Super-enhancer driven SOX2 promotes tumor formation by chromatin re-organization in nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant head and neck cancer with a high incidence in Southern China and Southeast Asia. Patients with remote metastasis and recurrent NPC have poor prognosis. Thus, a better understanding of NPC pathogenesis may identify novel therapies to address the unmet clinical needs. METHODS: H3K27ac ChIP-seq and HiChIP was applied to understand the enhancer landscapes and the chromosome interactions. Whole genome sequencing was conducted to analyze the relationship between genomic variations and epigenetic dysregulation. CRISPRi and JQ1 treatment were used to evaluate the transcriptional regulation of SOX2 SEs. Colony formation assay, survival analysis and in vivo subcutaneous patient-derived xenograft assays were applied to explore the function and clinical relevance of SOX2 in NPC. FINDINGS: We globally mapped the enhancer landscapes and generated NPC enhancer connectomes, linking NPC specific enhancers and SEs. We found five overlapped genes, including SOX2, among super-enhancer regulated genes, survival related genes and NPC essential genes. The mRNA expression of SOX2 was repressed when applying CRISPRi targeting different SOX2 SEs or JQ1 treatment. Next, we identified a genetic variation (Chr3:181422197, G > A) in SOX2 SE which is correlated with higher expression of SOX2 and poor survival. In addition, SOX2 was highly expressed in NPC and is correlated with short survival in patients with NPC. Knock-down of SOX2 suppressed tumor growth in vitro and in vivo. INTERPRETATION: Our study demonstrated the super-enhancer landscape with chromosome interactions and identified super-enhancer driven SOX2 promotes tumorigenesis, suggesting that SOX2 is a potential therapeutic target for patients with NPC. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

ZIC2 induces pro-tumor macrophage polarization in nasopharyngeal carcinoma by activating the JUNB/MCSF axis.

Nasopharyngeal carcinoma (NPC) is a common malignant epithelial tumor of the head and neck that often exhibits local recurrence and distant metastasis. The molecular mechanisms are understudied, and effective therapeutic targets are still lacking. In our study, we found that the transcription factor ZIC2 was highly expressed in NPC. Although ZIC family members play important roles in neural development and carcinogenesis, the specific mechanism and clinical significance of ZIC2 in the tumorigenesis and immune regulation of NPC remain elusive. Here, we first reported that high expression of ZIC2 triggered the secretion of MCSF in NPC cells, induced M2 polarization of tumor-associated macrophages (TAMs), and affected the secretion of TAM-related cytokines. Mechanistically, ChIP-seq and RNA-seq analyses identified JUNB as a downstream target of ZIC2. Furthermore, ZIC2 was significantly enriched in the promoter site of JUNB and activated JUNB promoter activity, as shown by ChIP-qPCR and luciferase assays. In addition, JUNB and MCSF participated in ZIC2-induced M2 TAMs polarization. Thus, blocking JUNB and MCSF could reverse ZIC2-mediated M2 TAMs polarization. Moreover, Kaplan-Meier survival analyses indicated that high expression of ZIC2, JUNB, and CD163 was positively associated with a poor prognosis in NPC. Overexpression of ZIC2 induced tumor growth in vivo, with the increase of JUNB, MCSF secretion, and CD163. In summary, our study implies that ZIC2 induces M2 TAM polarization, at least in part through regulation of JUNB/MCSF and that ZIC2, JUNB, and CD163 can be utilized as prognostic markers for NPC and as therapeutic targets for cancer immunotherapy.

Epigenetic Regulation of MAP3K8 in EBV-Associated Gastric Carcinoma.

