Investigating cellular similarities and differences between upper tract urothelial carcinoma and bladder urothelial carcinoma using single-cell sequencing.

Background: Upper tract urothelial carcinoma (UTUC) and bladder urothelial carcinoma (BLCA) both originate from uroepithelial tissue, sharing remarkably similar clinical manifestations and therapeutic modalities. However, emerging evidence suggests that identical treatment regimens may lead to less favorable outcomes in UTUC compared to BLCA. Therefore, it is imperative to explore molecular processes of UTUC and identify biological differences between UTUC and BLCA. Methods: In this study, we performed a comprehensive analysis using single-cell RNA sequencing (scRNA-seq) on three UTUC cases and four normal ureteral tissues. These data were combined with publicly available datasets from previous BLCA studies and RNA sequencing (RNA-seq) data for both cancer types. This pooled analysis allowed us to delineate the transcriptional differences among distinct cell subsets within the microenvironment, thus identifying critical factors contributing to UTUC progression and phenotypic differences between UTUC and BLCA. Results: scRNA-seq analysis revealed seemingly similar but transcriptionally distinct cellular identities within the UTUC and BLCA ecosystems. Notably, we observed striking differences in acquired immunological landscapes and varied cellular functional phenotypes between these two cancers. In addition, we uncovered the immunomodulatory functions of vein endothelial cells (ECs) in UTUC, and intercellular network analysis demonstrated that fibroblasts play important roles in the microenvironment. Further intersection analysis showed that MARCKS promote UTUC progression, and immunohistochemistry (IHC) staining revealed that the diverse expression patterns of MARCKS in UTUC, BLCA and normal ureter tissues. Conclusion: This study expands our multidimensional understanding of the similarities and distinctions between UTUC and BLCA. Our findings lay the foundation for further investigations to develop diagnostic and therapeutic targets for UTUC.

LCP1-mediated cytoskeleton alterations involve in arsenite-triggered malignant phenotype of human immortalized prostate stromal cells.

The connection between continuous arsenic exposure and prostate cancer is already established. However, the exact mechanisms of arsenic tumorigenesis are far from clear. Here, we employed human prostate stromal immortalized cells (WPMY-1) continuous exposure to 1 and 2 µM arsenite for 29 weeks to identify the malignant phenotype and explore the underlying molecular mechanism. As expected, continuous low-dose arsenite exposure led to the malignant phenotype of WPMY-1 cells. Quantitative proteomics identified 517 differentially expressed proteins (DEPs), of which the most remarkably changed proteins (such as LCP1 and DDX58, etc.) and the bioinformatic analysis were focused on the regulation of cytoskeleton, cell adhesion, and migration. Further, cell experiments showed that continuous arsenite exposure altered cytoskeleton structure, enhanced cell adhesive capability, and raised the levels of reactive oxygen species (ROS), ATM, p-ATM, p-ERK1/2, and LCP1 proteins. N-acetylcysteine (NAC) treatment antagonized the increase of LCP1 proteins, and LCP1 knockdown partially restored F-actin organization caused by arsenic. Overall, the results demonstrated that ROS-ATM-ERK1/2 signaling pathway was involved in the activation of LCP1, leading to cytoskeleton alterations. These alterations are believed to play a significant role in arsenite-triggered tumor microenvironment cell-acquired malignant phenotype, which could provide potential biomarkers with therapeutic implications for prostate cancer.

Loss of ACOX1 in clear cell renal cell carcinoma and its correlation with clinical features.

