Laryngeal Squamous Cell Carcinoma Specimens Research Articles

High hsa_circ_0081621 expression indicates a poor prognosis of laryngeal squamous cell carcinoma: A bioinformatics analysis and retrospective clinical study.

Laryngeal squamous cell carcinoma (LSCC) is the second most common malignant tumour of the head and neck with a low 5-year survival rate. There is need to identify novel biomarkers for diagnosis and treatment of LSCC. The present study identified differentially expressed circular RNAs (circRNAs/circs) in LSCC and larynx adjacent non-carcinoma epithelial specimens by analysing the circRNA microarray dataset GSE117001. hsa_circ_0081621 had highest expression among three circRNAs (hsa_circ_0015211, hsa_circ_0023326 and hsa_circ_0081621) in LSCC specimens by reverse transcription-quantitative PCR. The expression levels of hsa_circ_0081621 in 67 LSCC specimens were detected by fluorescence in situ hybridization (FISH). Expression levels of hsa_circ_0081621 were analysed in relation to clinicopathological parameters and prognosis of patients with LSCC. According to FISH results, 59.7% of LSCC specimens exhibited high hsa_circ_0081621 expression. In LSCC specimens, hsa_circ_0081621 high expression was associated with lymph node metastasis and high clinical stage. High expression levels of hsa_circ_0081621 were associated with a poor 5-year overall survival rate in patients with LSCC. In addition, high hsa_circ_0081621 expression was an independent prognostic factor for patients with LSCC. hsa_circ_0081621 may participate in malignant progression of LSCC, and its high expression could be used for prognostic assessment of patients with LSCC.

Correlation between expression of Lin28B and C-myc in patients with laryngeal squamous cell carcinoma and clinicopathological features and prognosis.

Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor of head and neck. Screening of target genes for malignant tumor therapy is one of the focuses of cancer research, with proto-oncogene and tumor suppressor gene as the breakthrough. It has become an urgent need to find the target gene related to the treatment and prognosis of LSCC.This study aims to explore the role of Lin28B and C-myc in LSCC by detecting the expressions of these two proteins and analyze the correlation between the expression of Lin28B and C-myc and clinicopathological features and prognosis of LSCC. We detected the expression of Lin28B and C-myc proteins in 102 specimens of LSCC and 90 specimens of adjacent tissues by immunochemistry, and analyzed the correlation between Lin28B and C-myc protein expressions in LSCC as well as the correlation between the expressions of the two proteins and the clinicopathological features of LSCC. At the same time, the Kaplan-Meier method was used to analyze the relation between Lin28B and C-myc protein levels with the postoperative survival rate of LSCC patients. The protein levels of Lin28B and C-myc in the LSCC tissnes were significantly higher than those in the adjacent tissues (both P<0.05),and there was a positive correlation between the expression of Lin28B and C-myc in LSCC (r＝0.476, P<0.05). The expression of Lin28B protein was closely related to age, lymph node metastasis, clinical stage, tumor size, and pathological differentiation of LSCC patients (all P<0.05). while the expression of C-myc protein was closely related to lymph node metastasis, clinical stage, tumor size, and pathological differentiation of LSCC patients (all P<0.05). A relevant survival analysis showed that in patients with higher level of Lin28B (P＝0.001) or C-myc protein (P<0.001), the postoperative survival rate was relatively low. Lin28B and C-myc proteins are highly expressed in LSCC with a positive correlation. Furthermore, they are closely related to lymph node metastasis, clinical stage, tumor size, pathological differentiation and prognosis, suggesting that both Lin28B and C-myc might be involved in the occurrence and development of LSCC.

Identification of Critical miRNAs as Novel Diagnostic Markers for Laryngeal Squamous Cell Carcinoma.

