Search

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.

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/β-catenin, receptor tyrosine kinase cascade, Notch and TGF-β 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.

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.

Supplementary Data from Efficacy of Systemically Administered Mutant Vesicular Stomatitis Virus (VSVΔ51) Combined with Radiation for Nasopharyngeal Carcinoma

Purpose: Nasopharyngeal carcinoma (NPC) is a malignancy of the head and neck region that is associated with EBV latency. Curative treatments for NPC achieve modest survival rates, underscoring a need to develop novel therapies. We evaluated the therapeutic potential of a mutant vesicular stomatitis virus (VSVΔ51) as single treatment modality or in combination with ionizing radiation (RT) in NPC.Experimental Design: MTS assay was used to assess cell viability in vitro; apoptosis was measured using propidium iodide staining and caspase activation. In vivo experiments were conducted using tumor-bearing nude mice with or without local RT (4 Gy). Apoptosis was assessed in excised tumor sections with terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining.Results: Our data showed that NPC cells are exquisitely sensitive to VSVΔ51 oncolysis, which correlated with the presence of EBV. Efficacy of VSVΔ51 against NPC cells was further augmented when combined with RT. A single systemic injection of VSVΔ51 achieved 50% survival in treated mice, which increased to 83% when combined with local tumor RT. In addition to induction of apoptosis, an antiangiogenic effect of VSVΔ51 was observed in vivo, suggesting a novel tumoricidal mechanism for VSVΔ51. This virus also prevented growth of NPC sphere-forming cells in vitro, showing potential utility in targeting NPC-initiating cells.Conclusions: Our data represent the first report showing that EBV-positive NPC cells are exquisitely sensitive to VSVΔ51 oncolysis and documenting the successful utilization of this combinatorial regimen as a novel curative therapeutic strategy for NPC.

Array CGH data set

Supplementary Figure S1 from Array-Based Comparative Genomic Hybridization Analysis Identified Cyclin D1 as a Target Oncogene at 11q13.3 in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma is highly prevalent in Southern China and Southeast Asia. To unveil the molecular basis of this endemic disease, high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in 26 nasopharyngeal carcinoma samples. A comprehensive picture of genetic lesions associated with tumorigenesis of nasopharyngeal carcinoma was generated. Consistent chromosomal gains were frequently found on 1q, 3q, 8q, 11q, 12p, and 12q. High incidences of nonrandom losses were identified on chromosomes 3p, 9p, 11q, 14q, and 16q. In addition to previously characterized regions, we have identified several novel minimal regions of gains, including 3q27.3-28, 8q21-24, 11q13.1-13.3, and 12q13, which may harbor candidate nasopharyngeal carcinoma–associated oncogenes. In this study, gain of 11q13.1-13.3 was the most frequently detected chromosomal aberration and a 5.3-Mb amplicon was delineated at this region. Within this 11q13 amplicon, concordant amplification and overexpression of cyclin D1 (CCND1) oncogene was found in nasopharyngeal carcinoma cell lines, xenografts, and primary tumors. Knockdown of cyclin D1 by small interfering RNA in nasopharyngeal carcinoma cell lines led to significant decrease of cell proliferation. The findings suggest that cyclin D1 is a target oncogene at 11q13 in nasopharyngeal carcinoma and its activation plays a significant role in nasopharyngeal carcinoma tumorigenesis.

Supplementary Tables S1& S2 from Array-Based Comparative Genomic Hybridization Analysis Identified Cyclin D1 as a Target Oncogene at 11q13.3 in Nasopharyngeal Carcinoma

