Nicotine downregulates miR-375-3p via neurotrophic tyrosine receptor kinase 2 to enhance the malignant behaviors of laryngopharyngeal squamous epithelial cells.

Nicotine exposure from smoking constitutes a significant global public health concern. Furthermore, smoking represents a pivotal risk factor for head and neck squamous cell carcinoma (HNSCC). However, the influence of nicotine on HNSCC remains relatively underexplored. Our aim was to unravel the molecular mechanisms that underlie the effect of nicotine on the metastatic cascade of HNSCC. In this study, we discovered a significant association between smoking and HNSCC metastasis and prognosis. Nicotine significantly enhanced HNSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro. Analysis of TCGA-HNSCC and FDEENT-HNSCC cohorts revealed reduced miR-375-3p levels in HNSCC tumor tissues, particularly among current smokers. Additionally, miR-375-3p level was strongly correlated with both lymph node metastasis and tumor stage. By downregulating miR-375-3p, nicotine promotes HNSCC cell metastasis in vitro and hematogenous metastatic capacity in vivo. Utilizing transcriptomic sequencing, molecular docking, dual-luciferase reporter assay, and fluorescence in situ hybridization (FISH), we demonstrated that miR-375-3p specifically binds to 3' untranslated region (3'UTR) of NTRK2 mRNA. Thus, this study uncovers a novel nicotine-induced mechanism involving miR-375-3p-mediated NTRK2 targeting, which promotes HNSCC metastasis. These findings have implications for improving the prognosis of patients with HNSCC, especially in smokers.

Correlation Between the NLRP3 Inflammasome and the Prognosis of Patients With LSCC.

Background: NLRP3 inflammasome is an inflammatory mediator. The expression of NLRP3 inflammasome is associated with the development of various tumors and is closely related to the prognosis of tumors. However, the role of NLRP3 inflammasome in laryngeal squamous cell carcinoma (LSCC) remains unclear. This study aim to investigate the influence of NLPR3 inflammasome expression in LSCC, and especially the NLRP3 inflammasome expression level and the prognosis of LSCC after surgery in a Chinese population. Methods: We used quantitative real-time PCR and immunohistochemical (IHC) staining to calculate the mRNA (20 patients, fresh tissue) and protein expression (104 patients, paraffin tissue microarray) levels of the NLRP3 inflammasome (NLRP3/IL-18/IL-1ß/ASC/caspase-1), respectively. We also analyzed the relationship between NLRP3 inflammasome expression levels and LSCC cancer tissues compared with adjacent normal tissues and the clinical features of LSCC. Kaplan-Meier survival curves of overall survival (OS) and disease-free survival (DFS) in LSCC patients were compared and analyzed under different expression levels of the NLRP3 inflammasome. Results: Our results indicated that the mRNA expression of the NLRP3 inflammasome was higher in LSCC cancer tissues compared with adjacent normal tissues (p < 0.001). The IHC staining score also demonstrated that the expression of the NLRP3 inflammasome was higher than in the adjacent normal tissues (p < 0.001). The NLRP3 inflammasome expression also exhibited a close relationship with the clinicopathological characteristics (especially the stage of LSCC) of LSCC. Univariate Cox regression analysis and multivariate Cox regression analysis revealed that both NLRP3 and IL-1ß had an increased risk of LSCC progression (p < 0.05). The Kaplan-Meier log rank test (OS and DFS) demonstrated that high expression of NLRP3/IL-18/IL-1ß/ASC was statistically different than the low expression group (p < 0.05) of LSCC patients after surgery. Conclusion: The high expression group of the NLRP3 inflammasome (NLRP3/IL-18/IL-1ß/ASC) had a poorer prognosis (OS and DFS) than the low expression group of LSCC patients 5 years after surgery. The NLRP3 inflammasome (NLRP3/IL-18/IL-1ß/ASC) may be used as an auxiliary indicator to predict LSCC patient prognosis after surgery.

Hypoxia promotes stem-like properties of laryngeal cancer cell lines by increasing the CD133+ stem cell fraction.

