Primary Laryngeal Squamous Cell Carcinoma Research Articles

Integrated Analysis of Long Noncoding RNA and mRNA Expression Profile in Advanced Laryngeal Squamous Cell Carcinoma.

Long non-coding RNA (lncRNA) plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC) are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 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. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies.

Alterations of p14 ARF , p15 INK4b , and p16 INK4a Genes in Primary Laryngeal Squamous Cell Carcinoma.

The 9p21 gene cluster, harboring growth suppressive genes p14 ARF , p15 INK4b , and p16 INK4a , is one of the major aberration hotspots in head and neck cancers. We try to elucidate specific aberrations affecting this region, throughout methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Methylation of the gene was investigated by MS-MLPA in a well-characterized series of 27 laryngeal squamous cell carcinomas and 20 samples of healthy mucosa. Aberrant promoter hypermethylation was confirmed using and methylation-specific. All samples studied except 3 (11%) presented losses at 9p21 segment. The most common finding was the small deletion (exon 1α) of the p16 INK4a locus (44%). Deletion of the 9p21 gene cluster was identified in 5 cases (18%). We only found methylation in 8 samples (30%) for p15 IK4b -exon 1. Promoter methylation of p14 ARF , p15 IK4b and p16 INK4a was not detected in any tumor sample. Methylation-specific polymerase chain reaction confirmed the results. Our data indicate that there may be a subgroup of patients in which epigenetic regulation of 9p21 segment might have little relevance. Nevertheless, MS-MLPA could not be suitable for the study of methylation at this region and further research is required.

High BAX/BCL2 mRNA ratio predicts favorable prognosis in laryngeal squamous cell carcinoma, particularly in patients with negative lymph nodes at the time of diagnosis

Objectives: Laryngeal squamous cell carcinoma (LSCC), a common type of head and neck cancer, is associated with high rates of metastasis and recurrence. Therefore, accurate prognostic stratification of LSCC patients based on molecular prognostic tumor biomarkers would definitely lead to a better clinical management of this malignancy. The aim of this study was the investigation of the potential combinatorial prognostic value of BCL2 and BAX mRNA expression in LSCC.Design and methods: Total RNA was isolated from 105 cancerous laryngeal tissue specimens obtained from patients having undergone surgical treatment for primary LSCC. After cDNA preparation, a low-cost, in-house developed, sensitive and accurate real-time quantitative PCR (qPCR) methodology was applied for the quantification of BCL2 and BAX mRNA levels. Then, we carried out a biostatistical analysis to assess the prognostic value of the BAX/BCL2 mRNA expression ratio.Results: High BAX/BCL2 mRNA expression constitutes a favorable prognosticator in LSCC, predicting significantly longer disease-free survival (P=0.011) and overall survival (P=0.014) of patients. More importantly, the significant prognostic value of the BAX/BCL2 mRNA expression appeared to be independent of the histological grade and size of the malignant laryngeal tumor as well as TNM stage, as revealed by the multivariate bootstrap Cox regression analysis. Kaplan–Meier survival analysis demonstrated also that the BAX/BCL2 ratio can stratify node-negative (N0) LSCC patients into two subgroups with significantly different DFS and OS (P=0.021 and P=0.009, respectively).Conclusions: The BAX/BCL2 mRNA ratio is a putative molecular tissue biomarker in CLL and hence deserves further validation in larger cohorts of LSCC patients.

Proteomic profiling identifies the inorganic pyrophosphatase (PPA1) protein as a potential biomarker of metastasis in laryngeal squamous cell carcinoma.

Relapse and metastasis are the main causes of unfavorable outcome in head and neck cancers. Whereas, understanding of the molecular background of these processes is far from being complete. Therefore, in this study we aimed to identify potential biomarker candidates of relapse and metastasis in laryngeal squamous cell carcinoma (LSCC) by combining the 2D electrophoresis based protein screen and immunohistochemical analysis of candidate proteins. We screened three groups of LSCC cell lines derived from primary tumors, recurrent tumors and metastases and identified seven proteins that differed significantly in relative abundance between the analyzed groups. Among the identified proteins were the heat shock proteins HSP60 and HSP70 that were significantly downregulated both in recurrences- and metastases-derived cell lines but not in primary tumor-derived cell lines. Moreover, we identified significant upregulation of the annexin V, calreticulin and the inorganic pyrophosphatase (PPA1) exclusively in the metastases-derived cell lines. As these upregulated proteins could potentially become novel biomarkers of metastasis, we have compared their abundance in primary tumor LSCC N(0) cases, primary tumor LSCC N(+) cases as well as in LSCC metastases N(+). Our results show an intense increase of cytoplasmic PPA1 abundance in the N(+) (p = 0.000042) compared to the N(0) group. In summary, we show a group of proteins deregulated in recurrences and metastases of LSCC. Moreover, we suggest the PPA1 protein as a potential new biomarker for metastasis in this cancer.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-016-2201-8) contains supplementary material, which is available to authorized users.

