Notch signaling genes and CD8+ T-cell dynamics: Their contribution to immune-checkpoint inhibitor therapy in oral squamous cell carcinoma: A retrospective study.

BACKGROUND: Aberrant Notch signaling pathway has been related with the tumorigenesis in head and neck region, involving oral cavity. Here, we report the correlation between mutations in the Notch signaling pathway and CD8+ T-cell infiltration via PD-L1, which lead to enhanced antitumor immunity and may target for immune-checkpoint inhibitors (ICIs) therapy. METHODS: This retrospective study analyzed the results of immunohistochemical staining for PD-L1 and CD8+ T-cell infiltration in 10 patients and whole-exome sequencing (WES) was conducted on five of these patients to identify frequently mutated genes. RESULTS: Four of 10 patients were positive for PD-L1 and CD8+ T. By analyzing WES in three of these four patients, we notably identified the mutations of NOTCH1, FBXW7, and noncoding RNA intronic mutation in NOTCH2NLR in two of these three patients. This study may enable better selection of ICI therapy with CD8+ T-cell infiltration via PD-L1 expression for oral squamous cell carcinoma patients with mutations in Notch signaling pathway.

ACLP Activates Cancer-Associated Fibroblasts and Inhibits CD8+ T-Cell Infiltration in Oral Squamous Cell Carcinoma.

We previously showed that upregulation of adipocyte enhancer-binding protein 1 (AEBP1) in vascular endothelial cells promotes tumor angiogenesis. In the present study, we aimed to clarify the role of stromal AEBP1/ACLP expression in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis showed that ACLP is abundantly expressed in cancer-associated fibroblasts (CAFs) in primary OSCC tissues and that upregulated expression of ACLP is associated with disease progression. Analysis using CAFs obtained from surgically resected OSCCs showed that the expression of AEBP1/ACLP in CAFs is upregulated by co-culture with OSCC cells or treatment with TGF-ß1, suggesting cancer-cell-derived TGF-ß1 induces AEBP1/ACLP in CAFs. Collagen gel contraction assays showed that ACLP contributes to the activation of CAFs. In addition, CAF-derived ACLP promotes migration, invasion, and in vivo tumor formation by OSCC cells. Notably, tumor stromal ACLP expression correlated positively with collagen expression and correlated inversely with CD8+ T cell infiltration into primary OSCC tumors. Boyden chamber assays suggested that ACLP in CAFs may attenuate CD8+ T cell migration. Our results suggest that stromal ACLP contributes to the development of OSCCs, and that ACLP is a potential therapeutic target.

Inflammation and malnutrition as markers of poor outcomes in head and neck cancer patients treated with nivolumab.

BACKGROUND: Although immune checkpoint inhibitors (ICIs) are approved for the treatment of recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC), the response to ICIs remains unclear. AIMS/OBJECTIVES: To summarize the clinical outcomes of patients with HNSCC treated with nivolumab (Nivo) in our institution, and provide a basis for research on biomarkers that can predict the efficacy of ICIs. MATERIAL AND METHODS: Forty-four patients with R/M HNSCC who received Nivo (2017-2022) were retrospectively analysed. RESULTS: Despite the older age of this cohort (median age of 72 years), we observed favourable long-term outcomes, with an overall survival of 24.1 months, which could be attributed to our aggressive nutritional intervention. Older age, poor performance status (≥1), and higher Glasgow Prognostic Scores, reflecting the chronic inflammation and malnutrition of patients, were associated with poor prognoses, with hazard ratios for death of 2.63 (95% confidence interval [CI]; 1.07-6.46, p = .016), 3.50 (95% CI; 1.28-9.55, p = .001), and 2.69 (95% CI; 1.17-6.21, p = .029), respectively. Peripheral blood biomarker analysis revealed that systemic inflammation may negatively affect the efficacy of Nivo. CONCLUSIONS AND SIGNIFICANCE: Our results suggest that nutrition and inflammation must be the focus of future studies aiming to identify novel biomarkers.

Immunohistological evaluation of patients treated with intra-arterial chemoradiotherapy and surgery for oral cancer.