Radiotherapy (RT) is the standard-of-care for Epstein–Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), where the post-RT clearance of plasma EBV DNA is prognostic. Currently, it is not known whether the post-RT clearance of plasma EBV DNA is related to the presence of circulating T-cell subsets. Blood samples from NPC patients were used to assess the frequency of T-cell subsets relating to differentiation, co-signaling and chemotaxis. Patients with undetectable versus detectable plasma EBV DNA levels post-RT were categorized as clearers vs. non-clearers. Clearers had a lower frequency of PD1+CD8+ T cells as well as CXCR3+CD8+ T cells during RT compared to non-clearers. Clearers exclusively showed a temporal increase in chemo-attractant receptors CCR1, 4 and/or 5, expressing CD8+ T cells upon RT. The increase in CCR-expressing CD8+ T cells was accompanied by a drop in naïve CD8+ T cells and an increase in OX40+CD8+ T cells. Upon stratifying these patients based on clinical outcome, the dynamics of CCR-expressing CD8+ T cells were in concordance with the non-recurrence of NPC. In a second cohort, non-recurrence associated with higher quantities of circulating CCL14 and CCL15. Collectively, our findings relate plasma EBV DNA clearance post-RT to T-cell chemotaxis, which requires validation in larger cohorts.

Backgroud: Cancer-associated fibroblasts (CAFs), a component of the tumor microenvironment, play a critical role in cancer progression, either pro- or anti-tumorigenic functions. Due to the original, phenotypic, and functional heterogeneity, CAFs can be subgrouped into several subpopulations. So far, no molecular classifications of CAFs based on a single-cell pan-cancer scale have been provided. Methods: This study employs a pan-cancer single-cell transcriptional atlas on 9 types of solid tumors (breast cancer, cholangiocarcinoma, colon adenocarcinoma, hepatocellular carcinoma, lung adenocarcinoma, neuroendocrine prostate cancer, pancreatic adenocarcinoma, prostate adenocarcinoma, and stomach adenocarcinoma) to provide a novel molecular classification, elucidate the CAF evolution. The function of each CAF subtype was analyzed by single-cell regulatory network inference and clustering (SCENIC) and single-cell GSEA, and the clinical significance was assessed using survival curves. Furthermore, we used molecular docking to screen small molecules targeting matCAF and conducted in vivo experiments to verify. Results: We distinguished CAFs in the solid tumor as 4 molecular clusters: progenitor CAF (proCAF), inflammatory CAF (iCAF), myofibroblastic CAF (myCAF), and matrix-producing CAF (matCAF) based on the prominent molecular features. The classification is universally applied in all the 9 solid tumors. The 4 CAF subtypes exhibit distinct evolutionary trajectories, functional roles, and clinical significance in different solid tumors. Besides, the matCAF signatures were found to have poor prognoses among multiple cancer types. Targeting matCAF by a screened small molecule, Procyanidin C1, exerted anti-tumor effects in suppressing tumor growth. Conclusions: Together, CAF subtypes play essential roles in cancer initiation and progression, especially mat CAF. Targeting matAF in solid tumors has tumor therapeutic potential.

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.

Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they were 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. This article is protected by copyright. All rights reserved.

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer., The genomic characterisation of nasopharyngeal carcinoma (NPC) remains crucial. Here, the authors perform whole-genome sequencing for 70 NPCs with EBV gene expression, report the somatic alterations and EBV-mediated effects converging on NF-κB activation and immune escape and identify targetable homozygous MTAP deletions.

The past three decades have borne witness to many advances in the understanding of the molecular biology and treatment of nasopharyngeal carcinoma (NPC), an Epstein–Barr virus (EBV)-associated cancer endemic to southern China, southeast Asia and north Africa. In this Review, we provide a comprehensive, interdisciplinary overview of key research findings regarding NPC pathogenesis, treatment, screening and biomarker development. We describe how technological advances have led to the advent of proton therapy and other contemporary radiotherapy approaches, and emphasize the relentless efforts to identify the optimal sequencing of chemotherapy with radiotherapy through decades of clinical trials. Basic research into the pathogenic role of EBV and the genomic, epigenomic and immune landscape of NPC has laid the foundations of translational research. The latter, in turn, has led to the development of new biomarkers and therapeutic targets and of improved approaches for individualizing immunotherapy and targeted therapies for patients with NPC. We provide historical context to illustrate the effect of these advances on treatment outcomes at present. We describe current preclinical and clinical challenges and controversies in the hope of providing insights for future investigation. Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV)-associated malignancy endemic to southern China, southeast Asia and north Africa. The authors of this Review present a comprehensive overview of advances from the past three decades on the pathogenic role of EBV, and the genomic, epigenomic and immune landscape of NPC, which have led to the development of new biomarkers, therapeutic targets and improved treatment approaches for patients with NPC.