Evaluation of PD-L1 Expression and Associated Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma.

Programmed death-ligand 1 (PD-L1; also known as CD274 or B7-H1) expression represents a mechanism of immune escape for cancer. Our purpose was to characterize tumor PD-L1 expression and associated T-cell infiltration in primary laryngeal squamous cell carcinomas (SCC). A well-annotated cohort of 260 operable primary laryngeal SCCs [formalin-fixed paraffin-embedded (FFPE) specimens] was morphologically characterized for stromal tumor-infiltrating lymphocytes (TIL), on hematoxylin/eosin-stained whole sections and for PD-L1 mRNA expression by qRT-PCR in FFPE specimens. For PD-L1 protein expression, automated quantitative protein analysis (AQUA) was applied on tissue microarrays consisting of two cores from these tumors. In addition, PD-L1 mRNA expression in fresh-frozen tumors and normal adjacent tissue specimens was assessed in a second independent cohort of 89 patients with primary laryngeal SCC. PD-L1 mRNA levels were upregulated in tumors compared with surrounding normal tissue (P = 0.009). TILs density correlated with tumor PD-L1 AQUA levels (P = 0.021). Both high TILs density and high PD-L1 AQUA levels were significantly associated with superior disease-free survival (DFS; TILs: P = 0.009 and PD-L1: P = 0.044) and overall survival (OS; TILs: P = 0.015 and PD-L1: P = 0.059) of the patients and retained significance in multivariate analysis. Increased TILs density and PD-L1 levels are associated with better outcome in laryngeal squamous cell cancer. Assessment of TILs and PD-L1 expression could be useful to predict response to immune checkpoint inhibitors.

Abstract 4001: Array profiling identified upregulated miRNAs and target genes and pathways in laryngeal squamous cell carcinoma

Abstract Background: Laryngeal squamous cell carcinoma (LSCC) is prevalent in the Chinese population, where smoking is a major carcinogen. MicroRNAs (miRNAs) and their gene targets potentially contribute to the multistep regulatory processes of LSCC development. Objective: The purpose of this study is to investigate and validate aberrant expressed miRNAs in LSCC, and analyze the putative target genes of the screened miRNAs. Methods: Six pairs of primary Stage II-IV LSCC and adjacent non-neoplastic tissues were surgically removed for miRNA array analysis using the Affymetrix GeneChip miRNA Array 3.0, containing probes for 5683 miRNAs, and the significantly altered miRNAs were validated by the qRT-PCR in an independent cohort of 36 LSCC patients. MiRNA gene targets were predicted using TargetScan, PITA and microRNAorg in GeneSpring 12.5 software. The functional categories and pathways of the target genes were analyzed by GO category and KEGG pathway database. Results: MiRNAs arrays identified seven up-regulated miRNAs, including hsa-miR-224, hsa-miR-183, hsa-miR-23a, hsa-miR-675, hsa-miR-27a, hsa-miR-503 and hsa-miR-4800-3p in LSCC by the SAM method, and qRT-PCR was performed to validate the screened miRNAs. 72 putative target genes were identified by GeneSpring 12.5 software. These putative target genes are involved in biological processes, such as metabolic, biological regulation, membrane, protein binding, ion binding by GO category analysis, and are the molecular components in MAPK signaling, adherence, and junction pathways by KEGG pathway database analysis. Conclusions: Seven upregulated miRNAs were identified in LSCC tissues by microarrays, and validated by qRT-PCR. The putative target genes were also identified. These up-regulated miRNAs could serve as potential biomarkers for LSCC diagnosis and therapy. Citation Format: Meng Lian, Xiaoqin Liu, Demin Han, Jugao Fang, Zhong Chen. Array profiling identified upregulated miRNAs and target genes and pathways in laryngeal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4001. doi:10.1158/1538-7445.AM2015-4001

Overexpression of Tiam1 promotes the progression of laryngeal squamous cell carcinoma.