Clear cell renal cell carcinoma (ccRCC) is a major pathological type of kidney cancer with a poor prognosis due to a lack of biomarkers for early diagnosis and prognosis prediction of ccRCC. In this study, we investigated the aberrant expression of Acyl-coenzyme A oxidase 1 (ACOX1) in ccRCC and evaluated its potential in diagnosis and prognosis. ACOX1 is the first rate-limiting enzyme in the peroxidation ß-oxidation pathway and is involved in the regulation of fatty acid oxidative catabolism. The mRNA and protein levels of ACOX1 were significantly downregulated in ccRCC, and its downregulation was closely associated with the tumor-node-metastasis stage of patients. The ROC curves showed that ACOX1 possesses a high diagnostic value for ccRCC. The OS analysis suggested that lower expression of ACOX1 was closely related to the worse outcome of patients. In addition, gene set enrichment analysis suggested that expression of ACOX1 was positively correlated with CDH1, CDH2, CDKL2, and EPCAM, while negatively correlated with MMP9 and VIM, which strongly indicated that ACOX1 may inhibit the invasion and migration of ccRCC by reversing epithelial-mesenchymal transition. Furthermore, we screened out that miR-16-5p is upregulated at the mRNA transcript level in ccRCC and negatively correlated with ACOX1. In conclusion, our results showed that ACOX1 is abnormally low expressed in ccRCC, suggesting that it could serve as a diagnostic and prognostic biomarker for ccRCC. Overexpression of miR-16-5p may be responsible for the inactivation of ACOX1.

The role of EBV-encoded miRNA in EBV-associated gastric cancer.

Epstein-Barr virus (human herpesvirus 4, EBV) is a linear double-stranded DNA virus that infects over 90% of the population worldwide. However, our understanding of EBV's contribution to tumorigenesis of EBV-associated GC (EBVaGC) remains incomplete. Recent advancements in EBVaGC research have highlighted that EBV-encoded microRNAs (miRNAs) play prominent roles in critical cellular processes such as migration, cell cycle, apoptosis, cell proliferation, immune response, and autophagy. Notably, the largest group of EBV-encoded miRNAs, known as BamHI-A rightward transcripts (BARTs), exhibit bidirectional effects in EBVaGC. For instance, they present both anti-apoptotic and pro-apoptotic functions and enhance chemosensitivity while also conferring resistance to 5-fluorouracil. Despite these findings, the comprehensive mechanisms through which miRNAs contribute to EBVaGC are yet to be fully elucidated. In this work, we summarize the current evidence of the roles of miRNA in EBVaGC, particularly with the application of multi-omic techniques. Additionally, we discuss the application of miRNA in EBVaGC in retrospective analyses and provide novel perspectives on the use of miRNA in EBVaGC in translational medicine.

Inactivation of epithelial sodium ion channel molecules serves as effective diagnostic biomarkers in clear cell renal cell carcinoma.

BACKGROUND: Non-voltage-gated sodium channel, also known as the epithelial sodium channel (ENaC), formed by heteromeric complexes consisting of SCNN1A, SCNN1B, and SCNN1G, is responsible for maintaining sodium ion and body fluid homeostasis in epithelial cells. However, no systematic study of SCNN1 family members has been conducted in renal clear cell carcinoma (ccRCC) to date. OBJECTIVE: To investigate the abnormal expression of SCNN1 family in ccRCC and its potential correlation with clinical parameters. METHODS: The transcription and protein expression levels of SCNN1 family members in ccRCC were analyzed based on the TCGA database, and were confirmed by quantitative RT-PCR and immunohistochemical staining assays, respectively. The area under curve (AUC) was used to evaluate the diagnostic value of SCNN1 family members for ccRCC patients. RESULTS: The mRNA and protein expression of SCNN1 family members was significantly downregulated in ccRCC compared with normal kidney tissues, which might be due to DNA hypermethylation in the promoter region. It is worth noting that the AUC of SCNN1A, SCNN1B, and SCNN1G were 0.965, 0.979, and 0.988 based on the TCGA database (p < 0.0001), respectively. The diagnostic value was even higher when combing these three members together (AUC = 0.997, p < 0.0001). Intriguingly, the mRNA level of SCNN1A was significantly lower in females compared with males, while SCNN1B and SCNN1G were increased with the progression of ccRCC and remarkably associated with a worse outcome for patients. CONCLUSION: The aberrantly decrease of SCNN1 family members might serve as valuable biomarkers for the diagnosis of ccRCC.

Ablation of EWS-FLI1 by USP9X inhibition suppresses cancer cell growth in Ewing sarcoma.

