ΔNp63-restricted viral mimicry response impedes cancer cell viability and remodels tumor microenvironment in esophageal squamous cell carcinoma.

Tumor protein p63 isoform ΔNp63 plays roles in the squamous epithelium and squamous cell carcinomas (SCCs), including esophageal SCC (ESCC). By integrating data from cell lines and our latest patient-derived organoid cultures, derived xenograft models, and clinical sample transcriptomic analyses, we identified a novel and robust oncogenic role of ΔNp63 in ESCC. We showed that ΔNp63 maintains the repression of cancer cell endogenous retrotransposon expression and cellular double-stranded RNA sensing. These subsequently lead to a restricted cancer cell viral mimicry response and suppressed induction of tumor-suppressive type I interferon (IFN-I) signaling through the regulations of Signal transducer and activator of transcription 1, Interferon regulatory factor 1, and cGAS-STING pathway. The cancer cell ΔNp63/IFN-I signaling axis affects both the cancer cell and tumor-infiltrating immune cell (TIIC) compartments. In cancer cells, depletion of ΔNp63 resulted in reduced cell viability. ΔNp63 expression is negatively associated with the anticancer responses to viral mimicry booster treatments targeting cancer cells. In the tumor microenvironment, cancer cell TP63 expression negatively correlates with multiple TIIC signatures in ESCC clinical samples. ΔNp63 depletion leads to increased cancer cell antigen presentation molecule expression and enhanced recruitment and reprogramming of tumor-infiltrating myeloid cells. Similar IFN-I signaling and TIIC signature association with ΔNp63 were also observed in lung SCC. These results support the potential application of ΔNp63 as a therapeutic target and a biomarker to guide candidate anticancer treatments exploring viral mimicry responses.

Establishment and Characterization of an Epstein-Barr Virus-positive Cell Line from a Non-keratinizing Differentiated Primary Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC), a cancer that is etiologically associated with the Epstein-Barr virus (EBV), is endemic in Southern China and Southeast Asia. The scarcity of representative NPC cell lines owing to the frequent loss of EBV episomes following prolonged propagation and compromised authenticity of previous models underscores the critical need for new EBV-positive NPC models. Herein, we describe the establishment of a new EBV-positive NPC cell line, designated NPC268 from a primary non-keratinizing, differentiated NPC tissue. NPC268 can undergo productive lytic reactivation of EBV and is highly tumorigenic in immunodeficient mice. Whole-genome sequencing revealed close similarities with the tissue of origin, including large chromosomal rearrangements, while whole-genome bisulfite sequencing and RNA sequencing demonstrated a hypomethylated genome and enrichment in immune-related pathways, respectively. Drug screening of NPC268 together with six other NPC cell lines using 339 compounds, representing the largest high-throughput drug testing in NPC, revealed biomarkers associated with specific drug classes. NPC268 represents the first and only available EBV-positive non-keratinizing differentiated NPC model, and extensive genomic, methylomic, transcriptomic, and drug response data should facilitate research in EBV and NPC, where current models are limited. SIGNIFICANCE: NPC268 is the first and only EBV-positive cell line derived from a primary non-keratinizing, differentiated nasopharyngeal carcinoma, an understudied but important subtype in Southeast Asian countries. This model adds to the limited number of authentic EBV-positive lines globally that will facilitate mechanistic studies and drug development for NPC.

Nasopharyngeal carcinoma cells promote regulatory T cell development and suppressive activity via CD70-CD27 interaction.