T-lymphoma invasion and metastasis‑inducing factor1 (Tiam1) has been reported in various types of human cancer, which play important roles in facilitating the meta-stasis of malignant tumor. However, the investigation of Tiam1 in laryngeal squamous-cell carcinoma is extremely rare. The aim of the present study was to assess Tiam1 expression and examine its function in tumorigenesis and the metastasis of laryngeal squamous cell carcinoma (LSCC) invitro. Tiam1 expression in 98 primary LSCC tissue specimens was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patients' survival. To investigate the effects of Tiam1 on the progression of LSCC, Tiam1/C1199 plasmid was transfected into LSCC, and proliferation, apoptosis, migration and invasion of transfected cells were examined using MTT, flow cytometry, wound-healing and Transwell assay, respectively. The results showed that, Tiam1 was detected in all primary LSCC samples. Additionally, Tiam1 overexpression was closely correlated with tumor progression and patient survival. Tiam1 overexpression was statistically significant, and served as an independent predictor of prognosis for patients with LSCC. The upregulation of Tiam1 by Tiam1/C1199 plasmid had no effect on the prolife-ration of transfected cells, but decreased the apoptotic rate of transfected cells, while the ability of migration and invasion was increased. These results suggested that Tiam1 overexpression in LSCC is possibly involved in the promotion of migration and invasion, and is a promising therapeutic target in the prevention of the progression of LSCC.

Laryngeal cancer in Finland: A 5-year follow-up study of 366 patients.

The purpose of this study was to acquire nationwide data on the management and outcome of laryngeal squamous cell carcinoma (SCC) in Finnish university hospitals over a 5-year posttreatment follow-up. All records of patients diagnosed and treated for primary laryngeal SCC during 2001 to 2005 were reviewed. Three hundred sixty-six patients with laryngeal cancer were identified, 360 of whom had laryngeal SCC. Three hundred forty-two patients with laryngeal SCC (95%) were treated with curative intent. Five-year disease-specific survival (DSS) for T1a, T1b, T2, T3, and T4 glottic SCC was 100%, 95%, 78%, 79%, and 53%, respectively. The corresponding figures for T1 to T4 supraglottic SCC were 68%, 54%, 72%, and 59%. Results of this nationwide study give a general overview of the outcome of unselected patients treated with unified guidelines. Patients with T2 tumors, usually treated with radiotherapy (RT), had a worse prognosis than expected. This patient group warrants further investigation and possibly treatment intensification.

Long-distance communication between laryngeal carcinoma cells.

Tunneling nanotubes and epithelial bridges are recently discovered new forms of intercellular communication between remote cells allowing their electrical synchronization, transfer of second messengers and even membrane vesicles and organelles. In the present study, we demonstrate for the first time in primary cell cultures prepared from human laryngeal squamous cell carcinoma (LSCC) samples that these cells communicate with each other over long distances (up to 1 mm) through membranous tunneling tubes (TTs), which can be open-ended or contain functional gap junctions formed of connexin 43. We found two types of TTs, containing F-actin alone or F-actin and α-tubulin. In the LSCC cell culture, we identified 5 modes of TT formation and performed quantitative assessment of their electrical properties and permeability to fluorescent dyes of different molecular weight and charge. We show that TTs, containing F-actin and α-tubulin, transport mitochondria and accommodate small DAPI-positive vesicles suggesting possible transfer of genetic material through TTs. We confirmed this possibility by demonstrating that even TTs, containing gap junctions, were capable of transmitting double-stranded small interfering RNA. To support the idea that the phenomenon of TTs is not only typical of cell cultures, we have examined microsections of samples obtained from human LSCC tissues and identified intercellular structures similar to those found in the primary LSCC cell culture.

ASAP1 mediates the invasive phenotype of human laryngeal squamous cell carcinoma to affect survival prognosis.

