CD4+ T cell activation distinguishes response to anti-PD-L1+anti-CTLA4 therapy from anti-PD-L1 monotherapy.

Cancer patients often receive a combination of antibodies targeting programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA4). We conducted a window-of-opportunity study in head and neck squamous cell carcinoma (HNSCC) to examine the contribution of anti-CTLA4 to anti-PD-L1 therapy. Single-cell profiling of on- versus pre-treatment biopsies identified T cell expansion as an early response marker. In tumors, anti-PD-L1 triggered the expansion of mostly CD8+ T cells, whereas combination therapy expanded both CD4+ and CD8+ T cells. Such CD4+ T cells exhibited an activated T helper 1 (Th1) phenotype. CD4+ and CD8+ T cells co-localized with and were surrounded by dendritic cells expressing T cell homing factors or antibody-producing plasma cells. T cell receptor tracing suggests that anti-CTLA4, but not anti-PD-L1, triggers the trafficking of CD4+ naive/central-memory T cells from tumor-draining lymph nodes (tdLNs), via blood, to the tumor wherein T cells acquire a Th1 phenotype. Thus, CD4+ T cell activation and recruitment from tdLNs are hallmarks of early response to anti-PD-L1 plus anti-CTLA4 in HNSCC.

Single-Cell Transcriptomic Analysis of Primary and Metastatic Tumor Ecosystems in Head and Neck Cancer.

The diverse malignant, stromal, and immune cells in tumors affect growth, metastasis, and response to therapy. We profiled transcriptomes of ∼6,000 single cells from 18 head and neck squamous cell carcinoma (HNSCC) patients, including five matched pairs of primary tumors and lymph node metastases. Stromal and immune cells had consistent expression programs across patients. Conversely, malignant cells varied within and between tumors in their expression of signatures related to cell cycle, stress, hypoxia, epithelial differentiation, and partial epithelial-to-mesenchymal transition (p-EMT). Cells expressing the p-EMT program spatially localized to the leading edge of primary tumors. By integrating single-cell transcriptomes with bulk expression profiles for hundreds of tumors, we refined HNSCC subtypes by their malignant and stromal composition and established p-EMT as an independent predictor of nodal metastasis, grade, and adverse pathologic features. Our results provide insight into the HNSCC ecosystem and define stromal interactions and a p-EMT program associated with metastasis.

The Hippo pathway noncanonically drives autophagy and cell survival in response to energy stress.

The Hippo pathway is known for its crucial involvement in development, regeneration, organ size control, and cancer. While energy stress is known to activate the Hippo pathway and inhibit its effector YAP, the precise role of the Hippo pathway in energy stress response remains unclear. Here, we report a YAP-independent function of the Hippo pathway in facilitating autophagy and cell survival in response to energy stress, a process mediated by its upstream components MAP4K2 and STRIPAK. Mechanistically, energy stress disrupts the MAP4K2-STRIPAK association, leading to the activation of MAP4K2. Subsequently, MAP4K2 phosphorylates ATG8-family member LC3, thereby facilitating autophagic flux. MAP4K2 is highly expressed in head and neck cancer, and its mediated autophagy is required for head and neck tumor growth in mice. Altogether, our study unveils a noncanonical role of the Hippo pathway in energy stress response, shedding light on this key growth-related pathway in tissue homeostasis and cancer.

RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

The evolving landscape of salivary gland tumors.

Salivary gland cancers are a rare, histologically diverse group of tumors. They range from indolent to aggressive and can cause significant morbidity and mortality. Surgical resection remains the mainstay of treatment, but radiation and systemic therapy are also critical parts of the care paradigm. Given the rarity and heterogeneity of these cancers, they are best managed in a multidisciplinary program. In this review, the authors highlight standards of care as well as exciting new research for salivary gland cancers that will strive for better patient outcomes.

The contemporary management of cancers of the sinonasal tract in adults.

Sinonasal malignancies make up <5% of all head and neck neoplasms, with an incidence of 0.5-1.0 per 100,000. The outcome of these rare malignancies has been poor, whereas significant progress has been made in the management of other cancers. The objective of the current review was to describe the incidence, causes, presentation, diagnosis, treatment, and recent developments of malignancies of the sinonasal tract. The diagnoses covered in this review included sinonasal undifferentiated carcinoma, sinonasal adenocarcinoma, sinonasal squamous cell carcinoma, and esthesioneuroblastoma, which are exclusive to the sinonasal tract. In addition, the authors covered malignances that are likely to be encountered in the sinonasal tract-primary mucosal melanoma, NUT (nuclear protein of the testis) carcinoma, and extranodal natural killer cell/T-cell lymphoma. For the purpose of keeping this review as concise and focused as possible, sarcomas and malignancies that can be classified as salivary gland neoplasms were excluded.

