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Supplementary Table S1. Sanger validated variants. Supplementary Table S2. Mutations in HSTL driver genes. Supplementary Table S3. Other mutations identified by exome sequencing. Supplementary Table S4. Copy number of HSTL patients and cell lines. Supplementary Table S5. Clinical and pathological characteristics of HSTL patients. Supplementary Tables S6, S7. Comparison to HSTL clinical data in the literature. Supplementary Table S8. Mutational frequencies in other lymphomas. Supplementary Table S9. HSTL mutations in other lymphoma driver genes. Supplementary Table S10. Gene set enrichment analysis of DERL2 SETD2 shRNA.

Supplementary Figures S1-S3 from Monochromosome Transfer and Microarray Analysis Identify a Critical Tumor-Suppressive Region Mapping to Chromosome 13q14 and THSD1 in Esophageal Carcinoma

Supplementary Figure S1. Sanger sequencing chromatograms. Supplementary Figure S2. Cancer cell fraction for driver genes. Supplementary Figure S3. Ideogram with chromosome 7 alterations. Supplementary Figure S4. Examples of Exome Copy Number. Supplementary Figure S5. Exploratory Kaplan-Meier plots for clinical covariates. Supplementary Figure S6. Exploratory Kaplan-Meier plots for molecular covariates. Supplementary Figure S7. Sanger and exome sequencing validation of SETD2 biallelic mutation in one HSTL case. Supplementary Figure S8. SETD2 expression in mutant vs. wildtype cases. Supplementary Figure S9. Mutual exclusivity of STAT5B, PIK3CD, and STAT3 mutations.

Loss of chromosome 13q regions in esophageal squamous cell carcinoma (ESCC) is a frequent event. Monochromosome transfer approaches provide direct functional evidence for tumor suppression by chromosome 13 in SLMT-1, an ESCC cell line, and identify critical regions at 13q12.3, 13q14.11, and 13q14.3. Differential gene expression profiles of three tumor-suppressing microcell hybrids (MCH) and their tumorigenic parental SLMT-1 cell line were revealed by competitive hybridization using 19k cDNA oligonucleotide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including RB1, showed down-regulation in SLMT-1, compared with NE1, an immortalized normal esophageal epithelial cell line; their average gene expression was restored in MCHs compared with SLMT-1. Reverse transcription-PCR validated gene expression levels in MCHs and a panel of ESCC cell lines. Results suggest that the tumor-suppressing effect is not attributed to RB1, but instead likely involves thrombospondin type I domain-containing 1 (THSD1), a novel candidate TSG mapping to 13q14. Quantitative reverse transcription-PCR detected down-regulation of THSD1 expression in 100% of ESCC and other cancer cell lines. Mechanisms for THSD1 silencing in ESCC involved loss of heterozygosity and promoter hypermethylation, as analyzed by methylation-specific PCR and clonal bisulfite sequencing. Transfection of wild-type THSD1 into SLMT-1 resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth-suppressive activity. These findings suggest that THSD1 is a good candidate TSG. (Mol Cancer Res 2008;6(4):592–603)

Purpose: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of aggressive lymphomas with the activated B-cell–like subtype characterized by constitutive NF-κB activation. Activating mutations of CARD11 and inactivating mutations of A20 are frequent events in DLBCL. However, the full extent of genetic alterations in the NF-κB pathway regulators and their potential prognostic value in DLBCL remain to be investigated. We investigated the genetic abnormalities of CARD11, A20, and ABIN-1/2/3 (the A20 binding inhibitor of NF-κB) and their clinicopathologic correlation in gastrointestinal DLBCL.Experimental Design: The somatic mutation and copy number changes of CARD11, A20, and ABIN-1/2/3 were investigated in 71 gastrointestinal DLBCLs by PCR/sequencing, and interphase FISH/array comparative genomic hybridization, respectively. The mutations identified were functionally characterized by NF-κB reporter assays and immunoprecipitation experiments.Results: Recurrent somatic mutations were found in CARD11 (10%), A20 (17%), ABIN-1 (4%), and ABIN-2 (3%), but not in ABIN-3. In comparison with the wild-type, all CARD11 mutants were potent NF-κB activators in vitro. On the basis of the destructive nature of the observed mutations, and the findings by reporter assays and immunoprecipitation studies, most if not all of the somatic mutations that were seen in A20, ABIN-1, and ABIN-2 could impair their normal functions. Among these genetic abnormalities, A20 somatic mutation was significantly associated with both poor overall survival and event-free survival.Conclusions: We show further evidence of NF-κB pathway genetic abnormalities in DLBCL, which are potentially valuable in the prognosis and design of future therapeutic strategies. Clin Cancer Res; 17(6); 1440–51. ©2011 AACR.

Supplementary Figures S1-S2; Supplementary Tables S1-S2.

Supplementary Figure 3 from CMTM3, Located at the Critical Tumor Suppressor Locus 16q22.1, Is Silenced by CpG Methylation in Carcinomas and Inhibits Tumor Cell Growth through Inducing Apoptosis

Zinc finger transcription factors are involved broadly in development and tumorigenesis. Here, we report that the little studied zinc finger transcription factor ZNF382 functions as a tumor suppressor in multiple carcinomas. Although broadly expressed in normal tissues, ZNF382 expression was attenuated in multiple carcinoma cell lines due to promoter CpG methylation. ZNF382 was also frequently methylated in multiple primary tumors (nasopharyngeal, esophageal, colon, gastric, and breast). Ectopic expression of ZNF382 in silenced tumor cells significantly inhibited their clonogenicity and proliferation and induced apoptosis. We further found that ZNF382 inhibited NF-κB and AP-1 signaling and downregulated the expression of multiple oncogenes including MYC, MITF, HMGA2, and CDK6, as well as the NF-κB upstream factors STAT3, STAT5B, ID1, and IKBKE, most likely through heterochromatin silencing. ZNF382 could suppress tumorigenesis through heterochromatin-mediated silencing, as ZNF382 was colocalized and interacted with heterochromatin protein HP1 and further changed the chromatin modifications of ZNF382 target oncogenes. Our data show that ZNF382 is a functional tumor suppressor frequently methylated in multiple carcinomas. Cancer Res; 70(16); 6516–26. ©2010 AACR.

