Tetraspanin CD63 reduces the progression and metastasis of head and neck squamous cell carcinoma via KRT1-mediated cell cycle arrest.

Despite the fact that metastasis is the leading cause of death in patients with head and neck squamous cell carcinoma, fundamental questions about the mechanisms that enable or inhibit metastasis remain unanswered. Tetraspanin CD63 has been linked to tumor progression and metastasis. However, few studies have examined the role of CD63 in HNSCC. In this study, we discovered that CD63 levels were abnormally altered in HNSCC tissue compared to adjacent tissue (n = 69 pairs), and that this was linked to prognosis. Through functional in vitro and in vivo experiments, the roles of CD63 in HNSCC were confirmed. Overexpression of CD63 inhibited the progression and metastasis of HNSCC cells. Using mass spectrometry and co-immunoprecipitation assays, we discovered that KRT1 could be a direct interacting partner of CD63. Furthermore, both CD63 and KRT1 expression was significantly decreased in metastatic tissue compared with primary tumor tissue (n = 13 pairs), suggesting that CD63 and KRT1 play a role in reducing the metastasis of HNSCC. In summary, we reveal a previously unrecognized role of CD63 in regulating KRT1-mediated cell cycle arrest in HNSCC cells, and our findings contribute to defining an important mechanism of HNSCC progression and metastasis.

Small extracellular vesicle-packaged TGFß1 promotes the reprogramming of normal fibroblasts into cancer-associated fibroblasts by regulating fibronectin in head and neck squamous cell carcinoma.

Tumor development and progression hinge upon ongoing coevolution and crosstalk with the tumor microenvironment. In particular, fibroblasts in the tumor stroma are coopted to support tumor growth and survival through interactions with tumor cells. Despite their significant importance, there is no consensus on the origin of cancer-associated fibroblasts (CAFs) in head and neck squamous cell carcinoma (HNSCC). In this study, we demonstrated that small extracellular vesicle (sEV)-packaged TGFß1 can reprogram normal fibroblasts (NFs) into CAFs both in vitro and in vivo. Mechanistically, TGFß1 in sEV activated NFs by regulating fibronectin, rather than modulating the canonical TGFß-Smad signal pathway. Furthermore, TGFß1 and fibronectin are related to HNSCC clinicopathologic features. Plasma sEV TGFß1 may serve as a potential diagnostic biomarker for HNSCC. This hitherto unknown mechanism of reprogramming of NFs into CAFs by a unique pathway has major implications for underlying cancer-recruited stroma responses.

Management of the N0 neck in patients with laryngeal squamous cell carcinoma.

Background: Neck lymph node status is the chief prognostic index in patients with head and neck squamous cell carcinoma (SCC), yet the management of a clinically negative neck in this setting is still controversial, especially in patients with laryngeal SCC (LSCC). Objectives: To evaluate the efficacy of selective neck dissection (SND) to control occult disease in patients with LSCC and clinically negative (cN0) necks. Materials and methods: Medical records of 1476 patients with cN0 LSCC were analyzed. In conjunction with primary treatment, 126 (8.5%) underwent at least unilateral elective neck dissection, whereas most 1350 (91.5%) followed a wait-and-see protocol. Prognostic significance was indicated by the Kaplan-Meier survival estimates. Results: The rate of occult neck disease was 15%. Five-year overall and disease-free survival rates were 74.4% and 66.7%, respectively. Prognosis was closely related to T stage, preoperative tracheotomy, and postoperative recurrence. There was no significant correlation with age, sex, or preoperative neck dissection; but in patients with supraglottic LSCC, the relation between prognosis and preoperative neck dissection was significant, with fewer neck and local recurrences than the wait-and-see group (p < .05). Conclusions and significance: Selective neck dissection is serving as an accurate prognostic tool in patients with supraglottic laryngeal cancers.

Dickkopf-1 is a novel prognostic biomarker for laryngeal squamous cell carcinoma.

CONCLUSIONS: Dickkopf-1 (DKK1) is a novel prognostic biomarker for laryngeal squamous cell carcinoma (LSCC). DKK1 may be a promising strategy for the future treatment of LSCC metastasis and recurrence. OBJECTIVES: DKK1 is reportedly involved in the metastasis and invasion of several tumor types. This study aimed to investigate the prognostic value of DKK1 in LSCC. METHODS: DKK1 expression was measured in Hep-2 cell lines, as well as in tumor and peritumoral tissues, using quantitative real-time PCR and western blot analyses. The role of DKK1 in LSCC was investigated by depleting DKK1 using small interfering RNAs. Tissue microarrays of 102 LSCC patient samples were employed to immunohistochemically detect expression of DKK1, vascular endothelial growth factor C (VEGF-C), and ß-catenin. Prognostic significance was assessed using Kaplan-Meier survival estimates. RESULTS: DKK1 expression was elevated in the Hep-2 cell line and tumor samples. DKK1 depletion decreased cell proliferation, migration, and invasiveness. High DKK1 expression was significantly associated with T and clinical stage, lymph node metastasis, and tumor size (p < 0.05). Increased DKK1 levels in LSCC tissues correlated with elevated VEGF-C and ß-catenin. Multivariate analyses revealed that DKK1 was an unfavorable predictor of overall survival.

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.

The Composition of Microbiome in Larynx and the Throat Biodiversity between Laryngeal Squamous Cell Carcinoma Patients and Control Population.

