Accurate identification of structural variations from cancer samples.

Structural variations (SVs) are commonly found in cancer genomes. They can cause gene amplification, deletion and fusion, among other functional consequences. With an average read length of hundreds of kilobases, nano-channel-based optical DNA mapping is powerful in detecting large SVs. However, existing SV calling methods are not tailored for cancer samples, which have special properties such as mixed cell types and sub-clones. Here we propose the Cancer Optical Mapping for detecting Structural Variations (COMSV) method that is specifically designed for cancer samples. It shows high sensitivity and specificity in benchmark comparisons. Applying to cancer cell lines and patient samples, COMSV identifies hundreds of novel SVs per sample.

Quantifying full-length circular RNAs in cancer.

Circular RNAs (circRNAs) are abundantly expressed in cancer. Their resistance to exonucleases enables them to have potentially stable interactions with different types of biomolecules. Alternative splicing can create different circRNA isoforms that have different sequences and unequal interaction potentials. The study of circRNA function thus requires knowledge of complete circRNA sequences. Here we describe psirc, a method that can identify full-length circRNA isoforms and quantify their expression levels from RNA sequencing data. We confirm the effectiveness and computational efficiency of psirc using both simulated and actual experimental data. Applying psirc on transcriptome profiles from nasopharyngeal carcinoma and normal nasopharynx samples, we discover and validate circRNA isoforms differentially expressed between the two groups. Compared with the assumed circular isoforms derived from linear transcript annotations, some of the alternatively spliced circular isoforms have 100 times higher expression and contain substantially fewer microRNA response elements, showing the importance of quantifying full-length circRNA isoforms.

Latent membrane protein 1 and macrophage-derived TNFα synergistically activate and mobilize invadopodia to drive invasion of nasopharyngeal carcinoma.

Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they have been visualized as localized and dot-like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live-cell imaging and molecular assays revealed the interaction of macrophage-released TNFα and EBV-encoded latent membrane protein 1 (LMP1) in co-activating the EGFR/Src/ERK/cortactin and Cdc42/N-WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot-like digestion patterns on a 2D gelatin to a dendrite-like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot-like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα-rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cancer-associated fibroblasts potentiate colorectal cancer progression by crosstalk of the IGF2-IGF1R and Hippo-YAP1 signaling pathways.

Colorectal cancer (CRC) is one of the most common cancers worldwide. The tumor microenvironment exerts crucial effects in driving CRC progression. Cancer-associated fibroblasts (CAFs) serve as one of the most important tumor microenvironment components promoting CRC progression. This study aimed to elucidate the novel molecular mechanisms of CAF-secreted insulin-like growth factor (IGF) 2 in colorectal carcinogenesis. Our results indicated that IGF2 was a prominent factor upregulated in CAFs compared with normal fibroblasts. CAF-derived conditioned media (CM) promoted tumor growth, migration, and invasion of HCT 116 and DLD-1 cells. IGF1R expression is significantly increased in CRC, serving as a potent receptor in response to IGF2 stimulation and predicting unfavorable outcomes for CRC patients. Apart from the PI3K-AKT pathway, RNA-seq analysis revealed that the YAP1-target signature serves as a prominent downstream effector to mediate the oncogenic signaling of IGF2-IGF1R. By single-cell RNA sequencing (scRNA-seq) and immunohistochemical validation, IGF2 was found to be predominantly secreted by CAFs, whereas IGF1R was expressed mainly by cancer cells. IGF2 triggers the nuclear accumulation of YAP1 and upregulates YAP1 target signatures; however, these effects were abolished by either IGF1R knockdown or inhibition with picropodophyllin (PPP), an IGF1R inhibitor. Using CRC organoid and in vivo studies, we found that cotargeting IGF1R and YAP1 with PPP and verteporfin (VP), a YAP1 inhibitor, enhanced antitumor effects compared with PPP treatment alone. In conclusion, this study revealed a novel molecular mechanism by which CAFs promote CRC progression. The findings highlight the translational potential of the IGF2-IGF1R-YAP1 axis as a prognostic biomarker and therapeutic target for CRC. © 2022 The Pathological Society of Great Britain and Ireland.