The prognosis of laryngeal squamous cell carcinoma (LSCC) patients remains poor, and early diagnosis can distinctly improve the long-term survival of LSCC patients. MicroRNAs (miRs) are a group of endogenous, noncoding, 18-24 nucleotide length single-strand RNAs and have been demonstrated to regulate the expression of many genes, thus modulating various cellular biological processes. In this study, we aimed to identify critical diagnostic miRNAs based on two machine learning algorithms. The GSE133632 dataset was acquired from the Gene Expression Omnibus (GEO) datasets, comprising LSCC tissular samples (57 specimens) and matched neighboring healthy mucosa tissular samples (57 specimens). Differentially expressed miRNAs (DEMs) were screened between 57 LSCC specimens and 57 normal specimens. The LASSO regression model and SVM-RFE analysis were carried out for the identification of critical miRNAs. ROC assays were applied to evaluate discriminatory ability. We identified 32 DEMs between LSCC specimens and normal specimens. Two machine learning algorithms confirmed that hsa-miR-615-3p, hsa-miR-4652-5p, hsa-miR-450a-5p, hsa-miR-196a-5p, hsa-miR-21-3p, hsa-miR-139-5p, and hsa-miR-424-5p were critical diagnostic factors. According to the ROC assays, seven miRNAs had an AUC value of >0.85 for LSCC. Taken together, our findings identified seven critical miRNAs in LSCC patients which can be used to diagnose LSCC patients with high sensitivity and specificity. These results must be verified by large-scale prospective studies.

Circular RNA CircSHKBP1 accelerates the proliferation, invasion, angiogenesis, and stem cell-like properties via modulation of microR-766-5p/high mobility group AT-hook 2 axis in laryngeal squamous cell carcinoma

ABSTRACT Laryngeal squamous cell carcinoma (LSCC) is a common malignancy in head and neck. Circular SHKBP1 (circSHKBP) exerts momentous functions in the occurrence of many cancers including LSCC. Thus, we investigated the oncogenic capacities of circSHKBP1 in LSCC, and revealed the underlying mechanism as a competing endogenous RNA. The expression levels of circSHKBP1, miR-766-5p, and high mobility group AT-hook 2 (HMGA2) were examined by quantitative real-time PCR and their influences on the overall survival were measured by Kaplan–Meier method. The correlations between circSHKBP1 and miR-766-5p or HMGA2 were detected by Spearman’s rank correlation analysis. In vitro, the influences of circSHKBP1/miR-766-5p/HMGA2 axis on the tumorigenesis of LSCC were examined by CCK-8, transwell, sphere formation, and angiogenesis assays, respectively. circSHKBP1 expression was up-regulated in the LSCC specimens and cell lines. And elevated circSHKBP1 expression was closely linked to poor prognosis. Silencing circSHKBP1 expression restrained cell proliferation, invasion, angiogenesis, stem cell-like properties and tumor growth. We observed that miR-766-5p was down-regulated and negatively correlated to circSHKBP1 in LSCC samples. However, HMGA2 was highly expressed and positively associated with circSHKBP1 in these specimens. Importantly, the levels of circSHKBP1, miR-766-5p, and HMGA2 were closely associated with patients’ clinical parameters including lymph nodes metastasis and TNM stages. Mechanistic analysis clarified that circSHKBP1 sponged miR-766-5p to regulate HMGA2, the target of miR-766-5p. Moreover, miR-766-5p inhibition and overexpression of HMGA2 rescued the tumor-suppressing roles of circSHKBP1 downregulation in LSCC. In conclusion, circSHKBP1 accelerated the tumorigenesis of LSCC via modulating HMGA2 by targeting miR-766-5p.

TMPO-AS1 is an independent prognostic factor for patients with laryngeal squamous cell carcinoma.