Preoperative intra-arterial chemoradiotherapy (IACRT) can improve the outcome and reduce the extent of surgery in patients with advanced oral cancer. However, the response to this regimen varies among patients, which may be related to the immune status of the tumor. We investigated the effects of proteins involved in tumor immunity on the outcomes of combined IACRT and surgery for oral cancer. We examined CD8 + and FoxP3 + tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) expression on immune cells and tumor cells in pretreatment biopsy samples from 69 patients diagnosed with oral cancer treated with IACRT at our institution during 2000-2020. Patients with abundant CD8 + TILs had significantly better 5-year disease-specific survival (DSS) compared to that of patients with less infiltration of these cells (P = 0.016). Patients with higher FoxP3 + T-cells invasion had significantly better DSS compared to that of less FoxP3 (P = 0.005). Patients with high PD-L1 expression in tumor cells and immune cells had significantly better DSS than that of patients with low PD-L1 expression in these cells (P = 0.009 and P = 0.025, respectively). Collectively, these results suggest that the tumor immune microenvironment could affect outcomes of IACRT treatment in oral cancer.

Exploring olfactory receptor family 7 subfamily C member 1 as a novel oral cancer stem cell target for immunotherapy.

The mortality rate of oral cancer has not improved over the past three decades despite remarkable advances in cancer therapies. Oral cancers contain a subpopulation of cancer stem cells (CSCs) that share characteristics associated with normal stem cells, including self-renewal and multi-differentiation potential. CSCs are tumorigenic, play a critical role in cancer infiltration, recurrence, and distant metastasis, and significantly contribute to drug resistance to current therapeutic strategies, including immunotherapy. Cytotoxic CD8+ T lymphocytes (CTLs) are key immune cells that effectively recognize peptide antigens presented by the major histocompatibility complex class I molecules. Increasing evidence suggests that cancer antigen-specific targeting by CTLs effectively regulates CSCs that drive cancer progression. In this study, we utilized data from public domains and performed various bioassays on human oral squamous cell carcinoma clinical samples and cell lines, including HSC-2 and HSC-3, to investigate the potential role of olfactory receptor family 7 subfamily C member 1 (OR7C1), a seven transmembrane G-protein-coupled olfactory receptor that is also expressed in nonolfactory tissues and was previously reported as a novel marker and target of colon cancer initiating cell-targeted immunotherapy, in CSC-targeted treatment against oral cancer. We found that the OR7C1 gene was expressed only in oral CSCs, and that CTLs reacted with human leukocyte antigen-A24-restricted OR7C1 oral CSC-specific peptides. Taken together, our findings suggest that OR7C1 represents a novel target for potent CSC-targeted immunotherapy in oral cancer.

Three-Dimensional Spheroid Configurations and Cellular Metabolic Properties of Oral Squamous Carcinomas Are Possible Pharmacological and Pathological Indicators.

The objective of the current study was to elucidate the clinicopathological significance and appearance of in vitro three-dimension (3D) spheroid models of oral malignant tumors that were prepared from four pathologically different squamous cell carcinoma (OSCC; low-grade; SSYP and MO-1000, intermediate-grade; LEM2) and oral adenosquamous carcinoma (OASC; high-grade; Mesimo) obtained from patients with different malignant stages. To characterize the biological significance of these cell lines themselves, two-dimensional (2D) cultured cells were subjected to cellular metabolic analysis by a Seahorse bioanalyzer alongside the measurement of the cytotoxicity of cisplatin (CDDP). The appearance of their 3D spheroids was then observed by phase contrast microscopy, and both 2D and 3D cultured cells were subject to trypsin digestion and qPCR analysis of factors related to oncogenic signaling and other related analyses. ATP-linked respiration and proton leaking were significantly different among the four cell lines, and the malignant stages of these cultures were significantly associated with increased ATP-linked respiration and decreased proton leakage. Alternatively, the appearances of these 3D spheroids were also significantly diverse among them, and their differences increased in the order of LEM2, MO-1000, SSYP, and Mesimo. Interestingly, these orders were exactly the same in that the efficacies of CDDP-induced cytotoxicity increased in the same order. qPCR analysis indicated that the levels of expression of oncogenic signaling-related factors varied among these four cell lines, and the values for fibronectin and a key regulator of mitochondrial biogenesis, PGC-1α, were prominently elevated in cultures of the worst malignant Mesimo cells. In addition, although 0.25% trypsin-induced destruction was comparable among all four 2D cultured cells, the values for the 3D spheroids were also substantially varied among these cultures. The findings reported herein indicate that cellular metabolic functions and 3D spheroid architectures may be valuable and useful indicators for estimating the pathological and drug-sensitive aspects of OSCC and OASC malignancies.