ADAMTS metalloprotease family member ADAMTS9 maps to 3p14.2 and shows significant associations with the aerodigestive tract cancers esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). However, the functional impact of ADAMTS9 on cancer development has not been explored. In this study, we evaluated the hypothesized antiangiogenic and tumor-suppressive functions of ADAMTS9 in ESCC and NPC, in stringent tumorigenicity and Matrigel plug angiogenesis assays. ADAMTS9 activation suppressed tumor formation in nude mice. Conversely, knockdown of ADAMTS9 resulted in clones reverting to the tumorigenic phenotype of parental cells. In vivo angiogenesis assays revealed a reduction in microvessel numbers in gel plugs injected with tumor-suppressive cell transfectants. Similarly, conditioned medium from cell transfectants dramatically reduced the tube-forming capacity of human umbilical vein endothelial cells. These activities were associated with a reduction in expression levels of the proangiogenic factors MMP9 and VEGFA, which were consistently reduced in ADAMTS9 transfectants derived from both cancers. Taken together, our results indicate that ADAMTS9 contributes an important function in the tumor microenvironment that acts to inhibit angiogenesis and tumor growth in both ESCC and NPC. Cancer Res; 70(13); 5567–76. ©2010 AACR.

Supplementary Table 1 from Extracellular Protease ADAMTS9 Suppresses Esophageal and Nasopharyngeal Carcinoma Tumor Formation by Inhibiting Angiogenesis

Supplementary Figure 1 from CMTM3, Located at the Critical Tumor Suppressor Locus 16q22.1, Is Silenced by CpG Methylation in Carcinomas and Inhibits Tumor Cell Growth through Inducing Apoptosis

Supplementary Table 1 from KRAB Zinc Finger Protein ZNF382 Is a Proapoptotic Tumor Suppressor That Represses Multiple Oncogenes and Is Commonly Silenced in Multiple Carcinomas

Supplementary Figure 2 from CMTM3, Located at the Critical Tumor Suppressor Locus 16q22.1, Is Silenced by CpG Methylation in Carcinomas and Inhibits Tumor Cell Growth through Inducing Apoptosis

Supplementary Table 2 from KRAB Zinc Finger Protein ZNF382 Is a Proapoptotic Tumor Suppressor That Represses Multiple Oncogenes and Is Commonly Silenced in Multiple Carcinomas

Supplementary Figures Legends 1-3 from CMTM3, Located at the Critical Tumor Suppressor Locus 16q22.1, Is Silenced by CpG Methylation in Carcinomas and Inhibits Tumor Cell Growth through Inducing Apoptosis

INTRODUCTION : Diffuse large B cell lymphoma (DLBCL) is a malignant non-Hodgkin's lymphoma with approximately one third of patients respond to front-line treatment such as R-CHOP. However, most of the patients often relapse within 2-3 years, and their 3-year progression free survival rate is dropped from 70%-10%. Therefore, understanding of the molecular mechanisms that associated with DLBCL relapse to develop the novel therapeutic approach is in an urgent need. METHODS : Diagnosis (n=34) and paired relapse samples (n=37) from 34 patients with DLBCL treated with R-CHOP were retrieved from the pathology archives under a protocol approved by the Institutional Review Board. Of these, matched normal germline controls are also available in 10 cases. We performed whole exome sequencing (WES) using the illumine Hiseq2000 platform. Short sequencing reads were aligned to human genome assembly GRCh37/hg19 using the BWA aligner. Variant calls were made and determined to be either 'diagnosis-specific", "relapse-specific" or shared based on variant allele frequency (VAF) in the diagnosis, relapsed and normal controls. To identify relapse specific variants that may play a driver role in relapse, the potential pathogenicity of all relapse specific variants with VAF > 20 is assessed by the UMD predictor and all predicted pathogenic variants are further annotated by Ingenuity variant analysis. Genes with mutations in 2 relapse samples and none of the diagnostic samples, or mutations in >3 relapse sample and RESULTS: Analysis of the paired DLBCL samples with matched normal controls demonstrates that relapsed DLBCL develop via divergent evolution from a common precursor as the pre-treatment DLBCL. A total of 661 relapse-specific variants from 595 genes are identified from the above pairwise analysis. 548 of them are missense mutations; 69 are nonsense mutations, and 29 are frameshift mutations. Recurrently mutated genes include TP53 (19% of cases), TNFRSF14 (13.5%), ACTG1 (11.4%), CELSR3 (11.4%), LPR1B (11.4%), GUCY2F (11.4%); ARHGEF10L, DNAH9, DSP, EHD2, HIST1H1B, HIVEP1, KIAA2022, MYC, SKI, SLC29A4, SLIT3, EPHB6, PCDHGA6, BRWD1, C17ORF70 (each 8.6%), and 21 other genes (each 5.7%). Notably, a recurrent missense mutation (p.E50V) in EIF4A2 was identified in 2 cases. About 50% of these recurrently mutated genes are more frequently mutated (p CONCLUSION: WES study on a large cohort of relapsed DLBCL with matched pre-treatment tumors provides a comprehensive view of the mutation landscape of relapsed DLBCL. Novel drivers for relapsed tumors are identified; and alterations in pathways previously not implicated in DLBCL pathogenesis are discovered. shRNA and CRISPR screening using a subset of our candidate genes is underway to functionally validate genes that may mediate chemo-resistance. Our study provides a framework for identifying the genetic mechanisms in DLBCL therapeutic resistance and relapse formation. Disclosures Tam: Takeda: Consultancy; Paragon Genomics: Consultancy.

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.

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/β-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.