Histone methylation antagonism drives tumor immune evasion in squamous cell carcinomas.

How cancer-associated chromatin abnormalities shape tumor-immune interaction remains incompletely understood. Recent studies have linked DNA hypomethylation and de-repression of retrotransposons to anti-tumor immunity through the induction of interferon response. Here, we report that inactivation of the histone H3K36 methyltransferase NSD1, which is frequently found in squamous cell carcinomas (SCCs) and induces DNA hypomethylation, unexpectedly results in diminished tumor immune infiltration. In syngeneic and genetically engineered mouse models of head and neck SCCs, NSD1-deficient tumors exhibit immune exclusion and reduced interferon response despite high retrotransposon expression. Mechanistically, NSD1 loss results in silencing of innate immunity genes, including the type III interferon receptor IFNLR1, through depletion of H3K36 di-methylation (H3K36me2) and gain of H3K27 tri-methylation (H3K27me3). Inhibition of EZH2 restores immune infiltration and impairs the growth of Nsd1-mutant tumors. Thus, our work uncovers a druggable chromatin cross talk that regulates the viral mimicry response and enables immune evasion of DNA hypomethylated tumors.

Immune Landscape of Viral- and Carcinogen-Driven Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) arises through exposure to environmental carcinogens or malignant transformation by human papillomavirus (HPV). Here, we assessed the transcriptional profiles of 131,224 single cells from peripheral and intra-tumoral immune populations from patients with HPV- and HPV+ HNSCC and healthy donors. Immune cells within tumors of HPV- and HPV+ HNSCC displayed a spectrum of transcriptional signatures, with helper CD4+ T cells and B cells being relatively divergent and CD8+ T cells and CD4+ regulatory T cells being relatively similar. Transcriptional results were contextualized through multispectral immunofluorescence analyses and evaluating putative cell-cell communication based on spatial proximity. These analyses defined a gene expression signature associated with CD4+ T follicular helper cells that is associated with longer progression-free survival in HNSCC patients. The datasets and analytical approaches herein provide a resource for the further study of the impact of immune cells on viral- and carcinogen-induced cancers.

Targeting KDM4A epigenetically activates tumor-cell-intrinsic immunity by inducing DNA replication stress.

Developing strategies to activate tumor-cell-intrinsic immune response is critical for improving tumor immunotherapy by exploiting tumor vulnerability. KDM4A, as a histone H3 lysine 9 trimethylation (H3K9me3) demethylase, has been found to play a critical role in squamous cell carcinoma (SCC) growth and metastasis. Here we report that KDM4A inhibition promoted heterochromatin compaction and induced DNA replication stress, which elicited antitumor immunity in SCC. Mechanistically, KDM4A inhibition promoted the formation of liquid-like HP1γ puncta on heterochromatin and stall DNA replication, which activated tumor-cell-intrinsic cGAS-STING signaling through replication-stress-induced cytosolic DNA accumulation. Moreover, KDM4A inhibition collaborated with PD1 blockade to inhibit SCC growth and metastasis by recruiting and activating CD8+ T cells. In vivo lineage tracing demonstrated that KDM4A inhibition plus PD1 blockade efficiently eliminated cancer stem cells. Altogether, our results demonstrate that targeting KDM4A can activate anti-tumor immunity and enable PD1 blockade immunotherapy by aggravating replication stress in SCC cells.

Noncanonical HPV carcinogenesis drives radiosensitization of head and neck tumors.