ASAP1 helps regulate cellular structures such as actin cytoskeletal remodeling and focal adhesions that have a pivotal function in tumor progression. Overexpression of ASAP1 has proven to be a malignant indicator for a variety of tumors. To further determine the potential involvement of ASAP1 in laryngeal squamous cell carcinoma (LSCC), we evaluated the expression levels of ASAP1 by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry in tissue samples of 64 LSCC patients. We then analyzed and correlated the results with clinicopathological features. Furthermore, we used small interfering RNA (siRNA) to inhibit ASAP1 expression invitro. The potential function of ASAP1 in invasiveness was evaluated in the Hep-2 LSCC cell line. Kaplan-Meier method was utilized to determine the association of ASAP1 expression with survival of patients. We showed that ASAP1 was upregulated in primary LSCC tumors and was correlated with lymph node metastasis and clinical tumor stage. Similarly, higher levels of ASAP1 were detected in the Hep-2 cell line compared to the 16 human bronchial epithelial (16HBE) cell line. ASAP1 expression was downregulated by lentiviral vector transfection containing siRNA invitro. The invasive potential of these cells was found to be significantly suppressed, while expression levels of Rac1 and Cdc42 positively correlated with the inhibition of ASAP1 expression. In Kaplan-Meier overall survival curves, higher ASAP1 mRNA levels were found to be associated with a shorter progression-free survival trend. Based on these results, ASAP1 appears to contribute to the malignant mechanism of LSCC and may represent a significant prognostic marker for LSCC patients.

Low mRNA expression levels of kallikrein-related peptidase 4 (KLK4) predict short-term relapse in patients with laryngeal squamous cell carcinoma

Several members of the family of tissue kallikrein and kallikrein-related peptidases have been suggested as promising tumor biomarkers with important prognostic significance. However, only one (KLK11) has already been studied in laryngeal squamous cell carcinoma (LSCC) as a potential biomarker for LSCC diagnosis and/or prognosis. Our study investigated the prognostic value of kallikrein-related peptidase-4 (KLK4) mRNA expression as a molecular tissue biomarker in LSCC. For this purpose, KLK4 mRNA expression analysis was performed in 116 cancerous and 74 paired non-cancerous laryngeal tissue specimens obtained from patients that had undergone surgical treatment for primary LSCC. A remarkable downregulation of KLK4 mRNA expression was discovered in laryngeal tumors, compared to non-cancerous laryngeal tissue specimens. KLK4 mRNA expression was also shown to distinguish LSCC from non-cancerous laryngeal tissues. Furthermore, low KLK4 mRNA expression was shown to predict poor disease-free survival, independently of the histological grade and size of the malignant tumor as well as patient TNM stage. According to Kaplan-Meier survival analysis, low KLK4 mRNA expression predicts short-term relapse even among patients with well-differentiated tumors or those at an early TNM stage. Thus, KLK4 mRNA positivity could be regarded as a novel independent indicator of favorable prognosis for the disease-free survival of LSCC patients.

Utility of MS-MLPA in DNA methylation profiling in primary laryngeal squamous cell carcinoma

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay is a method that has rarely been exploited in DNA methylation profiling of laryngeal squamous cell carcinoma (LSCC). Methylation of the gene was investigated by MS-MLPA in a well-characterized series of 53 LSCC and 30 samples of healthy mucosa. Aberrant promoter hypermethylation was confirmed using bisulfite pyrosequencing, and methylation-specific. Promoter hypermethylation was observed in 36 of the 53 patients (68%). CDKN2B (28%), APC (17%), RARβ (15%), DAPK1 (11%) and CHFR (11%) were most frequently hypermethylated. Aberrant methylation of CHFR was mainly a late-stage event. Methylation-specific polymerase chain reaction and bisulfite pyrosequencing confirmed aberrant methylation for CDKN2B, APC and DAPK1. Promoter methylation profiling of LSCC using MS-MLPA identified CDKN2B, DAPK1, RARβ, APC, and CHFR as frequent epigenetic events. The clinical implications of these genes as biomarkers are highly relevant as attractive targets for cancer therapy, given the reversible nature of epigenetic gene silencing.