Enteropathy-associated T cell lymphoma (EATL) is the most common oncologic complication of celiac disease. Moffitt and colleagues identify novel EATL-defining mutations in SETD2, as well as clinically relevant mutations in the JAK-STAT pathway., Enteropathy-associated T cell lymphoma (EATL) is a lethal, and the most common, neoplastic complication of celiac disease. Here, we defined the genetic landscape of EATL through whole-exome sequencing of 69 EATL tumors. SETD2 was the most frequently silenced gene in EATL (32% of cases). The JAK-STAT pathway was the most frequently mutated pathway, with frequent mutations in STAT5B as well as JAK1, JAK3, STAT3, and SOCS1. We also identified mutations in KRAS, TP53, and TERT. Type I EATL and type II EATL (monomorphic epitheliotropic intestinal T cell lymphoma) had highly overlapping genetic alterations indicating shared mechanisms underlying their pathogenesis. We modeled the effects of SETD2 loss in vivo by developing a T cell–specific knockout mouse. These mice manifested an expansion of γδ T cells, indicating novel roles for SETD2 in T cell development and lymphomagenesis. Our data render the most comprehensive genetic portrait yet of this uncommon but lethal disease and may inform future classification schemes.

Hepatosplenic T-cell lymphoma (HSTL) is a rare and lethal lymphoma; the genetic drivers of this disease are unknown. Through whole-exome sequencing of 68 HSTLs, we define recurrently mutated driver genes and copy-number alterations in the disease. Chromatin-modifying genes, including SETD2, INO80, and ARID1B, were commonly mutated in HSTL, affecting 62% of cases. HSTLs manifest frequent mutations in STAT5B (31%), STAT3 (9%), and PIK3CD (9%), for which there currently exist potential targeted therapies. In addition, we noted less frequent events in EZH2, KRAS, and TP53. SETD2 was the most frequently silenced gene in HSTL. We experimentally demonstrated that SETD2 acts as a tumor suppressor gene. In addition, we found that mutations in STAT5B and PIK3CD activate critical signaling pathways important to cell survival in HSTL. Our work thus defines the genetic landscape of HSTL and implicates gene mutations linked to HSTL pathogenesis and potential treatment targets. Significance: We report the first systematic application of whole-exome sequencing to define the genetic basis of HSTL, a rare but lethal disease. Our work defines SETD2 as a tumor suppressor gene in HSTL and implicates genes including INO80 and PIK3CD in the disease. Cancer Discov; 7(4); 369–79. ©2017 AACR. See related commentary by Yoshida and Weinstock, p. 352. This article is highlighted in the In This Issue feature, p. 339

Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.

Emerging evidence has indicated a role of the bone morphogenetic proteins (BMP) in the pathogenesis of certain cancers. The signaling of BMP family members is tightly regulated by their antagonists, including noggin and SOST, which are, in turn, positively regulated by BMP, thereby forming a negative feedback loop. Consequently, the expression of these antagonists should be taken into account in studies on the prognostic significance of BMP. In the present paper, we correlated protein and mRNA expression levels of BMP6, noggin and SOST, alone or in combination, with patient survival in various types of cancer. We found that BMP6 alone was not significantly correlated with esophageal squamous cell carcinoma patient survival. Instead, a high level of inhibitor of differentiation 1, a downstream factor of BMP6, was associated with shorter survival in patients whose tumors stained strongly for BMP6. Knockdown of noggin in esophageal cancer cell line EC109, which expresses BMP6 strongly and SOST weakly, enhanced the non‐adherent growth of the cells. Noggin and SOST expression levels, when analyzed alone, were not significantly correlated with patient survival. However, high BMP6 activity, defined by strong BMP6 expression coupled with weak noggin or SOST expression, was significantly associated with shorter survival in esophageal squamous cell carcinoma patients. We further confirmed that BMP6 activity could be used as a prognostic indicator in prostate, bladder and colorectal cancers, using publicly available data on BMP6, noggin and SOST mRNA expression and patient survival. Our results strongly suggest that BMP6, noggin and SOST could be used in combination as a prognostic indicator in cancer progression. (Cancer Sci 2012; 103: 1145–1154)

To examine the correlation of survivin (both total and nuclear survivin) with clinicopathological parameters of esophageal squamous cell carcinoma (ESCC) patients. Tumors and non-tumor tissues near the proximal resection margins were resected from ESCC patients undergone esophagectomy. Quantitative polymerase chain reaction (qPCR) was performed to detect survivin mRNA expression level in the 10 paired tumor and adjacent non-tumor tissues. To confirm with the clinical situation, survivin mRNA and protein expression were measured by qPCR and immunoblot, respectively, in 5 ESCC cell lines and a non-neoplastic esophageal epithelial cell line. Immunohistochemistry was employed to reveal the cellular localization of survivin in tumor tissues isolated from the 64 ESCC patients undergone surgery alone. Up-regulation of survivin mRNA and protein was found in 5 ESCC lines (HKESC-1, HKESC-2, HKESC-3, HKESC-4, and SLMT-1) when compared to a non-neoplastic esophageal epithelial cell line NE-1. In particular, HKESC-3, HKESC-4, and SLMT-1 cells demonstrated ~50-fold increase in survivin mRNA. High level of survivin mRNA in tumor tissues when compared to non-tumor tissues was found in 70 % (7 of 10) of clinical cases. The increase in expression ranged from ~twofold to ~16-fold. Immunohistochemistry results showed that survivin was found at the cell nuclei in all specimens examined. Nuclear expression of survivin was inversely associated with the likelihood of developing nodal metastasis (p = 0.021) and significantly associated with early-stage ESCC (p = 0.039). Nuclear survivin could serve as a marker for indicating disease status in ESCC patients. © 2012 The Author(s)., published_or_final_version