We analyzed transcriptional data from 104 HPV+ (Human papillomavirus) HNSCC (head and neck squamous cell carcinoma) tumors together with two publicly available sources to identify highly robust transcriptional programs (modules) which could be detected consistently despite heterogeneous sequencing and quantification methodologies. Among 22 modules identified, we found a single module that naturally subclassifies HPV+ HNSCC tumors based on a bimodal pattern of gene expression, clusters all atypical features of HPV+ HNSCC biology into a single subclass, and predicts patient outcome in four independent cohorts. The subclass-defining gene set was strongly correlated with Nuclear factor kappa B (NF-κB) target expression. Tumors with high expression of this NF-κB module were rarely associated with activating PIK3CA alterations or viral integration, and also expressed higher levels of HPHPV E2 and had decreased APOBEC mutagenesis. Alternatively, they harbored inactivating alterations of key regulators of NF-κB, TNF receptor associated factor 3 (TRAF3), and cylindromatosis (CYLD), as well as retinoblastoma protein (RB1). HPV+ HNSCC cells in culture with experimental depletion of TRAF3 or CYLD displayed increased expression of the subclass-defining genes, as well as robust radio-sensitization, thus recapitulating both the tumor transcriptional state and improved treatment response observed in patient data. Across all gene sets investigated, methylation to expression correlations were the strongest for the subclass-defining, NF-κB-related genes. Increased tumor-infiltrating CD4+ T cells and increased Estrogen receptors alpha (ERα) expression were identified in NF-κB active tumors. Based on the relatively high rates of cure in HPV+ HNSCC, deintensification of therapy to reduce treatment-related morbidity is being studied at many institutions. Tumor subclassification based on oncogenic subtypes may help guide the selection of therapeutic intensity or modality for patients with HPV+ HNSCC.

Roles of ZEB1 and ZEB2 in E-cadherin expression and cell aggressiveness in head and neck cancer.

Zinc finger E-box binding homeobox 1 (ZEB1) has been identified as a key factor in cancer cell differentiation and metastasis, and has been well studied in the field of cancer cell biology. ZEB2 has a highly similar conformation to ZEB1, but its role in head and neck squamous cell carcinoma (HNSCC) cells is not fully understood. Here, we separately overexpressed ZEB1 and ZEB2 in C57BL/6 mouse oral cancer (MOC) cells and investigated their cellular characteristics, including E-cadherin levels, motile properties, chemoresistance, and metastatic ability in immunocompetent mice. Both ZEB1 and ZEB2 overexpression reduced epithelial traits and converted cells to an aggressive phenotype. Surprisingly, ZEB1 overexpression increased the endogenous level of ZEB2 in MOC cells, and vice versa. The molecular mechanisms underlying these findings remain unclear. However, the in vitro anchorage-independent growth of MOC cells overexpressing ZEB2 was considerably greater than that of MOC cells overexpressing ZEB1. These findings suggest that ZEB2, like ZEB1, has the ability to induce the differentiation of cancer cells into those with highly aggressive traits.

Human papillomavirus 16 replication converts SAMHD1 into a homologous recombination factor and promotes its recruitment to replicating viral DNA.

We have demonstrated that SAMHD1 (sterile alpha motif and histidine-aspartic domain HD-containing protein 1) is a restriction factor for the human papillomavirus 16 (HPV16) life cycle. Here, we demonstrate that in HPV-negative cervical cancer C33a cells and human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), SAMHD1 is recruited to E1-E2 replicating DNA. Homologous recombination (HR) factors are required for HPV16 replication, and viral replication promotes phosphorylation of SAMHD1, which converts it from a dNTPase to an HR factor independent from E6/E7 expression. A SAMHD1 phospho-mimic (SAMHD1 T592D) reduces E1-E2-mediated DNA replication in C33a cells and has enhanced recruitment to the replicating DNA. In HFK+HPV16 cells, SAMHD1 T592D is recruited to the viral DNA and attenuates cellular growth, but does not attenuate growth in isogenic HFK cells immortalized by E6/E7 alone. SAMHD1 T592D also attenuates the development of viral replication foci following keratinocyte differentiation. The results indicated that enhanced SAMHD1 phosphorylation could be therapeutically beneficial in cells with HPV16 replicating genomes. Protein phosphatase 2A (PP2A) can dephosphorylate SAMHD1, and PP2A function can be inhibited by endothall. We demonstrate that endothall reduces E1-E2 replication and promotes SAMHD1 recruitment to E1-E2 replicating DNA, mimicking the SAMHD1 T592D phenotypes. Finally, we demonstrate that in head and neck cancer cell lines with HPV16 episomal genomes, endothall attenuates their growth and promotes recruitment of SAMHD1 to the viral genome. The results suggest that targeting cellular phosphatases has therapeutic potential for the treatment of HPV infections and cancers. IMPORTANCE: Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. The development of anti-viral therapeutics depends upon an increased understanding of the viral life cycle. Here, we demonstrate that HPV16 replication converts sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) into a homologous recombination (HR) factor via phosphorylation. This phosphorylation promotes recruitment of SAMHD1 to viral DNA to assist with replication. A SAMHD1 mutant that mimics phosphorylation is hyper-recruited to viral DNA and attenuates viral replication. Expression of this mutant in HPV16-immortalized cells attenuates the growth of these cells, but not cells immortalized by the viral oncogenes E6/E7 alone. Finally, we demonstrate that the phosphatase inhibitor endothall promotes hyper-recruitment of endogenous SAMHD1 to HPV16 replicating DNA and can attenuate the growth of both HPV16-immortalized human foreskin keratinocytes (HFKs) and HPV16-positive head and neck cancer cell lines. We propose that phosphatase inhibitors represent a novel tool for combating HPV infections and disease.

