Multiple DNA methylation changes in the cancer methylome are associated with the acquisition of drug resistance; however it remains uncertain how many represent critical DNA methylation drivers of chemoresistance. Using isogenic, cisplatin-sensitive/resistant ovarian cancer cell lines and inducing resensitizaton with demethylating agents, we aimed to identify consistent methylation and expression changes associated with chemoresistance. Using genome-wide DNA methylation profiling across 27 578 CpG sites, we identified loci at 4092 genes becoming hypermethylated in chemoresistant A2780/cp70 compared with the parental-sensitive A2780 cell line. Hypermethylation at gene promoter regions is often associated with transcriptional silencing; however, expression of only 245 of these hypermethylated genes becomes downregulated in A2780/cp70 as measured by microarray expression profiling. Treatment of A2780/ cp70 with the demethylating agent 2-deoxy-5 0 -azacytidine induces resensitization to cisplatin and re-expression of 41 of the downregulated genes. A total of 13/41 genes were consistently hypermethylated in further independent cisplatin-resistant A2780 cell derivatives. CpG sites at 9 of the 13 genes (ARHGDIB, ARMCX2, COL1A, FLNA, FLNC, MEST, MLH1, NTS and PSMB9) acquired methylation in ovarian tumours at relapse following chemotherapy or chemoresistant cell lines derived at the time of patient relapse. Furthermore, 5/13 genes (ARMCX2, COL1A1, MDK, MEST and MLH1) acquired methylation in drug-resistant ovarian cancersustaining (side population) cells. MLH1 has a direct role in conferring cisplatin sensitivity when reintroduced into cells in vitro. This combined genomics approach has identified further potential key drivers of chemoresistance whose expression is silenced by DNA methylation that should be further evaluated as clinical biomarkers of drug resistance.

Platinum-based chemotherapy, with cytoreductive surgery, is the cornerstone of treatment of advanced ovarian cancer; however, acquired drug resistance is a major clinical obstacle. It has been proposed that subpopulations of tumor cells with stem cell-like properties, such as so-called side populations (SP) that overexpress ABC drug transporters, can sustain the growth of drug-resistant tumor cells, leading to tumor recurrence following chemotherapy. The histone methyltransferase EZH2 is a key component of the polycomb-repressive complex 2 required for maintenance of a stem cell state, and overexpression has been implicated in drug resistance and shorter survival of ovarian cancer patients. We observed higher percentage SP in ascites from patients that have relapsed following chemotherapy compared with chemonaive patients, consistent with selection for this subpopulation during platinum-based chemotherapy. Furthermore, ABCB1 (P-glycoprotein) and EZH2 are consistently overexpressed in SP compared with non-SP from patients' tumor cells. The siRNA knockdown of EZH2 leads to loss of SP in ovarian tumor models, reduced anchorageindependent growth, and reduced tumor growth in vivo. Together, these data support a key role for EZH2 in the maintenance of a drug-resistant, tumor-sustaining subpopulation of cells in ovarian cancers undergoing chemotherapy. As such, EZH2 is an important target for anticancer drug development.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy with a unique geographical distribution. The genomic abnormalities leading to NPC pathogenesis remain unclear. In total, 135 NPC tumors were examined to characterize the mutational landscape using whole-exome sequencing and targeted resequencing. An APOBEC cytidine deaminase mutagenesis signature was revealed in the somatic mutations. Noticeably, multiple loss-of-function mutations were identified in several NF-κB signaling negative regulators NFKBIA, CYLD, and TNFAIP3. Functional studies confirmed that inhibition of NFKBIA had a significant impact on NF-κB activity and NPC cell growth. The identified loss-of-function mutations in NFKBIA leading to protein truncation contributed to the altered NF-κB activity, which is critical for NPC tumorigenesis. In addition, somatic mutations were found in several cancer-relevant pathways, including cell cycle-phase transition, cell death, EBV infection, and viral carcinogenesis. These data provide an enhanced road map for understanding the molecular basis underlying NPC.nasopharyngeal carcinoma | somatic mutation landscape | NF-κB signaling | whole-exome sequencing | APOBEC-mediated signature

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

Nasopharyngeal carcinoma (NPC) is a unique epithelial malignancy that shows a remarkable geographical and ethic distribution. Multiple factors including predisposing genetic factors, environmental carcinogens, and Epstein-Barr virus (EBV) infection contribute to the accumulation of genetic and epigenetic alterations leading to NPC development. Emerging technologies now allow us to detailedly characterize and understand cancer genomes. Genome-wide studies show that typically NPC tumors are characterized as having comparatively low mutation rates, widespread hypermethylation, and frequent copy number alterations and chromosome abnormalities. In this review, we provide an updated overview of the genetic and epigenetic aberrations that likely drive nasopharyngeal tumor development and progression. We integrate the previous knowledge and novel findings from whole-exome sequencing (WES) and methylome studies in NPC, and further discuss the potential use of these findings to identify biomarkers for NPC diagnosis and prognosis.