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV)-associated malignancy ranking as the 23rd most common cancer globally, while its incidence rate ranked the 9th in southeast Asia. Tumor metastasis is the dominant cause for treatment failure in NPC and metastatic NPC is yet incurable. The Wnt/β-catenin signaling pathway plays an important role in many processes such as cell proliferation, differentiation, epithelial–mesenchymal transition (EMT), and self-renewal of stem cells and cancer stem cells (CSCs). Both the EMT process and CSCs are believed to play a critical role in cancer metastasis. We here investigated whether the specific CBP/β-catenin Wnt antagonist, IGC-001, affects the metastasis of NPC cells. We found that ICG-001 treatment could reduce the adhesion capability of NPC cells to extracellular matrix and to capillary endothelial cells and reduce the tumor cell migration and invasion, events which are closely associated with distant metastasis. Through a screening of EMT and CSC-related microRNAs, it was found that miR-134 was consistently upregulated by ICG-001 treatment in NPC cells. Very few reports have mentioned the functional role of miR-134 in NPC, except that the expression was found to be downregulated in NPC. Transient transfection of miR-134 into NPC cells reduced their cell adhesion, migration, and invasion capability, but did not affect the growth of CSC-enriched tumor spheres. Subsequently, we found that the ICG-001-induced miR-134 expression resulting in downregulation of integrin β1 (ITGB1). Such downregulation reduced cell adhesion and migration capability, as demonstrated by siRNA-mediated knockdown of ITGB1. Direct targeting of ITGB1 by miR-134 was confirmed by the 3′-UTR luciferase assay. Lastly, using an in vivo lung metastasis assay, we showed that ICG-001 transient overexpression of miR-134 or stable overexpression of miR-134 could significantly reduce the lung metastasis of NPC cells. Taken together, we present here evidence that modulation of Wnt/β-catenin signaling pathway could inhibit the metastasis of NPC through the miR-134/ITGB1 axis.

Introduction: EBV-associated nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer that is prevalence in southern China and Southeast Asia. While radiotherapy or concurrent chemo-radiotherapy are efficient for the patients with early disease, more than 60% of NPC patients are diagnosed at an advanced stage and frequently develop local failure and distant metastases. The lack of druggable targets is a challenge for controlling this common cancer. By whole-genome sequencing and FISH analysis, we have defined the frequent homozygous deletion of 9p21.3 in NPC. The loss of MTAP that encodes methylthioadenosine phosphorylase on this region was found in 32% and 34% of primary and recurrent NPC respectively, indicating vulnerability to targeted-therapies for MAT2A/PRMT5 axis. Methods: The preclinical efficacy of MAT2A inhibitors, FIDAS-5 and BT115386 was elucidated in a panel of MTAP-deleted EBV-positive NPC cell lines and patient-derived xenografts. In vitro and in vivo growth inhibition of NPC cells by these compounds were determined. In addition to the suppression of PRMT5 activity, the effects of MAT2A inhibitor treatment on activating p53 pathway, inducing EBV lytic genes and modulating expression of proteins involved in cellular differentiation, apoptosis and lipogenesis in NPC were accessed. Results: In vitro study has revealed that treatment of the MAT2A inhibitors markedly reduced PRMT5 activity and cell growth in all MTAP-deleted NPCs. Compared to the MTAP-wild type NPC cells, the MTAP-deleted cells, either with wild type or mutated TP53, showed heightened sensitivity (>9 fold more sensitive) to the MAT2A inhibition by FIDAS-5 and BT115386. The potent in vivo growth inhibitory effect of BT115386 were observed in the MTAP-deleted xenografts, but not in that with wild type MTAP. In the MTAP-deleted NPC treated with MAT2A inhibitors, activation of p53 pathway and induction of BAX apoptotic protein were consistently found. The treatment also promoted differentiation, suppressed stemness transcription factors and inhibited lipogenesis. Notably, the MAT2A inhibitors also disrupted EBV latency in the MTAP-deleted NPC, induction of multiple EBV lytic genes including BZLF1 and BMRF1 were demonstrated. Conclusion: Selective sensitivity of MTAP-deleted NPC to the treatment of MAT2A inhibitors including FIDAS-5 and BT115386 was demonstrated. To facility its growth inhibitory effects, the compounds targeted PRMT5 activity, subsequently modulated multiple cellular mechanisms and induced EBV lytic reactivation. The potent antitumor effect of BT115386 was demonstrated in the in vivo MTAP-deleted NPC mouse models. The findings warrant future assessments of MAT2A inhibitors in clinical studies for NPC, which might well impact a large subset of patients. Acknowledgment: Acknowledgment: The work was supported by Hong Kong RGC funding (GRF:14101721 and AoE/M-401/20) and HMRF grant (#09203176) Citation Format: YUK-Yu Chan, Chi-Man Tsang, Grace T.Y. Chung, Ka-Fai To, Yi Zhang, Xiaodong Zhang, Jun Tang, Kwok Wai Lo. Therapeutic vulnerability of MTAP-deleted nasopharyngeal carcinoma by MAT2A inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6226.

