Deciphering head and neck cancer microenvironment: Single-cell and spatial transcriptomics reveals human papillomavirus-associated differences.

Human papillomavirus (HPV) is a major causative factor of head and neck squamous cell carcinoma (HNSCC), and the incidence of HPV- associated HNSCC is increasing. The role of tumor microenvironment in viral infection and metastasis needs to be explored further. We studied the molecular characteristics of primary tumors (PTs) and lymph node metastatic tumors (LNMTs) by stratifying them based on their HPV status. Eight samples for single-cell RNA profiling and six samples for spatial transcriptomics (ST), composed of matched primary tumors (PT) and lymph node metastases (LNMT), were collected from both HPV- negative (HPV- ) and HPV-positive (HPV+ ) patients. Using the 10x Genomics Visium platform, integrative analyses with single-cell RNA sequencing were performed. Intracellular and intercellular alterations were analyzed, and the findings were confirmed using experimental validation and publicly available data set. The HPV+ tissues were composed of a substantial amount of lymphoid cells regardless of the presence or absence of metastasis, whereas the HPV- tissue exhibited remarkable changes in the number of macrophages and plasma cells, particularly in the LNMT. From both single-cell RNA and ST data set, we discovered a central gene, pyruvate kinase muscle isoform 1/2 (PKM2), which is closely associated with the stemness of cancer stem cell-like populations in LNMT of HPV- tissue. The consistent expression was observed in HPV- HNSCC cell line and the knockdown of PKM2 weakened spheroid formation ability. Furthermore, we found an ectopic lymphoid structure morphology and clinical effects of the structure in ST slide of the HPV+ patients and verified their presence in tumor tissue using immunohistochemistry. Finally, the ephrin-A (EPHA2) pathway was detected as important signals in angiogenesis for HPV- patients from single-cell RNA and ST profiles, and knockdown of EPHA2 declined the cell migration. Our study described the distinct cellular composition and molecular alterations in primary and metastatic sites in HNSCC patients based on their HPV status. These results provide insights into HNSCC biology in the context of HPV infection and its potential clinical implications.

Serum Human ß-Defensin 2 Is Increased in Angioedema Accompanying Chronic Spontaneous Urticaria.

INTRODUCTION: Chronic spontaneous urticaria (CSU) is a common cutaneous disease caused by mast-cell degranulation. Human ß-defensin 2 (HBD2) is a well-known antimicrobial peptide that is also a pruritogen inducing vascular permeability via non-IgE-mediated mast-cell degranulation. OBJECTIVE: We investigated the associations between serum HBD2 levels and the clinical characteristics of CSU patients. METHODS: Serum samples from 124 CSU patients and 56 healthy controls were screened for the levels of HBD2 and translationally controlled tumor protein (TCTP)_ by using ELISA. The urticaria activity score over 7 days (UAS7) was used to measure disease activity in CSU patients. Accompanying angioedema was self-reported. RESULTS: Serum HBD2 levels were higher in the CSU group than in healthy subjects (median [interquartile range], 84.1 [43.5, 142.5] vs. 59.5 [26.7, 121.5], p = 0.034). In CSU patients, serum HBD2 level was negatively correlated with the peripheral basophil percentages (Spearman's rho = -0.229, p = 0.01) and vitamin D levels (-0.262, p = 0.02), but positively correlated with TCTP levels (0.252, p = 0.006). In CSU patients, HBD2 level was higher in those with than without angioedema (101.7 [50.9, 184.2] vs. 66.7 [37.9, 132.0], p = 0.019). It did not differ by aspirin hypersensitivity or atopy status, or autologous serum skin test positivity. CONCLUSION: A known mast-cell degranulator, HBD2 was elevated in the sera from CSU patients compared to healthy controls and may be involved in the pathogenesis of accompanying angioedema.

The Dual Inhibition of Met and EGFR by ME22S, a Novel Met/EGFR Bispecific Monoclonal Antibody, Suppresses the Proliferation and Invasion of Laryngeal Cancer.