The neomorphic transcription factor EWS-FLI1 is a key driver of Ewing sarcoma. Ablation of EWS-FLI1 may present a promising therapeutic strategy for this malignancy. Here we found that the deubiquitinase, ubiquitin specific peptidase 9 X-linked (USP9X) stabilizes EWS-FLI1 protein expression in Ewing sarcoma. We show that USP9X binds the ETS domain of EWS-FLI1 in Ewing sarcoma cells and deubiquitinates EWS-FLI1 and that USP9X and EWS-FLI1 protein expression is correlated in clinical Ewing sarcoma specimens. We found that treatment of Ewing sarcoma cells with the USP9X inhibitor WP1130 mediates rapid EWS-FLI1 degradation in vitro and in vivo which coincides with reduced growth of Ewing sarcoma cells and tumors. Our results suggest that USP9X might be a potential therapeutic target to mediate EWS-FLI1 depletion in Ewing sarcoma.

Decreased expression of 3-hydroxybutyrate dehydrogenase 1 is a prognostic marker and promotes tumor progression in hepatocellular carcinoma.

Growing evidence indicates that altered metabolism represents the hallmark of hepatocellular carcinoma (HCC). The mitochondrial 3-hydroxybutyrate dehydrogenase 1 (BDH1) is a key catalytic enzyme in ketogenesis with unknown roles in HCC. Hundred and four tissue sample pairs (HCC tissues, n = 104; matched normal tissues, n = 104) were obtained and analyzed with immunohistochemical (IHC) staining to investigate the clinical and functional role and the diagnostic and prognostic value of BDH1 in HCC. In addition, RNA-Seq datasets from the Tumor Immune Estimation Resource (TIMER) (HCC group, n = 371; normal group, n = 50) and microarray datasets from the Gene Expression Omnibus (GEO) database (HCC tissues, n = 1671; normal tissues, n = 1479) were used to assess BDH1 expression in HCC. Several bioinformatic methods were performed to identify pathways regulated by BDH1. The IHC staining showed that BDH1 expression decreased in HCC tissues (n = 69) compared with that in adjacent normal tissues (n = 35, P < 0.001). Low BDH1 expression was associated with advanced clinical stage (P = 0.033) and vascular invasion (P = 0.007). Moreover, ectopic expression of BDH1 reduced tumor proliferation and suppressed the migration and invasion of HCC cells in vitro. Therefore, our data suggest that BDH1 is a potentially valuable diagnostic biomarker and therapeutic target for HCC.

Mechanisms of Anergic Inflammatory Response in Nasopharyngeal Carcinoma Cells Despite Ubiquitous Constitutive NF-κB Activation.

Commensal microbes cross talk with their colonized mucosa. We show that microbes and their cell wall components induce an inflammatory response in cultured human mucosal cells derived from the nonmalignant nasopharyngeal epithelium (NNE) cells in vitro. NNE cells show significant induction of NF-κB with nuclear shuttling and inflammatory gene response when exposed to Gram-positive bacteria (streptococci) or peptidoglycan (PGN), a component of the Gram-positive bacterial cell wall. This response is abrogated in nasopharyngeal carcinoma (NPC)-derived cell lines. The inflammatory response induced by NF-κB signaling was blocked at two levels in the tumor-derived cells. We found that NF-κB was largely trapped in lipid droplets (LDs) in the cytoplasm of the NPC-derived cells, while the increased expression of lysine-specific histone demethylase 1 (LSD1, a repressive nuclear factor) reduces the response mediated by remaining NF-κB at the promoters responding to inflammatory stimuli. This refractory response in NPC cells might be a consequence of long-term exposure to microbes in vivo during carcinogenic progression. It may contribute to the decreased antitumor immune responses in NPC, among others despite heavy T-helper cell infiltration, and thus facilitate tumor progression.

Epigenetic Inactivation of Acetyl-CoA Acetyltransferase 1 Promotes the Proliferation and Metastasis in Nasopharyngeal Carcinoma by Blocking Ketogenesis.