The throat is an ecological assemblage involved human cells and microbiota, and the colonizing bacteria are important factors in balancing this environment. However, this bacterial community profile has thus been poorly investigated. The purpose of this study was to investigate the microbial biology of the larynx and to analyze the throat biodiversity in laryngeal carcinoma patients compared to a control population in a case-control study. Barcoded pyrosequencing analysis of the 16S rRNA gene was used. We collected tissue samples from 29 patients with laryngeal carcinoma and 31 control patients with vocal cord polyps. The findings of high-quality sequence datasets revealed 218 genera from 13 phyla in the laryngeal mucosa. The predominant communities of phyla in the larynx were Firmicutes (54%), Fusobacteria (17%), Bacteroidetes (15%), Proteobacteria (11%), and Actinobacteria (3%). The leading genera were Streptococcus (36%), Fusobacterium (15%), Prevotella (12%), Neisseria (6%), and Gemella (4%). The throat bacterial compositions were highly different between laryngeal carcinoma subjects and control population (p = 0.006). The abundance of the 26 genera was significantly different between the laryngeal cancer and control groups by metastats analysis (p<0.05). Fifteen genera may be associated with laryngeal carcinoma by partial least squares discriminant analysis (p<0.001). In summary, this study revealed the microbiota profiles in laryngeal mucosa from tissue specimens. The compositions of bacteria community in throat were different between laryngeal cancer patients and controls, and probably were related with this carcinoma. The disruption of this bio-ecological niche might be a risk factor for laryngeal carcinoma.

Reduced expression of mutS homolog 2 and mutL homolog 1 affects overall survival in laryngeal squamous cell carcinoma patients: Investigation into a potential cause.

The risk factors affecting the survival rates of laryngeal carcinoma are not well understood. In this study, we investigated the expression status of mutS homolog 2 (MSH2) and mutL homolog 1 (MLH1) and examined the relationship between these two molecules and overall survival rates in laryngeal cancer. We also explored the potential reason for the altered expression of these two genes. Using real-time polymerase chain reaction and western blotting, we detected MSH2 and MLH1 expression in laryngeal cancer tissue samples. We collected a retrospective cohort with 180 laryngeal cancer patients, and inspected MSH2 and MLH1 staining with tissue microarray immunohistochemistry. Prognostic value of clinicopathological characteristics was evaluated by statistical analysis. Laryngeal carcinoma cells were co-cultured with Helicobacter pylori (H. pylori) bacteria. MSH2 and MLH1 were expressed at lower levels compared to those of adjacent tissues in 21 laryngeal carcinoma patients. Patients with negative expression of MSH2 and MLH1 tended to have a higher risk of mortality compared to patients with positive expression (HR=4.38; HR=3.0, respectively). Cigarette smoking rate was higher in the MLH1 expression positive group. H. pylori infection reduced the MSH2 and MLH1 expression levels of laryngeal carcinoma cell lines within co-culture conditions. It is suggested that the altered expression levels of MSH2 and MLH1 probably affect the overall survival of laryngeal carcinoma patients. H. pylori infection may have an effect on the expression of MSH2 and MLH1 in laryngeal carcinoma patients.

CD24: a novel cancer biomarker in laryngeal squamous cell carcinoma.

PURPOSE: To investigate the role of CD24 in tumor invasion and the clinical significance of its expression in laryngeal squamous cell carcinoma (LSCC). PROCEDURES: CD24 expression was measured in Hep-2 cell lines and tumor and peritumoral tissues by quantitative real-time PCR and Western blot analysis. The role of CD24 in LSCC was investigated by CD24 depletion using small interfering RNA. Tumor tissue microarrays with samples from 102 LSCC patients were used to detect expression of CD24 and proliferating cell nuclear antigen (PCNA). Prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. RESULTS: CD24 was overexpressed in the LSCC cell line and in tumor tissues. Depletion of CD24 caused a notable decrease in cell proliferation, migration and invasiveness in vitro. High CD24 expression was significantly associated with T clinic stage, lymph node metastasis and tumor size (p < 0.05). Patients suffering from LSCC recurrence had higher levels of CD24 protein than those without recurrence (p < 0.0001). The proportion of patients with high PCNA expression was significantly greater among patients with CD24+ LSCC than those with CD24- LSCC (p = 0.000). Univariate and multivariate analyses revealed that CD24 was a significant predictor for overall survival. CONCLUSIONS: Overexpression of CD24 in LSCC is associated with invasiveness, metastatic potential and high tumor proliferation status. CD24 may be a promising strategy for the future treatment of LSCC metastasis and recurrence.

[Helicobacter pylori infection: a potential pathogenic factor for laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the difference in Helicobacter pylori (Hp) infection rate between the patients with laryngeal squamous cell carcinoma and the patients with benign laryngeal lesions and to explore the role of Hp infection in the pathogenesis of laryngeal squamous carcinoma. METHODS: Nested polymerase chain reaction (nPCR) and Hp culture were used to identify the Hp in laryngeal mucosa in 30 patients with laryngeal carcinoma and 15 patients with benign laryngeal lesions including polyps of vocal cords or epiglottic cysts. RESULTS: nPCR showed that Hp-positive rate (73.3%) of patients with laryngeal carcinoma was significantly higher than that (20.0%) of control patients with benign laryngeal lesions (χ(2) = 11.520, P = 0.010). Regarding the 22 positive neoplastic cases that have 44 tissues, out of the 22 tumor tissues, 10 were positive with Hp (45.4%), and among 22 tissues beside the tumor, 19 were positive (86.4%). This event also indicated a statistical significance (χ(2) = 4.697, P = 0.030). Hp culture showed that Hp was negative in all specimens. CONCLUSIONS: Hp exist in the laryngeal mucosa, but with the higher rate of Hp infection in the patients with laryngeal squamous cell carcinoma than that in the patients with benign laryngeal lesions, which suggests Hp may be one of pathogenic factors of laryngeal squamous cell carcinoma.