OBJECTIVE Long noncoding RNA (lncRNAs) are frequently abnormally expressed in tumors and involved in the occurrence and progression of human cancer. Recently, a disease-related lncRNA, TMPO antisense RNA 1 (TMPO-AS1), was identified to be dysregulated in several tumors. Hence, we aimed to demonstrate whether TMPO-AS1 could be a promising prognostic marker for patients with laryngeal squamous cell carcinoma (LSCC). METHODS RT-PCR was performed to test TMPO-AS1 expressions in 187 LSCC specimens compared with matched normal specimens. Chi-squared tests were used to determine the associations between TMPO-AS1 expressions and the clinicopathological characteristics of LSCC patients. Then, the clinical outcome of LSCC patients who had lower or higher TMPO-AS1 expression was analyzed using Kaplan-Meier assays. Finally, a Cox proportional hazards model was carried out to evaluate the prognostic values of TMPO-AS1 and other clinical features. RESULTS We found that TMPO-AS1 was distinctly upregulated in human LSCC tissues compared with corresponding normal specimens (p < 0.01). Higher expressions of TMPO-AS1 were observed to be positively associated with the clinical stage (p = 0.020) and lymph node metastasis (p = 0.027). A clinical study in 187 patients revealed that patients with TMPO-AS1 low expressions had poorer survival than those with TMPO-AS1 high expressions (p = 0.0012). In addition, the result of multivariate assays demonstrated TMPO-AS1 expression is an independent predictor for the overall survival of LSCC patients. CONCLUSIONS TMPO-AS1 might be considered a novel molecule involved in LSCC progression, which provides a possible prognostic biomarker.

MicroRNA-330-3p represses the proliferation and invasion of laryngeal squamous cell carcinoma through downregulation of Tra2β-mediated Akt signaling

MicroRNAs (miRNAs), a type of post-transcriptional regulators, exert a crucial role in the malignant progression of laryngeal squamous cell carcinoma (LSCC). MicroRNA-330-3p (miR-330-3p), a recently identified tumor-associated miRNA, is implicated in multiple cancers. Yet, the relevance of miR-330-3p in LSCC remains unexplored. The findings of our study demonstrated a lower expression of miR-330-3p in LSCC. Functional assays revealed that upregulation of miR-330-3p marked restricted the proliferation, colony formation and invasion of LSCC cells. Transformer-2 protein homolog beta (Tra2β) was identified as a target gene of miR-330-3p. An inverse correlation between miR-330-3p and Tra2β mRNA expression was evidenced in LSCC specimens. The upregulation of miR-330-3p significantly repressed Tra2β expression and the phosphorylation of the Akt protein. In addition, Tra2β overexpression markedly abrogated the tumor suppressive role of miR-330-3p in LSCC cells. Overall, our results uncovered that miR-330-3p exerted a tumor-inhibition function in LSCC through targeting Tra2β to inhibit Akt activation.

Investigation of Pepsin in Laryngeal Squamous Cell Carcinoma Specimens

Objective: Squamous cell carcinoma (SCC) composes 95% of the carcinoma of the larynx. One of the factor recognized in the past two decades as increasing the risk of SCC is laryngopharyngeal reflux (LPR). The role and mechanism of LPR in laryngeal cancer remain unclear. The aim of this study was to assess the presence of pepsin in laryngeal formalin-fixed, paraffin-embedded (FFPE) squamous cell carcinoma (SCC) specimens in order to test the hypothesis that LPR plays a role in the development of laryngeal SCC. Method: A total of 29 pathology specimens of advanced-stage laryngeal cancer patients who had undergone total laryngectomy were evaluated. The specimens were confirmed histopathologically as SCC and maintained in paraffin blocks in the pathology department. The western blot (WB) or the enzyme-linked immunosorbent assay (ELISA) analyses were used to measure pepsin enzyme levels. Results: According to the WB and ELISA analyses, there was no presence of the pepsin enzyme in any of the 29 formalin-fixed, paraffin-embedded (FFPE) laryngeal SCC specimens. However, pepsin was detected with both the WB and the ELISA tests in positive controls and fresh rat stomach specimen. Conclusion: Pepsin was not present in any of the malignant laryngeal squamous cell carcinoma specimens in this study. Relationship between LPR and laryngeal SCC still remains unclear and further studies are needed to verify the role of pepsin and LPR in laryngeal SCC.