P63 and Ki-67 immunostainings in laryngeal squamous cell carcinoma are related to survival

To examine the prognostic significance of the immunohistochemical expression of p63 and Ki-67 oncoproteins in patients with laryngeal squamous cell carcinoma, a retrospective evaluation was carried out on a cohort of 108 patients with primary laryngeal squamous cell carcinoma (LSCC) treated by primary surgery. For the immunohistochemical evaluation, tissue section obtained by formalin-fixed and paraffin-embedded tissue blocks from resection of each patient was used. Clinicopathologic data were associated with the immunostaining results. The association among the considered variables was assessed by Fisher's exact test, Mann-Whitney test, non-parametric χ(2) test, and Spearman's rho rank test was used to assess the relations among them. Differences in p63 and Ki-67 immunoreactivity among the different groups were compared via Kruskal-Wallis test and post hoc tests were performed using Mann-Whitney test with Bonferroni correction. The overall survival rate was estimated via Kaplan-Meier method, and the cumulative incidence functions for different groups were compared using log-rank statistics. Cox proportional hazard model was employed in a multivariate analysis to assess the effect of prognostic factors in the overall survival rate. Furthermore, taking into account death due to other causes, we estimated LSCC-related survival and disease-free survival rates using competing risk analysis. The results of immunohistochemical examination showed a statistically significant relationship between the up-regulation of P63 and Ki-67, an increase in histological grading, and primary tumours associated with lymph node metastases. p63 and Ki-67 up-regulation was related to a shorter disease-free survival and a significant association was found between p63 and Ki-67 percentage of positive cells and patient survival. Finally, we noticed a significant relation between p63 and Ki-67 (ρ = 0.87). On the other hand, no statistically significant associations were found between p63 and Ki-67 down-regulation and clinicopathologic data. Our findings suggest that abnormal p63 and Ki-67 immunoreactivity may be involved in the early phases of laryngeal tumorigenesis and may become a significant prognostic predictor for both overall and disease-free survivals. These biomarkers could thus help in the selection of high-risk patients with LSCC who may benefit from more aggressive therapy or chemoprevention.

MiR-375 suppresses IGF1R expression and contributes to inhibition of cell progression in laryngeal squamous cell carcinoma.

MicroRNAs (miRNAs) are small noncoding RNA molecules which are involved in tumorigenesis and development. To investigate their role in primary laryngeal squamous cell carcinoma (LSCC), miRNA GeneChips were used to screen the differentially expressed miRNA, and then validated by real-time quantitative PCR in LSCC samples, we found that miR-375 was frequently downregulated in primary LSCC tissues. The tumor-suppressive effect of miR-375 was determined by in vitro assays; through gain-of-function studies we demonstrated that miR-375 can inhibit LSCC cell (SNU-48 and SNU-899) proliferation, motility, and invasion, and promote their apoptosis. In addition, bioinformatics tools TargetScan, PicTar, and Miranda were used to investigate the potential target of miR-375; bioinformatics analysis and dual-luciferase reporter assay indicated that IGF1R was a novel direct target of miR-375. Ectopic transfection of miR-375 led to a significant reduction in IGF1R and its downstream signaling molecule AKT at both the mRNA and protein levels in LSCC cells. Our results suggested that downregulation of miR-375 is one of the molecular mechanisms for the development and progression of LSCC by directly targeting IGF1R and affecting its downstream AKT signaling pathways. Furthermore, miR-375 and IGF1R may serve as a novel therapeutic target for LSCC.

Microarray Gene Expression Analysis of Tumorigenesis and Regional Lymph Node Metastasis in Laryngeal Squamous Cell Carcinoma

BackgroundLaryngeal squamous cell carcinoma (LSCC) is the most common type in head and neck squamous cell carcinoma (HNSCC), and the development and progression of LSCC are multistep processes accompanied by changes of molecular biology.ObjectiveThe purpose of this study was to investigate the molecular basis of tumorigenesis and regional lymph node metastasis in LSCC, and provide a set of genes that may be useful for the development of novel diagnostic markers and/or more effective therapeutic strategies.MethodsA total number of 10 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for microarray analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by Illumina mRNA microarrays, and LSCC tissues with regional lymph node metastasis and LSCC tissues without regional lymph node metastasis were analyzed in the same manner. The most frequently differently expressed genes screened by microarrays were also validated by qRT-PCR in another 42 patients diagnosed for LSCC.ResultsAnalysed by Illumina mRNA microarrays, there were 361 genes significantly related to tumorigenesis while 246 genes significantly related to regional lymph node metastasis in LSCC. We found that the six genes (CDK1, CDK2, CDK4, MCM2, MCM3, MCM4) were most frequently differently expressed functional genes related to tumorigenesis while eIF3a and RPN2 were most frequently differently expressed functional genes related to regional lymph node metastasis in LSCC. The expressions of these genes were also validated by qRT-PCR.ConclusionsThe research revealed a gene expression signature of tumorigenesis and regional lymph node metastasis in laryngeal squamous cell carcinoma. Of the total, the deregulation of several genes (CDK1, CDK2, CDK4, MCM2, MCM3, MCM4, EIF3a and RPN2) were potentially associated with disease development and progression. The result will contribute to the understanding of the molecular basis of LSCC and help to improve diagnosis and treatment.