The E2F1-HOXB9/PBX2-CDK6 axis drives gastric tumorigenesis and serves as a therapeutic target in gastric cancer.

Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.

Targeting YAP1/TAZ in nonsmall-cell lung carcinoma: From molecular mechanisms to precision medicine.

Accumulating evidence has underscored the importance of the Hippo-YAP1 signaling in lung tissue homeostasis, whereas its deregulation induces tumorigenesis. YAP1 and its paralog TAZ are the key downstream effectors tightly controlled by the Hippo pathway. YAP1/TAZ exerts oncogenic activities by transcriptional regulation via physical interaction with TEAD transcription factors. In solid tumors, Hippo-YAP1 crosstalks with other signaling pathways such as Wnt/ß-catenin, receptor tyrosine kinase cascade, Notch and TGF-ß to synergistically drive tumorigenesis. As YAP1/TAZ expression is significantly correlated with unfavorable outcomes for the patients, small molecules have been developed for targeting YAP1/TAZ to get a therapeutic effect. In this review, we summarize the recent findings on the deregulation of Hippo-YAP1 pathway in nonsmall cell lung carcinoma, discuss the molecular mechanisms of its dysregulation in leading to tumorigenesis, explore the therapeutic strategies for targeting YAP1/TAZ, and provide the research directions for deep investigation. We believe that detailed delineation of Hippo-YAP1 regulation in tumorigenesis provides novel insight for accurate therapeutic intervention.

A three-dimensional spheroid-specific role for Wnt-ß-catenin and Eph-ephrin signaling in nasopharyngeal carcinoma cells.

One of the greatest unmet needs hindering the successful treatment of nasopharyngeal carcinomas (NPCs) is for representative physiological and cost-effective models. Although Epstein-Barr virus (EBV) infection is consistently present in NPCs, most studies have focused on EBV-negative NPCs. For the first time, we established and analyzed three-dimensional (3D) spheroid models of EBV-positive and EBV-negative NPC cells and compared these to classical two-dimensional (2D) cultures in various aspects of tumor phenotype and drug responses. Compared to 2D monolayers, the 3D spheroids showed significant increases in migration capacity, stemness characteristics, hypoxia and drug resistance. Co-culture with endothelial cells, which mimics essential interactions in the tumor microenvironment, effectively enhanced spheroid dissemination. Furthermore, RNA sequencing revealed significant changes at the transcriptional level in 3D spheroids compared to expression in 2D monolayers. In particular, we identified known (VEGF, AKT and mTOR) and novel (Wnt-ß-catenin and Eph-ephrin) cell signaling pathways that are activated in NPC spheroids. Targeting these pathways in 3D spheroids using FDA-approved drugs was effective in monoculture and co-culture. These findings provide the first demonstration of the establishment of EBV-positive and EBV-negative NPC 3D spheroids with features that resemble advanced and metastatic NPCs. Furthermore, we show that NPC spheroids have potential use in identifying new drug targets.

Accurate reconstruction of viral genomes in human cells from short reads using iterative refinement.

BACKGROUND: After an infection, human cells may contain viral genomes in the form of episomes or integrated DNA. Comparing the genomic sequences of different strains of a virus in human cells can often provide useful insights into its behaviour, activity and pathology, and may help develop methods for disease prevention and treatment. To support such comparative analyses, the viral genomes need to be accurately reconstructed from a large number of samples. Previous efforts either rely on customized experimental protocols or require high similarity between the sequenced genomes and a reference, both of which limit the general applicability of these approaches. In this study, we propose a pipeline, named ASPIRE, for reconstructing viral genomes accurately from short reads data of human samples, which are increasingly available from genome projects and personal genomics. ASPIRE contains a basic part that involves de novo assembly, tiling and gap filling, and additional components for iterative refinement, sequence corrections and wrapping. RESULTS: Evaluated by the alignment quality of sequencing reads to the reconstructed genomes, these additional components improve the assembly quality in general, and in some particular samples quite substantially, especially when the sequenced genome is significantly different from the reference. We use ASPIRE to reconstruct the genomes of Epstein Barr Virus (EBV) from the whole-genome sequencing data of 61 nasopharyngeal carcinoma (NPC) samples and provide these sequences as a resource for EBV research. CONCLUSIONS: ASPIRE improves the quality of the reconstructed EBV genomes in published studies and outperforms TRACESPipe in some samples considered.

