Laryngeal Squamous Cell Carcinoma Tumor Tissues Research Articles

Isolation of tumor stem-like cells from primary laryngeal squamous cell carcinoma cells (FD-LS-6).

The development of efficient treatments for laryngeal squamous cell carcinoma (LSCC) is hindered by the lack of applicable tumor cell lines and animal models of the disease, especially those related to cancer stem-like cells (CSCs). CSCs play critical roles in tumor propagation and pathogenesis whereas no CSCs lines have been developed to date. In this study, we establish an LSCC cell line (FD-LS-6) from primary LSCC tumor tissue (not experienced single-cell cloning) and adapted a culturing condition for the expansion of potential stem cells (EPSCs) to isolate CSCs from FD-LS-6. We successfully derived novel CSCs and named them as LSCC sphere-forming cells (LSCSCs) which were subsequently characterized for their CSC properties. We showed that LSCSCs shared many properties of CSCs, including CSC marker, robust self-renewal capacity, tumorigenesis ability, potential to generate other cell types such as adipocytes and osteoblasts, and resistance to chemotherapy. Compared to parental cells, LSCSCs were significantly more potent in forming tumors in vivo in mice and more resistant to chemotherapy. LSCSCs have higher expressions of epithelial-mesenchymal transition proteins and chemotherapy resistance factors, and exhibit an activated COX2/PEG2 signaling pathway. Altogether, our work establishes the first CSCs of LSCC (FD-LS-6) and provides a tool to study tumorigenesis and metastasis of LSCC and help the development of anticancer therapies.

The Novel Circular RNA circTRIO Silence Inhibits the Progression of Laryngeal Squamous Cell Carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is the most common and prevalent malignant tumor in head and neck squamous cell carcinoma. Recent studies have shown that circular RNAs (circRNAs) play a vital role in cancer development, but their specific role in the tumorigenesis and development of LSCC remains unclear. We selected five pairs of LSCC tumor tissues and paracancerous tissues for RNA sequencing. Reverse transcription-quantitative PCR (RT-qPCR), Sanger sequencing, and fluorescence in situ hybridization were utilized to study the expression, localization, and clinical significance of circTRIO in LSCC tissues, and TU212 and TU686 cell lines. Furthermore, cell counting Kit-8, colony-forming assay, Transwell, and flow cytometry assays were assessed to illustrate the crucial role played by the circTRIO in proliferation, colony-forming ability, migration, and apoptosis in LSCC cells. Finally, the molecule's role as a microRNA (miRNA) sponge was analyzed. In the results, we screened out a promising upregulated novel circRNA-circTRIO in LSCC tumor tissues compared with paracancerous tissues using RNA sequencing. Then, we used qPCR to evaluate the expression of circTRIO in 20 additional paired LSCC tissues and two cells, and the results showed that circTRIO was highly expressed in LSCC and that this high expression was closely related to the malignant progression of LSCC. Furthermore, we examined circTRIO expression in the Gene Expression Omnibus data sets GSE142083 and GSE27020, and circTRIO expression was considerably higher in tumor tissues compared with the adjacent tissues. Kaplan-Meier survival analysis showed that the expression of circTRIO was associated with worse disease-free survival. The Gene Set Enrichment Analysis biological pathway evaluation results demonstrated that circTRIO was mainly enriched in cancer pathways. Moreover, we confirmed that silencing circTRIOs can help to significantly inhibit LSCC cell proliferation and migration while triggering apoptosis. Upregulated circTRIO expression levels may play an important role in the tumorigenesis and development of LSCC.

