Simultaneous disturbance of NHE1 and LOXL2 decreases tumorigenicity of head and neck squamous cell carcinoma.

OBJECTIVE: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na+ / H+ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line. RESULTS: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells. CONCLUSION: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.

Elevated exosomal lysyl oxidase like 2 is a potential biomarker for head and neck squamous cell carcinoma.

OBJECTIVES: The secretory enzyme lysyl oxidase like 2 (LOXL2) is speculated to contribute to tumor progression through its functions in the remodeling of extracellular matrix and epithelial-mesenchymal transition. We previously identified elevated expression of LOXL2 in metastatic human head and neck squamous cell carcinoma (HNSCC) cells in a mouse lymph node metastases model. Here we performed a case series study examining LOXL2 expression levels in human serum from HNSCC patients to evaluate whether LOXL2 is worth evaluation in a large cohort study. METHODS: LOXL2 protein levels in three serum samples from HNSCC patients were assessed by immunoblotting and LOXL2 tissue expression was examined in one human tongue squamous cell carcinoma (SCC) tissue by immunohistochemistry as a representative of HNSCC tissue. Serum samples were further fractionated in exosomes and supernatants by ultracentrifugation, which were then subjected to immunoblot and in vitro LOX activity analyses. Exosomal LOXL2 levels of 36 serum samples from HNSCC patients and seven healthy volunteers were measured using polymer sedimentation exosome preparation followed by ELISA measurement and subjected to statistical analyses. RESULTS: Immunoblot analyses revealed that LOXL2 was present in serum exosomal fractions from three HNSCC patients, and we observed approximately threefold higher levels of LOXL2 in HNSCC patients compared with three healthy volunteers. Immunohistochemical LOXL2 staining was detected in HNSCC cells in addition to non-cancerous lipid tissues and some muscles in human tongue HNSCC tissue. Further measurements of exosomal LOXL2 by ELISA showed over ninefold higher mean LOXL2 levels in patients compared with controls. Statistical analysis revealed a correlation between elevated serum exosomal LOXL2 levels and low-grade, but not high-grade, HNSCC. CONCLUSIONS: Our case series study that elevated serum exosomal LOXL2 levels exhibited a correlation with low-grade HNSCCs. A follow-up large cohort clinical study will be required to determine the potential clinical utility of LOXL2 as a new biomarker and/or therapy target for HNSCCs. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E327-E334, 2020.

Elevated Na+/H+ exchanger-1 expression enhances the metastatic collective migration of head and neck squamous cell carcinoma cells.

Cancer cells can migrate as collectives during invasion and/or metastasis; however, the precise molecular mechanisms of this form of migration are less clear compared with single cell migration following epithelial-mesenchymal transition. Elevated Na+/H+ exchanger1 (NHE1) expression has been suggested to have malignant roles in a number of cancer cell lines and in vivo tumor models. Furthermore, a metastatic human head and neck squamous cell carcinoma (HNSCC) cell line (SASL1m) that was isolated based on its increased metastatic potential also exhibited higher NHE1 expression than its parental line SAS. Time-lapse video recordings indicated that both cell lines migrate as collectives, although with different features, e.g., SASL1m was much more active and changed the direction of migration more frequently than SAS cells, whereas locomotive activities were comparable. SASL1m cells also exhibited higher invasive activity than SAS in Matrigel invasion assays. shRNA-mediated NHE1 knockdown in SASL1m led to reduced locomotive and invasive activities, suggesting a critical role for NHE1 in the collective migration of SASL1m cells. SASL1m cells also exhibited a higher metastatic rate than SAS cells in a mouse lymph node metastasis model, while NHE1 knockdown suppressed in vivo SASL1m metastasis. Finally, elevated NHE1 expression was observed in human HNSCC tissue, and Cariporide, a specific NHE1 inhibitor, reduced the invasive activity of SASL1m cells, implying NHE1 could be a target for anti-invasion/metastasis therapy.

[A retrospective study on parotid carcinoma].

We retrospectively analyzed the clinicopathological factors affecting survival in patients with previously untreated parotid carcinoma. The subjects were 50 patients treated in our department from 1987 through 2011. The T stage was T1, T2, T3, and T4 in 4 patients, 11 patients, 9 patients, and 26 patients, respectively. The N stage was N0, N1, and N2 in 36 patients, 3 patients, and 11 patients, respectively. The clinical stage was I, II, III, and IV in 4 patients, 10 patients, 7 patients, and 29 patients, respectively. Histopathologically, eleven tumor types were observed; mucoepidermoid carcinoma was the most common. The overall 5-year survival rate was 72.1%, and the disease-specific 5-year survival rate was 74.0% in 42 patients who received radical surgery. Twelve patients relapsed; the site of relapse was the primary site alone in 2, in the neck alone in 3 patients, in the neck with distant metastases in 2 patients, and in distant metastatic site (s) alone in 5 patients. Univariate analysis showed that significant prognostic factors for overall survival rates were the T stage, cervical lymph node metastasis, clinical stage, grade, facial nerve palsy, and tumor size. We concluded that patients at high risk of recurrence should receive adjuvant therapy to improve the therapeutic outcomes.