Despite the intense CD8+ T-cell infiltration in the tumor microenvironment of nasopharyngeal carcinoma, anti-PD-1 immunotherapy shows an unsatisfactory response rate in clinical trials, hindered by immunosuppressive signals. To understand how microenvironmental characteristics alter immune homeostasis and limit immunotherapy efficacy in nasopharyngeal carcinoma, here we establish a multi-center single-cell cohort based on public data, containing 357,206 cells from 50 patient samples. We reveal that nasopharyngeal carcinoma cells enhance development and suppressive activity of regulatory T cells via CD70-CD27 interaction. CD70 blocking reverts Treg-mediated suppression and thus reinvigorate CD8+ T-cell immunity. Anti-CD70+ anti-PD-1 therapy is evaluated in xenograft-derived organoids and humanized mice, exhibiting an improved tumor-killing efficacy. Mechanistically, CD70 knockout inhibits a collective lipid signaling network in CD4+ naïve and regulatory T cells involving mitochondrial integrity, cholesterol homeostasis, and fatty acid metabolism. Furthermore, ATAC-Seq delineates that CD70 is transcriptionally upregulated by NFKB2 via an Epstein-Barr virus-dependent epigenetic modification. Our findings identify CD70+ nasopharyngeal carcinoma cells as a metabolic switch that enforces the lipid-driven development, functional specialization and homeostasis of Tregs, leading to immune evasion. This study also demonstrates that CD70 blockade can act synergistically with anti-PD-1 treatment to reinvigorate T-cell immunity against nasopharyngeal carcinoma.

DNTTIP1 promotes nasopharyngeal carcinoma metastasis via recruiting HDAC1 to DUSP2 promoter and activating ERK signaling pathway.

BACKGROUND: Distant metastasis remains the leading cause of treatment failure in patients with nasopharyngeal carcinoma (NPC), making it critical to identify efficient therapeutic targets for metastatic NPC. Previous studies have demonstrated that deoxynucleotidyltransferase terminal-interacting protein 1 (DNTTIP1) is associated with the development of various types of cancer. However, its role and mechanism in NPC have not been explored. METHODS: RNA-seq profiling was performed for three pairs of NPC and normal nasopharynx tissues. DNTTIP1 expression in NPC specimens was detected by immunohistochemistry. In vitro and in vivo assays were used to investigate the function of DNTTIP1. The molecular mechanism was determined using RT-qPCR, western blotting, RNA-seq, luciferase reporter assays, ChIP assays, and co-IP assays. FINDINGS: DNTTIP1 was found to be significantly upregulated in NPC tissues. Furthermore, DNTTIP1 promoted NPC growth and metastasis in vitro and in vivo. Upregulation of DNTTIP1 in NPC indicated poor clinical outcomes. Mechanistically, DNTTIP1 suppressed DUSP2 gene expression via recruiting HDAC1 to its promoter and maintaining a deacetylated state of histone H3K27. The downregulation of DUSP2 resulted in aberrant activation of the ERK signaling and elevated MMP2 levels, promoting NPC metastasis. Chidamide, an HDAC inhibitor, was shown to suppress NPC metastasis by regulating the DNTTIP1/HDAC1-DUSP2 axis. INTERPRETATION: Our findings demonstrate that DNTTIP1 not only regulates NPC metastasis but also independently predicts NPC prognosis. Furthermore, targeting DNTTIP1/HDAC1 by Chidamide may benefit NPC patients with metastasis. FUNDING: This work was supported by the National Natural Science Foundation of China (No. 81872464, 82073243).

Prognostic Biomarkers for Survival in Nasopharyngeal Carcinoma: A Systematic Review of the Literature.

This systematic review aims to identify prognostic molecular biomarkers which demonstrate strong evidence and a low risk of bias in predicting the survival of nasopharyngeal carcinoma (NPC) patients. The literature was searched for on PubMed to identify original clinical studies and meta-analyses which reported associations between molecular biomarkers and survival, including ≥150 patients with a survival analysis, and the results were validated in at least one independent cohort, while meta-analyses must include ≥1000 patients with a survival analysis. Seventeen studies fulfilled these criteria-two studies on single nucleotide polymorphisms (SNPs), three studies on methylation biomarkers, two studies on microRNA biomarkers, one study on mutational signature, six studies on gene expression panels, and three meta-analyses on gene expressions. The comparison between the hazard ratios of high-risk and low-risk patients along with a multivariate analysis are used to indicate that these biomarkers have significant independent prognostic values for survival. The biomarkers also indicate a response to certain treatments and whether they could be used as therapeutic targets. This review highlights that patients' genetics, epigenetics, and signatures of cancer and immune cells in the tumor microenvironment (TME) play a vital role in determining their survival.

Comprehensive single-cell sequencing reveals the stromal dynamics and tumor-specific characteristics in the microenvironment of nasopharyngeal carcinoma.