PURPOSE: It has been reported that the abnormal activation of receptor tyrosine kinases is associated with the development of many human carcinomas and the high activation of EGFR and Met mediates the tumorigenicity of laryngeal carcinoma. In this study, we have done the therapeutic efficacy of ME22S (a novel EGFR/Met bispecific antibody) in laryngeal carcinoma in vitro and in vivo was thoroughly evaluated. METHODS: The effects of ME22S on cell viability was assessed through MTT assays, and then Western blotting and immunocytochemistry were used to determine the expression of EGFR and Met. Also, wound healing and invasion assays were performed to observe the inhibitory effects of ME22S. RESULTS: We found the ability of ME22S reducing the expression of both EGFR and Met and significantly inhibiting the cell migration, invasion, and proliferation of SNU899 and HN3 in vitro. Also, the notably reduced levels of p-Met, p-ERK, and p-AKT were found when the cells were treated with only ME22S alone or with HGF together. Meanwhile, ME22S, interestingly enough, caused caspase-3-dependent apoptotic cell death when HN3 cells were treated with ME22S for 72 h, decreased the HGF-induced Slug expression, and also inhibited the tumor growth of HN3 cells in a xenograft model in vivo. CONCLUSIONS: Taken together, our findings suggest that the dual inhibition of EGFR and Met through ME22S largely suppresses the invasion and growth of laryngeal carcinoma both in vitro and in vivo, hence, can be a practical approach as a novel therapeutic strategy for the treatment of laryngeal carcinoma.

Downregulation of Nrf2 by the combination of TRAIL and Valproic acid induces apoptotic cell death of TRAIL-resistant papillary thyroid cancer cells via suppression of Bcl-xL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents an effective agent for the treatment of many cancers, though the majority of thyroid cancers are found to be resistant. Therefore it would be necessary to identify agents capable of increasing the sensitivity of these cancers to TRAIL-mediated cell death. Here, we examined the therapeutic effect and its underlying mechanism of combination treatment of TRAIL and histone deacetylase inhibitor, Valproic acid (VPA) in vitro using human papillary thyroid cancer (PTC) cells and in vivo using an orthotopic mouse model of PTC. TRAIL-VPA combination therapy synergistically induced apoptotic cell death in TRAIL-resistant PTC through caspase activation. In addition, downregulation of antioxidant transcription factor, Nrf2 by co-treatment of TRAIL-VPA induces cell death via suppression of Bcl-xL in vitro and in vivo; these effects were further enhanced following siRNA inhibition of these proteins in combination with TRAIL or TRAIL-VPA. Taken together, VPA sensitized TRAIL-resistant PTC cells to apoptotic cell death through involvement of Nrf2 and Bcl-xL. Thus, the combination of VPA and TRAIL may be a promising therapy for TRAIL-resistant PTC.

Anti-cancer Effect of Luminacin, a Marine Microbial Extract, in Head and Neck Squamous Cell Carcinoma Progression via Autophagic Cell Death.

PURPOSE: The purpose of this study is to determine whether luminacin, a marine microbial extract from the Streptomyces species, has anti-tumor effects on head and neck squamous cell carcinoma (HNSCC) cell lines via autophagic cell death. MATERIALS AND METHODS: Inhibition of cell survival and increased cell death was measured using cell viability, colony forming, and apoptosis assays. Migration and invasion abilities of head and cancer cells were evaluated using wound healing, scattering, and invasion assays. Changes in the signal pathway related to autophagic cell death were investigated. Drug toxicity of luminacin was examined in in vitro HaCaT cells and an in vivo zebrafish model. RESULTS: Luminacin showed potent cytotoxicity in HNSCC cells in cell viability, colony forming, and fluorescence-activated cell sorting analysis. In vitro migration and invasion of HNSCC cells were attenuated by luminacin treatment. Combined with Beclin-1 and LC3B, Luminacin induced autophagic cell death in head and neck cancer cells. In addition, in a zebrafish model and human keratinocyte cell line used for toxicity testing, luminacin treatment with a cytotoxic concentration to HNSCC cells did not cause toxicity. CONCLUSION: Taken together, these results demonstrate that luminacin induces the inhibition of growth and cancer progression via autophagic cell death in HNSCC cell lines, indicating a possible alternative chemotherapeutic approach for treatment of HNSCC.