Super-enhancers (SEs) regulate gene expressions, which are critical for cell type-identity and tumorigenesis. Although genome wide H3K27ac profiling have revealed the presence of SE-associated genes in gastric cancer (GC), their roles remain unclear. In this study, ChIP-seq and HiChIP-seq experiments revealed mitogen-activated protein kinase 8 (MAP3K8) to be an SE-associated gene with chromosome interactions in Epstein-Barr virus-associated gastric carcinoma (EBVaGC) cells. CRISPRi mediated repression of the MAP3K8 SEs attenuated MAP3K8 expression and EBVaGC cell proliferation. The results were validated by treating EBVaGC cells with bromodomain and the extra-terminal motif (BET) inhibitor, OTX015. Further, functional analysis of MAP3K8 in EBVaGC revealed that silencing MAP3K8 could inhibit the cell proliferation, colony formation, and migration of EBVaGC cells. RNA-seq and pathway analysis indicated that knocking down MAP3K8 obstructed the notch signaling pathway and epithelial-mesenchymal transition (EMT) in EBVaGC cells. Further, analysis of the cancer genome atlas (TCGA) and GSE51575 databases exhibited augmented MAP3K8 expression in gastric cancer and it was found to be inversely correlated with the disease-free progression of GC. Moreover, Spearman's correlation revealed that MAP3K8 expression was positively correlated with the expressions of notch pathway and EMT related genes, such as, Notch1, Notch2, C-terminal binding protein 2 (CTBP2), alpha smooth muscle actin isotype 2 (ACTA2), transforming growth factor beta receptor 1 (TGFßR1), and snail family transcriptional repressors 1/2 (SNAI1/SNAI2) in GC. Taken together, we are the first to functionally interrogate the mechanism of SE-mediated regulation of MAP3K8 in EBVaGC cell lines.

The genomic architecture of EBV and infected gastric tissue from precursor lesions to carcinoma.

BACKGROUND: Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs) present unique molecular signatures, but the tumorigenesis of EBVaGCs and the role EBV plays during this process remain poorly understood. METHODS: We applied whole-exome sequencing, EBV genome sequencing, and whole-genome bisulfite sequencing to multiple samples (n = 123) derived from the same patients (n = 25), which covered saliva samples and different histological stages from morphologically normal epithelial tissues to dysplasia and EBVaGCs. We compared the genomic landscape between EBVaGCs and their precursor lesions and traced the clonal evolution for each patient. We also analyzed genome sequences of EBV from samples of different histological types. Finally, the key molecular events promoting the tumor evolution were demonstrated by MTT, IC50, and colony formation assay in vitro experiments and in vivo xenograft experiments. RESULTS: Our analysis revealed increasing mutational burden and EBV load from normal tissues and low-grade dysplasia (LD) to high-grade dysplasia (HD) and EBVaGCs, and oncogenic amplifications occurred late in EBVaGCs. Interestingly, within each patient, EBVaGCs and HDs were monoclonal and harbored single-strain-originated EBV, but saliva or normal tissues/LDs had different EBV strains from that in EBVaGCs. Compared with precursor lesions, tumor cells showed incremental methylation in promotor regions, whereas EBV presented consistent hypermethylation. Dominant alterations targeting the PI3K-Akt and Wnt pathways were found in EBV-infected cells. The combinational inhibition of these two pathways in EBV-positive tumor cells confirmed their synergistic function. CONCLUSIONS: We portrayed the (epi) genomic evolution process of EBVaGCs, revealed the extensive genomic diversity of EBV between tumors and normal tissue sites, and demonstrated the synergistic activation of the PI3K and Wnt pathways in EBVaGCs, offering a new potential treatment strategy for this disease.

Thermally Stable Bioinert Zwitterionic Sulfobetaine Interfaces Tolerated in the Medical Sterilization Process.

This work introduces a thermally stable zwitterionic structure able to withstand steam sterilization as a general antifouling medical device interface. The sulfobetaine methacrylate (SBMA) monomer and its polymer form are among the most widely used zwitterionic materials. They are easy to synthesize and have good antifouling properties. However, they partially lose their properties after steam sterilization, a common procedure used to sterilize biomedical interfaces. In this study, ultrahigh-performance liquid chromatography/mass spectrometry (UHPLC-MS) was used to analyze and discuss the molecular structure of SBMA before and after a steam sterilization procedure, and a strategy to address the thermal stability issue proposed, using sulfobetaine methacrylamide (SBAA) instead of SBMA. Interestingly, it was found that the chemical structure of SBAA material can withstand the medical sterilization process at 121 °C while maintaining good antifouling properties, tested with proteins (fibrinogen), bacteria (Escherichia coli), and whole blood. On the other hand, SBMA gels failed at maintaining their excellent antifouling properties after sterilization. This study suggests that the SBAA structure can be used to replace SBMA in the bioinert interface of sterilizable medical devices, such as rayon fiber membranes used for disease control.