Esophageal cancer is a deadly cancer with esophageal squamous cell carcinoma (ESCC) as the major type. Until now there has been a lack of reliable prognostic markers for this malignancy. This study aims to investigate the clinical correlation between Forkhead box M1 (FoxM1) and patients' parameters in ESCC. Methods: Immunohistochemistry was performed to investigate the expression and localization of FoxM1 in 64 ESCC tissues and 10 nontumor esophageal tissues randomly selected from 64 patients before these data were used for clinical correlations. Results: Cytoplasmic and nuclear expressions of FoxM1 were found in 63 and 16 of the 64 ESCC tissues, respectively. Low cytoplasmic expression of FoxM1 was correlated with early pathological stage in ESCC (P = 0.018), while patients with nuclear FoxM1 were younger in age than those without nuclear expression (P, published_or_final_version, Springer Open Choice, 21 Feb 2012

Suppressive effects of DUSP6 in tumorigenesis and EMT-associated properties were observed. Dual-specificity phosphatase (DUSP6) is a MAP kinase phosphatase (MKP) negatively regulating the activity of ERK, one of the major molecular switches in the MAPK signaling cascade propagating the signaling responses during malignancies. The impact of DUSP6 in EMT and its contribution to tumor dissemination has not yet been characterized. Due to differences in tumor microenvironments affecting cell signaling during cancer progression, DUSP6 may play varying roles in tumor development. We sought to examine the potential role of DUSP6-mediated tumorigenesis and EMT-associated properties in two aerodigestive tract cancers, namely, esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). Significant loss of DUSP6 was observed in 100% of 11 ESCC cell lines and 71% of seven NPC cell lines. DUSP6 expression was down-regulated in 40% of 30 ESCC tumor tissues and 75% of 20 NPC tumor tissues compared to their respective normal counterparts. Suppressive effects of DUSP6 in tumor formation and cancer cell mobility are seen in in vivo tumorigenicity assay and in vitro colony formation, three-dimensional Matrigel culture, cell migration and invasion chamber tests. Notably, overexpression of DUSP6 impairs EMT-associated properties. Furthermore, tissue microarray analysis reveals a clinical association of DUSP6 expression with better patient survival. Taken together, our study provides a novel insight into understanding the functional impact of DUSP6 in tumorigenesis and metastasis of ESCC and NPC.

Natural killer (NK) cell lymphomas/leukemias are rare neoplasms with an aggressive clinical behavior. The majority of the cases belong to extranodal NK/T-cell lymphoma, nasal type (ENKTL) in the current WHO classification scheme. Gene-expression profiling (GEP) of 21 ENKTL and NK-cell lymphoma/leukemia patients, 17 NK- and T-cell lines and 5 indolent NK-cell large-granular-lymphocytic proliferation was performed and compared with 125 peripheral T-cell lymphoma (PTCL) patients previously studied. The molecular classifier derived for ENKTL patients was comprised of 84 transcripts with the majority of them contributed by the neoplastic NK cells. The classifier also identified a set of gamma delta-PTCLs both in the ENKTL cases as well as in cases initially classified as PTCL-not otherwise specified. These gamma delta-PTCLs expressed transcripts associated with the T-cell receptor (TCR)/CD3 complex, suggesting T cell rather than NK-cell lineage. They were very similar to NK-cell tumors by GEP, but were distinct from cytotoxic (alpha beta)-PTCL and hepatosplenic T-cell lymphoma, indicating derivation from an ontogenically and functionally distinct subset of gamma delta T cells. They showed distinct expression of V gamma 9, V delta 2 transcripts and were positive for TCR gamma, but negative for TCR beta by immuno-histochemistry. Targeted inhibition of two oncogenic pathways (AURKA and NOTCH-1) by small-molecular inhibitors induced significant growth arrest in NK-cell lines, thus providing a rationale for clinical trials of these inhibitors in NK-cell malignancies. Leukemia (2011) 25, 348-358; doi:10.1038/leu.2010.255; published online 5 November 2010

Zinc finger transcription factors are involved broadly in development and tumorigenesis. Here, we report that the little studied zinc finger transcription factor ZNF382 functions as a tumor suppressor in multiple carcinomas. Although broadly expressed in normal tissues, ZNF382 expression was attenuated in multiple carcinoma cell lines due to promoter CpG methylation. ZNF382 was also frequently methylated in multiple primary tumors (nasopharyngeal, esophageal, colon, gastric, and breast). Ectopic expression of ZNF382 in silenced tumor cells significantly inhibited their clonogenicity and proliferation and induced apoptosis. We further found that ZNF382 inhibited NF-κB and AP-1 signaling and downregulated the expression of multiple oncogenes including MYC, MITF, HMGA2, and CDK6, as well as the NF-κB upstream factors STAT3, STAT5B, ID1, and IKBKE, most likely through heterochromatin silencing. ZNF382 could suppress tumorigenesis through heterochromatin-mediated silencing, as ZNF382 was colocalized and interacted with heterochromatin protein HP1 and further changed the chromatin modifications of ZNF382 target oncogenes. Our data show that ZNF382 is a functional tumor suppressor frequently methylated in multiple carcinomas. Cancer Res; 70(16); 6516–26. ©2010 AACR.

ADAMTS metalloprotease family member ADAMTS9 maps to 3p14.2 and shows significant associations with the aerodigestive tract cancers esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). However, the functional impact of ADAMTS9 on cancer development has not been explored. In this study, we evaluated the hypothesized antiangiogenic and tumor-suppressive functions of ADAMTS9 in ESCC and NPC, in stringent tumorigenicity and Matrigel plug angiogenesis assays. ADAMTS9 activation suppressed tumor formation in nude mice. Conversely, knockdown of ADAMTS9 resulted in clones reverting to the tumorigenic phenotype of parental cells. In vivo angiogenesis assays revealed a reduction in microvessel numbers in gel plugs injected with tumor-suppressive cell transfectants. Similarly, conditioned medium from cell transfectants dramatically reduced the tube-forming capacity of human umbilical vein endothelial cells. These activities were associated with a reduction in expression levels of the proangiogenic factors MMP9 and VEGFA, which were consistently reduced in ADAMTS9 transfectants derived from both cancers. Taken together, our results indicate that ADAMTS9 contributes an important function in the tumor microenvironment that acts to inhibit angiogenesis and tumor growth in both ESCC and NPC. ©2010 AACR., link_to_OA_fulltext