POTENTIAL OF microRNAs AS A BIOMARKER FOR LARYNGEAL CANCER

Objective: Laryngeal malignancy known as the most frequently diagnosed cancer between head and neck tumor, covering 2.4% of all tumors and is the 11th most occurrence and deadliest tumor in the world. Research has shown that microRNAs (miRNAs) play a considerable role in the development of laryngeal malignancy. Moreover, studies also focus on the relationship between clinical relevance and miRNA deregulation in cancer illness. According to recent evidence, miR-205, miR-126, miR-302, and miR-132 have possible roles in the carcinogenesis process of the LSCC. Therefore, in this research, we aimed to investigate whether there are any considerable changes in the microRNAs expression level and also if they have any remarkable potential as a prognostic or diagnostic biomarker for laryngeal squamous cell carcinoma (LSCC). Methods: Thirty LSCC patients were included in this research, and tumor tissues and healthy normal tissues were collected during laryngectomy. Total RNA extraction from the normal and cancer tissues was accomplished and after the quality control, cDNA was synthesized, and finally, target miRNAs expression was determined by qPCR. The expression level of genes and their relationship with the patient’s clinicopathological characteristics were analyzed using appropriate statistical tests. Results: According to the study results, miR-302 (fold change: 1.o5, P-value = 0.0001) and miR-132 (fold change: 1.28, P-value = 0.0001), is considerably overexpressed in cancer tissues compare to normal peripheral tissues. We also found that miR-126 (fold change: 0.251, P-value = 0.0001) and miR-205 (fold change: 0.241, P-value<0.0001) is down-regulated in LSCC tumor tissue. Among these microRNAs, only mir-205 expression had no association with clinical-pathological features in tumor tissue. Conclusion: The results of this research revealed the potential of the miR-302, miR-132, miR-126, and miR-205 as prognostic or diagnostic biomarkers in LSCC.

Elevated ITGA5 facilitates hyperactivated mTORC1-mediated progression of laryngeal squamous cell carcinoma via upregulation of EFNB2.

Background: Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of the head and neck, and it has shown increasing incidence and mortality. The mechanistic target of rapamycin complex 1 (mTORC1) is frequently dysregulated in LSCC, but its underlying mechanisms remain unclear. Methods: Establishment of a novel LSCC cell line using primary LSCC tumor tissues with dysregulated mTORC1 activity and then stable knockdown of Raptor (an mTORC1 specific component) in this cell line. Transcriptomic sequencing, quantitative real-time PCR, western blot analysis, and immunofluorescence assays were used to identify the crucial downstream effector of mTORC1. A series of experiments were conducted to investigate the functions and underlying mechanisms of the mTORC1 target gene in LSCC progression. Clinical LSCC samples were used to evaluate the association of mTORC1 and its downstream targets with clinicopathological features and patient prognosis. Finally, the influence on cisplatin (CDDP) sensitivity upon depletion of the mTORC1 target gene was assessed using a cell culture system, a cell line-derived xenograft (CDX) model, and a patient-derived xenograft (PDX) model. Results: We successfully established a novel LSCC cell line with hyperactivated mTORC1 activity and then identified integrin subunit alpha 5 (ITGA5) as a novel functional downstream effector of mTORC1 in the progression of LSCC. Elevated ITGA5 promotes LSCC progression through augmentation of ephrin-B2 (EFNB2). Clinical data analysis indicated that the activation of the mTORC1-ITGA5-EFNB2 signaling pathway is associated with malignant progression and poor prognosis of LSCC patients. Inhibition of ITGA5 significantly sensitized LSCC cells to CDDP. Conclusions: Our findings highlight a novel molecular mechanism for the tumorigenesis driven by deregulated mTORC1 signaling in LSCC, suggesting that the ITGA5-EFNB2 axis may be a therapeutic target for the treatment of mTORC1-related LSCC.

Laryngeal carcinoma experimental model suggests the possibility of tumor seeding to gastrostomy site

Some studies state that laryngeal squamous cell carcinoma (LSCC) is associated with possible direct tumor cell seeding to percutaneous endoscopic gastrostomy (PEG) site. However, there is a lack of experimental proof that LSCC tumor tissue can adhere and grow in distant sites. Therefore, we aimed to investigate the growth pattern of LSCC implants on chicken embryo chorioallantoic membrane (CAM) and evaluate possible associations between clinical course of the disease and behavior of experimentally implanted LSCC tumors. Our results show that implanted LSCC tissue survives on CAMs in 95% of cases while retaining essential morphologic characteristics and proliferative capacity of the original tumor. We identified the increased CAM vascularization, an infiltrative growth pattern of the implant and formation of distant isolated metastatic nodes on the CAMs. LSCC tumors with worse differentiation degree (G2 or G3) adhered to the experimental CAMs significantly better than G1. These results facilitate the understanding of tumor biology and allow hypothetisezing that dissemination and direct implantation of LSCC cells into the stomal wall during the pull PEG procedure might be possible.