Single-cell transcriptomic analysis defines the interplay between tumor cells, viral infection, and the microenvironment in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy with a complex tumor ecosystem. How the interplay between tumor cells, EBV, and the microenvironment contributes to NPC progression and immune evasion remains unclear. Here we performed single-cell RNA sequencing on ~104,000 cells from 19 EBV+ NPCs and 7 nonmalignant nasopharyngeal biopsies, simultaneously profiling the transcriptomes of malignant cells, EBV, stromal and immune cells. Overall, we identified global upregulation of interferon responses in the multicellular ecosystem of NPC. Notably, an epithelial-immune dual feature of malignant cells was discovered and associated with poor prognosis. Functional experiments revealed that tumor cells with this dual feature exhibited a higher capacity for tumorigenesis. Further characterization of the cellular components of the tumor microenvironment (TME) and their interactions with tumor cells revealed that the dual feature of tumor cells was positively correlated with the expression of co-inhibitory receptors on CD8+ tumor-infiltrating T cells. In addition, tumor cells with the dual feature were found to repress IFN-γ production by T cells, demonstrating their capacity for immune suppression. Our results provide new insights into the multicellular ecosystem of NPC and offer important clinical implications.

ZIC2 is downregulated and represses tumor growth via the regulation of STAT3 in breast cancer.

Although early detection and systemic therapies have improved the diagnosis and clinical cure rate of breast cancer, breast cancer remains the most frequently occurring malignant cancer in women due to a lack of sufficiently effective treatments. Thus, to develop potential targeted therapies and thus benefit more patients, it is helpful to understand how cancer cells work. ZIC family members have been shown to play important roles in neural development and carcinogenesis. In our study, we found that ZIC2 is downregulated in breast cancer tissues at both the mRNA and protein levels. Low expression of ZIC2 was correlated with poor outcome in breast cancer patients and serves as an independent prognostic marker. Furthermore, overexpression of ZIC2 repressed, whereas knockdown of ZIC2 promoted, cell proliferation and colony formation ability in vitro and tumor growth in vivo. Using ChIP-seq and RNA-seq analysis, we screened and identified STAT3 as a potential target for ZIC2. ZIC2 bound to the STAT3 promoter and repressed the promoter activities of STAT3. ZIC2 knockdown induced the expression of STAT3, increasing the level of phosphorylated STAT3. These results suggest that ZIC2 regulates the transcription of STAT3 by directly binding to the STAT3 promoter. Additionally, interfering STAT3 with siRNAs or inhibitors abrogated the oncogenic effects induced by decreased ZIC2. Taken together, our results indicate that ZIC2 serves as a useful prognostic marker in breast cancer and acts as a tumor suppressor by regulating STAT3, implying that STAT3 inhibitors might provide an alternative treatment option for breast cancer patients with ZIC2 downregulation.