Summary Blood transport and mammary metabolism of 1-14C-palmitate was studied in a lactating buffalo (Bos bubalis). From the labelling patterns of blood proteins and blood lipids, obtained after intravenous injection of 1-14C-palmitate, it appeared that free fatty acids from the blood are rapidly removed by the liver and released into the blood stream as phosphatidylcholine and to some extent as triglyceride components of high density lipoproteins for use by extrahepatic tissues like the mammary glands. The comparative study of the metabolism of 1-14C-palmitate given by intravenous and intramammary routes showed that the mammary gland elongates 1-14C-palmitate to higher fatty acids and also desaturates them. These metabolic changes appear to be independent of the action of the liver. Zusammenfassung Untersuchungen uber den Transport von 1-14C-Palmitat im Blut und seine Umwandlung in andere Fettsauren in der Milchdruse von Buffeln (Bos bubalis) Nach i. v. Injektion von 1-14C-Palmitat werden die freien Fettsauren durch die Leber rasch aus dem Blut entnommen. Von der Leber werden sie als Phosphatidylcholin und teilweise als Trigliceride in Form von Lipoprotein hoher Dichte (high density) in das Blut abgegeben und derart den extrahepatischen Geweben bzw. der Milchdruse zur Verfugung gestellt. Vergleichende Versuche mit intravenoser und intramammarer Applikation von 1-14C-Palmitat ergaben, das die Milchdruse dasselbe einerseits in Fettsauren mit langerer Kette, andererseits in ungesattigte Fettsauren verwandelt. Die letzteren Umwandlungen durften sich unabhangig von der Leber abspielen. Resume Recherches sur le transport de 1-14C-palmitate dans le sang et sa transformation en d'autres acides gras dans la glande mammaire de buffles (Bos bubalis) Apres une injection intraveineuse de 1-14C-palmitate, les acides gras libres sont rapidement retires du sang par le foie. A partir du foie, ils sont redonnes dans le sang sous la forme de phosphatidylcholine et en partie de triglyceride en lipoproteine de haute densite (high density) et mis de cette facon a disposition des tissus extrahepatiques et de la glande mammaire. Des recherches comparatives avec une application intraveineuse et intramammaire ont montre que la glande mammaire transforme elle-meme d'une part en acides gras a longues chaines et d'autre part en acides gras non satures. Ces dernieres transformations ne devraient pas dependre du foie. Resumen Estudios sobre el transporte de palmitato 1-14C en sangre y su transformacion en otros acidos grasos en la glandula mamaria de bufalas (Bos bubalis) Tras inyeccion iv. de palmitato 1-14C son recogidos rapidamente los acidos grasos de la sangre por el higado. El higado los cede como fosfatidilcolina y, en parte tambien, como trigliceridos en forma de lipoproteinas de densidad elevada a la sangre, poniendolas de tal modo a disposicion de los tejidos extrahepaticos resp. de la glandula mamaria. Ensayos comparados con aplicacion intravenosa e intramamaria de palmitato 1-14C indicaron que la ubre transforma el mismo por un lado en acidos grasos de cadena larga, por otro en acidos grasos no saturados. Estos cambios metabolicos parecen ser independientes de la accion del higado.

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 IGHSμ 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 IGHSμ/CD44 translocations substitute Sμ for the CD44 promoter and remove exon 1 of CD44, resulting in the overexpression of Iμ-CD44 hybrid mRNA transcripts activated from derivative 11 that encode a new CD44 variant lacking the leader peptide and with a unique C-terminus (CD44ΔEx1). When overexpressed in vitro in the CD44− GCB-DLBCL cell line BJAB, CD44ΔEx1–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 CD44ΔEx1 in BJAB cells enhanced their proliferation rate and clonogenic ability, indicating a possible pathogenic role of the translocation.

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.

Nasal NK/T-cell lymphoma (NL) is a rare but clinically important entity of lymphoma. Its preferential incidence in Orientals but not Caucasians suggests possible genetic predisposition. 11q deletion is common in NL, indicating certain tumor suppressor genes (TSGs) at this locus involved in its pathogenesis. We investigated the expression and methylation of an 11q23.2 TSG, CADM1 (or TSLC1), and its partner DAL-1 (or EPB41L3) in NL. Methylation and silencing of CADM1 were detected in 2 NL and 4 of 8 (50%) of non-Hodgkin lymphoma (NHL) cell lines, but not in normal NK cells and normal PBMC. Absence of CADM1 protein was also detected in NL cell lines. 5-aza-2′-deoxycytidine (Aza) demethylation or genetic knockout of both DNMT1 and 3B genes restored CADM1 and DAL-1 expression. CADM1 methylation was further detected in 36 of 45 (80%) of NL tumors. Concomitantly, DAL-1 was epigenetically inactivated in NL cell lines and virtually all the tumors with methylated CADM1. A significant correlation between the methylation of both genes was found (p < 0.0001). Homozygous deletion of CADM1 was detected in only 3 of 18 (17%) of tumors. The stress-response of CADM1 was abolished when its promoter becomes methylated. Our results demonstrate a frequent, predominant epigenetic silencing of CADM1 and DAL-1 in NL, which likely play a synergic role in NL pathogenesis. © 2008 Wiley-Liss, Inc.