The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

Diverse roles of dendritic cell and regulatory T cell crosstalk in controlling health and disease.

Dendritic cells (DCs) are specialized antigen-presenting cells for lymphocytes, including regulatory T (Treg) cells, a subset of CD4+ T cells expressing CD25 and Foxp3, a transcription factor. Treg cells maintain immunological self-tolerance in mice and humans, and suppress autoimmunity and other various immune responses such as tumor immunity, transplant rejection, allergy, responses to microbes, and inflammation. Treg cell proliferation is controlled by antigen-presenting DCs. On the other hand, Treg cells suppress the function of DCs by restraining DC maturation. Therefore, the interaction between DCs and Treg cells, DC-Treg crosstalk, could contribute to controlling health and disease. We recently found that unique DC-Treg crosstalk plays a role in several conditions. First, Treg cells are expanded in ultraviolet-B (UVB)-exposed skin by interacting with DCs, and the UVB-expanded Treg cells have a healing function. Second, manipulating DC-Treg crosstalk can induce effective acquired immune responses against SARS-CoV2 antigens without adjuvants. Third, Treg cells with a special feature interact with DCs in the tumor microenvironment of human head and neck squamous cell cancer, which may contribute to the prognosis. Understanding the underlying mechanisms of DC-Treg crosstalk may provide a novel strategy to control health and disease.

Single-cell sequencing of head and neck carcinoma: Transcriptional landscape and prognostic model based on malignant epithelial cell features.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, and the development of novel therapeutic strategies for HNSCC requires a profound understanding of tumor cells and the tumor microenvironment (TME). Additionally, HNSCC has a poor prognosis, necessitating the use of genetic markers for predicting clinical outcomes in HNSCC. In this study, we performed single-cell sequencing analysis on tumor tissues from seven HNSCC patients, along with one adjacent normal tissue. Firstly, the analysis of epithelial cell clusters revealed two clusters of malignant epithelial cells, characterized by unique gene expression patterns and dysregulated signaling pathways compared to normal epithelial cells. Secondly, the examination of the TME unveiled extensive crosstalk between fibroblasts and malignant epithelial cells, potentially mediated through ligand-receptor interactions such as COL1A1-SDC1, COL1A1-CD44, and COL1A2-SDC1. Furthermore, transcriptional heterogeneity was observed in immune cells present in the TME, including macrophages and dendritic cells. Finally, leveraging the gene expression profiles of malignant epithelial cells, we developed a prognostic model comprising six genes, which we validated using two independent datasets. These findings shed light on the heterogeneity within HNSCC tumors and the intricate interplay between malignant cells and the TME. Importantly, the developed prognostic model demonstrates high efficacy in predicting the survival outcomes of HNSCC patients.

FTO in oral diseases: Functions, mechanisms, and therapeutic potential.

Fat mass and obesity-associated protein (FTO) is the first identified N6-methyladenosine (m6A) demethylase widely distributed in various tissues in adults and children. It plays an essential role in diverse mRNA-associated processes including transcriptional stability, selective splicing, mRNA translocation, and also protein translation. Recently, emerging studies have shown that FTO is involved in the genesis and development of oral diseases. However, the correlation between FTO and oral diseases and its specific regulatory mechanism still needs further study. In this review, we will summarize the discovery, distribution, gene expression, protein structure, biological functions, inhibitors, and quantifying methods of FTO, as well as its regulatory role and mechanism in oral diseases. Notably, FTO genetic variants are strongly associated with periodontal diseases (PDs), temporomandibular joint osteoarthritis (TMJOA), and obstructive sleep apnea (OSA). Besides, the latest studies that describe the relationship between FTO and PDs, head and neck squamous cell carcinoma (HNSCCs), TMJOA, and OSA will be discussed. We elaborate on the regulatory roles of FTO in PDs, HNSCCs, and TMJOA, which are modulated through cell proliferation, cell migration, apoptosis, bone metabolism, and immune response. The review will enrich our understanding of RNA epigenetic modifications in oral diseases and present a solid theoretical foundation for FTO to serve as a novel diagnosis and prognostic biomarker for oral diseases.