The dysregulation of epigenetic modification and energy metabolism cooperatively contribute to the tumorigenesis of nasopharyngeal carcinoma (NPC). However, the detailed mechanisms underlying their joint contribution to NPC development and progression remain unclear. Here, we investigate the role of Acy1 Coenzyme A Acyltransferases1 (ACAT1), a key enzyme in the metabolic pathway of ketone bodies, in the proliferation and metastasis of NPC and to elucidate the underlying molecular mechanisms. Ketogenesis, plays a critical role in tumorigenesis. Previously, we reported two enzymes involved in ketone body metabolism mediate epigenetic silencing and act as tumor suppressor genes in NPC. Here, we identify another key enzyme, Acetyl-CoA acetyltransferase 1 (ACAT1), and show that its transcriptional inactivation in NPC is due to promoter hypermethylation. Ectopic overexpression of ACAT1 significantly suppressed the proliferation and colony formation of NPC cells in vitro. The migratory and invasive capacity of NPC cells was inhibited by ACAT1. The tumorigenesis of NPC cells overexpressing ACAT1 was decreased in vivo. Elevated ACAT1 in NPC cells was accompanied by an elevated expression of CDH1 and a reduced expression of vimentin and SPARC, strongly indicating that ACAT1 is involved in regulating epithelial-mesenchymal transition (EMT). We also found that ACAT1 contributes to increased intracellular levels of ß-hydroxybutyrate (ß-HB). Exogenously supplied ß-HB significantly inhibits the growth of NPC cells in a dose-dependent manner. In summary, ACAT1 may function as a tumor suppressor via modulation of ketogenesis and could thus serve as a potential therapeutic target in NPC. In summary, our data suggest that regulation of ketogenesis may serve as adjuvant therapy in NPC.

Inverse correlation of miR-27a-3p and CDH5 expression serves as a diagnostic biomarker of proliferation and metastasis of clear cell renal carcinoma.

BACKGROUND: Cadherin-5 (CDH5) is aberrantly expressed in a variety of human cancers and plays an important role in angiogenesis. The present study provides further insight into the role of miR-27a-3p in the regulation of CDH5 expression in renal clear cell carcinoma (ccRCC). METHODS: Thedysregulation of CDH5 expression in ccRCC and its association with clinicopathological characteristics were analyzed using the TCGA database. A meta-analysis was performed to verify the alteration of CDH5 expression in ccRCC using the GEO database. Quantitative RT-PCR and immunohistochemical staining were applied to assess the transcriptional and protein levels of CDH5. TargetScan and Tarbase were employed to predict the miRNAs with the potential to target mRNA of CDH5. RESULTS: The mRNA level of CDH5 in ccRCCwas significantly higher than in normal tissue. CDH5 mRNA expression could therefore serve as a potential diagnostic biomarker for ccRCC (AUC = 0.844). However, the reduced CDH5 transcription levels were significantly correlated with patients in the T3-4 stage, lymph node, and distant metastasis, as well as with a worse clinical outcome. We further observed that CDH5, at the protein level, was almost absent in ccRCC samples. In addition, a few databases screen showed that mir-27a-3p is a highly conserved miRNA targeting CDH5. The expression of mir-27a-3p was significantly elevated in ccRCC tissues in contrast to normal tissues. Importantly, it was positively associated with the T3-4 stage and M stage, respectively, suggesting that the expression level of mir-27a-3p could serve as a diagnostic biomarker for ccRCC (AUC = 0.775). CONCLUSION: Our data suggest that thereduced translational level of CDH5 in ccRCC was related to the overexpression of mir-27a-3p. The higher mir-27a-3p and lower CDH5 expression significantly correlated with advanced clinical stages for ccRCC patients.

Downregulation of SLC27A6 by DNA Hypermethylation Promotes Proliferation but Suppresses Metastasis of Nasopharyngeal Carcinoma Through Modulating Lipid Metabolism.