8 páginas, 6 figuras, 1 tabla -- PAGS nros. 2816-2823, Endoglin (ENG) has been identified as a candidate tumor-suppressor gene in esophageal squamous cell carcinoma (ESCC). Earlier microcell-mediated chromosome transfer (MMCT) studies of chromosome 9 in ESCC narrowed down a tumor-suppressive critical region to 9q33-34. ENG maps to 9q34-qter and encodes a transformation growth factor beta (TGFβ) superfamily auxiliary receptor. This study aims to identify the potential role for ENG in ESCC development. Significant downregulation of ENG was detected at frequencies of 87.5% in 16 ESCC cell lines, 39.1% directly in 23 ESCC tumor specimens from Hong Kong, and 33.4% in 18 ESCC tumor specimens from the high-risk ESCC region of Henan, China. By methylation-specific PCR, methylated sequences were detected in an ESCC cell line panel and in clinical specimens. Following demethylation treatment in 9 ESCC cell lines, ENG expression was obviously restored. Loss of heterozygosity (LOH) in a 4.7 Mb region on 9q32-q34, where ENG maps, was observed directly in ESCC tumor tissues. Both epigenetic methylation and allelic loss appear to contribute to ENG downregulation in tumor cells. In vitro and in vivo functional studies such as colony formation, Matrigel culture, invasion and tumorigenicity assays were performed. Colony formation efficiency was significantly reduced by overexpression of ENG. In addition, significantly smaller colonies of ENG stable transfectants were formed in Matrigel culture. Significant suppression of invasion efficiency and tumorigenicity were also observed, when comparing the ENG stable transfectants with the vector-alone transfectants. This study provides evidence supporting ENG, as a cell invasion and tumor-suppressing gene in ESCC. © 2008 Wiley-Liss, Inc.

Recent studies have revealed an oncogenic role of DJ-1 through its ability to transform normal cells, prevent oxidative damage, and inhibit apoptosis. However, its role in esophageal squamous cell carcinoma (ESCC) is unknown. In this study, by immunohistochemistry, we analyzed the expression of DJ-1 in 81 ESCC tumors, 31 paired nonneoplastic esophageal epithelia, and 19 paired ESCC lymph node metastases. We found that cytoplasmic DJ-1 expression was significantly higher in ESCC and ESCC lymph node metastases than in nonneoplastic esophageal epithelium. ESCC specimens with high distant metastatic potential also had a significantly higher level of nuclear DJ-1 expression (P = 0.018). By Kaplan-Meier analysis, we found that a high level of nuclear DJ-1 was significantly associated with poorer patient survival in our cohort (P = 0.028). To investigate whether DJ-1 promotes ESCC progression through phosphatidylinositol 3-kinase pathway and modulation of apoptosis, we performed immunohistochemistry of pAkt and Daxx. We found that DJ-1 expression was significantly associated with pAkt, whereas nuclear DJ-1 expression was significantly correlated with nuclear expression of Daxx. These results suggest that phosphatidylinositol 3-kinase pathway and Daxx-regulated apoptosis might be important in DJ-1-mediated ESCC progression. By using multivariate Cox regression, we further showed that T4 stage (P = 0.003) and DJ-1 (P = 0.034) are independent predictors of patient survival. In conclusion, our results suggest that DJ-1 plays a very important role in transformation and progression of ESCC and may be used as a prognostic marker in ESCC. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3593–602)

Esophageal cancer is the sixth leading causes of cancer-related death in the world. It is suggested that beta-adrenoceptor is involved in the control of cell proliferation, but its role in the pathogenesis of esophageal cancer remains unknown. We therefore studied the role of beta-adrenergic signaling in the regulation of growth of an esophageal squamous-cell carcinoma cell line HKESC-1. Results showed that both beta(1)- and beta(2)-adrenoceptors were expressed in HKESC-1 cells. Stimulation of beta-adrenoceptors with epinephrine significantly increased HKESC-1 cell proliferation accompanied by elevation of intracellular cyclic AMP levels, which were abolished by beta(1)- or beta(2)-selective antagonists. Epinephrine also increased extracellular signal-regulated kinase-1/2 (ERK1/2) phosphorylation as well as cyclooxygenase-2 (COX-2) and cytosolic phospholipase A(2) expression, which were blocked by beta(1)- or beta(2)-selective antagonists. Moreover, epinephrine increased cyclin D(1), cyclin E(2), cyclin-dependent kinase (CDK)-4, CDK-6, and E(2)F-1 expression and retinoblastoma protein phosphorylation at Ser807/811, all of which were abrogated by beta(1)-adrenoceptor antagonist. Furthermore, epinephrine increased the expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1 and -2 in a beta(2)-adrenoceptor-, mitogen-activated protein kinase/ERK kinase (MEK)-, and COX-2-dependent manner. MEK or COX-2 inhibitor also significantly inhibited HKESC-1 cell proliferation induced by epinephrine. Collectively, we demonstrate that epinephrine stimulates esophageal squamous-cell carcinoma cell proliferation via beta-adrenoceptor-dependent transactivation of ERK/COX-2 pathway. Stimulation of beta(1)- and beta(2)-adrenoceptors also elicits a differential response on the expression of cell cycle regulators. These novel findings may shed new light on the understanding of beta-adrenergic signaling in the control of esophageal cancer cell growth.

Summary Dysregulation of cell cycle control is an important mechanism in carcinogenesis. Gene promoter hypermethylation is an alternative mechanism of gene inactivation. We analyzed the methylation status of the tumor suppressor components of the INK4/Rb pathway in mantle cell lymphoma and follicular lymphoma by methylation-specific polymerase chain reaction for p15 , p16 , p18 , and Rb1 in 23 mantle cell lymphoma and 30 follicular lymphoma cases and lymphoma cell lines. The methylation-specific polymerase chain reaction results showed that in mantle cell lymphoma, frequent p16 (82%) but infrequent p15 (8.7%) or Rb1 (17.4%) hypermethylation occurred, with p16 and Rb1 hypermethylation being mutually exclusive ( P = .01). In follicular lymphoma, frequent hypermethylation of p15 (36.7%), p16 (56.7%), and Rb1 (43.3%) occurred, with p15 and Rb1 hypermethylation being mutually exclusive ( P = .05). Concurrent methylation of p15 and p16 occurred in 26.7% of patients with follicular lymphoma and 8.7% of patients with mantle cell lymphoma. Compared with mantle cell lymphoma, there was more frequent p15 ( P = .025) hypermethylation but comparable Rb1 ( P = .07) and p16 ( P = .07) hypermethylation in follicular lymphoma. In a patient with follicular lymphoma with sequential biopsies, Rb1 was unmethylated and expressed at diagnosis but became methylated and down-regulated at relapse. Moreover, methylation analysis of these 4 genes in an additional 8 patients with grade I follicular lymphoma showed that Rb , but not the other genes, was preferentially methylated in grade II ( P = .03). In summary, most patients with mantle cell lymphoma and follicular lymphoma had epigenetic aberrations targeting the INK4/Rb pathway. There is more frequent p16 hypermethylation in mantle cell lymphoma and p15 or Rb1 hypermethylation in follicular lymphoma. The role of Rb methylation in disease or histologic transformation in follicular lymphoma warrants further study.