Expression of long-chain non-coding RNA LINC00152 in laryngeal squamous cell carcinoma and its clinical significance

Objective:The aim of this study is to explore the relative expression level of LINC00152 in laryngeal squamous cell carcinoma（LSCC） and its clinical significance. Method:The relative expression levels of LINC00152 in LSCC cell lines and 36 paired LSCC specimens were measured by qRT-PCR method. And the correlations between the expression level of LINC00152 and the clinical features derived from LSCC patients were analyzed and compared through the independent sample t-test. Result:The relative expression level of LINC00152 was over-expressed in LSCC cell lines and cancerous tissues than that in paired adjacent normal tissues, and the difference was statistically significant（P=0.006）. Even the associations between LINC00152 expression level and clinicopathological features（P=0.044 for clinical stage, P=0.032 for pathological differentiation degree） were significantly. Conclusion:LINC00152 is highly expressed in LSCC and it may become a new tumor marker for the diagnosis and prognosis of LSCC.

MicroRNA-98-HMGA2-POSTN signal pathway reverses epithelial-to-mesenchymal transition in laryngeal squamous cell carcinoma

It has been widely considered that reversing epithelial-to-mesenchymal transition (EMT) is a potential access to restrain cancer progression and therapeutic resistance. Here, we aim to uncover the novel mechanisms by which we can reverse EMT and inhibit metastasis in laryngeal squamous cell carcinoma (LSCC). We show that miR-98 is significantly reduced in both LSCC specimens and cell lines. Over-expression of miR-98 inhibits the EMT-related gene expression and metastasis and invasive behavior in LSCC in vitro, as well as reduces lung metastasis in mouse model. In the mechanistically study, miR-98 directly targets HMGA2 in mediating EMT. HMGA2 knock down by si-RNA method declines several EMT-related genes expression and LSCC migration and invasion. In parallel, overexpression of HMGA2 transforms LSCC cells to acquire stem cell-like features. Furthermore, we reveal that HMGA2-mediated EMT is closely linked with the expression of POSTN that inhibits EMT, as a tumor suppressor, by gene profiling analyses. POSTN is transcriptionally repressed by HMGA2. In clinic, the HMGA2 mRNA level is negatively correlated with the miR-98 level in LSCC patient cohort. In conclusion, our study confers a powerful signal: miR-98-HMGA-POSTN in LSCC, which is able to reverse EMT and inhibit metastasis, underlining the therapeutic potential of this signal.

Epithelial cell transforming sequence 2 expression is associated with the progression of laryngeal squamous cell carcinoma.

Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor and its expression is associated with the development of malignant tumor types. However, to the best of our knowledge, there is no information on the role of ECT2 in the development and progression of laryngeal squamous cell carcinoma (LSCC). The present study aimed at investigating the expression pattern and potential role of ECT2 in the development and progression of LSCC. The expression of ECT2 in 81 pairs of LSCC and adjacent non-tumor tissues was characterized by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. ECT2 expression was upregulated in LSCC tissues and associated significantly with poor differentiation, higher stages, lymph node metastasis and poor survival in the sample population. The relative expression levels of ECT2 mRNA transcripts were correlated with the intensity of anti-ECT2 staining in 25 ECT2+LSCC specimens selected randomly. These results indicated that ECT2 expression was crucial for the progression of LSCC and may serve as a biomarker for the diagnosis and prognosis of LSCC.

DIAPH2 alterations increase cellular motility and may contribute to the metastatic potential of laryngeal squamous cell carcinoma.

