upregulation of B7 expression and 15.3% showed upregulation of TNFR expression in oral squamous cell carcinoma, with CD274 (PD-L1) upregulation being the most common alteration. Mutations and copy number alterations were shown to have little effect on B7 and TNFR expression. The mRNA levels of B7 and TNFR genes were negatively correlated with their methylation levels. Furthermore, oral squamous cell carcinoma patients with high expression levels of CD274 showed poor overall survival, while those with high expression levels of CD276 or HHLA2 showed good clinical outcomes. CONCLUSION: This study elucidated the molecular landscapes of the B7 and TNFR genes in oral squamous cell carcinoma, which could provide a novel strategy for clinical therapy.

TIPE3 hypermethylation correlates with worse prognosis and promotes tumor progression in nasopharyngeal carcinoma.

BACKGROUND: Increasing evidence recognizes that DNA methylation abnormalities play critical roles in cancer development. Our previous genome-wide methylation profile showed that tumor necrosis factor-alpha-induced protein 8 like 3 (TIPE3) was hypermethylated in nasopharyngeal carcinoma (NPC). However, the relationship between TIPE3 methylation and its mRNA expression, as well as its biological roles in NPC are unknown. METHODS: Bisulfite pyrosequencing and quantitative RT-PCR were performed to quantify the TIPE3 methylation and expression levels. Kaplan-Meier curves and Cox regression analysis were used to estimate the correlation between TIPE3 methylation levels and survival in two patient cohorts collected from two hospitals (n = 441). The MTT, colony formation, Transwell migration and invasion assays, and xenograft tumor growth and lung metastatic colonization models were used to identify the functions of TIPE3 on NPC cells. RESULTS: We found that TIPE3 CpG island (CGI) was hypermethylated and its mRNA levels were downregulated in many cancers, including NPC. TIPE3 downregulation was associated with its CGI hypermethylation. Furthermore, NPC patients with high TIPE3 CGI methylation levels had poorer clinical outcomes than those with low methylation levels. The TIPE3 CGI methylation level was an independent prognostic factor. Moreover, restoring TIPE3 expression significantly inhibited NPC cell proliferation, migration and invasion in vitro, and suppressed tumor growth and lung metastatic colonization in vivo, while silencing TIPE3 acted in an opposite way. CONCLUSIONS: TIPE3 downregulation correlates with its CGI hypermethylation in several solid cancers. TIPE3 acts as a tumor suppressor in NPC, providing a further insight into NPC progression and representing a potential prognostic biomarker for NPC.

MicroRNA-101 inhibits invasion and angiogenesis through targeting ITGA3 and its systemic delivery inhibits lung metastasis in nasopharyngeal carcinoma.

Clinically, distant metastasis after primary treatment remains a key problem in nasopharyngeal carcinoma (NPC), and the treatment outcome of metastatic NPC remains disappointing, so there is a pressing need to identify novel therapeutic strategies. In accordance with our previous microarray data, we found that miR-101 was downregulated in NPC clinical specimens and cell lines. Ectopic expression of miR-101 significantly suppressed NPC cell migration, invasion and angiogenesis in vitro and inhibited angiogenesis and metastasis in vivo using the chicken chorioallantoic membrane model. Furthermore, ITGA3 was identified and validated as a novel target of miR-101, and the restoration of ITGA3 expression potently rescued the suppressive effects of miR-101. In addition, NPC patients with high ITGA3 expression had poorer overall survival and distant metastasis-free survival than patients with low ITGA3 expression, and ITGA3 overexpression was an independent poor prognostic factor in NPC. More importantly, we demonstrated that the systemic delivery of lentivirus-mediated miR-101 abrogated the lung metastatic colonization formation of NPC cells without obvious toxicity. Our study elucidates the molecular mechanisms of miR-101/ITGA3 pathway in regulating NPC metastasis and angiogenesis, and the systemic delivery of miR-101 provides a potent evidence for the development of a novel microRNA-targeting anticancer strategy for NPC patients.

Prognostic implications of dynamic serum lactate dehydrogenase assessments in nasopharyngeal carcinoma patients treated with intensity-modulated radiotherapy.

The prognostic value of dynamic serum lactate dehydrogenase (LDH) levels in patients with nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiotherapy (IMRT) hasn't been explored. We retrospectively analyzed 1,428 cases of NPC treated with IMRT with or without chemotherapy. Elevated pre- and/or post-treatment LDH levels were found to be associated with unfavorable overall survival (OS), disease-free survival (DFS) and distant metastasis-free survival (DMFS), but not with local relapse-free survival (LRFS). The dynamic variations in LDH levels were prognostic factors for OS, DFS and DMFS, but not for LRFS. Multivariate analysis revealed that the N category, T category, post-treatment serum LDH level and age were independent prognostic factors for OS. Our results demonstrated that dynamic variations in LDH levels were associated with risk of distant failure and death, which may shed light on the dynamics of the disease and the response to therapy. We consider that LDH measurements will be of great clinical importance in the management of NPC, especially, when considering "decision points" in treatment algorithms. Therefore, we strongly recommend that LDH levels should be determined before and after treatment in NPC patients and the results integrated into decisions regarding treatment strategies.