Cancer-associated fibroblasts in nonsmall cell lung cancer: From molecular mechanisms to clinical implications.

Lung cancer is the common and leading cause of cancer death worldwide. The tumor microenvironment has been recognized to be instrumental in tumorigenesis. To have a deep understanding of the molecular mechanism of nonsmall cell lung carcinoma (NSCLC), cancer-associated fibroblasts (CAFs) have gained increasing research interests. CAFs belong to the crucial and dominant cell population in the tumor microenvironment to support the cancer cells. The interplay and partnership between cancer cells and CAFs contribute to each stage of tumorigenesis. CAFs exhibit prominent heterogeneity and secrete different kinds of cytokines and chemokines, growth factors and extracellular matrix proteins involved in cancer cell proliferation, invasion, metastasis and chemoresistance. Many studies focused on the protumorigenic functions of CAFs, yet many challenges about the heterogeneity of CAFS remain unresolved. This review comprehensively summarized the tumor-promoting role and molecular mechanisms of CAFs in NSCLC, including their origin, phenotypic changes and heterogeneity and their functional roles in carcinogenesis. Meanwhile, we also highlighted the updated molecular classifications based on the molecular features and functional roles of CAFs. With the development of cutting-edge platforms and further investigations of CAFs, novel therapeutic strategies for accurately targeting CAFs in NSCLC may be developed based on the increased understanding of the relevant molecular mechanisms.

Isolation of exosome from the culture medium of Nasopharyngeal cancer (NPC) C666-1 cells using inertial based Microfluidic channel.

Isolation of exosome from culture medium in an effective way is desired for a less time consuming, cost saving technology in running the diagnostic test on cancer. In this study, we aim to develop an inertial microfluidic channel to separate the nano-size exosome from C666-1 cell culture medium as a selective sample. Simulation was carried out to obtain the optimum flow rate for determining the dimension of the channels for the exosome separation from the medium. The optimal dimension was then brought forward for the actual microfluidic channel fabrication, which consisted of the stages of mask printing, SU8 mould fabrication and ended with PDMS microchannel curing process. The prototype was then used to verify the optimum flow rate with polystyrene particles for its capabilities in actual task on particle separation as a control outcome. Next, the microchip was employed to separate the selected samples, exosome from the culture medium and compared the outcome from the conventional exosome extraction kit to study the level of effectiveness of the prototype. The exosome outcome from both the prototype and extraction kits were characterized through zetasizer, western blot and Transmission electron microscopy (TEM). The microfluidic chip designed in this study obtained a successful separation of exosome from the culture medium. Besides, the extra benefit from this microfluidic channels in particle separation brought an evenly distributed exosome upon collection while the exosomes separated through extraction kit was found clustered together. Therefore, this work has shown the microfluidic channel is suitable for continuous separation of exosome from the culture medium for a clinical study in the future.

Reactivation of Epstein-Barr virus by a dual-responsive fluorescent EBNA1-targeting agent with Zn2+-chelating function.

Epstein-Barr nuclear antigen 1 (EBNA1) plays a vital role in the maintenance of the viral genome and is the only viral protein expressed in nearly all forms of Epstein-Barr virus (EBV) latency and EBV-associated diseases, including numerous cancer types. To our knowledge, no specific agent against EBV genes or proteins has been established to target EBV lytic reactivation. Here we report an EBNA1- and Zn2+-responsive probe (ZRL5P4) which alone could reactivate the EBV lytic cycle through specific disruption of EBNA1. We have utilized the Zn2+ chelator to further interfere with the higher order of EBNA1 self-association. The bioprobe ZRL5P4 can respond independently to its interactions with Zn2+ and EBNA1 with different fluorescence changes. It can selectively enter the nuclei of EBV-positive cells and disrupt the oligomerization and oriP-enhanced transactivation of EBNA1. ZRL5P4 can also specifically enhance Dicer1 and PML expression, molecular events which had been reported to occur after the depletion of EBNA1 expression. Importantly, we found that treatment with ZRL5P4 alone could reactivate EBV lytic induction by expressing the early and late EBV lytic genes/proteins. Lytic induction is likely mediated by disruption of EBNA1 oligomerization and the subsequent change of Dicer1 expression. Our probe ZRL5P4 is an EBV protein-specific agent that potently reactivates EBV from latency, leading to the shrinkage of EBV-positive tumors, and our study also suggests the association of EBNA1 oligomerization with the maintenance of EBV latency.