Prognostic Value of Lymphocyte Activation Gene-3 (LAG-3) Expression in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a malignant epithelial tumor with a high incidence in East Asia and the Middle East. The outcomes for ESCC patients are usually not optimal due to the recurrence and metastasis. This study is aim to examine the expression and the prognostic value of LAG-3 in ESCC. We applied immunohistochemistry analysis to examine the expression of LAG-3, CD4 and CD8 in 287 ESCC cohorts. Our study demonstrated that the decreased LAG-3 expression was significantly associated with CD4 tumor-infiltrated lymphocytes (TILs) (p=0.000), CD8 TILs (p=0.000), and the advanced clinical stages (p=0.041) by Chi-square analysis. Kaplan-Meier survival analysis revealed that higher LAG-3 expression were positively correlated with a better overall survival (OS) (p=0.010) and better progression free survival (PFS) (p=0.006), especially in the patients at stages T1-2 status (p=0.001, OS; p=0.001, PFS), N0 status (p=0.036, OS; p=0.050, PFS), and early stages (I-II) (p=0.006, OS; p=0.008, PFS). Both high of CD4 TIL /CD8 TIL ratio and LAG-3 expression were correlated with longer OS and PFS. Cox proportional hazards regression analysis showed that LAG-3 is an independent biomarker of survival (HR, 0.724; 95% CI 0.526-0.995; p = 0.047) (p=0.036). Taken together, we found that high expression of LAG-3 was correlated with an improved survival and LAG-3 is an independent predictor of survival, suggesting that LAG-3 may serve as a useful immune marker for the prognosis of ESCC.

Structure of Schlafen13 reveals a new class of tRNA/rRNA- targeting RNase engaged in translational control.

Cleavage of transfer (t)RNA and ribosomal (r)RNA are critical and conserved steps of translational control for cells to overcome varied environmental stresses. However, enzymes that are responsible for this event have not been fully identified in high eukaryotes. Here, we report a mammalian tRNA/rRNA-targeting endoribonuclease: SLFN13, a member of the Schlafen family. Structural study reveals a unique pseudo-dimeric U-pillow-shaped architecture of the SLFN13 N'-domain that may clamp base-paired RNAs. SLFN13 is able to digest tRNAs and rRNAs in vitro, and the endonucleolytic cleavage dissevers 11 nucleotides from the 3'-terminus of tRNA at the acceptor stem. The cytoplasmically localised SLFN13 inhibits protein synthesis in 293T cells. Moreover, SLFN13 restricts HIV replication in a nucleolytic activity-dependent manner. According to these observations, we term SLFN13 RNase S13. Our study provides insights into the modulation of translational machinery in high eukaryotes, and sheds light on the functional mechanisms of the Schlafen family.

Novel Magnet and Thermoresponsive Chemosensory Electrospinning Fluorescent Nanofibers and Their Sensing Capability for Metal Ions.

Novel multifunctional switchable chemosensors based on fluorescent electrospun (ES) nanofibers with sensitivity toward magnetism, temperature, and mercury ions (Hg2+) were prepared using blends of poly(N-isopropylacrylamide)-co-(N-methylolacrylamide)-co-(Acrylic acid), the fluorescent probe 1-benzoyl-3-[2-(2-allyl-1,3-dioxo-2,3-dihydro-1Hbenzo[de]isoquinolin-6-ylamino)-ethyl]-thiourea (BNPTU), and magnetite nanoparticles (NPs), and a single-capillary spinneret. The moieties of N-isopropylacrylamide, N-methylolacrylamide, acrylic acid, BNPTU, and Iron oxide (Fe3O4) NPs were designed to provide thermoresponsiveness, chemical cross-linking, Fe3O4 NPs dispersion, Hg2+ sensing, and magnetism, respectively. The prepared nanofibers exhibited ultrasensitivity to Hg2+ (as low as 10-3 M) because of an 80-nm blueshift of the emission maximum (from green to blue) and 1.6-fold enhancement of the emission intensity, as well as substantial volume (or hydrophilic to hydrophobic) changes between 30 and 60 °C, attributed to the low critical solution temperature of the thermoresponsive N-isopropylacrylamide moiety. Such temperature-dependent variations in the presence of Hg2+ engendered distinct on⁻off switching of photoluminescence. The magnetic ES nanofibers can be collected using a magnet rather than being extracted through alternative methods. The results indicate that the prepared multifunctional fluorescent ES nanofibrous membranes can be used as naked eye sensors and have the potential for application in multifunctional environmental sensing devices for detecting metal ions, temperature, and magnetism as well as for water purification sensing filters.