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.

Fibroblast growth factor receptor type 2 (FGFR2) has emerged as a key oncogenic factor that regulates gastric cancer (GC) progression, but the underlying mechanism of FGF–FGFR2 signaling pathway remains largely unknown. To identify the potential molecular mechanisms of the oncogenic FGFR2 in gastric carcinogenesis and convey a novel therapeutic strategy, we profiled the FGFR alterations and analyzed their clinical associations in TCGA and Hong Kong GC cohorts. We found that FGFR2 overexpression in GC cell lines and primary tumors predicted poor survival and was associated with advanced stages of GC. Functionally, growth abilities and cell cycle progression of GC were inhibited by inactivation of ERK–MAPK signal transduction after FGFR2 knockdown, while apoptosis was promoted. Meanwhile, the first-line anti-cancer drug sensitivity was enhanced. RNA-seq analysis further revealed that YAP1 signaling serves as a significant downstream modulator and mediates the oncogenic signaling of FGFR2. When stimulating FGFR2 by rhFGF18, we observed intensified F-actin, nuclear accumulation of YAP1, and overexpression of YAP1 targets, but these effects were attenuated by either FGFR2 depletion or AZD4547 administration. Additionally, the FGF18–FGFR2 signaling upregulated YAP1 expression through activating c-Jun, an effector of MAPK signaling. In our cohort, 28.94% of GC cases were characterized as FGFR2, c-Jun, and YAP1 co-positive and demonstrated worse clinical outcomes. Remarkably, we also found that co-targeting FGFR2 and YAP1 by AZD4547 and Verteporfin synergistically enhanced the antitumor effects in vitro and in vivo. In conclusion, we have identified the oncogenic FGF–FGFR2 regulates YAP1 signaling in GC. The findings also highlight the translational potential of FGFR2–c-Jun–YAP1 axis, which may serve as a prognostic biomarker and therapeutic target for GC.