Accurate estimation of skeletal muscle mass by comparison of computed tomographic images of the third lumbar and third cervical vertebrae in Japanese patients with oral squamous cell carcinoma.

OBJECTIVES: We evaluated the accuracy of estimating the cross-sectional area (CSA) at the third lumbar vertebra (L3) based on the CSA at the third cervical vertebra (C3) using computed tomographic images, and we identified the sources of error and bias using the evaluation of absolute reliability in 89 Japanese patients with oral squamous cell carcinoma. METHODS: Skeletal muscle CSA was measured at the C3 and L3 on pretreatment computed tomographic images. We used the CSA at the C3 to estimate CSA at the L3 in an existing prediction formula. Correlation coefficients were used to evaluate the relative reliability of the estimate, and Bland-Altman analysis and minimum detectable change (MDC) were used to evaluate its absolute reliability. RESULTS: Estimated and actual CSAs at L3 were strongly correlated (r = 0.885, p < 0.001). The mean difference between the estimated and actual CSAs was - 1.0887 cm2, the 95% confidence interval was - 4.09 to 1.91 cm2 (p = 0.472), and the 95% limits of agreement were - 29.0 and 26.8 cm2. The MDC at the 95% level of confidence in estimated and actual CSAs was 27.9 cm2. CONCLUSIONS: The estimation of CSA at the L3 from the existing prediction formula with the CSA at the C3 had no systematic biases, but it did have random errors. Random errors resulted from measurement errors and biological variation. Usefulness of the existing formula is limited by physical differences in populations.

CXCL12 is expressed by skeletal muscle cells in tongue oral squamous cell carcinoma.

BACKGROUND: The CXCL12/CXCR4 axis plays a pivotal role in the progression of various malignancies, including oral squamous cell carcinoma (OSCC). In this study, we aimed to clarify the biological and clinical significance of CXCL12 in the tumor microenvironment of OSCCs. METHODS: Publicly available single-cell RNA-sequencing (RNA-seq) datasets were used to analyze CXCL12 expression in head and neck squamous cell carcinomas (HNSCC). Immunohistochemical analysis of CXCL12, α-smooth muscle antigen (α-SMA), fibroblast activation protein (FAP) and CD8 was performed in a series of 47 surgically resected primary tongue OSCCs. Human skeletal muscle cells were co-cultured with or without OSCC cells, after which CXCL12 expression was analyzed using quantitative reverse-transcription PCR. RESULTS: Analysis of the RNA-seq data suggested CXCL12 is abundantly expressed in stromal cells within HNSCC tissue. Immunohistochemical analysis showed that in grade 1 primary OSCCs, CXCL12 is expressed in both tumor cells and muscle cells. By contrast, grade 3 tumors were characterized by disruption of muscle structure and reduced CXCL12 expression. Quantitative analysis of CXCL12-positive areas within tumors revealed that reduced CXCL12 expression correlated with poorer overall survival. Levels of CXCL12 expression tended to inversely correlate α-SMA expression and positively correlate with infiltration by CD8+ lymphocytes, though these relations did not reach statistical significance. CXCL12 was significantly upregulated in muscle cells co-cultured with OSCC cells. CONCLUSION: Our results suggest that tongue OSCC cells activate CXCL12 expression in muscle cells, which may contribute to tumor progression. However, CXCL12 is reduced in advanced OSCCs due to muscle tissue destruction.

Clinical and histopathologic effects of neoadjuvant intra-arterial chemoradiotherapy with cisplatin in combination with oral S-1 on stage III and IV oral cancer.

