Comprehensive Profiling of EBV Gene Expression and Promoter Methylation Reveals Latency II Viral Infection and Sporadic Abortive Lytic Activation in Peripheral T-Cell Lymphomas.

Epstein-Barr virus (EBV) latency patterns are well defined in EBV-associated epithelial, NK/T-cell, and B-cell malignancies, with links between latency stage and tumorigenesis deciphered in various studies. In vitro studies suggest that the oncogenic activity of EBV in T-cells might be somewhat different from that in EBV-tropic B lymphoid cells, prompting us to study this much less investigated viral gene expression pattern and its regulation in nine EBV+ peripheral T-cell lymphoma (PTCL) biopsies. Using frozen specimens, RT-PCR showed 6/7 cases with a latency II pattern of EBV gene expression. Analyses of EBNA1 promoter usage and CpG methylation status in these six cases showed that only Qp was used, while Cp, Wp, and Fp were all silent. However, the remaining case showed an exceptionally unique latency III type with lytic activation, as evidenced by EBV lytic clonality and confirmed by the full usage of Cp and Qp as well as weakly lytic Fp and Wp, fully unmethylated Cp and marginally unmethylated Wp. Further immunostaining of the eight cases revealed a few focally clustered LMP1+ cells in 7/8 cases, with rare isolated LMP1+ cells detected in another case. Double immunostaining confirmed that the LMP1+ cells were of the T-cell phenotype (CD3+). In 6/8 cases, sporadically scattered Zta+ cells were detected. Double staining of EBER-ISH with T-cell (CD45RO/UCHL1) or B-cell (CD20) markers confirmed that the vast majority of EBER+ cells were of the T-cell phenotype. Predominant type-A EBV variant and LMP1 30-bp deletion variant were present, with both F and f variants detected. In summary, the EBV gene expression pattern in PTCL was found to be mainly of latency II (BART+EBNA1(Qp)+LMP1+LMP2A+BZLF1+), similar to that previously reported in EBV-infected nasopharyngeal epithelial, NK/T-cell, and Hodgkin malignancies; however, fully lytic infection could also be detected in occasional cases. Rare cells with sporadic immediate-early gene expression were commonly detected in PTCL. These findings have implications for the future development of EBV-targeting therapeutics for this cancer.

Erratum: Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas: Erratum.

[This corrects the article DOI: 10.7150/thno.20893.].

Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas.

Rationale: Oncogenic STAT3 signaling activation and 3p22-21.3 locus alteration are common in multiple tumors, especially carcinomas of the nasopharynx, esophagus and lung. Whether these two events are linked remains unclear. Our CpG methylome analysis identified a 3p22.2 gene, DLEC1, as a methylated target in esophageal squamous cell (ESCC), nasopharyngeal (NPC) and lung carcinomas. Thus, we further characterized its epigenetic abnormalities and functions. Methods: CpG methylomes were established by methylated DNA immunoprecipitation. Promoter methylation was analyzed by methylation-specific PCR and bisulfite genomic sequencing. DLEC1 expression and clinical significance were analyzed using TCGA database. DLEC1 functions were analyzed by transfections followed by various cell biology assays. Protein-protein interaction was assessed by docking, Western blot and immunoprecipitation analyses. Results: We defined the DLEC1 promoter within a CpG island and p53-regulated. DLEC1 was frequently downregulated in ESCC, lung and NPC cell lines and primary tumors, but was readily expressed in normal tissues and immortalized normal epithelial cells, with mutations rarely detected. DLEC1 methylation was frequently detected in ESCC tumors and correlated with lymph node metastasis, tumor recurrence and progression, with DLEC1 as the most frequently methylated among the established 3p22.2 tumor suppressors (RASSF1A, PLCD1 and ZMYND10/BLU). DLEC1 inhibits carcinoma cell growth through inducing cell cycle arrest and apoptosis, and also suppresses cell metastasis by reversing epithelial-mesenchymal transition (EMT) and cell stemness. Moreover, DLEC1 represses oncogenic signaling including JAK/STAT3, MAPK/ERK, Wnt/ß-catenin and AKT pathways in multiple carcinoma types. Particularly, DLEC1 inhibits IL-6-induced STAT3 phosphorylation in a dose-dependent manner. DLEC1 contains three YXXQ motifs and forms a protein complex with STAT3 via protein docking, which blocks STAT3-JAK2 interaction and STAT3 phosphorylation. IL-6 stimulation enhances the binding of DLEC1 with STAT3, which diminishes their interaction with JAK2 and further leads to decreased STAT3 phosphorylation. The YXXQ motifs of DLEC1 are crucial for its inhibition of STAT3 phosphorylation, and disruption of these motifs restores STAT3 phosphorylation through abolishing DLEC1 binding to STAT3. Conclusions: Our study demonstrates, for the first time, predominant epigenetic silencing of DLEC1 in ESCC, and a novel mechanistic link of epigenetic DLEC1 disruption with oncogenic STAT3 signaling in multiple carcinomas.