MiR-139 targets CXCR4 and inhibits the proliferation and metastasis of laryngeal squamous carcinoma cells

Our previous studies have showed that chemokine receptor 4 (CXCR4) was over-expressed in laryngeal squamous cell carcinoma (LSCC). However, the mechanism underlying aberrant CXCR4 expression remains unclear. To investigate the roles played by miRNAs in CXCR4 over-expression in LSCC, putative miR-139 was predicted through computational algorithms, including TargetScan, PicTar and miRBase, and luciferase reporter assay was explored to confirm that whether CXCR4 was directly regulated by miR-139. Then, quantitative real-time PCR, immunohistochemistry and in situ hybridization methods were employed to detect the expression of miR-139 and CXCR4 in primary LSCC tissues, normal adjacent mucosal tissues and metastatic lesions derived from 40 LSCC patients in the Second Hospital, Xi'An JiaoTong University. Finally, gain- and loss-of-function assays were adopted to explore the effects of miR-139 and CXCR4 on proliferation, invasion and metastasis of the human LSCC cell line Hep-2 in vitro and in vivo. Our results showed that miR-139 dampened CXCR4 expression, and CXCR4 was directly targeted by miR-139. Additionally, the expression of miR-139 was reduced in alignment with the progression of primary to metastatic LSCC. Moreover, an inverse correlation was observed between miR-139 and CXCR4 protein levels in LSCC specimens. Functional analyses demonstrated that ectopic expression of miR-139 inhibited cell proliferation, migration and metastasis of Hep-2 cells in vitro and in vivo. Similar to the observations seen in restoring miR-139 expression, dampening of CXCR4 expression inhibited cell growth, migration and invasion, whereas miR-139 over-expression reversed the pro-metastatic effect of CXCR4. Taken together, we conclude that miR-139 targets CXCR4 and inhibits proliferation and metastasis of LSCC.

Down-Regulation of miR-129-5p Inhibits Growth and Induces Apoptosis in Laryngeal Squamous Cell Carcinoma by Targeting APC

miRNAs regulate gene expression and are key mediators of tumourigenesis. miR-129 has diverse effects in tumours, but its role in laryngeal squamous cell carcinoma (LSCC) remains unknown. This article focuses on the role of miR-129-5p in LSCC. We show miR-129-5p is upregulated in primary LSCC tumours and correlated with advanced disease. Down-regulating miR-129-5p suppressed cell proliferation and migration, and caused cell cycle arrest in Hep-2 cell lines. Downregulation of miR-129-5p alone is sufficient to induce apoptosis both in vivo and in vitro. Moreover, the growth of LSCC xenograft exposed to miR-129-5p antisense oligonucleotides (ASO) in BALB/c mice was markedly inhibited. In addition, we found that miR-129-5p targeted adenomatous polyposis coli (APC) to release inhibition of Wnt signalling causing cell growth and tumourigenesis. Our results suggest miR-129-5p functions as an oncogene in LSCC by repressing APC and is a potential therapeutic target for LSCC.

Prognostic Significance of Beclin-1 Expression in Laryngeal Squamous Cell Carcinoma

Beclin-1 plays a critical role in the regulation of autophagy, apoptosis, differentiation and the development and progression of cancer. The aim of the present investigation was to analyze the Beclin-1 protein expression and to assess its prognostic significance in tissue of laryngeal squamous cell carcinoma (LSCC). Beclin-1 protein expression in 82 primary laryngeal squamous cell carcinoma and 40 paracarcinoma non-tumor tissue samples was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patients' outcome. The expression of Beclin-1 in tumor tissues was significantly lower than that in non-tumor tissues (P = 0.035). Reduced Beclin-1 expression was significantly correlated with lymph node metastases (P = 0.021). Kaplan-Meier survival estimates showed a significant correlation between Beclin-1 expression and patient's survival rate (log-rank P < 0.05). Multivariate Cox proportional hazards model analysis confirmed that lymph node metastases (P = 0.048) and Beclin-1 expression (P = 0.029) were statistically significant, independent prognostic factors for LSCC. Our findings suggested that decreased Beclin-1 expression and lymph node metastases, as examined by immunohistochemistry, are both independent biomarker for poor prognosis of patients with LSCC.