OBJECTIVE: The aim of this study was to examine the clinical and histopathologic effects of neoadjuvant intra-arterial chemoradiotherapy (IACRT) using cisplatin in combination with oral S-1 (tegafur/gimeracil/oteracil potassium) on stage III and IV oral squamous cell carcinoma. STUDY DESIGN: Thirty patients received infusions of superselective intra-arterial cisplatin 60 mg/m2 by the Seldinger method and conventional external beam radiotherapy (total 40 Gy) combined with oral S-1 on the day of irradiation. Curative surgery and neck dissection were performed 4 to 6 weeks after IACRT. The clinical response of the primary lesion was evaluated approximately 4 weeks after IACRT. The surgically resected specimens were examined for histologic features according to the grading system for histologic evaluation and for residual tumor grade (RGrades). RESULTS: Histopathologic evaluation of the therapeutic effect was grade 2 in 10 patients and grade 3 in 16 patients. According to the distribution of RGrades, the remaining tumor cells were mostly in the central area of the primary lesion, as seen in 24 patients. CONCLUSIONS: These findings indicate that neoadjuvant IACRT with cisplatin and oral S-1 was an effective treatment, suggesting the possibility of reducing the extent of curative surgery based on RGrades.

The anatomy of the foramina and efferent nerve fibers from the pterygopalatine ganglion in posterolateral nasal wall.

Background: The advance of endoscopic surgery has enabled selective section of the postganglionic nerve branches from pterygopalatine ganglion (PPG) as a modification of the vidian neurectomy. Recent microanatomic studies have suggested that the nasal mucosa is also innervated by multiple efferent rami associated with the sphenopalatine artery (SPA) in the procedure "posterior nasal neurectomy." This anatomic cadaveric study aims to identify all postganglionic nerve fibers in the lateral nasal wall which should inform future surgical procedures aimed at interrupting these nerve fibers. Methods: Two cadaver heads, with a total of three individual sides, were dissected. All neurovascular structures penetrating the vertical plate of palatine bone were carefully identified following meticulous removal of the overlying mucosa layers. The efferent nerve fibers were identified and dissected back to their origin-the PPG or greater palatine nerve. Results: Several foramina with efferent PPG nerves were identified on the vertical plate of the palatine bone and medial pterygoid plate. The superior, middle, and inferior turbinates (IT) were innervated by efferent nerves from the PPG via the anterior region of the SPA. The IT was innervated from nerves originating from behind the SPA through bony foramina. The lateral wall of inferior meatus was innervated by efferent nerves that originated from greater palatine nerve and pharyngeal nerve. Conclusion: This study demonstrated the anatomical positions of the postganglionic nerves that innervate the lateral nasal wall. These nerves are located anterior to the SPA as well as posterior to the SPA, where they penetrate the palatine bone.Level of evidence: NA.

Retinoic acid promotes fibrinolysis and may regulate polyp formation.

BACKGROUND: Patients with aspirin-exacerbated respiratory disease (AERD) regularly exhibit severe nasal polyposis. Studies suggest that chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by excessive fibrin deposition associated with a profound decrease in epithelial tissue plasminogen activator (tPA). Retinoids, including vitamin A and its active metabolite retinoic acid (RA), are necessary for maintaining epithelial function and well-known inducers of tPA in endothelial cells. OBJECTIVES: This study sought to determine whether endogenous retinoids are involved in NP pathophysiology and disease severity in patients with CRSwNP and AERD. METHODS: NP tissue was collected from patients with AERD or CRSwNP, and concentrations of retinoids and fibrinolysis markers were measured using ELISA. Normal human bronchial epithelial cells were stimulated alone or in combination with RA and IL-13 for 24 hours. RESULTS: This study observed lower retinoid levels in nasal polyps of patients with AERD than those with CRSwNP or healthy controls (P < .01). Levels of the fibrin-breakdown product d-dimer were the lowest in AERD polyps (P < .01), which is consistent with lower tPA expression (P < .01). In vitro, all-trans RA upregulated tPA levels in normal human bronchial epithelial cells by 15-fold and reversed the IL-13-induced attenuation of tPA expression in cultured cells (P < .01). CONCLUSIONS: RA, a potent inducer of epithelial tPA in vitro, is reduced in tissue from patients with AERD, a finding that may potentially contribute to decreased levels of tPA and fibrinolysis in AERD. RA can induce tPA in epithelial cells and can reverse IL-13-induced tPA suppression in vitro, suggesting the potential utility of RA in treating patients with CRSwNP and/or AERD.

