A potential novel cancer immunotherapy: Agonistic anti-CD40 antibodies.

CD40, a novel immunomodulatory cancer therapy target, is expressed by B cells, macrophages, and dendritic cells (DCs) and mediates cytotoxic T cell priming through the CD40 ligand. Some tumors show promising responses to monotherapy or combination therapy with agonistic anti-CD40 antibodies. The development of improved anti-CD40 antibodies makes CD40 activation an innovative strategy in cancer immunotherapy. In this review, we trace the history of CD40 research and summarize preclinical and clinical findings. We emphasize the ongoing development of improved anti-CD40 antibodies and explore strategies for effective combination therapies. Guided by predictive biomarkers, future research should identify patient populations benefiting the most from CD40 activation.

Genomic and Transcriptomic Landscape of an Oral Squamous Cell Carcinoma Mouse Model for Immunotherapy.

The immune checkpoint inhibitor (ICI), anti-programmed death-1 (anti-PD-1), has shown moderate efficacy in some patients with head and neck squamous cell carcinoma (HNSCC). Because of this, it is imperative to establish a mouse tumor model to explore mechanisms of antitumor immunity and to develop novel therapeutic options. Here, we examined the 4-nitroquinoline-1-oxide (4NQO)-induced oral squamous cell carcinoma (OSCC) model for genetic aberrations, transcriptomic profiles, and immune cell composition at different pathologic stages. Genomic exome analysis in OSCC-bearing mice showed conservation of critical mutations found in human HNSCC. Transcriptomic data revealed that a key signature comprised of immune-related genes was increased beginning at the moderate dysplasia stages. We first identified that macrophage composition in primary tumors differed across pathologic stages, leading to an oncogenic evolution through a change in the M1/M2 macrophage ratio during tumorigenesis. We treated the 4NQO-induced OSCC-bearing mice with anti-PD-1 and agonistic anti-CD40, which modulated multiple immune responses. The growth of tumor cells was significantly decreased by agonistic anti-CD40 by promoting an increase in the M1/M2 ratio. By examining cross-species genomic conservation in human and mouse tumors, our study demonstrates the molecular mechanisms underlying the development of OSCC and the regulation of contributing immune-related factors, and aims to facilitate the development of suitable ICI-based treatments for patients with HNSCC.

SN-38, an active metabolite of irinotecan, enhances anti-PD-1 treatment efficacy in head and neck squamous cell carcinoma.

Anti-programmed cell death 1 (anti-PD-1) therapy shows definite but modest activity in patients with advanced/metastatic head and neck squamous cell carcinoma (HNSCC). Preliminary evidence suggests that SN-38, an activated form of irinotecan that increases expression of the transcription factor FoxO3a, can suppress programmed cell death ligand-1 (PD-L1) expression in breast and ovarian tumor models. We analyzed the SN-38-mediated activation of natural killer cells in vitro and explored the efficacy of SN-38 in combination with anti-PD-1 for treatment in vivo. In vitro, SN-38 enhanced the expression of FoxO3a and reduced the expression of c-Myc and PD-L1 dose-dependently in tumor cells. Low-dose SN-38 increased interferon-γ secretion by NK cells and promoted NK cell-mediated cytotoxicity in tumor cells. In vivo studies revealed that at non-cytotoxic drug concentrations, SN-38 significantly enhanced anti-PD-1 activity in suppressing murine tumor growth. We found increased NK cell and CD8+ T-cell infiltration in post-treatment tumors. RNA-seq analysis indicated that SN-38 increased the enrichment of immune cells and biological function genes related to the immune responses. SN-38 is a potentially beneficial adjunct to checkpoint inhibitor therapy in HNSCC. Further studies exploring its mechanism of action and possible applications are necessary. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Effects of Diet and Lifestyle on Audio-Vestibular Dysfunction in the Elderly: A Literature Review.