KIF7 attenuates prostate tumor growth through LKB1-mediated AKT inhibition.

This study investigated kinesin family member 7 (KIF7) expression and function in prostate cancer (PCa). Our results showed that KIF7 was significantly downregulated in PCa, compared with normal, benign prostatic hyperplasia and prostate intraepithelial neoplasia tissues, partially through promoter hypermethylation. We further investigated the effects of KIF7 coiled coil (CC) domain and motor domain (MD) on PCa development in vitro and in vivo. Our results showed that KIF7-CC but not KIF7-MD significantly attenuated proliferation and colony formation, impeded migration and invasion, induced apoptosis and sensitized PCa cells to paclitaxel. Further analysis revealed that KIF7-CC enhanced LKB1 expression and phosphorylation at Ser428, which induced PTEN phosphorylation at Ser380/Thr382/383 and consequently blocked AKT phosphorylation at Ser473. Downregulation of LKB1 significantly attenuated the suppressive effects of KIF7-CC on cell proliferation, colony formation and AKT phosphorylation. Furthermore, our in vivo studies showed that KIF7-CC reduced prostate tumorigenesis in cell-derived xenografts. Downregulation of LKB1 abrogated the anti-tumor effects of KIF7-CC in these xenografts. Taken together, these findings provide the first evidence to support the role of KIF7 as a negative regulator that inhibits PCa development partially through LKB1-mediated AKT inhibition.

Receptor-type tyrosine-protein phosphatase κ directly targets STAT3 activation for tumor suppression in nasal NK/T-cell lymphoma.

Nasal-type natural killer/T-cell lymphoma (NKTCL) is an aggressive disease characterized by frequent deletions on 6q, and constitutive activation of signal transducer and activator of transcription 3 (STAT3). Phosphorylation at Tyr705 activates STAT3, inducing dimerization, nuclear translocation, and DNA binding. In this study, we investigated whether receptor-type tyrosine-protein phosphatase κ (PTPRK), the only protein tyrosine phosphatase at 6q that contains a STAT3-specifying motif, negatively regulates STAT3 activation in NKTCL. PTPRK was highly expressed in normal NK cells but was underexpressed in 4 of 5 (80%) NKTCL cell lines and 15 of 27 (55.6%) primary tumors. Significantly, PTPRK protein expression was inversely correlated with nuclear phospho-STAT3(Tyr705) expression in NKTCL cell lines (P = .025) and tumors (P = .040). PTPRK restoration decreased nuclear phospho-STAT3(Tyr705) levels, whereas knockdown of PTPRK increased such levels in NKTCL cells. Phosphatase substrate-trapping mutant assays demonstrated the binding of PTPRK to STAT3, and phosphatase assays showed that PTPRK directly dephosphorylated phospho-STAT3(Tyr705). Restoration of PTPRK inhibited tumor cell growth and reduced the migration and invasion ability of NKTCL cells. Monoallelic deletion and promoter hypermethylation caused underexpression of PTPRK messenger RNA in NKTCL, and methylation of the PTPRK promoter significantly correlated with inferior overall survival (P = .049) in NKTCL patients treated with the steroid-dexamethasone, methotrexate, ifosfamide, l-asparaginase, and etoposide regimen. Altogether, our findings show that PTPRK underexpression leads to STAT3 activation and contributes to NKTCL pathogenesis.

Rab25 is a tumor suppressor gene with antiangiogenic and anti-invasive activities in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is a devastating disease characterized by distinctly high incidences and mortality rates. However, there remains limited understanding of molecular events leading to development and progression of the disease, which are of paramount importance to defining biomarkers for diagnosis, prognosis, and personalized treatment. By high-throughout transcriptome sequence profiling of nontumor and ESCC clinical samples, we identified a subset of significantly differentially expressed genes involved in integrin signaling. The Rab25 gene implicated in endocytic recycling of integrins was the only gene in this group significantly downregulated, and its downregulation was confirmed as a frequent event in a second larger cohort of ESCC tumor specimens by quantitative real-time PCR and immunohistochemical analyses. Reduced expression of Rab25 correlated with decreased overall survival and was also documented in ESCC cell lines compared with pooled normal tissues. Demethylation treatment and bisulfite genomic sequencing analyses revealed that downregulation of Rab25 expression in both ESCC cell lines and clinical samples was associated with promoter hypermethylation. Functional studies using lentiviral-based overexpression and suppression systems lent direct support of Rab25 to function as an important tumor suppressor with both anti-invasive and -angiogenic abilities, through a deregulated FAK-Raf-MEK1/2-ERK signaling pathway. Further characterization of Rab25 may provide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target in ESCC treatment.

Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells.

Deregulation of nuclear factor (NF)-kappaB signalling is common in cancers and is essential for tumourigenesis. Constitutive NF-kappaB activation in extranodal natural killer (NK)-cell lymphoma, nasal type (ENKL) is known to be associated with aberrant nuclear translocation of BCL10. Here we investigated the mechanisms leading to NF-kappaB activation and BCL10 nuclear localization in ENKLs. Given that ENKLs are dependent on T-cell-derived interleukin-2 (IL2) for cytotoxicity and proliferation, we investigated whether IL2 modulates NF-kappaB activation and BCL10 subcellular localization in ENKLs. In the present study, IL2-activated NK lymphoma cells were found to induce NF-kappaB activation via the PI3K/Akt pathway, leading to an increase in the entry of G(2)/M phase and concomitant transcription of NF-kappaB-responsive genes. We also found that BCL10, a key mediator of NF-kappaB signalling, participates in the cytokine receptor-induced activation of NF-kappaB. Knockdown of BCL10 expression resulted in deficient NF-kappaB signalling, whereas Akt activation was unaffected. Our results suggest that BCL10 plays a role downstream of Akt in the IL2-triggered NF-kappaB signalling pathway. Moreover, the addition of IL2 to NK cells led to aberrant nuclear translocation of BCL10, which is a pathological feature of ENKLs. We further show that BCL10 can bind to BCL3, a transcriptional co-activator and nuclear protein. Up-regulation of BCL3 expression was observed in response to IL2. Similar to BCL10, the expression and nuclear translocation of BCL3 were induced by IL2 in an Akt-dependent manner. The nuclear translocation of BCL10 was also dependent on BCL3 because silencing BCL3 by RNA interference abrogated this translocation. We identified a critical role for BCL10 in the cytokine receptor-induced NF-kappaB signalling pathway, which is essential for NK cell activation. We also revealed the underlying mechanism that controls BCL10 nuclear translocation in NK cells. Our findings provide insight into a molecular network within the NF-kappaB signalling pathway that promotes the pathogenesis of NK cell lymphomas.

CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation.

Using inverse polymerase chain reaction, we identified CD44, located on chromosome 11p13, as a novel translocation partner of IGH in 9 of 114 cases of gastric, nongastric extranodal, follicular, and nodal diffuse large B-cell lymphoma (DLBCL). Notably, these translocations involving IGHSmu were detected in follicular lymphomas and exclusively in germinal center B cell-ike (GCB)-DLBCLs. CD44 is not expressed in reactive GC B cells. The IGHSmu/CD44 translocations substitute Smu for the CD44 promoter and remove exon 1 of CD44, resulting in the overexpression of Imu-CD44 hybrid mRNA transcripts activated from derivative 11 that encode a new CD44 variant lacking the leader peptide and with a unique C-terminus (CD44DeltaEx1). When overexpressed in vitro in the CD44(-) GCB-DLBCL cell line BJAB, CD44DeltaEx1-green fluorescent protein localized to the cytoplasm and nucleus, whereas CD44s-green fluorescent protein (standard form) localized to the plasma membrane. The ectopic expression of CD44DeltaEx1 in BJAB cells enhanced their proliferation rate and clonogenic ability, indicating a possible pathogenic role of the translocation.

DNA fingerprinting tags novel altered chromosomal regions and identifies the involvement of SOX5 in the progression of prostate cancer.