Low 5-year survival rate in laryngeal squamous cell carcinoma (LSCC) is to large extent attributable to high rate of recurrences and metastases. Despite the importance of the latter process, its complex genetic background remains not fully understood. Recently, we identified two metastasis-related candidate genes, DIAPH2 and DIAPH3 to be frequently targeted by hemizygous/homozygous deletions, respectively, in LSCC cell lines. They physiologically regulate such processes as cell movement and adhesion, hence we found it as a rationale, to study if tumor LSCC specimens harbor mutations of these genes and whether the mutations are associated with metastasizing tumors. As a proof of concept, we sequenced both genes in five LSCC cell lines derived from lymph node metastases assuming there the highest probability of finding alterations. Indeed, we identified one hemizygous deletion (c.3116_3240del125) in DIAPH2 targeting the FH2 domain. Moreover, we analyzed 95 LSCC tumors (53 N0 and 42 N+) using the Illumina platform and identified three heterozygous single nucleotide variants in DIAPH2 targeting conserved domains exclusively in N+ tumors. By combining these results with cBioPortal data we showed significant enrichment of DIAPH2 mutations (P = 0.036) in N+ tumors. To demonstrate the consequences of DIAPH2 inactivation, CRISPR/Cas9 editing was used to obtain a heterozygous DIAPH2+/- mutant HEK-293T cell line. Importantly, the edited line shows a shift from 'proliferation' to 'migration' phenotype typically observed in metastasizing cells. In conclusion, we report that DIAPH2 alterations are present primarily in metastasizing specimens of LSCC and suggest that they may contribute to the metastatic potential of the tumor.

Study on the mechanism of miR-497-induced laryngeal squamous cell carcinoma growth inhibition by targeting CDK6

Objective: To study the effects of miR-497 and CDK6 on the growth of laryngeal squamous cell carcinoma (LSCC). Methods: The expressions of CDK6 mRNA in fresh LSCC specimens, the adjacent normal mucosa of LSCC, and cell lines of LSCC were detected with quantitative real time polymerase chain reaction, pcDNA3.1(+) CDK6 plasmids were respectively transfected into the LSCC cells, and MTT assay and clone formation assay were performed to evaluate the growth of LSCC cells. Flow cytometry was employed for cell cycle analysis. SPSS17.0 software was used to analyze the data. Results: CDK6 was highly expressed in LSCC(t=14.01, P=0.009) and the overall survival rate of the patients with high CDK6 expression was less than that with low CDK6 expression, with a significant difference (HR=3.236, P<0.001). Double luciferase reporter gene analysis showed that fluorescence activity in wild type CDK6 group was significantly different from that in control group (P<0.01), while there was no significant difference in the fluorescence activity between mutant CDK6 group and control group (P>0.05). A(490) values were respectively 0.42±0.14 (Mean±SD) in siRNA Hep-2 group, 0.51±0.13 in siRNA TU-212 group; 0.98±0.16 in control Hep-2 group and 1.17±0.20 in control TU-212 group. Colonies were 55±4 in siRNA Hep-2 group, 51±3 in siRNA TU-212 group, 108±6 in control Hep-2 group and 105±7 in control TU-212 group, namely, cell growth and clone formation ability in CDK6 siRNA group were significantly lower than those in the control group. Cells cycle was blocked in G0/G1 phase (G0/G1: 65.20%±10.12% in siRNA Hep-2 group; 63.42%±8.97% in siRNA TU-212 group; 45.31%±7.55% in control Hep-2 group; and 42.37%±7.28% in control TU-212 group), and cells decreased obviously in S phase (S: 25.39%±5.51% in siRNA Hep-2 group; 27.21%±5.43% in siRNA TU-212 group; 42.87%±6.85% in control Hep-2 group; and 44.76%±7.02% in control TU-212 group). Compared with miR-497 group, cell growth and clone formation ability in miR-497/CDK6 group were partly restored (all P<0.05). Conclusions: CDK6 expression in LSCC is upregulated, functioning as an oncogene. High expression of CDK6 is a predictor for poor prognosis. miR-497, functioning as a tumor suppressor gene, inhibits the growth of LSCC by targeting CDK6.

Mutational profiles of persistent/recurrent laryngeal squamous cell carcinoma.

