Loss of p53-DREAM-mediated repression of cell cycle genes as a driver of lymph node metastasis in head and neck cancer.

BACKGROUND: The prognosis for patients with head and neck cancer (HNC) is poor and has improved little in recent decades, partially due to lack of therapeutic options. To identify effective therapeutic targets, we sought to identify molecular pathways that drive metastasis and HNC progression, through large-scale systematic analyses of transcriptomic data. METHODS: We performed meta-analysis across 29 gene expression studies including 2074 primary HNC biopsies to identify genes and transcriptional pathways associated with survival and lymph node metastasis (LNM). To understand the biological roles of these genes in HNC, we identified their associated cancer pathways, as well as the cell types that express them within HNC tumor microenvironments, by integrating single-cell RNA-seq and bulk RNA-seq from sorted cell populations. RESULTS: Patient survival-associated genes were heterogenous and included drivers of diverse tumor biological processes: these included tumor-intrinsic processes such as epithelial dedifferentiation and epithelial to mesenchymal transition, as well as tumor microenvironmental factors such as T cell-mediated immunity and cancer-associated fibroblast activity. Unexpectedly, LNM-associated genes were almost universally associated with epithelial dedifferentiation within malignant cells. Genes negatively associated with LNM consisted of regulators of squamous epithelial differentiation that are expressed within well-differentiated malignant cells, while those positively associated with LNM represented cell cycle regulators that are normally repressed by the p53-DREAM pathway. These pro-LNM genes are overexpressed in proliferating malignant cells of TP53 mutated and HPV + ve HNCs and are strongly associated with stemness, suggesting that they represent markers of pre-metastatic cancer stem-like cells. LNM-associated genes are deregulated in high-grade oral precancerous lesions, and deregulated further in primary HNCs with advancing tumor grade and deregulated further still in lymph node metastases. CONCLUSIONS: In HNC, patient survival is affected by multiple biological processes and is strongly influenced by the tumor immune and stromal microenvironments. In contrast, LNM appears to be driven primarily by malignant cell plasticity, characterized by epithelial dedifferentiation coupled with EMT-independent proliferation and stemness. Our findings postulate that LNM is initially caused by loss of p53-DREAM-mediated repression of cell cycle genes during early tumorigenesis.

Oxidised IL-33 drives COPD epithelial pathogenesis via ST2-independent RAGE/EGFR signalling complex.

BACKGROUND: Epithelial damage, repair and remodelling are critical features of chronic airway diseases including chronic obstructive pulmonary disease (COPD). Interleukin (IL)-33 released from damaged airway epithelia causes inflammation via its receptor, serum stimulation-2 (ST2). Oxidation of IL-33 to a non-ST2-binding form (IL-33ox) is thought to limit its activity. We investigated whether IL-33ox has functional activities that are independent of ST2 in the airway epithelium. METHODS: In vitro epithelial damage assays and three-dimensional, air-liquid interface (ALI) cell culture models of healthy and COPD epithelia were used to elucidate the functional role of IL-33ox. Transcriptomic changes occurring in healthy ALI cultures treated with IL-33ox and COPD ALI cultures treated with an IL-33-neutralising antibody were assessed with bulk and single-cell RNA sequencing analysis. RESULTS: We demonstrate that IL-33ox forms a complex with receptor for advanced glycation end products (RAGE) and epidermal growth factor receptor (EGFR) expressed on airway epithelium. Activation of this alternative, ST2-independent pathway impaired epithelial wound closure and induced airway epithelial remodelling in vitro. IL-33ox increased the proportion of mucus-producing cells and reduced epithelial defence functions, mimicking pathogenic traits of COPD. Neutralisation of the IL-33ox pathway reversed these deleterious traits in COPD epithelia. Gene signatures defining the pathogenic effects of IL-33ox were enriched in airway epithelia from patients with severe COPD. CONCLUSIONS: Our study reveals for the first time that IL-33, RAGE and EGFR act together in an ST2-independent pathway in the airway epithelium and govern abnormal epithelial remodelling and muco-obstructive features in COPD.

Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA.

Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We developed an unsupervised machine learning algorithm, CELESTA, which identifies the cell type of each cell, individually, using the cell's marker expression profile and, when needed, its spatial information. We demonstrate the performance of CELESTA on multiplexed immunofluorescence images of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, and validate our findings in an independent cohort. By coupling our spatial analysis with single-cell RNA-sequencing data on proximal sections of the same specimens, we identify cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of CELESTA to facilitate identification of clinically relevant interactions.

Landscape of innate lymphoid cells in human head and neck cancer reveals divergent NK cell states in the tumor microenvironment.

Natural killer (NK) cells comprise one subset of the innate lymphoid cell (ILC) family. Despite reported antitumor functions of NK cells, their tangible contribution to tumor control in humans remains controversial. This is due to incomplete understanding of the NK cell states within the tumor microenvironment (TME). Here, we demonstrate that peripheral circulating NK cells differentiate down two divergent pathways within the TME, resulting in different end states. One resembles intraepithelial ILC1s (ieILC1) and possesses potent in vivo antitumor activity. The other expresses genes associated with immune hyporesponsiveness and has poor antitumor functional capacity. Interleukin-15 (IL-15) and direct contact between the tumor cells and NK cells are required for the differentiation into CD49a+CD103+ cells, resembling ieILC1s. These data explain the similarity between ieILC1s and tissue-resident NK cells, provide insight into the origin of ieILC1s, and identify the ieILC1-like cell state within the TME to be the NK cell phenotype with the greatest antitumor activity. Because the proportions of the different ILC states vary between tumors, these findings provide a resource for the clinical study of innate immune responses against tumors and the design of novel therapy.