Effect of Programmed Death-Ligand 1 in Cancer-Associated Fibroblasts on Advanced Laryngeal Squamous Cell Carcinoma.

This study aimed to explore the effect of programmed death-ligand 1 (PD-L1) in cancer-associated fibroblasts (CAFs) on advanced laryngeal squamous cell carcinoma (LSCC). The expression of PD-L1 in advanced LSCC tumor tissues was observed in 83 patients with LSCC by immunofluorescence microscopy and compared with that in normal laryngeal mucosa. The CAFs of LSCC and normal fibroblasts (NFs) were isolated, cultured, purified, and examined by fluorescence. The expression of PD-L1 in purified CAFs and NFs was measured by flow cytometry. The expression of PD-L1 in CAFs was downregulated through small interferring RNA (siRNA) transfection. The proliferation and migration capacities of CAFs were observed using proliferation and scratch tests, respectively. The proliferation of HEP-2 cells and T cells was measured after cocultured with CAFs. The secretion of interleukins IL-2 and IL-10 was detected using enzyme-linked immuno sorbent assay (ELISA). PD-L1 was expressed in 62 of 83 cases of the advanced LSCC tumor tissues. Also, CAFs expressed more PD-L1 compared with NFs. The proliferation and migration capacities of CAFs were significantly lower after transfection with PD-L1-siRNA. The proliferation rate of HEP-2 cells cocultured with CAFs decreased in PD-L1-siRNA-transfected cells. However, the proliferation rate of T cells increased in transfected cells. The ELISA results showed that the secretion of IL-2 increased and that of IL-10 decreased in PD-L1-siRNA-transfected cells. The expression of PD-L1 in CAFs of advanced LSCC was higher than that in NFs. The downregulation of PD-L1 reduced the proliferation and migration of CAFs and HEP-2 cells but enhanced the proliferation and pro-inflammatory function of T cells in the coculture experiment.

The deubiquitinase USP34 stabilizes SOX2 and induces cell survival and drug resistance in laryngeal squamous cell carcinoma.

Recent studies showed that the deubiquitinase ubiquitin-specific protease 34 (USP34) was involved in the tumorigenesis of several tumors, but its function and mechanism are still unclear in laryngeal squamous cell carcinoma (LSCC). In this study, we found that USP34 and SOX2 were elevated in LSCC tumor tissues, and we also found that USP34 expression was positively correlated with SOX2 expression. Our further studies showed that USP34 regulated the protein level of SOX2 in LSCC cells, but not the mRNA level, which suggested that USP34 stabilized SOX2. Moreover, USP34, as a deubiquitinase, could interact with SOX2, and reduce the polyubiquitination of SOX2. In addition, knockdown of USP34 could significantly inhibit LSCC cell growth, but overexpression of SOX2 could reverse this effect. Finally, we also found that USP34 and SOX2 were upregulated in cisplatin-resistant LSCC cells, but knockdown of USP34 could enhance the drug sensitivity of cisplatin in the resistant cells. Collectively, targeting USP34/SOX2 axis may be a promising strategy for the treatment of LSCC.