YPEL3 suppresses epithelial-mesenchymal transition and metastasis of nasopharyngeal carcinoma cells through the Wnt/ß-catenin signaling pathway.

BACKGROUND: Metastasis remains the major cause of death in nasopharyngeal carcinoma (NPC). Yippee-like 3 (YPEL3) plays an important role in tumorigenesis. However, its function and mechanism in NPC has not been systematically explored. METHODS: We evaluated YPEL3 expression in NPC cell lines and tissues using real-time PCR and western blotting. Then, we established NPC cell lines that stably overexpressed YPEL3 and knocked down YPEL3 expression to explore its function in NPC in vitro and in vivo. Additionally, we investigated the potential mechanism of YPEL3 action by identifying the Wnt/ß-catenin signaling pathway downstream genes using western blotting. RESULTS: YPEL3 was downregulated in NPC cell lines and tissue samples. Ectopic expression of YPEL3 inhibited NPC cell migration and invasion in vitro; while silencing of YPEL3 promoted NPC cell migration and invasion. Further study indicated that overexpression of YPEL3 inhibited NPC cell epithelial-mesenchymal transition (EMT) and that silencing it enhanced EMT. Overexpression of YPEL3 suppressed NPC cell lung metastasis in vivo. The mechanism study determined that YPEL3 suppressed the expression levels of Wnt/ß-catenin signaling pathway downstream genes and the nuclear translocation of ß-catenin. CONCLUSIONS: YPEL3 suppresses NPC EMT and metastasis by suppressing the Wnt/ß-catenin signaling pathway, which would help better understanding the molecular mechanisms of NPC metastasis and provide novel therapeutic targets for NPC treatment.

MicroRNA-93 promotes cell growth and invasion in nasopharyngeal carcinoma by targeting disabled homolog-2.

Dysregulation of microRNAs (miRNAs) has been demonstrated to contribute to malignant progression in nasopharyngeal carcinoma (NPC). We previously reported that miR-93 was significantly upregulated in NPC based on a microarray analysis. However, the potential role and mechanism of action of miR-93 in the initiation and progression of NPC remain largely unknown. Quantitative RT-PCR demonstrated that miR-93 was significantly upregulated in NPC cell lines and clinical specimens. The MTT assay, colony formation assay, anchorage-independent growth, and Transwell migration and invasion assays showed that depletion of miR-93 inhibited NPC cell growth, invasion and migration in vitro and suppressed tumor growth in vivo. Disabled homolog-2 (Dab2) was verified as a miR-93 target gene using Luciferase reporter assays, quantitative RT-PCR and Western blotting and was involved in miR-93-regulated NPC cell growth, invasion and migration. These results indicated that miR-93 plays an important role in the initiation and progression of NPC by targeting Dab2 and the miR-93/Dab2 pathway may contribute to the development of novel therapeutic strategies for NPC in the future.

MiR-145 inhibits metastasis by targeting fascin actin-bundling protein 1 in nasopharyngeal carcinoma.

BACKGROUND: Based on our recent microarray analysis, we found that miR-145 was obviously downregulated in nasopharyngeal carcinoma (NPC) tissues. However, little is known about its function and mechanism involving in NPC development and progression. METHODS: Quantitative RT-PCR was used to detect miR-145 expression in NPC cell lines and clinical samples. Wound healing, Transwell migration and invasion, three-dimension spheroid invasion assays, and lung metastasis model were performed to test the migratory, invasive, and metastatic ability of NPC cells. Luciferase reporter assay, quantitative RT-PCR, and Western blotting were used to verify the target of miR-145. RESULTS: MiR-145 was obviously decreased in NPC cell lines and clinical samples (P<0.01). Ectopic overexpression of miR-145 significantly inhibited the migratory and invasive ability of SUNE-1 and CNE-2 cells. In addition, stably overexpressing of miR-145 in SUNE-1 cells could remarkably restrain the formation of metastatic nodes in the lungs of mice. Furthermore, fascin actin-bundling protein 1 (FSCN1) was verified as a target of miR-145, and silencing FSCN1 with small RNA interfering RNA could suppress NPC cell migration and invasion. CONCLUSIONS: Our findings demonstrated that miR-145 function as a tumor suppressor in NPC development and progression via targeting FSCN1, which could sever as a potential novel therapeutic target for patients with NPC.

Low SFRP1 Expression Correlates with Poor Prognosis and Promotes Cell Invasion by Activating the Wnt/ß-Catenin Signaling Pathway in NPC.