Lipid is the building block and an important source of energy, contributing to the malignant behavior of tumor cells. Recent studies suggested that lipid droplets (LDs) accumulations were associated with nasopharyngeal carcinoma (NPC) progression. Solute carrier family 27 member 6 (SLC27A6) mediates the cellular uptake of long-chain fatty acid (LCFA), a necessary lipid component. However, the functions of SLC27A6 in NPC remain unknown. Here, we found a significant reduction of SLC27A6 mRNA in NPC tissues compared with normal nasopharyngeal epithelia (NNE). The promoter methylation ratio of SLC27A6 was greater in NPC than in non-cancerous tissues. The demethylation reagent 5-aza-2'-deoxycytidine (5-aza-dC) remarkably restored the mRNA expression of SLC27A6, suggesting that this gene was downregulated in NPC owing to DNA promoter hypermethylation. Furthermore, SLC27A6 overexpression level in NPC cell lines led to significant suppression of cell proliferation, clonogenicity in vitro, and tumorigenesis in vivo. Higher SLC27A6 expression, on the other hand, promoted NPC cell migration and invasion. In particular, re-expression of SLC27A6 faciliated epithelial-mesenchymal transition (EMT) signals in xenograft tumors. Furthermore, we observed that SLC27A6 enhanced the intracellular amount of triglyceride (TG) and total cholesterol (T-CHO) in NPC cells, contributing to lipid biosynthesis and increasing metastatic potential. Notably, the mRNA level of SLC27A6 was positively correlated with cancer stem cell (CSC) markers, CD24 and CD44. In summary, DNA promoter hypermethylation downregulated the expression of SLC27A6. Furthermore, re-expression of SLC27A6 inhibited the growth capacity of NPC cells but strengthened the CSC markers. Our findings revealed the dual role of SLC27A6 in NPC and shed novel light on the link between lipid metabolism and CSC maintenance.

Combination of RERG and ZNF671 methylation rates in circulating cell-free DNA: A novel biomarker for screening of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia, hence, identifying easily detectable biomarkers for NPC screening is essential for better diagnosis and prognosis. Using genome-wide and targeted analyses based on next-generation sequencing approaches, we previously showed that gene promoters are hypermethylated in NPC tissues. To confirm whether DNA methylation rates of genes could be used as biomarkers for NPC screening, 79 histologically diagnosed NPC patients and 29 noncancer patients were recruited. A convenient quantitative analysis of DNA methylation using real-time PCR (qAMP) was carried out, involving pretreatment of tissue DNA, and circulating cell-free DNA (ccfDNA) from nonhemolytic plasma, with methylation-sensitive and/or methylation-dependent restriction enzymes. The qAMP analyses revealed that methylation rates of RERG, ZNF671, ITGA4, and SHISA3 were significantly higher in NPC primary tumor tissues compared to noncancerous tissues, with sufficient diagnostic accuracy of the area under receiver operating characteristic curves (AUC). Interestingly, higher methylation rates of RERG in ccfDNA were statistically significant and yielded a very good AUC; however, those of ZNF671, ITGA4, and SHISA3 were not significant. Furthermore, the combination of methylation rates of RERG and ZNF671 in ccfDNA showed higher diagnostic accuracy than either of them individually. In conclusion, the methylation rates of specific genes in ccfDNA can serve as novel biomarkers for early detection and screening of NPC.

Knockdown Rab11-FIP2 inhibits migration and invasion of nasopharyngeal carcinoma via suppressing Rho GTPase signaling.

Rab11 family interacting protein 2 (Rab11-FIP2) is a conserved protein and effector molecule for the small GTPase Rab11. By interacting with Rab11 and MYO5B, Rab11-FIP2 regulates endosome trafficking of plasma membrane proteins, promoting cellular motility. The endosomal trafficking system in nasopharyngeal carcinoma (NPC) remains unclear. Here, an outlier analysis using the Oncomine database suggested that Rab11-FIP2 but not Rab11 and MYO5B was overexpressed in NPC. We confirmed that the transcription of Rab11-FIP2 was upregulated in NPC cell lines and primary tumor tissues as compared with a normal nasopharyngeal epithelial cell line and normal nasopharynx tissues. We further confirmed the elevated protein expression level of Rab11-FIP2 in NPC biopsies. Instead of regulating the epithelial-mesenchymal transition or Akt signaling pathway, knockdown of Rab11-FIP2 inhibited the migration and invasion ability of NPC cell lines by decreasing the expression of Rac and Cdc42. In summary, Rab11-FIP2 could be an oncogene in NPC, mainly contributing to metastatic capacity by activating Rho GTPase signaling.