Link between CIITA rs3087456 polymorphism and the risk of laryngeal squamous cell carcinoma in a Chinese population

The class II trans-activator (CIITA) is the master regulator of the major histocompatibility complex (MHC) gene expression. CIITA mutations have been previously associated with several kinds of tumors, while the role of CIITA polymorphisms (rs3087456) in laryngeal squamous cell carcinoma (LSCC) is little known. We evaluate the link between CIITA polymorphisms and the existence of LSCC in patients. This study was conducted with 200 Chinese Han patients (LSCC) and 200 healthy control subjects.The association of CIITA genetic polymorphism rs3087456 with the risk of LSCC was assessed through pyrosequencing. The CIITA expression in LSCC tumor tissue and adjacent normal tissue was detected by immunohistochemistry (IHC) staining. The relationship between the genotype of rs3087456 in controls and in clinical pathology features in LSCC were analyzed, and in-silico analysis was also used for the CIITA gene. The in-silico analysis results showed that the CIITA gene is closely related to genes such as RFX5 and RFXAP. The IHC results showed that CIITA was highly expressed in LSCC tumor tissues, compared with the corresponding adjacent normal tissues. The AG, AG + AA, and A genotypes of rs3087456 of CIITA gene notably increased the risk of LSCC compared to the controls. Our study suggests that CIITA polymorphism (rs3087456) is associated with a higher risk of developing LSCC in a Chinese cohort.

High Mobility Group A2 (HMGA2) and Matrix Metalloproteinase 12 (MMP12) Genes Expression in Egyptian Patients with Laryngeal Squamous Cell Carcinoma

Background: genetic studies of laryngeal squamous cell carcinoma (LSCC) are rare in Egypt. Increased expression of some relevant genes may have diagnostic and therapeutic implications.Aim of the study: to compare the expression of HMGA2 and MMP12 genes in LSCC tumor tissue with their expression in adjacent non-tumor tissue.Type of the study: Descriptive studySetting: Tertiary referral hospital-Ismailia-EgyptPatients and Methods: 24 male patients (age range 49-78 years) with LSCC were included in the study. Under general anesthesia, two biopsies were taken: one from the tumor tissue and the other from the adjacent normal tissue and they were examined histopathologically and then analyzed for level of expression of MMP12 and HMGA2 genes.Results: Median expression levels of HMGA2 and MMP12 in the whole group were 0.98 and .8 (fold change) in normal tissue and 6.49 and 122.6 (fold change) in tumor tissue respectively. There was higher expression of the HMGA2 and MMP12 genes in tumor tissue compared to normal tissue (P <0.0001). Also there was positive correlation of level of expression of the two genes with the degree of tumor differentiation (P <.0001 and p<.0008)Conclusion: Expression of HMGA2 and MMP12 genes is higher in LSCC compared to normal tissue samples. The higher the level of expression of the two genes, the poorer the degree of differentiation of the tumor Further studies should be pursued to determine the diagnostic, therapeutic and prognostic implications of this in management of LSCC.

MicroRNA-4497 functions as a tumor suppressor in laryngeal squamous cell carcinoma via negatively modulation the GBX2

ObjectiveMicroRNAs (miRNAs) are aberrantly expressed in various tumors and play a critical role in the progression and development of tumors. However, there is little information about the role of miR-4497 in laryngeal squamous cell carcinoma (LSCC). The aim of this study is to investigate the role of miR-4497 in LSCC. MethodsMiR-4497 expression in tumor tissues and adjacent normal tissues was measured by RT-PCR. The effects of miR-4497 on cell viability and apoptosis were evaluated by the MTT assay, Flow cytometry and caspase-3 activity assay. Western blot analysis was used to measure the expression of various proteins. Bioinformatic analysis and luciferase reporter assay were applied to investigate the relationship between miR-4497 and GBX2. ResultsWe found that miR-4497 expression was downregulated in LSCC tumor tissues and cell lines compared to the normal counterparts. Overexpression of miR-4497 inhibits the proliferation and induces apoptosis of LSCC cells accompanied by the down-regulation of anti-apoptotic Bcl-2 proteins. Mechanisms investigation revealed that GBX2 is a direct target of miR-4497. miR-4497 expression was inversely correlated with GBX2 expression in LSCC tissues. Moreover, overexpression of miR-4497 leads to the activation of ERK, JNK but not p38. Inhibition of ERK by specific inhibitor SCH772984 could interfere the apoptosis induced by overexpression of miR-4497. ConclusionTherefore, our results indicate that miR-4497 may play a suppressive role in LSCC by targeting GBX2, which offer new insights into the tumorigenesis of LSCC.