Therapeutic effect of hepatocyte growth factor-secreting mesenchymal stem cells in a rat model of liver fibrosis.

Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to be beneficial for the treatment of liver fibrosis. Here, we investigated the use of genetically engineered MSCs that overexpress hepatocyte growth factor (HGF) as a means to improve their therapeutic effect in liver fibrosis. Liver fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. HGF-secreting MSCs (MSCs/HGF) were prepared by transducing MSCs with an adenovirus carrying HGF-encoding cDNA. MSCs or MSCs/HGF were injected directly into the spleen of fibrotic rats. Tissue fibrosis was assessed by histological analysis 12 days after stem cell injection. Although treatment with MSCs reduced fibrosis, treatment with MSCs/HGF produced a more significant reduction and was associated with elevated HGF levels in the portal vein. Collagen levels in the liver extract were decreased after MSC/HGF therapy, suggesting recovery from fibrosis. Furthermore, liver function was improved in animals receiving MSCs/HGF, indicating that MSC/HGF therapy resulted not only in reduction of liver fibrosis but also in improvement of hepatocyte function. Assessment of cell and biochemical parameters revealed that mRNA levels of the fibrogenic cytokines PDGF-bb and TGF-ß1 were significantly decreased after MSC/HGF therapy. Subsequent to the decrease in collagen, expression of matrix metalloprotease-9 (MMP-9), MMP-13, MMP-14 and urokinase-type plasminogen activator was augmented following MSC/HGF, whereas tissue inhibitor of metalloprotease-1 (TIMP-1) expression was reduced. In conclusion, therapy with MSCs/HGF resulted in an improved therapeutic effect compared with MSCs alone, probably because of the anti-fibrotic activity of HGF. Thus, MSC/HGF represents a promising approach toward a cell therapy for liver fibrosis.

Valproic acid sensitizes TRAIL-resistant anaplastic thyroid carcinoma cells to apoptotic cell death.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor associated with a median survival of 2-6 months. TRAIL, as a ligand of death receptors, is known to induce apoptotic cell death in several cancer cells. However, TRAIL treatment alone is not effective against TRAIL-resistant cancer cells. This study was designed to investigate whether valproic acid (VPA) enhances apoptotic cell death of TRAIL-resistant ATC cells and to identify the mechanism of cell death of ATC cells by combination treatment with VPA and TRAIL. METHODS: To evaluate the cytotoxic effect of TRAIL and/or VPA on ATC cells, we used the MTT assay. The effects of VPA and TRAIL on apoptosis were assessed using FACS analysis (Annexin-V/PI stain) and Western blotting. RESULTS: The combination of VPA with TRAIL significantly induced apoptotic cell death compared with 8505C and ARO cells treated with TRAIL alone. The protein levels of cleaved caspase-8, -3, and PARP were increased in VPA and TRAIL co-treated ARO cells. The combination induced the activation of JNK and the phosphorylation of FADD and c-Jun but not p38. However, pretreatment with caspase inhibitors reduced the expression of cleaved caspase-8, -3, and PARP in co-treated ARO cells. SP600125 remarkably reduced the expression of cleaved caspase-8, -3, and PARP and the phosphorylation of FADD and c-Jun, as well as apoptotic cell death. CONCLUSIONS: VPA sensitized TRAIL-resistant ATC cells to apoptotic cell death through involvement of the JNK pathway. Thus, the combination of VPA and TRAIL may be a promising therapy for ATC.

AY4, an agonistic anti-death receptor 4 MAB, induces apoptotic cell death in anaplastic thyroid cancer cells via downregulation of Bcl-xL with reactive oxygen species generation.