BACKGROUND: The world's age-related health concerns continue to rise. Audio-vestibular disorders, such as hearing loss, tinnitus, and vertigo, are common complaints in the elderly and are associated with social and public health burdens. Various preventative measures can ease their impact, including healthy food consumption, nutritional supplementation, and lifestyle modification. We aim to provide a comprehensive summary of current possible strategies for preventing the age-related audio-vestibular dysfunction. METHODS: A PubMed, Embase, and Cochrane review databases search was conducted to identify the relationship between diet, lifestyle, and audio-vestibular dysfunction. "Diet", "nutritional supplement", "lifestyle", "exercise", "physical activity", "tinnitus", "vertigo" and "age-related hearing loss" were used as keywords. RESULTS: Audio-vestibular dysfunction develops and progresses as a result of age-related inflammation and oxidative stress. Diets with anti-inflammatory and antioxidant effects have been proposed to alleviate this illness. A high-fat diet may induce oxidative stress and low protein intake is associated with hearing discomfort in the elderly. Increased carbohydrate and sugar intake positively correlate with the incidence of audio-vestibular dysfunction, whereas a Mediterranean-style diet can protect against the disease. Antioxidants in the form of vitamins A, C, and E; physical activity; good sleep quality; smoking cessation; moderate alcohol consumption; and avoiding noise exposure are also beneficial. CONCLUSIONS: Adequate diet or nutritional interventions with lifestyle modification may protect against developing audio-vestibular dysfunction in elderly individuals.

Mouse Models for Immune Checkpoint Blockade Therapeutic Research in Oral Cancer.

The most prevalent oral cancer globally is oral squamous cell carcinoma (OSCC). The invasion of adjacent bones and the metastasis to regional lymph nodes often lead to poor prognoses and shortened survival times in patients with OSCC. Encouraging immunotherapeutic responses have been seen with immune checkpoint inhibitors (ICIs); however, these positive responses to monotherapy have been limited to a small subset of patients. Therefore, it is urgent that further investigations into optimizing immunotherapies are conducted. Areas of research include identifying novel immune checkpoints and targets and tailoring treatment programs to meet the needs of individual patients. Furthermore, the advancement of combination therapies against OSCC is also critical. Thus, additional studies are needed to ensure clinical trials are successful. Mice models are advantageous in immunotherapy research with several advantages, such as relatively low costs and high tumor growth success rate. This review paper divided methods for establishing OSCC mouse models into four categories: syngeneic tumor models, chemical carcinogen induction, genetically engineered mouse, and humanized mouse. Each method has advantages and disadvantages that influence its application in OSCC research. This review comprehensively surveys the literature and summarizes the current mouse models used in immunotherapy, their advantages and disadvantages, and details relating to the cell lines for oral cancer growth. This review aims to present evidence and considerations for choosing a suitable model establishment method to investigate the early diagnosis, clinical treatment, and related pathogenesis of OSCC.

Immunomodulatory Effects of Current Targeted Therapies on Hepatocellular Carcinoma: Implication for the Future of Immunotherapy.

Multikinase inhibitors with antiangiogenic properties used to be standard therapy for patients with advanced hepatocellular carcinoma (HCC). Recently, several antiangiogenic agents (lenvatinib, cabozantinib, and ramucirumab) have demonstrated antitumor activity for advanced HCC in randomized controlled trials. However, the landscape of drug development for HCC may change dramatically with the advent of immune checkpoint inhibitor therapy, particularly the anti-programmed cell death-1 (anti-PD1) agents. In addition, early-phase clinical trials of combination of anti-PD-1 and antiangiogenic agents have shown very promising anti-tumor activity in patients with advanced HCC. Therefore, the critical research questions at present are whether this combination strategy will be the next generation of standard therapy and which antiangiogenic agents will be the optimal partner for the combination. All of the 4 multikinase inhibitors for HCC (sorafenib, regorafenib, lenvatinib, and cabozantinib) have been reported to have immune modulatory effects. The authors systematically reviewed the pre-clinical evidence of their immune modulatory effects to explore whether these effects were mediated by angiogenesis inhibition or by other "off-target" effects on the tumor microenvironment. Studies of sorafenib comprised the majority (58 of the 71) of the research articles reviewed. Potentially beneficial effects on anti-tumor immunity may result from increased M1 polarization of macrophages and stimulation of CD8 T cell function. On the other hand, high dosage of the kinase inhibitors in pre-clinical models and hypoxia associated with angiogenesis may contribute to immune suppression in the tumor microenvironment. Sorafenib and other multikinase inhibitors may promote anti-tumor immunity through modulation of multiple immune cell types as well as the tumor microenvironment. The optimal immune modulatory dosage should be defined to facilitate design of future combination regimens.