LZTS2 promoter hypermethylation: a potential biomarker for the diagnosis and prognosis of laryngeal squamous cell carcinoma

BackgroundLZTS2 (leucine zipper tumor suppressor 2), a candidate tumor suppressor gene, suppresses cell growth and plays a vital role in the carcinogenesis and development of tumors. No studies to date have described methylation of the LZTS2 promoter in human cancers, including LSCC (laryngeal squamous cell carcinoma). Therefore, the aim of this study was to explore the relationship between LZTS2 promoter methylation and risk of LSCC.MethodsIn our study, LZTS2 promoter methylation levels in LSCC tumor and adjacent normal tissues from 96 patients were measured using quantitative methylation-specific polymerase chain reaction (qMSP) assays.ResultsThe qMSP analyses revealed that LZTS2 promoter methylation levels in the LSCC tumor samples were significantly higher than those in paired adjacent healthy tissue samples. Furthermore, LZTS2 methylation levels were elevated in smokers, advanced T classified, and clinically staged patients, as well as in patients with lymph node metastases. In addition, Kaplan-Meier survival curves results showed that overall survival of LSCC patients with hypomethylated LZTS2 promoters was significantly higher than that in patients with hyper-methylated LZTS2 promoters (log-rank test P = 0.028). Meanwhile, the area under the receiver operating characteristic curve was 0.920. The diagnostic threshold value for LZTS2 methylation was 11.63% (94.7% sensitivity and 80.4% specificity).ConclusionsLZTS2 promoter hypermethylation is associated with risk, progression, and prognosis of LSCC in a cohort of 96 human subjects; LZTS2 promoter hypermethylation is a candidate diagnostic and prognostic biomarker for LSCC.

Preliminary findings for metabolite profiles of papillary thyroid carcinoma and laryngeal squamous cell carcinoma

Objective:To identify distinct metabolite profiles of papillary thyroid cancer (PTC) and laryngeal squamous cell carcinoma (LSCC).Method:Tumor and adjacent non-tumor specimens were collected from 57 PTC and 33 LSCC patients. Distinct metabolite profiles of tissues were examined using a combination of gas chromatography-time-of-flight mass spectrometry and ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. The data were analyzed with multivariate statistical analysis to compare the distinct metabolite profiles and related pathways of these three tumors.Result:A panel of 46 and 41 differentially expressed metabolites were identified in tumor and adjacent non-tumor tissues of PTC and LSCC, respectively. Increased glycolysis, amino acids metabolism, one carbon metabolism and tryptophan metabolism were found in these two types of tumor tissues compared to adjacent non tumor tissues. Purine and pyrimidine metabolism was significantly elevated in PTC and LSCC tumor tissues, while taurine and hypotaurine were only higher in PTC tumor tissues. The fatty acid metabolism was detected at lower level in both PTC and LSCC tumor tissue.Conclusion:PTC and LSCC tumor tissues not only have common metabolic signatures characterized by increased glycolysis, amino acids metabolism, one carbon metabolism and tryptophan metabolism, but also have distinct metabolic signatures. It is helpful to understand the nature of these two tumors.

Expression Levels and Clinical Significance of miR-21-5p, miR-let-7a, and miR-34c-5p in Laryngeal Squamous Cell Carcinoma

Objective Altered microRNAs (miRNAs) expression has been found in many cancer types, including laryngeal squamous cell carcinoma (LSCC). The aim of this study was to determine the role and clinical value of three LSCC-related miRs, such as miR-21-5p, miR-let-7a, and miR-34c-5p in a homogeneous cohort of patients with primary LSCC treated by primary surgery. Methods Expression levels of miR-21-5p, miR-let-7a, and miR-34c-5p were detected in 43 pairs of LSCC and adjacent normal tissues by reverse-transcription quantitative PCR. Overall survival and disease-free survival were evaluated using the Kaplan–Meier method, and multivariate analysis was performed using the Cox proportional hazard analysis. Results miR-21-5p is significantly upregulated, while miR-let-7a is significantly downregulated in LSCC tumor tissues compared with the corresponding adjacent normal tissues. The downregulation of miR-34c-5p expression significantly correlated with a shorter disease-free survival and, in the multivariate analysis, low miR-34c-5p expression was associated with an increased risk of recurrence. Conclusions miR-21-5p, miR-let-7a, and miR-34c-5p seem to play a critical role in LSCC carcinogenesis and might have a diagnostic and prognostic clinical value. The miR-let-7a levels could have a predictive role for lymph node metastases and miR-34c-5p might be a promising biomarker of patient outcome.