The tumor microenvironment (TME) of nasopharyngeal carcinoma (NPC) harbors a heterogeneous and dynamic stromal population. A comprehensive understanding of this tumor-specific ecosystem is necessary to enhance cancer diagnosis, therapeutics, and prognosis. However, recent advances based on bulk RNA sequencing remain insufficient to construct an in-depth landscape of infiltrating stromal cells in NPC. Here we apply single-cell RNA sequencing to 66,627 cells from 14 patients, integrated with clonotype identification on T and B cells. We identify and characterize five major stromal clusters and 36 distinct subpopulations based on genetic profiling. By comparing with the infiltrating cells in the non-malignant microenvironment, we report highly representative features in the TME, including phenotypic abundance, genetic alternations, immune dynamics, clonal expansion, developmental trajectory, and molecular interactions that profoundly influence patient prognosis and therapeutic outcome. The key findings are further independently validated in two single-cell RNA sequencing cohorts and two bulk RNA-sequencing cohorts. In the present study, we reveal the correlation between NPC-specific characteristics and progression-free survival. Together, these data facilitate the understanding of the stromal landscape and immune dynamics in NPC patients and provides deeper insights into the development of prognostic biomarkers and therapeutic targets in the TME.

Clinical Outcome-Related Mutational Signatures Identified by Integrative Genomic Analysis in Nasopharyngeal Carcinoma.

PURPOSE: Investigation of biological mechanisms underlying genetic alterations in cancer can assist the understanding of etiology and identify the potential prognostic biomarkers. EXPERIMENTAL DESIGN: We performed an integrative genomic analysis for a total of 731 nasopharyngeal carcinoma cases from five independent nasopharyngeal carcinoma cohorts to identify the genetic events associated with clinical outcomes. RESULTS: In addition to the known mutational signatures associated with aging, APOBEC and mismatch repair (MMR), a new signature for homologous recombination deficiency (BRCAness) was discovered in 64 of 216 (29.6%) cases in the discovery set including three cohorts. This signature appeared more frequently in the recurrent and metastatic tumors and significantly correlated with shorter overall survival (OS) in the primary tumors. Independent prognostic value of MMR and BRCAness signatures was revealed by multivariable Cox analysis after adjustment for clinical parameters and stratification by studies. The cases with both signatures had much worse clinical outcome than those without these signatures [hazard ratio (HR), 12.4; P = 0.002]. This correlation was confirmed in the validation set (HR, 8.9; P = 0.003). The BRCAness signature is highly associated with BRCA2 pathogenic germline or somatic alterations (7.8% vs. 0%; P = 0.002). Targeted sequencing results from a prospective nasopharyngeal carcinoma cohort (N = 402) showed that the cases carrying BRCA2 germline rare variants are more likely to have poor OS and progression-free survival. CONCLUSIONS: Our study highlights importance of defects of DNA repair machinery in nasopharyngeal carcinoma pathogenesis and their prognostic values for clinical implications. These signatures will be useful for patient stratification to evaluate conventional and new treatment for precision medicine in nasopharyngeal carcinoma.

CR2Cancer: a database for chromatin regulators in human cancer.

Chromatin regulators (CRs) can dynamically modulate chromatin architecture to epigenetically regulate gene expression in response to intrinsic and extrinsic signalling cues. Somatic alterations or misexpression of CRs might reprogram the epigenomic landscape of chromatin, which in turn lead to a wide range of common diseases, notably cancer. Here, we present CR2Cancer, a comprehensive annotation and visualization database for CRs in human cancer constructed by high throughput data analysis and literature mining. We collected and integrated genomic, transcriptomic, proteomic, clinical and functional information for over 400 CRs across multiple cancer types. We also built diverse types of CR-associated relations, including cancer type dependent (CR-target and miRNA-CR) and independent (protein-protein interaction and drug-target) ones. Furthermore, we manually curated around 6000 items of aberrant molecular alterations and interactions of CRs in cancer development from 5007 publications. CR2Cancer provides a user-friendly web interface to conveniently browse, search and download data of interest. We believe that this database would become a valuable resource for cancer epigenetics investigation and potential clinical application. CR2Cancer is freely available at http://cis.hku.hk/CR2Cancer.