Histone Methyltransferase G9a Drives Chemotherapy Resistance by Regulating the Glutamate-Cysteine Ligase Catalytic Subunit in Head and Neck Squamous Cell Carcinoma.

Transient chemotherapeutic response is a major obstacle to treating head and neck squamous cell carcinomas (HNSCC). Histone methyltransferase G9a has recently been shown to be abundantly expressed in HNSCC, and is required to maintain the malignant phenotype. In this study, we found that high G9a expression is significantly associated with poor chemotherapeutic response and disease-free survival in HNSCC patients. Similarly, G9a expression and enzymatic activity were elevated in cisplatin-resistant HNSCC cells. Genetic or pharmacologic inhibition of G9a sensitized the resistant cells to cisplatin, increasing cellular apoptosis. Mechanistic investigations indicated that G9a contributes to transcriptional activation of the glutamate-cysteine ligase catalytic subunit (GCLC), which results in upregulation of cellular glutathione (GSH) and drug resistance. In addition, we observed a significant positive correlation between G9a and GCLC expression in tumors of HNSCC patients. Taken together, our findings provide evidence that G9a protects HNSCC cells against chemotherapy by increasing the synthesis of GSH, and imply G9a as a promising target for overcoming cisplatin resistance in HNSCC. Mol Cancer Ther; 16(7); 1421-34. ©2017 AACR.

G9a/RelB regulates self-renewal and function of colon-cancer-initiating cells by silencing Let-7b and activating the K-RAS/ß-catenin pathway.

Epigenetic reprogramming has been associated with the functional plasticity of cancer-initiating cells (CICs); however, the regulatory pathway has yet to be elucidated. A siRNA screen targeting known epigenetic genes revealed that G9a profoundly impairs the chemo-resistance, self-renewal and metastasis of CICs obtained from patients with colorectal cancer (CRC). Patients with elevated G9a were shown to face a high risk of relapse and poor survival rates. From a mechanistic perspective, G9a binds with and stabilizes RelB, thereby recruiting DNA methyltransferase 3 on the Let-7b promoter and repressing its expression. This leads to the activation of the K-RAS/ß-catenin pathway and regulates self-renewal and function of CICs. These findings indicate that the G9a/RelB/Let-7b axis acts as a critical regulator in the maintenance of CIC phenotypes and is strongly associated with negative clinical outcomes. Thus, these findings may have diagnostic as well as therapeutic implications for the treatment of chemotherapy-resistant or metastatic CRC.