Promoter hypermethylation of miR-34a contributes to the risk, progression, metastasis and poor survival of laryngeal squamous cell carcinoma

MiR-34a is a direct transcriptional target of p53, which induces cell cycle arrest, senescence, and apoptosis. Recently, we and others identified abnormal expression of miR-34a in laryngeal squamous cell carcinoma (LSCC). The aim of our present study was to investigate the contribution of miR-34a promoter methylation to LSCC. Bisulfite pyrosequencing technology was applied to measure DNA methylation levels of six CpG sites in the miR-34a promoter from 104 LSCC tumor tissues and their corresponding adjacent tissues. Our results showed that the methylation levels of the miR-34a promoter were significantly higher in cancer tissues compared with the adjacent tissues (adjusted P=5.05E−10). A breakdown analysis for cigarette smoking behavior indicated a significantly elevated tendency of miR-34a methylation level in LSCC patients with smoking behavior but not in LSCC patients without smoking behavior (Smoking: Tumor vs Normal, adjusted P=3.12E−9; Non-smoking: Tumor vs Normal, adjusted P=0.073). In addition, miR-34a promoter methylation frequency remarkably increased in the advanced stage patients (adjusted P=0.003) and advanced T classified tumors (adjusted P=0.015). Moreover, significant association of miR-34a promoter hypermethylation with LSCC lymph metastasis was observed (adjusted P=0.002). Meanwhile, Kaplan-Meier survival curves results showed that high methylation of miR-34a promoter were associated with poor overall survival (log-rank test, P=0.023). Our study revealed that miR-34a promoter hypermethylation was a risk factor for LSCC, played a critical role in the disease progression and metastasis, and could serve as a poor prognostic factor for LSCC.

Abstract 978: Integrated analysis of long noncoding RNA and mRNA expression profile in laryngeal squamous cell carcinoma

Abstract Objective: The purpose of this study is to investigate the aberrant expressed lncRNA and mRNA in laryngeal squamous cell carcinoma (LSCC), and identify the potential lncRNA correlated with functional mRNA in LSCC. Methods: A total number of 9 pairs of primary Stage IV LSCC and adjacent non-neoplastic tissues were surgically removed for integrated microarray analysis of lncRNA and mRNA using the Aglient GeneChip with 46506 lncRNA probes and 30656 mRNA probes. Random Variance Model(RVM) t-test was applied to filter the differentially expressed genes. Hierarchical clustering analysis was used to reveal the expression patterns of lncRNA and mRNA. GO analysis was applied to analyze the main function of the differentially expressed genes according to the Gene Ontology. Pathway analysis was used to find out the significant pathway of the differentially expressed genes according to KEGG, BioCarta and Reactome. Gene-gene interaction network was constructed based on the data of differentially expressed genes to build and analyze molecular networks. LncRNA-mRNA-network was built according to the normalized signal intensity of specific expression in mRNA and lncRNA to identify the interactions between mRNA and lncRNA. Then the significantly altered lncRNA and its correlated mRNA were validated by the qRT-PCR in an independent cohort of 30 pairs of LSCC tumor tissue and adjacent normal tissue samples. Results: The integrated microarray analysis of lncRNA and mRNA identified 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs(fold change≥2, p&amp;lt;0.05). To identify typical mRNA with core functions from the identified mRNAs, the GO, KEGG pathway analysis and Gene-gene interaction network selected three mRNAs HIF1A, ITGB1, and DDIT4, which mainly involved in biological processes, such as pathways in cancer, HIF-1 signaling pathway, Focal adhesion, ECM-receptor interaction, and PI3K-Akt signaling pathway. QRT-PCR validated that the three mRNAs exhibited higher expression in cancer tissues. According to lncRNA-mRNA-network,we found that lncRNA MIR31HG was positive correlated with HIF1A, lncRNA NR-027340 was positive correlated with ITGB1, lncRNA SOX2-OT-015 was positive correlated with DDIT4. QRT-PCR further validated that the 3 lncRNAs were overexpressed in cancer tissues compared to adjacent non-neoplastic tissues of the 30 samples. Conclusions: lncRNA MIR31HG, NR-027340, SOX2-OT-015 and their correlated mRNAs were identified in LSCC tissues by microarrays, and further validated by qRT-PCR. These up-regulated lncRNAs may act as novel biomarkers in LSCC and may be potential therapeutic targets for LSCC patients. Citation Format: Ru Wang, Jugao Fang, Ling Feng. Integrated analysis of long noncoding RNA and mRNA expression profile in laryngeal squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 978.