Translational genomics of nasopharyngeal cancer.

Nasopharyngeal carcinoma (NPC), also named the Cantonese cancer, is a unique cancer with strong etiological association with infection of the Epstein-Barr virus (EBV). With particularly high prevalence in Southeast Asia, the involvement of EBV and genetic aberrations contributive to NPC tumorigenesis have remained unclear for decades. Recently, genomic analysis of NPC has defined it as a genetically homogeneous cancer, driven largely by NF-κB signaling caused by either somatic aberrations of NF-κB negative regulators or by overexpression of the latent membrane protein 1 (LMP1), an EBV viral oncoprotein. This represents a landmark finding of the NPC genome. Exome and RNA sequencing data from new EBV-positive NPC models also highlight the importance of PI3K pathway aberrations in NPC. We also realize for the first time that NPC mutational burden, mutational signatures, MAPK/PI3K aberrations, and MHC Class I gene aberrations, are prognostic for patient outcome. Together, these multiple genomic discoveries begin to shape the focus of NPC therapy development. Given the challenge of NF-κB targeting in human cancers, more innovative drug discovery approaches should be explored to target the unique atypical NF-κB activation feature of NPC. Our next decade of NPC research should focus on further identification of the -omic landscapes of recurrent and metastatic NPC, development of gene-based precision medicines, as well as large-scale drug screening with the newly developed and well-characterized EBV-positive NPC models. Focused preclinical and clinical investigations on these major directions may identify new and effective targeting strategies to further improve survival of NPC patients.

EBV-encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy. The high expression of BART-miRNAs (miR-BARTs) during latent EBV infection in NPC strongly supports their pathological importance in cancer progression. Recently, we found that several BART-miRNAs work co-operatively to modulate the DNA damage response (DDR) by reducing Ataxia-telangiectasia-mutated (ATM) activity. In this study, we further investigated the role of miR-BARTs on DDR. The immunohistochemical study showed that the DNA repair gene, BRCA1, is consistently down-regulated in primary NPCs. Using computer prediction programs and a series of reporter assays, we subsequently identified the negative regulatory role of BART2-3p, BART12, BART17-5p and BART19-3p in BRCA1 expression. The ectopic expression of these four miR-BARTs suppressed endogenous BRCA1 expression in EBV-negative epithelial cell lines, whereas BRCA1 expression was enhanced by repressing endogenous miR-BARTs activities in C666-1 cells. More importantly, suppressing BRCA1 expression in nasopharyngeal epithelial cell lines using miR-BART17-5p and miR-BART19-3p mimics reduced the DNA repair capability and increased the cell sensitivity to the DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. Our findings suggest that miR-BARTs play a novel role in DDR and may facilitate the development of effective NPC therapies.

Contaminated and misidentified cell lines commonly use in cancer research.

Cell line authentication is important for credibility concern and scientific reproducibility. Authenticated cancer cell lines retain the properties of the cancers of origin and serve valuable resources for medical research. Experimental results commonly will be validated in more than one cell line to avoid specific cell line effect not generalizable to the disease on the whole. The use of appropriate and verified cell lines would therefore be very important in preclinical studies of translational research, bridging basic research to clinics.

Role of miR-96/EVI1/miR-449a Axis in the Nasopharyngeal Carcinoma Cell Migration and Tumor Sphere Formation.