We sought to describe targeted DNA sequencing data of persistent/recurrent laryngeal squamous cell carcinoma (LSCC) and to compare gene-specific alteration frequencies with that of primary, untreated LSCC specimens from The Cancer Genome Atlas (TCGA). The tumors of 21 patients with persistent/recurrent LSCC were subjected to targeted DNA sequencing using the Ion AmpliSeq Comprehensive Cancer Panel. Gene-specific alteration frequencies were compared (Chi-Square test) to primary, untreated LSCC sequencing data from TCGA using the cBioPortal platform. Persistent/recurrent LSCC was characterized by a high rate of inactivating alterations in TP53 (38.1%) and CDKN2A (33%), amplification events of CCND1 (19.1%), and ERBB2 (14.3%), and NOTCH1 (19.1%) mutations. Comparison of primary vs persistent/recurrent LSCC revealed significant differences in alteration frequencies of eight critical genes: BAP1, CDKN2A, DCUN1D1, MSH2, MTOR, PIK3CA, TET2, and TP53. Our results provide preliminary support for a distinct mutational profile of persistent/recurrent LSCC that requires validation in larger cohorts.

Immunohistochemical Study of Epithelial Mesenchymal Transition Markers Slug and E-Cadherin in Laryngeal Squamous Cell Carcinoma

Background: laryngeal carcinoma is the most common head and neck cancer, with a high rate of metastasis and poor prognosis. It has a global incidence of 3.2 cases per 100,000 and a death rate of 1.1 per 100,000 per annum. Squamous cell carcinomas constitute more than 95% of primary cancer larynx. Despite of the recent advances in the treatment modalities for laryngeal carcinoma, the survival rate of patients with this malignancy has not improved significantly. Therefore, there is an urgent need to identify novel biomarkers for the prediction of tumour progression and metastasis. Epithelial-mesenchymal transition (EMT) process promotes the aggressive behaviour of cancer through downregulation of epithelial markers and upregulation of mesenchymal markers of cancer cells in order to become motile and invasive. Several transcription factors are key regulators of EMT, including the Snail/Slug family, Twist, ZEB1 and ZEB2. Aim of the work: this study aimed to evaluate the immunohistochemical (IHC) expression of the transcription factor Slug as an inducer of EMT and E-cadherin as an epithelial protein in 50 Egyptian cases of laryngeal squamous cell carcinoma (LSCC) and investigation of the relationship between both markers and the clinicopathologic parameters. Material and Method: the material of this study included 50 specimens of LSCC. All specimens were embedded in paraffin and stained with Hematoxilin and Eosin (H & E) for histopathological evaluation. Other Sections were immunostained with antibodies to Slug and E-Cadherin. The results were tabled, statistically analyzed and discussed. Results: high tumor grade and high TNM stage were positively correlated with Slug IHC expression (P-value= 0.02 and 0.001) but inversely correlated with E-cadherin IHC expression (pvalue= 0.006 and 0.03) respectively. Conclusion: LSCC patients with Slug IHC overexpression and reduced Ecadherin IHC expression are at higher risk of metastatic carcinoma and might benefit from more aggressive treatment after surgery.

MiR-613 suppressed the laryngeal squamous cell carcinoma progression through regulating PDK1.

MicroRNAs (miRNAs) are aberrantly expressed in several tumors and play important role in tumorigenesis. However, little is known about the role of miR-613 in laryngeal squamous cell carcinoma (LSCC). We determined the expression of miR-613 in a panel of 30 LSCC specimens. Compared with the adjacent normal samples, 20 cases of LSCC tissues exhibited decreased expression of miR-613. The average expression of miR-613 in LSCC tissues was lower than in normal samples. Moreover, we demonstrated that exogenous expression of miR-613 suppressed LSCC cell proliferation, invasion, and blocked G1/S phase transition. We identified that 3-phosphoinositide-dependent protein kinase-1 (PDK1) was a direct target gene of miR-613 in LSCC cell. Overexpression of miR-613 suppressed PDK1 expression in LSCC cell. Furthermore, we demonstrated that PDK1 was upregulated in LSCC tissues. MiR-613 expression was inversely correlated with the expression of PDK1 in LSCC tissues. Moreover, we showed that PDK1 was involved in the miR-613-mediated cancer suppression of LSCC cell. These results suggested that miR-613 played as a tumor suppressor gene in LSCC partly by inhibiting PDK1 expression.