Tumor necrosis factor superfamily member 13 is a novel biomarker for diagnosis and prognosis and promotes cancer cell proliferation in laryngeal squamous cell carcinoma.

Tumor necrosis factor superfamily member 13 (TNFSF13) modulates cell proliferation and apoptosis and participates in the pathogenesis of solid tumors, but its role in laryngeal cancer development is not clearly defined. In order to investigate whether TNFSF13 can be used as a biomarker for diagnosis and prognosis in laryngeal squamous cell carcinoma (LSCC) and the role of TNFSF13 in laryngeal cancer carcinogenesis, we conducted immunohistochemistry and ELISA assays to evaluate the expression level of TNFSF13 in laryngeal cancer patients and the contrast. We also conducted experiments on the functional study of TNFSF13 in vitro. We found that the expression levels of TNFSF13, ki-67, and NF-κB p65 in LSCC tumor tissues were higher than those in vocal polyp and para-carcinoma tissues. The Spearman rank correlation analysis showed that the expression of TNFSF13 had a positive correlation with the expression of ki-67 and NF-κB p65. Cox regression analysis and Kaplan-Meier plots confirmed the expression level of TNFSF13 was a prognostic factor for LSCC. Moreover, the serum TNFSF13 level was significantly higher in LSCC patients than in the controls, and the serum expression level of TNFSF13 can distinguish LSCC from healthy people, precancerosis, or laryngeal benign tumor. In addition, functional study of TNFSF13 in vitro revealed that knockdown of TNFSF13 inhibited cell proliferation by inducing G1 phase cell cycle arrest in Hep-2 cells. In conclusion, TNFSF13 may be a potential novel molecular target for diagnosis and prognosis in human LSCC, and therapies that target TNFSF13 may have clinical significance for the treatment of LSCC.

Role of miR-145 in human laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (SCC), being an aggressive malignancy, is one of the most commonly diagnosed malignant types of head and neck SCC worldwide. Incidences of laryngeal SCC have been reported to increase recently. In this study, we aimed to explore the biological effects of miR-145 on laryngeal cancer cells. The relative miR-145 level in laryngeal SCC tumor tissues and normal samples was investigated. Then, Hep-2 cells were utilized for functional analysis of miR-145. The proliferation abilities of transfected cells were measured using MTS assay. Scratch assay and single colony migration assay were performed to observe the alterations in migration behavior of transfected cells. Caspase assay and cell cycle analysis were used to investigate the underlying reasons of proliferative inhibition in cells in which miR-145 is overexpressed. Moreover, expression of SOX2 was analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis in Hep-2 cells upon miR-145 transfection and its expression was evaluated in tumor and normal tissue sample of the larynx. The miR-145 expression in laryngeal SCC tumor samples has been shown to be downregulated. The miR-145 overexpression caused inhibition of proliferation and migration in Hep-2 cells through induction of apoptosis and cell cycle arrest. The SOX2 level was demonstrated to be overexpressed in tumor samples and its expression was significantly decreased in miR-145 overexpressed Hep-2 cells. We have demonstrated the deregulation of miR-145 and SOX2 in laryngeal SCC. Based on these results, we propose that miR-145, as an important regulator of SOX2, carries crucial roles in laryngeal SCC tumorigenesis.