Anaplastic thyroid carcinoma (ATC) is an aggressive human tumor with a median survival of 6 months. We previously developed an agonistic anti-death receptor 4 MAB, AY4, and demonstrated the antitumor effects of AY4 in head and neck cancer cells. Presently, we show that ATC cells are sensitive to AY4 and that the sensitivity correlates with the reduced expression level of Bcl-xL and reactive oxygen species (ROS) generation. AY4 induced death of C-643, U-HTH 7, HTH83, and SW1736 cells. To elucidate the role of ROS generation in AY4-induced apoptosis of ATC cells, U-HTH 7 and SW1736 cells were pretreated with an antioxidant (N-acetyl cysteine, NAC) followed by AY4 treatment. The cell death was blocked by NAC. AY4-induced cell death was accompanied by the downregulation of the anti-apoptotic protein, Bcl-xL (BCL2L1). To examine the link between the apoptotic response and Bcl-xL protein expression, U-HTH 7 cells were transfected with Bcl-xL plasmid. The consequence of the overexpression of Bcl-xL appeared to decrease AY4-mediated cell death by blocking ROS generation in U-HTH 7 cells. By contrast, Bcl-xL knockdown using small interfering RNA of Bcl-xL enhanced AY4 sensitivity in HTH83 and C-643 cells and rendered the cells sensitive to AY4-induced cell death. The results support the conclusion that the expression level of Bcl-xL is important in the AY4-induced apoptosis of ATC cells through ROS generation. AY4 may be a promising tool for ATC therapy.

Epigallocatechin-3-gallate inhibits paracrine and autocrine hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion.

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the cell-based screening assay, (-)epigallocatechin-3-gallate (EGCG) inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering and uPA activation (IC50=15.8 microgram/ml). Further analysis revealed that EGCG at low doses specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met but not epidermal growth factor (EGF)-induced phosphorylation of EGF receptor (EGFR). On the other hand, high-dose EGCG decreased both Met and EGFR proteins. We also found that EGCG did not act on the intracellular portion of Met receptor tyrosine kinase, i.e., it inhibited InlB-dependent activation of Met but not NGF-induced activation of Trk-Met hybrid receptor. This inhibition decreased HGF-induced migration and invasion by parental or HGF/SF-transfected B16F10 melanoma cells in vitro in either a paracrine or autocrine manner. Furthermore, EGCG inhibited the invasion/metastasis of HGF/SF-transfected B16F10 melanoma cells in mice. Our data suggest the possible use of EGCG in human cancers associated with dysregulated paracrine or autocrine HGF/SF-Met signaling.

Hepatocyte growth factor induces delayed STAT3 phosphorylation through interleukin-6 expression.

Met receptor tyrosine kinase mediates pleiotropic cellular responses following its activation by hepatocyte growth factor or scatter factor (HGF/SF). STAT3 was reported to be one of direct downstream molecules in HGF/SF-Met signaling. In the present study, however, we observed that Tyr705 of STAT3 was phosphorylated from 2 h or 6 h in NIH3T3 and Chang liver cells, respectively, after HGF/SF treatment. Blocking of the phosphorylation by cycloheximide or actinomycin D and the rapid STAT3 phosphorylation with the conditioned medium from HGF/SF-treated NIH3T3 cells suggested that a newly synthesized secretory protein was responsible for the delayed STAT3 phosphorylation. Among the known mediators to induce STAT3 phosphorylation, interleukin-6 (IL-6) mRNA and protein were induced by HGF/SF, and the released IL-6 was accumulated in the conditioned medium after HGF/SF treatment. Furthermore, the neutralizing IL-6 antibody abolished the STAT3 phosphorylation. Treatment with LY294002, a PI3 kinase inhibitor, but not with other signal inhibitors, resulted in the loss of delayed STAT3 phosphorylation by HGF/SF, showing the involvement of PI3 kinase pathway. Collectively, these results demonstrate that HGF/SF-Met signal cascade stimulates IL-6 production via PI3 kinase pathway, leading to STAT3 phosphorylation as a secondary effect.

Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation.

Pervanadate, a complex of vanadate and H(2)O(2), has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.