DNA damage stress-induced translocation of mutant FUS proteins into cytosolic granules and screening for translocation inhibitors.

Fused in sarcoma/translated in liposarcoma (FUS) is an RNA-binding protein, and its mutations are associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), through the DNA damage stress response, aberrant stress granule (SG) formation, etc. We previously reported that translocation of endogenous FUS into SGs was achieved by cotreatment with a DNA double-strand break inducer and an inhibitor of DNA-PK activity. In the present study, we investigated cytoplasmic SG formation using various fluorescent protein-tagged mutant FUS proteins in a human astrocytoma cell (U251) model. While the synergistic enhancement of the migration of fluorescent protein-tagged wild-type FUS to cytoplasmic SGs upon DNA damage induction was observed when DNA-PK activity was suppressed, the fluorescent protein-tagged FUSP525L mutant showed cytoplasmic localization. It migrated to cytoplasmic SGs upon DNA damage induction alone, and DNA-PK inhibition also showed a synergistic effect. Furthermore, analysis of 12 sites of DNA-PK-regulated phosphorylation in the N-terminal LC region of FUS revealed that hyperphosphorylation of FUS mitigated the mislocalization of FUS into cytoplasmic SGs. By using this cell model, we performed screening of a compound library to identify compounds that inhibit the migration of FUS to cytoplasmic SGs but do not affect the localization of the SG marker molecule G3BP1 to cytoplasmic SGs. Finally, we successfully identified 23 compounds that inhibit FUS-containing SG formation without changing normal SG formation. Highlights Characterization of DNA-PK-dependent FUS stress granule localization.A compound library was screened to identify compounds that inhibit the formation of FUS-containing stress granules.

DLEU1 promotes oral squamous cell carcinoma progression by activating interferon-stimulated genes.

Long noncoding RNAs (lncRNAs) are deeply involved in cancer development. We previously reported that DLEU1 (deleted in lymphocytic leukemia 1) is one of the lncRNAs overexpressed in oral squamous cell carcinoma (OSCC) cells, where it exhibits oncogenic activity. In the present study, we further clarified the molecular function of DLEU1 in the pathogenesis of OSCC. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that DLEU1 knockdown induced significant changes in the levels of histone H3 lysine 4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27ac) in OSCC cells. Notably, DLEU1 knockdown suppressed levels of H3K4me3/ H3K27ac and expression of a number of interferon-stimulated genes (ISGs), including IFIT1, IFI6 and OAS1, while ectopic DLEU1 expression activated these genes. Western blot analysis and reporter assays suggested that DLEU1 upregulates ISGs through activation of JAK-STAT signaling in OSCC cells. Moreover, IFITM1, one of the ISGs induced by DLUE1, was frequently overexpressed in primary OSCC tumors, and its knockdown inhibited OSCC cell proliferation, migration and invasion. These findings suggest that DLEU1 exerts its oncogenic effects, at least in part, through activation of a series ISGs in OSCC cells.

Remote Training of Functional Endoscopic Sinus Surgery With Advanced Manufactured 3D Sinus Models and a Telemedicine System.