Inactivation of 3-hydroxybutyrate dehydrogenase type 2 promotes proliferation and metastasis of nasopharyngeal carcinoma by iron retention.

BACKGROUND: 3-Hydroxybutyrate dehydrogenase type 2 (BDH2) is known to catalyse a rate-limiting step in the biogenesis of the mammalian siderophore and regulate intracellular iron metabolism. Here we aim to explore the expression and possible function of BDH2 in nasopharyngeal carcinoma (NPC). METHODS: The transcription and protein expression of BDH2 in NPC were determined by both real-time RT-PCR and immunohistochemistry staining assays. Cell proliferation, migration and invasion were evaluated by MTT assay, wound-healing assay and Transwell assay, respectively. The profile of genes regulated by restoring BDH2 expression in NPC cells was analysed by cDNA microarray. The level of iron in NPC cells was detected by iron colorimetric assay. RESULTS: The expression of BDH2 was significantly downregulated in NPC. Ectopic expression of BDH2 inhibited NPC cell proliferation and colony formation. Meanwhile, BDH2 suppressed the migration and invasion of NPC cells by reversing the epithelial-mesenchymal transition (EMT). In addition, a higher level of BDH2 decreased the growth and metastasis of NPC cells via reducing intracellular iron level. CONCLUSIONS: Our findings suggest that BDH2 may be a candidate tumour-suppressor gene in NPC. Decreasing intracellular iron could be an effective therapeutic approach for NPC.

Anti-Cancer Mechanisms of Taurine in Human Nasopharyngeal Carcinoma Cells.

Taurine displays anti-tumor activity in some kinds of human cancers. However, the underlying mechanisms are poorly understood. Epstein-Barr virus-related nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck cancer in Southeast Asia with the highest incidence in South China. We examined an apoptosis-inducing effect of taurine against NPC cells (HK1 and HK1-EBV) to clarify the mechanisms of anti-tumor effects of taurine by immunocytochemical methods. We observed that taurine induced cleavage of caspase-9/3 in a concentration-dependent manner, suggesting the involvement of mitochondrial apoptotic signals. Both PTEN and p53 activation were detected in a dose-dependent manner after taurine treatment in NPC cells. In conclusion, taurine may play an anti-tumor role by activating tumor suppressor PTEN and p53.

Dysregulation of Ketone Body Metabolism Is Associated With Poor Prognosis for Clear Cell Renal Cell Carcinoma Patients.

Kidney is an important organ for ketone body metabolism. However, the role of abnormal ketone metabolism and its possible function in tumorigenesis of clear cell renal cell carcinoma (ccRCC) have not yet been elucidated. Three differentially expressed key enzymes involved in ketone body metabolism, ACAT1, BDH2, and HMGCL, were screened out between ccRCC and normal kidney tissues using the GEO and TCGA databases.We confirmed that the transcription and protein expression of ACAT1, BDH2, and HMGCL were significantly lower in ccRCC by real-time RT-PCR and IHC assays. Those patients with lower expression of these three genes have a worse outcome. In addition, we demonstrated that ectopic expression of each of these genes inhibited the proliferation of ccRCC cells. The overexpressed ACAT1 and BDH2 genes remarkably impeded the migratory and invasive capacity of ccRCC cells. Furthermore, exogenous ß-hydroxybutyrate suppressed the growth of ccRCC cells in vitro in a dose-dependent manner. Our findings suggest that ACAT1, BDH2, and HMGCL are potential tumor suppressor genes, and constitute effective prognostic biomarkers for ccRCC. Ketone body metabolism might thus be a promising target in a process for developing novel therapeutic approaches to treat ccRCC.

Inactivation of HMGCL promotes proliferation and metastasis of nasopharyngeal carcinoma by suppressing oxidative stress.