The Wnt signaling pathway is one of the major signaling pathways used by cancer stem cells (CSC). Ecotropic Viral Integration Site 1 (EVI1) has recently been shown to regulate oncogenic development of tumor cells by interacting with multiple signaling pathways, including the Wnt signaling. In the present study, we found that the Wnt modulator ICG-001 could inhibit the expression of EVI1 in nasopharyngeal carcinoma (NPC) cells. Results from loss-of-function and gain-of-function studies revealed that EVI1 expression positively regulated both NPC cell migration and growth of CSC-enriched tumor spheres. Subsequent studies indicated ICG-001 inhibited EVI1 expression via upregulated expression of miR-96. Results from EVI1 3'UTR luciferase reporter assay confirmed that EVI1 is a direct target of miR-96. Further mechanistic studies revealed that ICG-001, overexpression of miR-96, or knockdown of EVI1 expression could restore the expression of miR-449a. The suppressive effect of miR-449a on the cell migration and tumor sphere formation was confirmed in NPC cells. Taken together, the miR-96/EVI1/miR-449a axis is a novel pathway involved in ICG-001-mediated inhibition of NPC cell migration and growth of the tumor spheres.

Activation of sterol regulatory element-binding protein 1 (SREBP1)-mediated lipogenesis by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) promotes cell proliferation and progression of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded latent membrane protein 1 (LMP1), which is commonly expressed in NPC, engages multiple signaling pathways that promote cell growth, transformation, and metabolic reprogramming. Here, we report a novel function of LMP1 in promoting de novo lipogenesis. LMP1 increases the expression, maturation and activation of sterol regulatory element-binding protein 1 (SREBP1), a master regulator of lipogenesis, and its downstream target fatty acid synthase (FASN). LMP1 also induces de novo lipid synthesis and lipid droplet formation. In contrast, small interfering RNA (siRNA) knockdown of LMP1 in EBV-infected epithelial cells diminished SREBP1 activation and lipid biosynthesis. Furthermore, inhibition of the mammalian target of rapamycin (mTOR) pathway, through the use of either mTOR inhibitors or siRNAs, significantly reduced LMP1-mediated SREBP1 activity and lipogenesis, indicating that LMP1 activation of the mTOR pathway is required for SREBP1-mediated lipogenesis. In primary NPC tumors, FASN overexpression is common, with high levels correlating significantly with LMP1 expression. Moreover, elevated FASN expression was associated with aggressive disease and poor survival in NPC patients. Luteolin and fatostatin, two inhibitors of lipogenesis, suppressed lipogenesis and proliferation of nasopharyngeal epithelial cells, effects that were more profound in cells expressing LMP1. Luteolin and fatostatin also dramatically inhibited NPC tumor growth in vitro and in vivo. Our findings demonstrate that LMP1 activation of SREBP1-mediated lipogenesis promotes tumor cell growth and is involved in EBV-driven NPC pathogenesis. Our results also reveal the therapeutic potential of utilizing lipogenesis inhibitors in the treatment of locally advanced or metastatic NPC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Establishment of a nasopharyngeal carcinoma cell line capable of undergoing lytic Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV) infects more than 90% of the adult human population. Undifferentiated nasopharyngeal carcinoma (NPC) is common in Southeast Asia, with a particularly high incidence among southern Chinese. The EBV genome can be detected in practically all cancer cells in undifferentiated NPC. The role of EBV in pathogenesis of undifferentiated NPC remains elusive. NPC cell lines are known to be difficult to establish in culture. The EBV+ve NPC cell lines, even if established in culture, rapidly lost their EBV episomes upon prolonged propagation. At present, the C666-1 NPC cell line, which is defective in lytic EBV reactivation, is the only EBV+ve NPC cell line available for NPC and EBV research. The need to establish new and representative NPC cell lines is eminent for NPC and EBV research. In this study, we report the use of the Rho-associated kinase inhibitor (Y-27632) has facilitated the establishment of a new EBV+ve NPC cell line from an earlier established NPC xenograft, C17. The C17 cell line was tumorigenic in immune-deficient mice (NOD/SCID). It retained the EBV episomes and could be induced to undergo productive lytic reactivation of EBV to generate infectious virus particles. The C17 cell line represents a new investigative tool for NPC and EBV studies. The ability of C17 to undergo lytic reactivation is unique and opens up the opportunity to examine regulation of latent and lytic infection of EBV and their contributions to NPC pathogenesis.