Inhibition of G9a induces DUSP4-dependent autophagic cell death in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a common cancer worldwide. Emerging evidence indicates that alteration of epigenetics might be a key event in HNSCC progression. Abnormal expression of histone methyltransferase G9a, which contributes to transcriptional repression of tumor suppressors, has been implicated in promoting cancerous malignancies. However, its role in HNSCC has not been previously characterized. In this study, we elucidate the function of G9a and its downstream mechanism in HNSCC. METHODS: We investigated the clinical relevance of G9a in HNSCC using immunohistochemistry (IHC) staining. In vitro cell proliferation and tumorigenesis ability of G9a-manipulated HNSCC cells were examined with MTT assays, clonogenic assays, and soft agar assays. We examined different routes of cell death in HNSCC cells induced by G9a-depletion or enzymatic inhibition by immunoblot, flow cytometry, fluorescent and transmission electron microscopy analysis. Specific targets of G9a were identified by affymetrix microarray and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Lastly, functions of G9a in vivo were confirmed with a xenograft tumor model. RESULTS: G9a expression is positively correlated to proliferation marker Ki-67 and to poor prognosis in HNSCC patients. Genetic or pharmacological inhibition of G9a reduced cell proliferation without inducing necrosis or apoptosis. Instead, autophagic cell death was the major consequence, and our investigation of mechanisms suggested it is mediated via the dual specificity phosphatase-4 (DUSP4) dependent ERK inactivation pathway. An orthotopic tumor model further confirmed the growth inhibiting effect and induction of autophagy that followed suppression of G9a. CONCLUSIONS: In this study, we provide evidence that G9a confers the survival advantage of HNSCC. Genetic or pharmacological inhibition of G9a induces autophagic cell death; this finding provides a basis for new therapeutic targets for treating HNSCC.

Photodynamic detection of oral cancers with high-performance chitosan-based nanoparticles.

Oral cancer, a subtype of head and neck cancer, is one of the leading causes of cancer death and is difficult to detect in the early stages. Improved methods of detecting primary oral lesions during endoscopy would significantly improve cancer survival rates. Here we report a high-performance nanoparticle for photodynamic detection of oral cancer. Succinate-modified chitosan (SCHI) is physically complexed with folic-acid-modified chitosan to form nanoparticles with a high drug loading efficiency and to improve drug release in the cellular lysosome. The z-average diameter and zeta potential of the prepared nanoparticles (fSCN) were 110.0 nm and 18.6 mV, respectively, enough to keep the nanoparticles stable in aqueous suspension without aggregating. When loaded with 5-aminolaevulinic acid (5-ALA; 72.8% loading efficiency) in the prepared fSCNA, there were no significant differences between the fSCN and fSCNA in particle size or zeta potential. Moreover, the fSCNA nanoparticles were readily engulfed by oral cancer cells via folate-receptor-mediated endocytosis. The release of loaded 5-ALA in the lysosome was promoted by the reduced attraction intensity between chitosan and 5-ALA via the deprotonated SCHI molecules, which resulted in a higher accumulation of intracellular protoporphyrin IX (PpIX) for photodynamic detection. These results demonstrate that N-succinyl-chitosan-incorporated and folic-acid-conjugated chitosan nanoparticles are an excellent vector for oral-specific delivery of 5-ALA for fluorescent endoscopic detection.

Induction of chemokine receptor CXCR4 expression by transforming growth factor-ß1 in human basal cell carcinoma cells.

BACKGROUND: Higher CXCR4 expression enhances basal cell carcinoma (BCC) invasion and angiogenesis. The underlying mechanism of increased CXCR4 expression in invasive BCC is still not well understood. OBJECTIVE: To investigate the mechanisms involved in the regulation of CXCR4 expression in invasive BCC. METHODS: We used qRT-PCR, RT-PCR, Western blot, and flow cytometric analyses to examine different CXCR4 levels among the clinical samples, co-cultured BCC cells and BCC cells treated with recombinant transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF). Immunohistochemical studies were used to demonstrate the correlation between TGF-ß1 and CXCR4 expressions. The signal transduction pathway and transcriptional regulation were confirmed by treatments with chemical inhibitors, neutralizing antibodies, or short interfering RNAs, as well as luciferase reporter activity. RESULTS: Invasive BCC has higher TGF-ß1 and CTGF levels compared to non-invasive BCC. Non-contact dermal fibroblasts co-culture with human BCC cells also increases the expression of CXCR4 in BCC cells. Treatment with recombinant human TGF-ß1, but not CTGF, enhanced the CXCR4 levels in time- and dose-dependent manners. The protein level and surface expression of CXCR4 in human BCC cells was increased by TGF-ß1 treatment. TGF-ß1 was intensely expressed in the surrounding fibroblasts of invasive BCC and was positively correlated with the CXCR4 expression of BCC cells. The transcriptional regulation of CXCR4 by TGF-ß1 is mediated by its binding to the TGF-ß receptor II and phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2)-ETS-1 pathway. CONCLUSION: TGF-ß1 induces upregulation of CXCR4 in human BCC cells by phosphorylation of ERK1/2-ETS-1 pathway.