Evidence indicates that a hypoxic micro-environment plays an essential role in the regulation of cancer stem cells (CSCs). However, whether hypoxia is able to regulate the stem-like biological properties of laryngeal cancer cells remains unknown. In this study, we investigated the influence of hypoxia on the stemness of two laryngeal cancer cell lines, Hep-2 and AMC-HN-8. We cultured the two cell lines under hypoxia and normoxia and examined the influence of hypoxia on the expression of hypoxia-inducible factors (HIFs) and the cancer stem-like properties of these cells, including cell cycle distribution, expression of stem cell genes (OCT4, SOX2 and NANOG) and laryngeal CSC surface marker (CD133), proliferation, invasion, colony formation and sphere formation capacity. We determined that both of these cell lines, when maintained under hypoxic conditions, showed expanded cells in the G0/G1 phase, exhibited preferential expression of stem cell genes and CD133, and manifested upregulation of HIFs. When treated with hypoxia followed by normoxia exposure, the two cell lines exhibited enhanced capacities for proliferation, invasion, and sphere and colony formation compared with cells maintained consistently under normoxia. Our findings indicate that a hypoxic microenvironment may upgrade the stem-like biological properties of laryngeal cancer cell lines by the expansion of the CD133(+) stem cell fraction.

Establishment and characterization of a novel HPV-negative laryngeal squamous cell carcinoma cell line, FD-LSC-1, with missense and nonsense mutations of TP53 in the DNA-binding domain.

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy in China; however, publically available LSCC cell lines are few and not established from Chinese populations. Hence, novel and well-characterized LSCC cell lines of Chinese origin are urgently needed to provide researchers with a comprehensive database for LSCC research. From 40 cases of LSCC, we established a novel cell line that was maintained for more than 100 passages in vitro and was found to have typical epithelial morphology and ultrastructure. In-depth characterization analysis revealed polyploidy in DNA content; a doubling time of some 24h; high tumorigenicity in immunodeficient mice; higher invasive potential and more sensitive to radiation and cisplatin compared with HeLa cell line; upregulated Ki67, Notch1, EGFR, and CK5 protein levels; negative infection of human papillomavirus (HPV) and mycoplasma; expression of head and neck squamous cell carcinoma (HNSCC) biomarkers; mutations of TP53 in exons 5 and 8; a near-triploid karyotype with complex structural aberrations; and dozens of dysregulated genes and miRNAs. Cell authentication testing by the American Type Culture Collection (ATCC) confirmed the human origin of this cell line. Our findings indicate that a novel and well-differentiated LSCC cell line recapitulating the primary tumor's malignant characteristics is established and well characterized. It does not match any cell lines within the ATCC database and helps to elucidate the molecular pathogenesis of LSCC.

Identification of cancer stem-like side population cells in purified primary cultured human laryngeal squamous cell carcinoma epithelia.

Cancer stem-like side population (SP) cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC) cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs) were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07%) was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC)-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP) LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.

Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line.

CONCLUSIONS: Carcinoma-associated fibroblasts (CAFs) can influence the biological characteristics of a laryngeal carcinoma cell line. These results could lay the foundation for further studies on the role of CAFs in the laryngeal tumor-host microenvironment. OBJECTIVE: CAFs are important contributors to the microenvironment in determining the fate of tumors. The aim of this study was to separate, culture, and identify laryngeal CAFs and investigate their biological influence on the laryngeal carcinoma cell line. METHODS: The primary CAFs and normal fibroblasts (NFs) of the larynx were obtained by tissue culture. The cells were verified according to immunohistochemical and immunofluorescence staining of certain proteins. Conditioned medium (CM) from CAFs and NFs was obtained. Functional assays were performed to test the influence of each CM on laryngeal carcinoma cell lines. RESULTS: Third-passage purified laryngeal CAFs and NFs were successfully attained. The CAFs showed positive staining for vimentin, α-smooth muscle actin (α-SMA), and fibroblast activation protein (FAP). The migration ability of the CAFs increased significantly compared with that of NFs (p < 0.05). CM from CAFs (compared with CM from NFs) stimulated proliferation, migration, and invasion to a greater extent.