RASAL2 promotes tumor progression through LATS2/YAP1 axis of hippo signaling pathway in colorectal cancer.

BACKGROUND: Patients with colorectal cancer (CRC) have a high incidence of regional and distant metastases. Although metastasis is the main cause of CRC-related death, its molecular mechanisms remain largely unknown. METHODS: Using array-CGH and expression microarray analyses, changes in DNA copy number and mRNA expression levels were investigated in human CRC samples. The mRNA expression level of RASAL2 was validated by qRT-PCR, and the protein expression was evaluated by western blot as well as immunohistochemistry in CRC cell lines and primary tumors. The functional role of RASAL2 in CRC was determined by MTT proliferation assay, monolayer and soft agar colony formation assays, cell cycle analysis, cell invasion and migration and in vivo study through siRNA/shRNA mediated knockdown and overexpression assays. Identification of RASAL2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays. RESULTS: Integrated genomic analysis identified copy number gains and upregulation of RASAL2 in metastatic CRC. RASAL2 encodes a RAS-GTPase-activating protein (RAS-GAP) and showed increased expression in CRC cell lines and clinical specimens. Higher RASAL2 expression was significantly correlated with lymph node involvement and distant metastasis in CRC patients. Moreover, we found that RASAL2 serves as an independent prognostic marker of overall survival in CRC patients. In vitro and in vivo functional studies revealed that RASAL2 promoted tumor progression in both KRAS/NRAS mutant and wild-type CRC cells. Knockdown of RASAL2 promoted YAP1 phosphorylation, cytoplasm retention and ubiquitination, therefore activating the hippo pathway through the LATS2/YAP1 axis. CONCLUSIONS: Our findings demonstrated the roles of RASAL2 in CRC tumorigenesis as well as metastasis, and RASAL2 exerts its oncogenic property through LATS2/YAP1 axis of hippo signaling pathway in CRC.

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Upregulates Glucose Transporter 1 Transcription via the mTORC1/NF-κB Signaling Pathways.

Accumulating evidence indicates that oncogenic viral protein plays a crucial role in activating aerobic glycolysis during tumorigenesis, but the underlying mechanisms are largely undefined. Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a transmembrane protein with potent cell signaling properties and has tumorigenic transformation property. Activation of NF-κB is a major signaling pathway mediating many downstream transformation properties of LMP1. Here we report that activation of mTORC1 by LMP1 is a key modulator for activation of NF-κB signaling to mediate aerobic glycolysis. NF-κB activation is involved in the LMP1-induced upregulation of glucose transporter 1 (Glut-1) transcription and growth of nasopharyngeal carcinoma (NPC) cells. Blocking the activity of mTORC1 signaling effectively suppressed LMP1-induced NF-κB activation and Glut-1 transcription. Interfering NF-κB signaling had no effect on mTORC1 activity but effectively altered Glut-1 transcription. Luciferase promoter assay of Glut-1 also confirmed that the Glut-1 gene is a direct target gene of NF-κB signaling. Furthermore, we demonstrated that C-terminal activating region 2 (CTAR2) of LMP1 is the key domain involved in mTORC1 activation, mainly through IKKß-mediated phosphorylation of TSC2 at Ser939 Depletion of Glut-1 effectively led to suppression of aerobic glycolysis, inhibition of cell proliferation, colony formation, and attenuation of tumorigenic growth property of LMP1-expressing nasopharyngeal epithelial (NPE) cells. These findings suggest that targeting the signaling axis of mTORC1/NF-κB/Glut-1 represents a novel therapeutic target against NPC.IMPORTANCE Aerobic glycolysis is one of the hallmarks of cancer, including NPC. Recent studies suggest a role for LMP1 in mediating aerobic glycolysis. LMP1 expression is common in NPC. The delineation of essential signaling pathways induced by LMP1 in aerobic glycolysis contributes to the understanding of NPC pathogenesis. This study provides evidence that LMP1 upregulates Glut-1 transcription to control aerobic glycolysis and tumorigenic growth of NPC cells through mTORC1/NF-κB signaling. Our results reveal novel therapeutic targets against the mTORC1/NF-κB/Glut-1 signaling axis in the treatment of EBV-infected NPC.