IQGAP1 silencing suppresses the malignant characteristics of laryngeal squamous cell carcinoma cells.

Laryngeal squamous cell carcinoma (LSCC) has a poor prognosis due to recurrence and metastasis. IQ-domain GTPase-activating protein 1 (IQGAP1), a scaffold protein, plays an important role in tumorigenesis and malignant development. In this study, we aimed to explore the role of IQGAP1 in LSCC. Expression of IQGAP1 in human LSCC specimens was assessed by immunohistochemistry. We also evaluated the roles of IQGAP1 in cell proliferation, migration and invasion and epithelial-to-mesenchymal transition (EMT) in Hep-2 cells. The expression of IQGAP1 protein was significantly up-regulated in LSCC tissues compared with normal laryngeal tissues (p = 0.002). Furthermore, the knockdown of IQGAP1 in Hep-2 cells inhibited cell growth, migration and invasion. Moreover, we found that IQGAP1 silencing reversed EMT. These results show for the first time that IQGAP1 is up-regulated in LSCC tissues and plays an important role in LSCC cell proliferation and invasiveness, which indicates that IQGAP1 could work as an oncogene and may serve as a promising molecular target for treatment of LSCC.

The primary growth of laryngeal squamous cell carcinoma cells in vitro is effectively supported by paired cancer-associated fibroblasts alone.

Most primarily cultured laryngeal squamous cell carcinoma cells are difficult to propagate in vitro and have a low survival rate. However, in our previous work to establish a laryngeal squamous cell carcinoma cell line, we found that laryngeal cancer-associated fibroblasts appeared to strongly inhibit the apoptosis of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In this study, we investigated whether paired laryngeal cancer-associated fibroblasts alone can effectively support the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In all, 29 laryngeal squamous cell carcinoma specimens were collected and primarily cultured. The laryngeal squamous cell carcinoma cells were separated from cancer-associated fibroblasts by differential trypsinization and continuously subcultured. Morphological changes of the cultured laryngeal squamous cell carcinoma cells were observed. Immunocytofluorescence was used to authenticate the identity of the cancer-associated fibroblasts and laryngeal squamous cell carcinoma cells. Flow cytometry was used to quantify the proportion of apoptotic cells. Western blot was used to detect the protein levels of caspase-3. Enzyme-linked immunosorbent assay was used to detect the levels of chemokine (C-X-C motif) ligand 12, chemokine (C-X-C motif) ligand 7, hepatocyte growth factor, and fibroblast growth factor 1 in the supernatants of the laryngeal squamous cell carcinoma and control cells. AMD3100 (a chemokine (C-X-C motif) receptor 4 antagonist) and an anti-chemokine (C-X-C motif) ligand 7 antibody were used to block the tumor-supporting capacity of cancer-associated fibroblasts. Significant apoptotic changes were detected in the morphology of laryngeal squamous cell carcinoma cells detached from cancer-associated fibroblasts. The percentage of apoptotic laryngeal squamous cell carcinoma cells and the protein levels of caspase-3 increased gradually in subsequent subcultures. In contrast, no significant differences in the proliferation capacity of laryngeal squamous cell carcinoma cells cocultured with cancer-associated fibroblasts were detected during subculturing. High level of chemokine (C-X-C motif) ligand 12 was detected in the culture supernatant of cancer-associated fibroblasts. The tumor-supporting effect of cancer-associated fibroblasts was significantly inhibited by AMD3100. Our findings demonstrate that the paired laryngeal cancer-associated fibroblasts alone are sufficient to support the primary growth of laryngeal squamous cell carcinoma cells in vitro and that the chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 axis is one of the major contributors.