An epidermal growth factor receptor-targeting immunotoxin based on IgG shows potent antitumor activity against head and neck cancer.

The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.

A radiomics model to predict γδ T-cell abundance and overall survival in head and neck squamous cell carcinoma.

γδ T cells are becoming increasingly popular because of their attractive potential for antitumor immunotherapy. However, the role and assessment of γδ T cells in head and neck squamous cell carcinoma (HNSCC) are not well understood. We aimed to explore the prognostic value of γδ T cell and predict its abundance using a radiomics model. Computer tomography images with corresponding gene expression data and clinicopathological data were obtained from online databases. After outlining the volumes of interest manually, the radiomic features were screened using maximum melevance minimum redundancy and recursive feature elimination algorithms. A radiomics model was developed to predict γδ T-cell abundance using gradient boosting machine. Kaplan-Meier survival curves and univariate and multivariate Cox regression analyses were used for the survival analysis. In this study, we confirmed that γδ T-cell abundance was an independent predictor of favorable overall survival (OS) in patients with HNSCC. Moreover, a radiomics model was built to predict the γδ T-cell abundance level (the areas under the operating characteristic curves of 0.847 and 0.798 in the training and validation sets, respectively). The calibration and decision curves analysis demonstrated the fitness of the model. The high radiomic score was an independent protective factor for OS. Our results indicated that γδ T-cell abundance was a promising prognostic predictor in HNSCC, and the radiomics model could discriminate its abundance levels and predict OS. The noninvasive radiomics model provided a potentially powerful prediction tool to aid clinical judgment and antitumor immunotherapy.

USP18 promotes the proliferation, invasion, and migration of head and neck squamous cell carcinoma by deubiquitinating PLK1.

The ubiquitin specific peptidase 18 (USP18), a well-established deubiquitinase, has been extensively implicated in the malignant progression of various human tumors. However, its role in head and neck squamous cell carcinoma (HNSC) requires further investigation. Here, we revealed that USP18 was significantly upregulated in HNSC and knockdown of USP18 markedly suppressed tumor growth in vivo. Furthermore, silencing USP18 attenuated HNSC cell proliferation, invasion, and migration, while overexpression of USP18 exerted converse effects. Mechanistically, USP18 diminished K48-linked ubiquitination of polo-like kinase 1 (PLK1) to stabilize the protein through its deubiquitinase activity. Subsequently, we validated that USP18 modulated PLK1 to activate the mTORC1 pathway, thereby facilitating HNSC cell proliferation, invasion, and migration. In conclusion, our findings demonstrate that elevated expression of USP18 in HNSC cells promotes tumorigenesis by regulating the PLK1-mTORC1 pathway.

Stroma modulation of radiation response in head and neck squamous cell carcinoma: Insights from zebrafish larvae xenografts.

BACKGROUND: The tumour microenvironment (TME) of head and neck squamous cell carcinoma (HNSCC) consists of different subtypes of cells that interact with the tumour or with each other. This study investigates the possibility of co-culturing HNSCC cells with different stroma cells in a zebrafish xenograft model, focusing on the effect of stroma cells on HNSCC growth and response to irradiation. MATERIAL AND METHOD: HNSCC metastatic cell line HSC-3 was used along with five types of stroma cells: normal gingival fibroblasts (NOF), cancer associated fibroblasts (CAF), macrophages, CD4+ T cells, and human umbilical vein endothelial cells (HUVEC). The mixture of HSC-3 cells and each-stroma cell type-was injected into 2-day post-fertilization zebrafish embryos, and the effect of stroma cells on tumour growth was tested. The study also aimed to mimic the HNSCC tumour by injecting a mixture of HSC-3 cells, CAFs, macrophages, and HUVECs into zebrafish embryos and testing the effect of these stroma cells on the cancer cells' response to irradiation compared to HSC-3-only tumours. RESULTS: CAFs had a significant inducement effect on tumour size, while HUVECs showed the opposite effect. The irradiated group of HSC-3-only tumour had a significantly smaller tumor cell area compared to the control, while the group with stroma cells and HSC-3 cells showed cancer cells being resistant to irradiation. CONCLUSION: This is the first report of co-culturing cancer cells with several types of stroma cells using a zebrafish xenograft model. This study also highlighted the role of stroma cells in turning the cancer cells from radioresponsive to radioresistant.