Altered metabolism is considered as a hallmark of cancer. Here we investigated expression of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 2 lyase (HMGCL), an essential enzyme in ketogenesis, which produces ketone bodies by the breakdown of fatty acids to supply energy, in nasopharyngeal carcinoma (NPC). The expression of HMGCL was silenced in NPC tissue. Downregulation of HMGCL in NPC was associated with low intracellular ß-hydroxybutyrate (ß-HB) production, thereby reducing reactive oxygen species (ROS) generation. Ectopic expression of HMGCL restored ß-HB level, associated with suppressed proliferation and colony formation of NPC cells in vitro and decreased tumorigenicity in vivo. HMGCL suppressed the migration and invasion of NPC cells in vitro via mesenchymal-epithelial transition. Furthermore, extracellular ß-HB supply suppressed the proliferation and migration of NPC cells. Both intra- and extracellular ß-HB exerting a suppressive role in NPC depends on ROS generation. Ketogenesis may be impaired in NPC cells due to lack of HMGCL expression, suggesting that it may be a promising target in NPC therapy.

Degradation of cofilin is regulated by Cbl, AIP4 and Syk resulting in increased migration of LMP2A positive nasopharyngeal carcinoma cells.

Expression of cofilin is directly associated with metastatic activity in many tumors. Here, we studied the role of Latent Membrane Protein 2 A (LMP2A) of Epstein-Barr Virus (EBV) in the accumulation of cofilin observed in nasopharyngeal cancer (NPC) tumor cells. We used LMP2A transformed NPC cell lines to analyze cofilin expression. We used mutation analysis, ectopic expression and down-regulation of Cbl, AIP4 and Syk in these cell lines to determine the effect of the LMP2A viral protein on cofilin degradation and its role in the assembly of a cofilin degrading protein complex. The LMP2A of EBV was found to interfer with cofilin degradation in NPC cells by accelerating the proteasomal degradation of Cbl and Syk. In line with this, we found significantly higher cofilin expression in NPC tumor samples as compared to the surrounding epithelial tissues. Cofilin, as an actin severing protein, influences cellular plasticity, and facilitates cellular movement in response to oncogenic stimuli. Thus, under relaxed cellular control, cofilin facilitates tumor cell movement and dissemination. Interference with its degradation may enhance the metastatic potential of NPC cells.

Quantitation of DNA methylation in Epstein-Barr virus-associated nasopharyngeal carcinoma by bisulfite amplicon sequencing.

BACKGROUND: Epigenetic changes, including DNA methylation, disrupt normal cell function, thus contributing to multiple steps of carcinogenesis. Nasopharyngeal carcinoma (NPC) is endemic in southern China and is highly associated with Epstein-Barr virus (EBV) infection. Significant changes of the host cell methylome are observed in EBV-associated NPC with cancer development. Epigenetic marks for NPC diagnosis are urgently needed. In order to explore DNA methylation marks, we investigated DNA methylation of candidate genes in EBV-associated nasopharyngeal carcinoma. METHODS: We first employed methyl-capture sequencing and cDNA microarrays to compare the genome-wide methylation profiles of seven NPC tissues and five non-cancer nasopharyngeal epithelium (NNE) tissues. We found 150 hypermethylated CpG islands spanning promoter regions and down-regulated genes. Furthermore, we quantified the methylation rates of seven candidate genes using bisulfite amplicon sequencing for nine NPC and nine NNE tissues. RESULTS: All seven candidate genes showed significantly higher methylation rates in NPC than in NNE tissues, and the ratios (NPC/NNE) were in descending order as follows: ITGA4 > RERG > ZNF671 > SHISA3 > ZNF549 > CR2 > RRAD. In particular, methylation levels of ITGA4, RERG, and ZNF671 could distinguish NPC patients from NNE subjects. CONCLUSIONS: We identified the DNA methylation rates of previously unidentified NPC candidate genes. The combination of genome-wide and targeted methylation profiling by next-generation sequencers should provide useful information regarding cancer-specific aberrant methylation.