Increased expression of metadherin protein predicts worse disease‐free and overall survival in laryngeal squamous cell carcinoma

Metadherin (MTDH) is involved in tumourigenesis and cancer progression in multiple human malignancies. However, the MTDH protein has rarely been reported in laryngeal squamous cell carcinoma (LSCC). The expression pattern of the MTDH protein in 176 primary archival LSCC and 27 corresponding adjacent noncarcinoma specimens was detected by immunohistochemistry and further correlated with clinicopathological parameters. The results demonstrated that 161 (91.48%) primary LSCC samples stained positive for MTDH; however, staining was barely detectable in all adjacent noncarcinoma samples. Moreover, the expression of the MTDH protein was significantly associated with the primary tumour site (p = 0.021), T classification (p = 0.002), clinical stage (I + II/III + IV; p < 0.001), lymph node metastasis (p < 0.001) and postoperational recurrence (p < 0.001). Kaplan-Meier analysis revealed that MTDH expression was significantly associated with worse disease-free survival (DFS) and overall survival (OS) rates in patients with LSCC (both p < 0.001). When lymph node metastasis and MTDH expression were considered together, patients with lymph node metastasis and high MTDH expression had both poorer DFS and OS rates than others (both p < 0.001). Finally, multivariate analysis demonstrated that MTDH expression was an independent prognostic factor for both DFS and OS rates in patients with LSCC. Strong MTDH expression was negatively correlated with a canonical epithelial-mesenchymal transition molecule E-cadherin (p < 0.001) and positively associated with proangiogenic protein vascular endothelial growth factor (p < 0.001). MTDH overexpression was tightly associated with more aggressive tumour behaviour and a poor prognosis, indicating that MTDH is a valuable molecular biomarker for LSCC progression.

Low expression of PTK6/Brk predicts poor prognosis in patients with laryngeal squamous cell carcinoma

BackgroundProtein tyrosine kinase 6 (PTK6), also known as breast tumor kinase (Brk), was a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. The deregulated expression of PTK6 was observed in various human cancers. However, little was known about PTK6 expression and its clinicopathological significance in human laryngeal squamous cell carcinoma (LSCC).MaterialsPTK6 expression was evaluated in 7 pairs of surgically resectable laryngeal tissues by Western blotting and in 13 pairs of surgically resectable laryngeal tissues by reverse transcription-PCR (RT-PCR). Using immunohistochemistry, we performed a retrospective study of the PTK6 expression levels on 134 archival LSCC paraffin-embedded samples. Prognostic outcomes correlated with PTK6 were examined using Kaplan–Meier analysis and Cox proportional hazards model.ResultsThe PTK6 expression level was lower in LSCC tissues than in the adjacent noncancerous epithelial laryngeal tissues by Western blots and RT-PCR. By immunohistochemical analysis, we observed high expression of PTK6 in 25 of 76 (32.9%) adjacent noncancerous epithelial laryngeal tissues and in 39 of 134 (29.1%) of LSCC, respectively. Multivariate analysis demonstrated that pN status and the expression level of PTK6 (P < 0.05) were independent and significant prognostic factors. In the primary LSCC category, median DFS (disease free survival) of high, medium and low PTK6 expression patients were 88.5 months ,74.5 months and 49.0 months (log-rank test, P = 0.002); median OS (overall survival) of high, medium and low PTK6 expression patients were 88.5 months ,76.3 months and 65.7 months (log-rank test, P = 0.002). Reduced cytoplasmic PTK6 expression in LSCC was significantly associated with late pN status (P =0.005, r = 0.27), advanced pTNM stages (III and IV) (P =0.027, r = 0.147), and poor differentiated LSCC (P <0.0001, r = 0.486). In adjacent paracancerous laryngeal epithelial samples, median DFS of high, medium and low PTK6 expression patients were 92.6 months ,75.6 months and 48.5 months (log-rank test, P = 0.020); median OS of high, medium and low PTK6 expression patients were 92.9 months ,78.9 months and 74.6 months (log-rank test, P = 0.042).ConclusionThe present findings indicated that cytoplasmic PTK6 expression is a potential prognostic factor for survival in LSCC patients. High expression of PTK6 was associated with favorable OS and DFS in LSCC patients.