Id protein family consists of four members namely Id-1 to Id-4. Different from other basic helix–loop–helix transcription factors, they lack the DNA binding domain. Id proteins have been shown to be dysregulated in many different cancer types and their prognostic value has also been demonstrated. Recently, Id-1 has been shown to be upregulated in oesophageal squamous cell carcinoma (ESCC). However, the prognostic implications of Id proteins in ESCC have not been reported. We examined the expression of the Id proteins in ESCC cell lines and clinical ESCC specimens and found that Id protein expressions were dysregulated in both the ESCC cell lines and specimens. By correlating the expression levels of Id proteins and the clinicopathological data of our patient cohort, we found that M1 stage tumours had significantly higher nuclear Id-1 expression (P=0.012) while high nuclear Id-1 expression could predict development of distant metastasis within 1 year of oesophagectomy (P=0.005). In addition, high levels of Id-2 expression in both cytoplasmic and nuclear regions predicted longer patient survival (P=0.041). Multivariate analysis showed that high-level expression of Id-2 in both cytoplasmic and nuclear regions and lower level of nuclear Id-1 expression were independent favourable predictors of survival in our ESCC patients. Our results suggest that Id-1 may promote distant metastasis in ESCC, and both Id-1 and Id-2 may be used for prognostication for ESCC patients.

A new human esophageal cancer cell line, HKESC-4, was established from a nude-mouse xenograft of a moderately differentiated esophageal squamous cell carcinoma (ESCC) developed from a 65-year-old Hong Kong Chinese man. The cellular characteristics (morphological, electron microscopic, and immunohistochemical studies), tumorigenicity in athymic nude mice, cytogenetic features, and DNA ploidy of the cell line were investigated. The cell line was maintained in vitro for 17 months and passaged 80 times. HKESC-4 grew as a monolayer, with a doubling time of 63 hours. The epithelial nature of HKESC-4 included the presence of cytokeratin intermediate filaments, as shown by antibodies (AE1/AF3, CAM5.2, and MAK 6), and the presence of the tonofilaments, as seen under electron microscopy. HKESC-4 was tumorigenic in nude mice and had DNA aneuploidy. The cytogenetic abnormalities of HKESC-4 included -1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -15, -16, -17, -18, -19, +20, -21, -22, +del(11)(p11), +i(11)(q10), and +21 marker chromosomes. Comparative genomic hybridization analysis demonstrated chromosomal gains at 1p36.13, 3q23 approximately q28, 5p15.33 approximately p15.1, 6p25.1 approximately p22.3, 7p21.3 approximately p11.2, 7q11.21 approximately q21.13, 8q23.3 approximately q23.3, 11p11.2, 11q12.1 approximately q13.2, 14q21.3 approximately q32.2, 17p13.3, 18p11.32 approximately p11.31, and 20p13 approximately p12.2 and chromosomal losses at 1q12, 2p25.1 approximately p24.3, 13p13 approximately p11.2, 21p, 22p13 approximately p11.2, and Y. The newly established cell line HKESC-4 promises to be a useful tool in future studies of molecular pathogenesis and therapeutics in ESCC.

Aberrant activation of the wingless-type- (Wnt)-signaling pathway is common in many cancers including nasopharyngeal (NPC) and esophageal squamous cell (ESCC) carcinomas, both prevalent in Southern China and Southeast Asia. However, the molecular mechanism leading to this abnormality is still obscure. Wnt inhibitory factor-1 (WIF1) is a secreted antagonist of the Wnt pathway, and is recently shown to be inactivated by epigenetic mechanism in some tumors. Here, we examined whether WIF1 is also inactivated epigenetically in NPC and ESCC. With semiquantitative reverse transcription-PCR and methylation-specific PCR, we detected WIF1 downregulation or silencing in 6/6 of NPC and 12/19 of ESCC cell lines, which is well correlated with its methylation status. Methylation was further confirmed by high-resolution bisulfite genomic sequencing. Methylation was also frequently observed in a large collection of primary tumors of NPC (85%, 55/65) and ESCC (27%, 25/92), with WIF1 expressed and unmethylated in normal NPC and esophageal cell lines and normal tissues. Treatment of 5-aza-2'-deoxycytidine demethylated WIF1 and induced its expression in NPC and ESCC cell lines, highlighting a direct role of epigenetic inactivation. Ectopic expression of WIF1 in NPC and ESCC tumor cells resulted in significant inhibition of tumor cell colony formation, similar to TP53, and also significant downregulation of beta-catenin protein level in NPC cells. Thus, WIF1 functions as a tumor suppressor for both NPC and ESCC through suppressing the Wnt-signaling pathway, but is frequently silenced by epigenetic mechanism in a tumor-specific way. Our study indicates that epigenetic inactivation of WIF1 contributes to the aberrant activation of Wnt pathway and is involved in the pathogenesis of both tumors. WIF1 methylation could also serve as a specific biomarker for these tumors.