Hippo signaling functions to limit cellular growth, but the aberrant nuclear accumulation of its downstream YAP1 leads to carcinogenesis. YAP1/TEAD complex activates the oncogenic downstream transcription, such as CTGF and c-Myc. How YAP1 is protected in the cytoplasm from ubiquitin-mediated degradation remains elusive. In this study, a member of Angiomotin (Motin) family, AMOTL1 (Angiomotin Like 1), was screened out as the only one to promote YAP1 nuclear accumulation by several clinical cohorts, which was further confirmed by the cellular functional assays. The interaction between YAP1 and AMOTL1 was suggested by co-immunoprecipitation and immunofluorescent staining. The clinical significance of the AMOTL1–YAP1–CTGF axis in gastric cancer (GC) was analyzed by multiple clinical cohorts. Moreover, the therapeutic effect of targeting the oncogenic axis was appraised by drug-sensitivity tests and xenograft-formation assays. The upregulation of AMOTL1 is associated with unfavorable clinical outcomes of GC, and knocking down AMOTL1 impairs its oncogenic properties. The cytoplasmic interaction between AMOTL1 and YAP1 protects each other from ubiquitin-mediated degradation. AMOTL1 promotes YAP1 translocation into the nuclei to activate the downstream expression, such as CTGF. Knocking down AMOTL1, YAP1, and CTGF enhances the therapeutic efficacies of the first-line anticancer drugs. Taken together, AMOTL1 plays an oncogenic role in gastric carcinogenesis through interacting with YAP1 and promoting its nuclear accumulation. A combination of AMOTL1, YAP1, and CTGF expression might serve as a surrogate of Hippo activation status. The co-activation of the AMOTL1/YAP1–CTGF axis is associated with poor clinical outcomes of GC patients, and targeting this oncogenic axis may enhance the chemotherapeutic effects.

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.

G-protein-coupled receptors (GPCRs) belong to a cell surface receptor superfamily responding to a wide range of external signals. The binding of extracellular ligands to GPCRs activates a heterotrimeric G protein and triggers the production of numerous secondary messengers, which transduce the extracellular signals into cellular responses. GPCR signaling is crucial and imperative for maintaining normal tissue homeostasis. High-throughput sequencing analyses revealed the occurrence of the genetic aberrations of GPCRs and G proteins in multiple malignancies. The altered GPCRs/G proteins serve as valuable biomarkers for early diagnosis, prognostic prediction, and pharmacological targets. Furthermore, the dysregulation of GPCR signaling contributes to tumor initiation and development. In this review, we have summarized the research progress of GPCRs and highlighted their mechanisms in gastric cancer (GC). The aberrant activation of GPCRs promotes GC cell proliferation and metastasis, remodels the tumor microenvironment, and boosts immune escape. Through deep investigation, novel therapeutic strategies for targeting GPCR activation have been developed, and the final aim is to eliminate GPCR-driven gastric carcinogenesis.

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.

Simple Summary Nasopharyngeal cancer (NPC) is a malignant epithelial tumor endemic to parts of Asia and associated with infection by the Epstein–Barr virus (EBV) in these regions. The cancer is often detected at a late stage which is associated with poor outcomes (63% 5-year survival). Advances for the management of this disease have remained largely stagnant and treatment relies primarily on radiotherapy and chemotherapy, as well as surgery when indicated. Nevertheless, our understanding of its underlying biology has grown rapidly in the past two decades, laying the foundation for the development of improved therapeutics which have the potential to improve outcomes. This review offers a comprehensive, up-to-date summary of this disease, with a focus on the role of somatostatin receptor 2 (SSTR2) in NPC and how this increased knowledge may lead to improved diagnosis and management of this disease. Abstract Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor, most commonly located in the pharyngeal recess and endemic to parts of Asia. It is often detected at a late stage which is associated with poor prognosis (5-year survival rate of 63%). Treatment for this malignancy relies predominantly on radiotherapy and/or systemic chemotherapy, which can be associated with significant morbidity and impaired quality of life. In endemic regions NPC is associated with infection by Epstein–Barr virus (EBV) which was shown to upregulate the somatostatin receptor 2 (SSTR2) cell surface receptor. With recent advances in molecular techniques allowing for an improved understanding of the molecular aetiology of this disease and its relation to SSTR2 expression, we provide a comprehensive and up-to-date overview of this disease and highlight the emergence of SSTR2 as a key tumor biomarker and promising target for imaging and therapy.