Objective: Traditionally, cadaveric courses have been an important tool in surgical education for Functional Endoscopic Sinus Surgery (FESS). The recent COVID-19 pandemic, however, has had a significant global impact on such courses due to its travel restrictions, social distancing regulations, and infection risk. Here, we report the world-first remote (Functional Endoscopic Sinus Surgery) FESS training course between Japan and Australia, utilizing novel 3D-printed sinus models. We examined the feasibility and educational effect of the course conducted entirely remotely with encrypted telemedicine software. Methods: Three otolaryngologists in Hokkaido, Japan, were trained to perform frontal sinus dissections on novel 3D sinus models of increasing difficulty, by two rhinologists located in Adelaide, South Australia. The advanced manufactured sinus models were 3D printed from the Computed tomography (CT) scans of patients with chronic rhinosinusitis. Using Zoom and the Quintree telemedicine platform, the surgeons in Adelaide first lectured the Japanese surgeons on the Building Block Concept for a three Dimensional understanding of the frontal recess. They in real time directly supervised the surgeons as they planned and then performed the frontal sinus dissections. The Japanese surgeons were asked to complete a questionnaire pertaining to their experience and the time taken to perform the frontal dissection was recorded. The course was streamed to over 200 otolaryngologists worldwide. Results: All dissectors completed five frontal sinusotomies. The time to identify the frontal sinus drainage pathway (FSDP) significantly reduced from 1,292 ± 672 to 321 ± 267 s (p = 0.02), despite an increase in the difficulty of the frontal recess anatomy. Image analysis revealed the volume of FSDP was improved (2.36 ± 0.00 to 9.70 ± 1.49 ml, p = 0.014). Questionnaires showed the course's general benefit was 95.47 ± 5.13 in dissectors and 89.24 ± 15.75 in audiences. Conclusion: The combination of telemedicine software, web-conferencing technology, standardized 3D sinus models, and expert supervision, provides excellent training outcomes for surgeons in circumstances when classical surgical workshops cannot be realized.

Trimellitic anhydride facilitates transepithelial permeability disrupting tight junctions in sinonasal epithelial cells.

Trimellitic anhydride (TMA) is a chemical agent classified as a low molecular weight (LMW) agent causing occupational rhinitis (OR) or asthma. Although TMA is recognized as a respiratory sensitizer, the direct and non-immunologic effects of TMA remain unclear. Air- liquid interface (ALI) cultured human nasal epithelial cells (HNECs) derived from control subjects were treated with TMA, followed by measurement of the transepithelial electrical resistance (TEER), paracellular permeability of fluorescein isothiocyanate (FITC)-dextran and immunofluorescence of tight junction proteins claudin-1 and zonula occludens-1 (ZO-1). The cytotoxicity of TMA was evaluated by lactate dehydrogenase (LDH) assay. TMA at concentrations of 2 and 4 mg/mL significantly reduced the TEER within 10 min (p = 0.0177 on 2 mg/mL; p < 0.0001 on 4 mg/mL). The paracellular permeability of FITC-dextran was significantly increased upon challenge with 4 mg/mL TMA for 3 h (p = 0.0088) and 6 h (p = 0.0004). TMA treatment induced a reduction in the fluorescence intensity of claudin-1 and ZO-1 in a dose-dependent manner. LDH assay revealed 4 mg/mL TMA induced cytotoxicity only after 6 h incubation, while 1 or 2 mg/mL TMA caused no cytotoxicity. Our results suggest that TMA has a potential to penetrate the epithelial barrier by disrupting claudin-1 and ZO-1, indicating an important role for sensitization and OR development.

Prognostic Value of CD45Ro+ T-Cell Expression in Patients With Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: The role of tumour-infiltrating CD45Ro+ T-cells in oral squamous cell carcinoma (OSCC) is unclear. This study aimed to evaluate prognostic biomarkers for OSCC. PATIENTS AND METHODS: We determined the density of tumour-infiltrating CD45Ro+ T cells in the parenchyma and stroma at the tumour centre (TCe) and invasive front (IF) and examined the association between the density of these cells and histopathological status in 142 patients. RESULTS: Five-year overall survival (OS) and recurrence-free survival were favourable in patients with high CD45Ro+ T-cell density in the TCe stroma. OS was favourable in patients with high CD45Ro+ T-cell density in the IF stroma. Stepwise Cox regression model analysis indicated that CD45Ro+ T-cells in the stroma of the IF and TCe were an independent prognostic factor for OS. CONCLUSION: CD45Ro+ T-cells in the stroma of the IF and TCe play a role in cancer immune surveillance and may be a useful prognostic factor.

ORMDL3 overexpression facilitates FcεRI-mediated transcription of proinflammatory cytokines and thapsigargin-mediated PERK phosphorylation in RBL-2H3 cells.