MicroRNA-17/20a functions to inhibit cell migration and can be used a prognostic marker in oral squamous cell carcinoma.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) accounts for>90% oral cancer which is a leading cause of cancer death worldwide. Early diagnosis may well offer an opportunity to increase survival to this neoplasm. Micro(mi)RNA-interfered cancer progression is crucial, yet its migration machinery of OSCC is still unknown. To access whether the possible miRNA prognostic markers and underlying mechanisms, we developed a highly migratory TW2.6 MS-10 cells from TW2.6 cells to investigate the issue. MATERIALS AND METHODS: miRNA profiling was performed on TW2.6 and TW2.6 MS-10. Target miRNA was correlated to pathological status in OSCC patients by real-time RT-PCR. A downstream effector was identified using a bioinformatics analysis, and a 3'-untranslated region (UTR) reporter assay was used. RESULTS: An miRNA cluster, miR-17-92, including miR-17, miR-19b, miR-20a, and miR-92a, was found to be significantly down-regulated in TW2.6 MS-10 compared to TW2.6 cells. Overexpression of this cluster decreased the migratory ability of OSCC cell lines. We further demonstrated that miR-17 and miR-20a are the main miRNAs of miR-17-92 cluster which modulate OSCC migration. Clinically, miR-17/20a showed negative correlation with TNM stage and lymphatic metastasis. Through a bioinformatics screening analysis and 3'UTR reporter assay, we confirmed the integrin (ITG) ß8 as a direct target of miR-17/20a, and knockdown of ITGß8 reduced cell migratory capability of OSCC. CONCLUSIONS: miR-17/20a acts as a prognostic predictor of OSCC patients' outcome and a tumor migration suppressor miRNA.

Angiopoietin-like protein 1 suppresses SLUG to inhibit cancer cell motility.

Angiopoietin-like protein 1 (ANGPTL1) is a potent regulator of angiogenesis. Growing evidence suggests that ANGPTL family proteins not only target endothelial cells but also affect tumor cell behavior. In a screen of 102 patients with lung cancer, we found that ANGPTL1 expression was inversely correlated with invasion, lymph node metastasis, and poor clinical outcomes. ANGPTL1 suppressed the migratory, invasive, and metastatic capabilities of lung and breast cancer cell lines in vitro and reduced metastasis in mice injected with cancer cell lines overexpressing ANGPTL1. Ectopic expression of ANGPTL1 suppressed the epithelial-to-mesenchymal transition (EMT) by reducing the expression of the zinc-finger protein SLUG. A microRNA screen revealed that ANGPTL1 suppressed SLUG by inducing expression of miR-630 in an integrin α(1)ß(1)/FAK/ERK/SP1 pathway-dependent manner. These results demonstrate that ANGPTL1 represses lung cancer cell motility by abrogating the expression of the EMT mediator SLUG.

The role of IL-8 in the SDF-1α/CXCR4-induced angiogenesis of laryngeal and hypopharyngeal squamous cell carcinoma.