Downregulated Chibby in laryngeal squamous cell carcinoma with increased expression in laryngeal carcinoma Hep-2 cells.

Chibby (Cby) inhibits Wnt/β-catenin-mediated transcriptional activation by competing with Lef-1 (the transcription factor and target of β-catenin) to bind to β-catenin. This suggests that Cby could be a tumor suppressor protein. In the present study, we examined Cby expression in laryngeal squamous cell carcinoma (LSCC) and its function and mechanism in laryngeal carcinoma cell lines. Cby expression levels were investigated by immunohistochemistry in a panel of 36 LSCC patient cases. The expression of β-catenin, c-myc and cyclinD1 in Hep-2 were determined through RT-PCR and western blot analysis. Activity of Wnt/β-catenin signaling pathway after overexpression of Cby was measured by TCF/LEF luciferase reporter gene assay. Proliferation, clone forming ability, cell cycle distribution and cell apoptosis of Hep-2 cells were detected by MTT assay, plate colony forming assay, flow cytometry and TUNEL assay, respectively. This study showed that expression of Cby protein was strongly downregulated in LSCC tumor tissues in comparison to normal laryngeal mucosa samples. No significant correlation was found between the expression of Cby in tumor tissue and gender, age, clinical stage and tumor differentiation of laryngeal cancer patients. When Cby was overexpressed in Hep-2 cells, the expression of cyclin D1 was reduced and β-catenin activity was inhibited. Proliferation and plate colony forming assays revealed a significant inhibitory effect of Cby on growth and colony formation ability of Hep-2 cells after Cby overexpression in comparison to control and mock-infected cells. In addition, we also found that upregulated expression of Cby resulted in accumulation of numbers of cells in G0/G1 phase with concomitant decrease in S phase by cell cycle assay. TUNEL staining demonstrated that, compared with the control group, the rate of apoptosis in the plv-cs2.0-Cby group was significantly increased. Taken together, downregulation of Cby was observed in LSCC, but with no significant correlation to the clinicopathological features of LSCC patients. Overexpression of Cby effectively suppressed laryngeal carcinoma cell growth and promoted its apoptosis. A better understanding of the mechanisms of Cby gene activation in LSCC could provide potential novel therapeutic targets for human laryngeal carcinoma.

The involvement of CHD5 hypermethylation in laryngeal squamous cell carcinoma

Chromodomain helicase DNA-binding protein 5 (CHD5) has been found to be a candidate tumor suppressor gene (TSG) in malignant neural tumors. In mice heterozygous for chd5 deficiency, the first tumor observed was pathological squamous cell carcinoma. More than 95% of primary laryngeal cancer is squamous cell carcinoma. Thus, we explored the expression of CHD5 in 65 patients with laryngeal squamous cell carcinoma (LSCC) using real-time PCR, immunohistochemistry and Western blotting. DNA methylation was detected using bisulfate-specific sequencing. The potential function of CHD5 was determined using MTT, apoptosis and transwell migration assays in CHD5-transfected Hep-2 cells. Our results revealed that the mRNA and protein expression levels of CHD5 in LSCC tissues were significantly lower than those in clear surgical margin tissues (p<0.05), and there is a significant correlation between the mRNA and protein expression levels of CHD5 (p<0.01). In addition, there were significant differences in CHD5 mRNA and protein levels with respect to the patient's clinical stage (p<0.05). Aberrant methylation of the CHD5 promoter was frequently found in the Hep-2 cell line and LSCC tumor tissues, especially tumor tissues from advanced TNM (p<0.05) or older patients (p<0.05). Finally, ectopic expression of CHD5 in laryngeal cancer cells led to significant inhibition of growth and invasiveness. Our data suggest that CHD5 is a tumor suppressor gene that is epigenetically downregulated in LSCC.