By comparative DNA fingerprinting, we identified a 357-bp DNA fragment frequently amplified in esophageal squamous cell carcinomas (ESCC). This fragment overlaps with an expressed sequence tag mapped to 7q22. Further 5' and 3'-rapid amplification of cDNA ends revealed that it is part of a novel, single-exon gene with full-length mRNA of 2052 bp and encodes a nuclear protein of 109 amino acids ( approximately 15 kDa). This gene, designated as gene amplified in esophageal cancer 1 (GAEC1), was located within a 1-2 Mb amplicon at 7q22.1 identified by high-resolution 1 Mb array-comparative genomic hybridization in 6/10 ESCC cell lines. GAEC1 was ubiquitously expressed in normal tissues including esophageal and gastrointestinal organs; with amplification and overexpression in 6/10 (60%) ESCC cell lines and 34/99 (34%) primary tumors. Overexpression of GAEC1 in 3T3 mouse fibroblasts caused foci formation and colony formation in soft agar, comparable to H-ras and injection of GAEC1-transfected 3T3 cells into athymic nude mice formed undifferentiated sarcoma in vivo, indicating that GAEC1 is a transforming oncogene. Although no significant correlation was observed between GAEC1 amplification and clinicopathological parameters and prognosis, our study demonstrated that overexpressed GAEC1 has tumorigenic potential and suggest that overexpressed GAEC1 may play an important role in ESCC pathogenesis.

Epigenetic silencing of tumour suppressor genes (TSG) inactivates TSG functions. Previously, we identified PCDH10 as a methylated TSG in carcinomas. Here, we detected its frequent silencing and methylation in lymphoma cell lines including 100% Burkitt, 100% diffuse large B cell, 86% Hodgkin, 100% nasal natural killer/T-cell lymphoma and 1/3 of leukaemia cell lines, and in primary tumours but not in normal peripheral blood mononuclear cells or lymph nodes. PCDH10 silencing could be reversed by demethylation with 5-aza-2'-deoxycytidine. Methylation was further detected in 14% of Hodgkin lymphoma sera. Thus, PCDH10 methylation is frequently involved in lymphomagenesis and could serve as a tumour-specific biomarker.

Protocadherins constitute the largest subgroup in the cadherin superfamily of cell adhesion molecules. Their major functions are poorly understood, although some are implicated in nervous system development. As tumor-specific promoter methylation is a marker for tumor suppressor genes (TSG), we searched for epigenetically inactivated TSGs using methylation-subtraction combined with pharmacologic demethylation, and identified the PCDH10 CpG island as a methylated sequence in nasopharyngeal carcinoma (NPC). PCDH10 is broadly expressed in all normal adult and fetal tissues including the epithelia, though at different levels. It resides at 4q28.3--a region with hemizygous deletion detected by array-CGH in NPC cell lines; however, PCDH10 itself is not located within the deletion. In contrast, its transcriptional silencing and promoter methylation were frequently detected in multiple carcinoma cell lines in a biallelic way, including 12/12 nasopharyngeal, 13/16 esophageal, 3/4 breast, 5/5 colorectal, 3/4 cervical, 2/5 lung and 2/8 hepatocellular carcinoma cell lines, but not in any immortalized normal epithelial cell line. Aberrant methylation was further frequently detected in multiple primary carcinomas (82% in NPC, 42-51% for other carcinomas), but not normal tissues. The transcriptional silencing of PCDH10 could be reversed by pharmacologic demethylation with 5-aza-2'-deoxycytidine or genetic demethylation with double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Ectopic expression of PCDH10 strongly suppressed tumor cell growth, migration, invasion and colony formation. Although the epigenetic and genetic disruptions of several classical cadherins as TSGs have been well documented in tumors, this is the first report that a widely expressed protocadherin can also function as a TSG that is frequently inactivated epigenetically in multiple carcinomas.

The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2′-deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45G inhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcriptional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11 of 13 (85%) non-Hodgkin's lymphoma, 3 of 6 (50%) Hodgkin's lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2′-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1 of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

B cell-activating factor (BAFF), a member of tumor necrosis factor family cytokines, has been shown to enhance the maturation and survival of peripheral B cells. While BAFF is implicated in regulating B cell function and autoimmunity, its role in the development of autoimmune arthritis has not been fully clarified. Using a collagen-induced arthritis (CIA) mouse model, we detected dysregulated expression of BAFF and its receptors in the peripheral lymphoid organs during arthritis induction. Elevated serum levels of BAFF were closely correlated with increased levels of anti-collagen antibodies during the CIA progression. Moreover, dendritic cells (DCs) and macrophages were found to express high amount of BAFF proteins at the acute and chronic stages of CIA, respectively. In cultures, recombinant BAFF suppressed apoptosis of splenic B cells from arthritic mice, and DC-induced B cell proliferation was specifically blocked by soluble decoy receptor B cell maturation antigen-Fc. These findings suggest that overproduction of BAFF by DCs and macrophages may play a crucial role in the pathogenesis of experimental arthritis.

Objective: The objectives of this study were: 1) to examine the expression of macrophage migration inhibitory factor (MIF) in esophageal squamous cell carcinoma (ESCC); 2) to see if a relationship exists between MIF expression, clinicopathologic features, and long-term prognosis; and 3) to ascertain the possible biologic function of MIF in angiogenesis. Summary Background Data: MIF has been linked to fundamental processes such as those controlling cell proliferation, cell survival, angiogenesis, and tumor progression. Its role in ESCC, and the correlation of MIF expression and tumor pathologic features in patients, has not been elucidated. Methods: The expression of MIF in tumor and nontumor tissues was examined by irnmunohistochemical staining. Concentrations of MIF, vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) in patients' sera and in the supernatant of tumor cells culture were examined by ELISA. Correlations with clinicopathologic factors were made. Results: In 72 patients with ESCC, intracellular MIF was overexpressed in esophagectomy specimens. The expression of MIF correlated with both tumor differentiation and lymph node status. The median survival in the low-MIF expression group (, published_or_final_version