Stromal cell-derived factor-1 (SDF-1) (CXCL12) has been observed to promote laryngeal and hypopharyngeal squamous cell carcinomas (LHSCCs) invasion through cooperation with its receptor CXCR4. Here, we further explore the angiogenesis mechanism induced by SDF-1/CXCR4 interaction in LHSCCs. Immunohistochemistry (IHC) reveals the significant correlation between CXCR4 and angiogenesis in tumors. After blocking the function of CXCR4 by specific inhibitor AMD3100 and neutralized antibody 12G5 or inhibiting the expression by siRNA, we were able to disrupt the HUVECs tube formation, demonstrating that SDF-1/CXCR4 indeed regulated the angiogenesis mechanism. The angiogenesis profiling from angiogenesis array and reverse transcription polymerase chain reaction indicates that IL-8 can be significantly triggered by SDF-1/CXCR4 interaction in LHSCCs. We also demonstrate that IL-8 secretion mechanism is regulated by Akt phosphorylation after SDF-1 stimulation. These results point out the importance of SDF-1/CXCR4 interaction in LHSCCs angiogenesis. The angiogenic factor IL-8 would be triggered by the cooperation of SDF-1 and CXCR4 through an Akt-dependent pathway. This provides a new targeting therapy utility, disrupting SDF-1/CXCR4 interaction combined with downstream-induced angiogenic factors in LHSCCs would be beneficial to improve clinical outcome.

N-α-acetyltransferase 10 protein suppresses cancer cell metastasis by binding PIX proteins and inhibiting Cdc42/Rac1 activity.

N-α-acetyltransferase 10 protein, Naa10p, is an N-acetyltransferase known to be involved in cell cycle control. We found that Naa10p was expressed lower in varieties of malignancies with lymph node metastasis compared with non-lymph node metastasis. Higher Naa10p expression correlates the survival of lung cancer patients. Naa10p significantly suppressed migration, tumor growth, and metastasis independent of its enzymatic activity. Instead, Naa10p binds to the GIT-binding domain of PIX, thereby preventing the formation of the GIT-PIX-Paxillin complex, resulting in reduced intrinsic Cdc42/Rac1 activity and decreased cell migration. Forced expression of PIX in Naa10-transfected tumor cells restored the migration and metastasis ability. We suggest that Naa10p functions as a tumor metastasis suppressor by disrupting the migratory complex, PIX-GIT- Paxillin, in cancer cells.

Retropalatal Müller grade is associated with the severity of obstructive sleep apnea in non-obese Asian patients. Retropalatal Müller grade and OSA in non-obese.

PURPOSE: The aim of this study was to investigate whether physical evaluations could be used for predicting the presence and severity of obstructive sleep apnea (OSA) in non-obese snoring patients. METHODS: This is a retrospective study, and a total of 244 non-obese (body mass index, BMI, <27 kg/m(2)) snoring patients (178 men and 66 women; mean age = 43.1 ± 12.1 years) were included. Each patient underwent polysomnography and a thorough physical examination, including flexible nasopharyngoscopy and Müller maneuver. Patients were divided into four groups based on apnea-hypopnea index (AHI) scores: normal (simple snoring), AHI < 5; mild OSA, 5 ≦ AHI < 15; moderate OSA, 15 ≦ AHI < 30; severe OSA, AHI ≧ 30. Logistic regression was used to identify risk factors for OSA severity. RESULTS: Fifty-nine patients (24%) were simple snorers. The prevalence of sleep apnea (mild, moderate, or severe OSA) for our non-obese snoring patients was 76%. Univariate logistic analyses showed that higher BMI, male gender and retropalatal Müller grades were significantly associated with OSA severity. Multivariate logistic regression analysis identified male gender and retropalatal Müller grade as risk factors for OSA in non-obese snoring patients. CONCLUSIONS: Physical examination may be useful for studying the upper airway in non-obese snoring patients. Flexible nasopharyngoscopy with Müller maneuver appears to be useful for evaluating the severity of OSA in non-obese patients. Retropalatal Müller grade is highly related to both the presence and severity of OSA, particularly in males.