Epstein-Barr virus (EBV) has been recently found to generate novel circular RNAs (circRNAs) through backsplicing. However, comprehensive catalogs of EBV circRNAs in other cell lines and their functional characterization are still lacking. In this study, we have identified a list of putative EBV circRNAs in GM12878, an EBV-transformed lymphoblastoid cell line, with a significant majority encoded from the EBV latent genes. A novel EBV circRNA derived from the exon 5 of LMP-2 gene which exhibited highest prevalence, was further validated using RNase R assay and Sanger sequencing. This circRNA, which we term circLMP-2_e5, can be universally detected in a panel of EBV-positive cell lines modelling different latency programs. It ranges from lower expression in nasopharyngeal carcinoma (NPC) cells to higher expression in B cells, and is localized to both the cytoplasm and the nucleus. We provide evidence that circLMP-2_e5 is expressed concomitantly with its cognate linear LMP-2 RNA upon EBV lytic reactivation, and may be produced as a result of exon skipping, with its circularization possibly occurring without the involvement of cis elements in the short flanking introns. Furthermore, we show that circLMP-2_e5 is not involved in regulating cell proliferation, host innate immune response, its linear parental transcripts, or EBV lytic reactivation. Taken together, our study expands the current repertoire of putative EBV circRNAs, broadens our understanding of the biology of EBV circRNAs, and lays the foundation for further investigation of their function in the EBV life cycle and disease development.

Here, we present the Northeast Asian Reference Database (NARD), including whole-genome sequencing data of 1779 individuals from Korea, Mongolia, Japan, China, and Hong Kong. NARD provides the genetic diversity of Korean (n = 850) and Mongolian (n = 384) ancestries that were not present in the 1000 Genomes Project Phase 3 (1KGP3). We combined and re-phased the genotypes from NARD and 1KGP3 to construct a union set of haplotypes. This approach established a robust imputation reference panel for Northeast Asians, which yields the greatest imputation accuracy of rare and low-frequency variants compared with the existing panels. NARD imputation panel is available at https://nard.macrogen.com/.

EBV infection of preinvasive nasopharyngeal epithelium is believed to be an initiation step during pathogenesis of nasopharyngeal carcinoma (NPC), but the mechanisms remain poorly understood. Here we report a novel mechanism driving NPC metastasis through the EBV-encoded LMP1-mediated metabolic reprogramming, via activation of IGF1-mTORC2 signaling and nuclear acetylation of the Snail promoter by the PDHE1α, an enzyme involved in glucose metabolism. Mechanistically, EBV-LMP1 increases the cellular secretion of IGF1 which promotes phosphorylation of IGF1R to activate mTORC2/AKT signaling linking glucose metabolism to cell motility. LMP1 expression facilitates translocation of mitochondrial PDHE1α into the nucleus in a phosphorylation-dependent manner at Ser293 residue. Functionally, nuclear PDHE1α promotes H3K9 acetylation on the Snail promoter to enhance cell motility, thereby driving cancer metastasis. Importantly, the IGF1/mTORC2/PDHE1α/Snail axis correlates significantly with disease progression and poor prognosis in NPC patients. This study highlights the functional importance of IGF1-mTORC2-PDHE1α signaling mediated by EBV-LMP1 in NPC pathogenesis.