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.

BackgroundMicroRNA-21 (miR-21) plays an important role in the pathogenesis and progression of liver fibrosis. Here, we determined the serum and hepatic content of miR-21 in patients with liver cirrhosis and rats with dimethylnitrosamine-induced hepatic cirrhosis and examined the effects of miR-21 on SPRY2 and HNF4α in modulating ERK1 signaling in hepatic stellate cells (HSCs) and epithelial-mesenchymal transition (EMT) of hepatocytes.MethodsQuantitative RT-PCR was used to determine miR-21 and the expression of SPRY2, HNF4α and other genes. Immunoblotting assay was carried out to examine the expression of relevant proteins. Luciferase reporter assay was performed to assess the effects of miR-21 on its predicted target genes SPRY2 and HNF4α. Primary HSCs and hepatocytes were treated with miR-21 mimics/inhibitors or appropriate adenoviral vectors to examine the relation between miR-21 and SPRY2 or HNF4α. ResultsThe serum and hepatic content of miR-21 was significantly higher in cirrhotic patients and rats. SPRY2 and HNF4α mRNA levels were markedly lower in the cirrhotic liver. MiR-21 overexpression was associated with enhanced ERK1 signaling and EMT in liver fibrosis. Luciferase assay revealed suppressed SPRY2 and HNF4α expression by miR-21. Ectopic miR-21 stimulated ERK1 signaling in HSCs and induced hepatocyte EMT by targeting SPRY2 or HNF4α. Downregulating miR-21 suppressed ERK1 signaling, inhibited HSC activation, and blocked EMT in TGFβ1-treated hepatocytes.ConclusionsMiR-21 modulates ERK1 signaling and EMT in liver fibrosis by regulating SPRY2 and HNF4α expression. MiR-21 may serve as a potentially biomarker as well as intervention target for hepatic cirrhosis.

Purpose: Wnt pathways control key biological processes that potentially impact on tumor progression and patient survival. We aimed to evaluate DNA methylation at promoter CpG islands (CGI) of Wnt pathway genes in ovarian tumors at presentation and identify biomarkers of patient progression-free survival (PFS).Experimental Design: Epithelial ovarian tumors (screening study n ¼ 120, validation study n ¼ 61), prospectively collected through a cohort study, were analyzed by differential methylation hybridization at 302 loci spanning 189 promoter CGIs at 137 genes in Wnt pathways. The association of methylation and PFS was examined by Cox proportional hazards model.Results: DNA methylation is associated with PFS at 20 of 302 loci (P < 0.05, n ¼ 111), with 5 loci significant at false discovery rate (FDR) less than 10%. A total of 11 of 20 loci retain significance in an independent validation cohort (n ¼ 48, P 0.05, FDR 10%), and 7 of these loci, at FZD4, DVL1, NFATC3, ROCK1, LRP5, AXIN1, and NKD1 genes, are independent from clinical parameters (adjusted P < 0.05). Increased methylation at these loci associates with increased hazard of disease progression. A multivariate Cox model incorporates only NKD1 and DVL1, identifying two groups differing in PFS [HR ¼ 2.09; 95% CI (1.39-3.15); permutation test P < 0.005]. Methylation at DVL1 and NFATC3 show significant association with response. Consistent with their epigenetic regulation, reduced expression of FZD4, DVL1, and ROCK1 is an indicator of early-disease relapse in an independent ovarian tumor cohort (n ¼ 311, adjusted P < 0.05).Conclusion: The data highlight the importance of epigenetic regulation of multiple promoter CGIs of Wnt pathway genes in ovarian cancer and identify methylation at NKD1 and DVL1 as independent predictors of PFS. Clin Cancer Res; 17(12); 4052-62. Ó2011 AACR.