Identification of genomic alterations associated with the progression of prostate cancer may facilitate the better understanding of the development of this highly variable disease. Matched normal, premalignant high-grade prostatic intraepithelial neoplasia and invasive prostate carcinoma cells were procured by laser capture microdissection (LCM) from human radical prostatectomy specimens. From these cells, comparative DNA fingerprints were generated by a modified PCR-based technique called scanning of microdissected archival lesion (SMAL)-PCR. Recurrent polymorphic fingerprint fragments were used in tagging altered chromosomal regions. Altered regions were found at cytobands 1p31.3, 1q44, 2p23.1, 3p26.3, 3q22.3, 4q22.3, 4q35.2, 5q23.2, 8q22.3, 8q24.13, 9q21.3, 9q22.32, 10q11.21, 11p13, 12p12.1, 13q12.1, 16q12.2 and 18q21.31. Candidate genes in the surrounding area that may possibly harbor mutations that change normal prostatic cells to progress into their tumor stages were proposed. Of these fragments, a 420 bp alteration, absent in all 26 normal samples screened, was observed in 2 tumors. This fragment was cloned, sequenced and localized to chromosome 12p12.1. Within this region, candidate gene sex determining region Y-box 5 (SOX5) was proposed. Further studies of SOX5 in cell lines, xenografts and human prostate specimens, at both the RNA and protein levels, found overexpression of the gene in tumors. This overexpression was then subsequently found by fluorescent in situ hybridization to be caused by amplification of the region. In conclusion, our results suggest LCM coupled with SMAL-PCR DNA fingerprinting is a useful method for the screening and identification of chromosomal regions and genes associated with cancer development. Further, overexpression of SOX5 is associated with prostate tumor progression and early development of distant metastasis.

Cell death by bortezomib-induced mitotic catastrophe in natural killer lymphoma cells.

The proteasome inhibitor bortezomib (PS-341/Velcade) is used for the treatment of relapsed and refractory multiple myeloma and mantle-cell lymphoma. We recently reported its therapeutic potential against natural killer (NK)-cell neoplasms. Here, we investigated the molecular mechanisms of bortezomib-induced cell death in NK lymphoma cells. NK lymphoma cell lines (SNK-6 and NK-YS) and primary cultures of NK lymphomas treated with bortezomib were examined for alterations in cell viability, apoptosis, cellular senescence, and cell cycle status. Bortezomib primarily induced mitochondrial apoptosis in NK-YS cells and in primary lymphoma cells at the same concentration as reported in myeloma cells. Unexpectedly, SNK-6 cells required a significantly higher median inhibitory concentration of bortezomib (23 nmol/L) than NK-YS and primary lymphoma cells (6-13 nmol/L). Apoptosis was limited in SNK-6 cells due to the extensively delayed turnover of Bcl-2 family members. These cells were killed by bortezomib, albeit at higher pharmacologic concentrations, via mitotic catastrophe-a mitotic cell death associated with M-phase arrest, cyclin B1 accumulation, and increased CDC2/CDK1 activity. Our results suggest that, in addition to cell death by apoptosis at lower bortezomib concentrations, NK lymphoma cells resistant to bortezomib-induced apoptosis can be killed via mitotic catastrophe, an alternative cell death mechanism, at higher pharmacologic concentrations of bortezomib. Hence, activating mitotic catastrophe by bortezomib may provide a novel therapeutic approach for treating apoptosis-resistant NK-cell malignancies and other cancers.

Aberrant promoter hypermethylation and silencing of the critical 3p21 tumour suppressor gene, RASSF1A, in Chinese oesophageal squamous cell carcinoma.

3p21 is an important locus harbouring critical tumour suppressor genes (TSG), which are implicated in the pathogenesis of multiple tumours, including oesophageal carcinoma. RASSF1A is a 3p21.3 candidate TSG frequently inactivated by promoter methylation in multiple tumours. We investigated RASSF1A promoter methylation and gene expression in Chinese oesophageal squamous cell carcinoma (ESCC) to compare it to data from Japanese patients. Methylation-specific PCR (MSP) showed that RASSF1A was partially methylated in 3/7 (43%) cell lines; 22/64 (34%) primary tumours and 3/64 (5%) corresponding non-tumour samples; and was not methylated in 2 immortalized normal oesophageal epithelial cell lines and 6 normal oesophageal epithelium samples. Bisulfite genome sequencing confirmed the MSP results. Promoter hypermethylation correlated well with RASSF1A mRNA down-regulation. Treatment of cell lines with 5-aza-2'-deoxycytidine activated RASSF1A mRNA expression along with promoter demethylation. RASSF1A hypermethylation in the Chinese cohort was much lower than in a published report of Japanese ESCC patients (52%) and cell lines (74%). Our own analysis of Japanese ESCC cell lines for direct comparison also detected a high frequency of RASSF1A hypermethylation (8/10; 80%) and high levels of hypermethylation at each CpG site. No significant association between RASSF1A hypermethylation and histological differentiation (p=0.953), tumour staging (p=0.117), or survival (p=0.7571) was found in Chinese ESCC, unlike the results of Japanese patients. The incidence of oesophageal cancer shows marked variation by geographic area and ethnic group; it is almost three times higher in China than in Japan, indicating possible different pathogenetic mechanisms. Our results show that RASSF1A hypermethylation in ESCC has epidemiological/ethnic differences, and suggest that Chinese ESCC may result from different pathogenetic mechanisms.