One of the key features of nasopharyngeal carcinoma (NPC) is dense lymphocyte infiltration. Despite elevated lymphocyte populations in tumor microenvironment, these lymphocytes have modest killing activity. One of the possible speculations is constitutive STAT3 overexpression within NPC microenvironment which results in immunosuppression of cytolytic immune cells. Natural killer (NK) cells act as first line defense of innate immunity through cytolytic killing of virus-infected cells and/or cancer cells. Unlike T cells, NK killing does not require antigen presentation and therefore would be effective even for MHC defective cancers. This makes NK therapy attractive in form of autologous and allogeneic transfer. Here we hypothesized that activated NK cells would show enhanced anti-NPC activity. We also sought to investigate the potential of combined NK with STAT3 inhibitor in NPC treatment. In this study, NK cells from peripheral blood mononuclear cells (PBMC) of healthy donors (N=3) were isolated and expanded. Three Epstein Barr Virus (EBV) positive NPC cell lines (NPC43, C666-1, C17) were employed in this study. Naïve and activated NK cells were subjected to NPC coculture at various effector: target (E:T) cell ratios. In combination treatment with NK cells, a small molecule inhibitor Napabucasin was employed for STAT3 treatment. NK killing activity was assessed through flow cytometry analysis of NPC cell death. The current study demonstrated significant elevation of NK activation marker expressions after 4 weeks of ex vivo expansion (CD69 34.7±2.3 fold, p=0.002; NKG2D 4.2±0.8 fold, p=0.018). Concomitantly, ex vivo activated NK cells demonstrated augmented killing activity against NPC cells at a dose-dependent manner, compared to their naïve status (p Citation Format: Shinyee Hui, Kwok Wai Lo, Mingjing Xu, Siu Tim Cheung. Therapeutic potential of activated natural killer cells in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5514.

The past three decades have borne witness to many advances in the understanding of the molecular biology and treatment of nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-associated cancer endemic to southern China, southeast Asia and north Africa. In this Review, we provide a comprehensive, interdisciplinary overview of key research findings regarding NPC pathogenesis, treatment, screening and biomarker development. We describe how technological advances have led to the advent of proton therapy and other contemporary radiotherapy approaches, and emphasize the relentless efforts to identify the optimal sequencing of chemotherapy with radiotherapy through decades of clinical trials. Basic research into the pathogenic role of EBV and the genomic, epigenomic and immune landscape of NPC has laid the foundations of translational research. The latter, in turn, has led to the development of new biomarkers and therapeutic targets and of improved approaches for individualizing immunotherapy and targeted therapies for patients with NPC. We provide historical context to illustrate the effect of these advances on treatment outcomes at present. We describe current preclinical and clinical challenges and controversies in the hope of providing insights for future investigation.

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 to 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, demonstrating the importance of quantifying full-length circRNA isoforms.

Background Recent genomic analyses revealed that druggable molecule targets were only detectable in approximately 6% of patients with nasopharyngeal carcinoma (NPC). However, a dependency on dysregulated CDK4/6–cyclinD1 pathway signaling is an essential event in the pathogenesis of NPC. In this study, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemotherapeutic drugs for the treatment of NPC by using newly established xenograft models and cell lines derived from primary, recurrent, and metastatic NPC. Methods We evaluated the efficacies of palbociclib monotherapy and concurrent treatment with palbociclib and cisplatin or suberanilohydroxamic acid (SAHA) in NPC cell lines and xenograft models. RNA sequencing was then used to profile the drug response–related pathways. Palbociclib-resistant NPC cell lines were established to determine the potential use of cisplatin as a second-line treatment after the development of palbociclib resistance. We further examined the efficacy of palbociclib treatment against cisplatin-resistant NPC cells. Results In NPC cells, palbociclib monotherapy was confirmed to induce cell cycle arrest in the G1 phase in vitro. Palbociclib monotherapy also had significant inhibitory effects in all six tested NPC tumor models in vivo, as indicated by substantial reductions in the total tumor volumes and in Ki-67 proliferation marker expression. In NPC cells, concurrent palbociclib treatment mitigated the cytotoxic effect of cisplatin in vitro. Notably, concurrent treatment with palbociclib and SAHA synergistically promoted NPC cell death both in vitro and in vivo. This combination also further inhibited tumor growth by inducing autophagy-associated cell death. NPC cell lines with induced palbociclib or cisplatin resistance remained sensitive to treatment with cisplatin or palbociclib, respectively. Conclusions Our study findings provide essential support for the use of palbociclib as an alternative therapy for NPC and increase awareness of the effective timing of palbociclib administration with other chemotherapeutic drugs. Our results provide a foundation for the design of first-in-human clinical trials of palbociclib regimens in patients with NPC.