INTRODUCTION: The chromosomal region 17q21 harbors the human orosomucoid-like 3 (ORMDL3) gene and has been linked to asthma and other inflammatory diseases. ORMDL3 is involved in the unfolded protein response (UPR), lipid metabolism, and inflammatory reactions. We investigated the effects of ORMDL3 overexpression in RBL-2H3 cells to determine the contribution of ORMDL3 to inflammatory disease development. METHODS: We generated ORMDL3 stably overexpressing RBL-2H3 cells to assess degranulation, transcriptional upregulation of interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and mitogen-activated protein kinase (MAPK) phosphorylation via FcεRI. In addition, we examined the effects of ORMDL3 overexpression on thapsigargin (TG)-mediated proinflammatory cytokine transcription and UPR by monitoring MAPK, protein kinase-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 (IRE1) phosphorylation. RESULTS: Overexpression of ORMDL3 enhanced IL-4, TNF-α, and MCP-1 expression after FcεRI cross-linking, whereas the sphingosine-1-phosphate (S1P) agonist FTY720 suppressed this enhancement. There was no significant difference in degranulation and MAPK phosphorylation via FcεRI-mediated activation between vector-transfected and ORMDL3-overexpressing cells. ORMDL3 overexpression accelerated TG-mediated PERK phosphorylation, while MAPK phosphorylation and proinflammatory cytokine expression showed no significant changes in ORMDL3-overexpressing cells. CONCLUSIONS: Our findings suggest that ORMDL3 plays an important role in regulating proinflammatory cytokine expression via the S1P pathway and selectively affects the UPR pathway in mast cells.

Involvement of ferroptosis in human motor neuron cell death.

Ferroptosis was recently defined as a novel type of programmed cell death depending on iron and lipid peroxidation. It is biologically different from other types of cell death such as apoptosis. While the involvement of ferroptosis in cancer, patient and animal model have been intensely studied, ferroptosis in human motor neuron model is still clearly unknown. Here we carefully assessed ferroptosis using human iPS cell-derived motor neuron (hiMNs). We found that almost all hiMNs died by the treatment of glutathione peroxidase 4 (GPX4) inhibitors. Importantly, the cell death was rescued by one antioxidant, vitamin E acetate, iron chelators and lipid peroxidase inhibitors with high dynamic ranges. Finally, these data clearly indicated that ferroptosis constitutively occurs in hiMNs, suggesting the possibility that it might play a biologically and pathologically important roles in motor neuron death such as motor neuron disease (MND)/Amyotrophic lateral sclerosis (ALS).

Identification of hub molecules of FUS-ALS by Bayesian gene regulatory network analysis of iPSC model: iBRN.

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.

Metallothionein-3 is a clinical biomarker for tissue zinc levels in nasal mucosa.

OBJECTIVE: Recently, depleted tissue zinc levels were found in nasal mucosa from patients with chronic rhinosinusitis (CRS) in correlation with tissue eosinophilia, however, no clinical biomarkers for tissue zinc levels have been identified. Metallothionein-3 (MT3) is an intracellular zinc chelator and previous data showed MT3 mRNA levels to be reduced in CRS patients with nasal polyps (CRSwNP). In this study, we examined the correlation between MT3 expression and zinc levels in nasal mucosa and primary human nasal epithelial cells (HNECs) to investigate whether MT3 could be a clinical biomarker for tissue zinc levels. METHOD: Tissue was harvested from 36 patients and mounted on tissue micro-array (TMA) slides. MT3 expression and tissue zinc fluorescence intensity were measured at different areas within the mucosa (surface epithelium and lamina propria) and compared between controls, CRSwNP and CRS without nasal polyps (CRSsNP) patients. MT3 mRNA and protein expression were examined in zinc-depleted HNECs by qPCR and immunofluorescence microscopy. RESULTS: MT3 expression in CRSwNP was significantly decreased in both surface epithelium (p<0.001 to controls) and lamina propria (p = 0.0491 to controls). There was a significant positive correlation between tissue zinc levels and MT3 expression in nasal mucosa (r = 0.45, p = 0.007). In zinc-deplete HNECs, MT3 expression was significantly decreased at mRNA (p = 0.02) and protein level (p<0.01). There was a significant positive correlation between tissue zinc levels and MT3 expression within individual HNECs (r = 0.59, p<0.001). CONCLUSIONS: MT3 expression reflects intramucosal zinc levels in both nasal mucosa and HNECs indicating MT3 could be used as a clinical biomarker for monitoring intracellular zinc levels in the nasal mucosa.