The expression and clinical significance of RhoA, Ezrin and CD44 in laryngeal squamous cell carcinoma

Objective:To investigate the expression of RhoA, Ezrin and CD44 proteins in laryngeal squamous cell carcinoma (LSCC), and analyze their roles and mutual relations in invasion and metastasis.Method:The expressions of RhoA, Ezrin and CD44 in 68 specimens of LSCC and 35 specimens of normal laryngeal mucosa tissues were detected by immunohistochemical SABC staining. The results were correlated with clinicopathological parameters.Result:The positive expression rates of RhoA, Ezrin and CD44 in LSCC were 80.9%,82,4% and 70.6%, and the positive expression rates of RhoA, Ezrin and CD44 in normal laryngeal mucosa tissues were 28.5%, 25.7% and 20%, respectively. The expressions of RhoA, Ezrin and CD44 were significantly correlated to depth of invasion (T), lymph node metastasis (N), clinical TNM stage, tumor grade respectively (P< 0.05), but not to age, gender, smoking, tumor location (P> 0.05). The expression of RhoA was positively correlated to that of Ezrin in LSCC, and the expression of Ezrin was positively correlated to that of CD44 in LSCC too (P< 0.05).Conclusion:Our findings suggested that high expressions of RhoA, Ezrin and CD44 might play an important role in the malignant progression of invasion and metastasis of LSCC, which indicated that they might have played synergistic effects in the process.

Folate deficiency and aberrant expression of cell adhesion molecule 1 are potential indicators of prognosis in laryngeal squamous cell carcinoma.

The etiology of laryngeal squamous cell carcinoma (LSCC) has not yet been adequately examined. Therefore, the present study aimed to investigate the association between serum folate deficiency and abnormal expression of the cell adhesion molecule 1 (CADM1) protein in the progression of LSCC. Samples were collected from 60 patients with LSCC and 30 healthy people. Radioimmunoassays and immunohistochemical staining were performed to measure serum folate levels and CADM1 protein expression, respectively. The results demonstrated that CADM1 expression in LSCC specimens was significantly lower than in adjacent normal tissues (χ2=28.229, P<0.001), which was associated with histological differentiation and clinical stage (P=0.010 and 0.020, respectively). Levels of serum folate in patients with LSCC were significantly lower than those observed in healthy individuals (P=0.002). Furthermore, TSLCl expression and serum folate levels were positively correlated in LSCC (r=0.642, P=0.001). Thus, the present study determined that decreased CADM1 protein expression and low levels of serum folate were correlated with an increased severity of LSCC.

Expression of MAGE-A1, -A9, -A11 in laryngeal squamous cell carcinoma and their prognostic significance: a retrospective clinical study

ABSTRACTConclusion The melanoma-associated antigens A1, -A9, -A11 (MAGE-A1, -A9, -A11) are relatively tumor-specific in laryngeal squamous cell carcinoma (LSCC), and could be ideal antigens for LSCC immunotherapy. In addition, MAGE-A9 probably is a poor prognostic marker for LSCC patients. Objective The MAGE-A family belongs to Cancer/testis antigens (CTA). However, the expression pattern of MAGE-A1, MAGE-A9, and MAGE-A11 in LSCC is still unclear. This study aims to evaluate the expression and possible prognostic role of MAGE-A1, MAGE-A9, and MAGE-A11 in LSCC patients. Methods The expression of MAGE-A1, MAGE-A9, and MAGE-A11 in LSCC specimens was investigated by immunohistochemistry, and the association of their expression and the clinical parameters and the survival of LSCC patients were analyzed by chi-square test, Kaplan-Meier survival and Cox regression analysis. Results The expression rates of MAGE-A1, MAGE-A9, and MAGE-A11 in LSCC were 54.7%, 46.2%, and 51.9%, respectively. The expression of MAGE-A1, MAGE-A9, and MAGE-A11 in LSCC was correlated with clinical stage, lymph node metastasis, and tumor size. The overall survival of LSCC patients with positive MAGE-A1, MAGE-A9, or MAGE-A11 expression was lower than the patients without MAGE-A1, MAGE-A9, or MAGE-A11 expression. Cox’s multivariable analysis showed that MAGE-A9 expression was an independently poor prognostic factor for LSCC patients.