Distant metastasis remains the predominant mode of treatment failure in nasopharyngeal carcinoma (NPC). Unfortunately, the molecular events underlying NPC metastasis remain poorly understood. Secreted frizzled-related protein 1 (SFRP1) plays an important role in tumorigenesis and progression. However, little is known about the function and mechanism of SFRP1 in NPC. Immunohistochemistry was used to determine SFRP1 expression levels in patients with NPC. SFRP1 function was evaluated using MTT, colony formation, wound-healing, Transwell assays, and in vivo models. The methylation level of SFRP1 in NPC cells was examined using bisulfate pyrosequencing; the Wnt/ß-catenin signaling pathway genes were studied using Western blotting. Compared with patients with high SFRP1 expression, patients with low SFRP1 expression had worse overall survival [HR, 2.32; 95% confidence interval (CI), 1.36-3.94; P = 0.002], disease-free survival (HR, 1.98; 95% CI, 1.23-3.18; P = 0.005), and distant metastasis-free survival (HR, 2.07; 95% CI, 1.19-3.59; P = 0.009). Multivariate Cox regression analysis indicated that SFRP1 was an independent prognostic factor. Furthermore, SFRP1 was significantly downregulated in NPC cell lines. SFRP1 overexpression suppressed NPC cell proliferation, migration, and invasion in vitro and lung colonization in vivo. SFRP1 expression was restored after treatment with a demethylation agent, and the SFRP1 promoter region was hypermethylated in NPC cells. ß-Catenin, c-Myc, and cyclin D1 were downregulated after SFRP1 restoration, which suggested that SFRP1 suppressed growth and metastasis by inhibiting the Wnt/ß-catenin signaling pathway in NPC. SFRP1 provides further insight into NPC progression and may provide novel therapeutic targets for NPC treatment.

Identification of miR-143 as a tumour suppressor in nasopharyngeal carcinoma based on microRNA expression profiling.

Recent evidence has indicated that miRNAs play important roles in carcinogenesis. The identification of dysregulated miRNAs and the target genes they regulate might enhance our understanding of the molecular mechanisms of nasopharyngeal carcinoma (NPC). A microarray analysis was performed to identify dysregulated miRNAs in NPC tissue samples, and protein-coding genes targeted by three or more downregulated miRNAs were selected using miRWalk and used in a pathway enrichment analysis. Nineteen KEGG pathways were selected by DAVID, including the MAPK, focal adhesion, gap junction, ECM-receptor interaction, TGF-beta, and p53 signalling pathways, most of which are involved in NPC carcinogenesis and progression. MiR-143 was significantly downregulated in NPC cell lines and clinical samples. The ectopic expression of miR-143 suppressed NPC cell viability, colony formation, and anchorage-independent growth in vitro, and it inhibited xenograft tumour growth in vivo. Furthermore, KRAS was confirmed as a direct target of miR-143, and silencing KRAS expression suppressed NPC cell viability and proliferation. The miR-143/KRAS pathway provides new insight into the molecular mechanisms that regulate the development and progression of NPC, and it provides novel therapeutic targets for NPC.

Genome-Wide Identification of a Methylation Gene Panel as a Prognostic Biomarker in Nasopharyngeal Carcinoma.

DNA methylation, the best known epigenetic marker, can be used as a prognostic biomarker in many cancers. We examined DNA methylation status and survival in nasopharyngeal carcinoma (NPC) patients. Aberrant DNA-methylated genes in 24 NPC tissues and 24 noncancer nasopharyngitis biopsy tissues (NCNBT) were identified using Illumina 450K BeadChip. Correlations between DNA methylation and clinical outcomes were evaluated using bisulfite pyrosequencing in 454 NPC patients. Genome-wide methylation analysis demonstrated that NPC tissues had distinct DNA methylation patterns compared with NCNBT. Among all significant CpG sites, 2,173 CpG sites with ß change ≥ 0.2 (1,880 hypermethylated, 293 hypomethylated) were identified (P < 0.05). A methylation gene panel comprising six hypermethylated genes was constructed with the average Z-score method. Patients in the training cohort with high methylation had poorer disease-free survival [DFS, HR, 2.26; 95% confidence interval (CI), 1.28-4.01; P, 0.005] and overall survival (OS, HR, 2.47; 95% CI, 1.30-4.71; P, 0.006) than those with low methylation. There were similar results in the validation (DFS, HR, 2.07; 95% CI, 1.17-3.67; P, 0.013; OS, HR, 1.83; 95% CI, 1.01-3.31; P, 0.046) and independent cohorts (DFS, HR, 1.94; 95% CI, 1.08-3.47; P, 0.026; OS, HR, 2.09; 95% CI, 1.10-3.98; P, 0.022). Analysis indicated that the methylation gene panel was an independent prognostic factor. Furthermore, patients with low methylation had a favorable response to concurrent chemotherapy with an improved DFS (P = 0.045) and OS (P = 0.031), whereas patients with high methylation did not benefit from concurrent chemotherapy. The six-hypermethylated gene panel was associated with poor survival in patients with NPC, demonstrating its potential usefulness as a prognostic biomarker to clinicians in NPC management.