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.

Monochromosome transfer and microarray analysis identify a critical tumor-suppressive region mapping to chromosome 13q14 and THSD1 in esophageal carcinoma.

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.

Establishment and characterization of a new xenograft-derived human esophageal squamous cell carcinoma cell line HKESC-4 of Chinese origin.

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.

Oncogenic properties of a novel gene JK-1 located in chromosome 5p and its overexpression in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) shows high frequency and mortality in Asian regions, including China. Previous analysis of genomic DNA of ESCC using comparative genomic hybridization indicated that amplification of the chromosome 5p regions is a common event in ESCC cell lines and patient cases of Hong Kong Chinese origin, and the results suggested that the genes located in the chromosome 5p regions may play crucial roles in the molecular pathogenesis of ESCC. Our previous studies on ESCC confirmed the tumorigenic and overexpression properties of a novel gene JS-1 located in chromosome 5p15.2 upstream to delta-catenin. In the present study, another novel gene JK-1 which is located at 5p15.1 downstream to delta-catenin was characterized for its roles in the pathogenesis of ESCC. Thirteen ESCC cell lines and 30 surgical specimens of esophageal tumors were studied for the overexpression of JK-1 using multiplex RT-PCR analysis. The transforming capacity of overexpression of JK-1 was also investigated by transfecting NIH 3T3 and HEK 293 cells with the expression vector cloned with JK-1, followed by the soft agar and foci formation assays. JK-1 was overexpressed in 9/13 (69%) of the ESCC cell lines and 9/30 (30%) of the ESCC patient cases. Both NIH 3T3 and HEK 293 cells acquired the properties of anchorage-dependent and -independent growth when JK-1 was overexpressed. Most significantly, subcutaneous sarcomas were formed in all (3/3) the athymic nude mice after NIH 3T3 cells overexpressing JK-1 were injected subcutaneously. Our results thus indicated that JK-1 is commonly overexpressed in ESCC and has a prominent capacity to transform normal cells. Our overall results thus provide the first evidence that the overexpression of JK-1 and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC.

Inhibitory effects of Gleditsia sinensis fruit extract on telomerase activity and oncogenic expression in human esophageal squamous cell carcinoma.

Previous studies have shown that the anomalous fruit extract of Gleditsia sinensis (GSE) exhibited apoptotic properties in various solid and non-solid tumors in vitro. However, the inhibitory actions of GSE on oncogenic expression and telomerase activity in esophageal squamous cell carcinoma (ESCC) have not been studied before. In the present study, the anti-cancer effects of GSE were demonstrated in three ESCC cell lines (HKESC-1, HKESC-2 and SLMT-1) by MTS and anchorage-independent clongen-icity assays, expression studies on oncogenes at 11q13 (CCND1, INT2, FGF4 and EMS1) and real-time quantitative telomeric repeat amplification protocol assay to show the inhibitory effect of GSE on telomerase in ESCC. The means of MTS50 of GSE for the ESCC cell lines and non-tumor NIH 3T3 cells were 21 and 163 microg/ml respectively. The anchorage-independent clongenicity assay showed that SLMT-1 cells lost their colony-forming potential which was dose-dependent to GSE. Moreover, GSE demonstrated dose-dependent suppression on the expression of INT2, EMS1 and FGF4, and inhibition of telomerase activity in the ESCC cell lines. Our overall results thus provide the first evidence that the anti-cancer effects of GSE on ESCC involve the suppression of oncogenic expression and inhibition of telomerase activity. Our findings also offer a new opportunity for the future development of GSE as a novel anti-cancer agent for ESCC and possibly for other cancers.

Tumor suppressive role of a 2.4 Mb 9q33-q34 critical region and DEC1 in esophageal squamous cell carcinoma.

The key genes involved in the development of esophageal squamous cell carcinoma (ESCC) remain to be elucidated. Previous studies indicate extensive genomic alterations occur on chromosome 9 in ESCC. Using a monochromosome transfer approach, this study provides functional evidence and narrows down the critical region (CR) responsible for chromosome 9 tumor suppressing activity to a 2.4 Mb region mapping to 9q33-q34 between markers D9S1798 and D9S61. Interestingly, a high prevalence of allelic loss in this CR is also observed in primary ESCC tumors by microsatellite typing. Allelic loss is found in 30/34 (88%) tumors and the loss of heterozygosity (LOH) frequency ranges from 67 to 86%. Absent to low expression of a 9q32 candidate tumor suppressor gene (TSG), DEC1 (deleted in esophageal cancer 1), is detected in four Asian ESCC cell lines. Stably expressing DEC1 transfectants provide functional evidence for inhibition of tumor growth in nude mice and DEC1 expression is decreased in tumor segregants arising after long-term selection in vivo. There is 74% LOH in the DEC1 region of ESCC primary tumors. This study provides the first functional evidence for the presence of critical tumor suppressive regions on 9q33-q34. DEC1 is a candidate TSG that may be involved in ESCC development.

Transforming capacity of two novel genes JS-1 and JS-2 located in chromosome 5p and their overexpression in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) has a high mortality rate and geographic differences in incidence. Previous studies of comparative genomic hybridization (CGH) showed that chromosomal 5p is frequently amplified in cell lines and primary ESCC of Hong Kong Chinese origin. In this report, attempt was made to study two novel genes, named as JS-1 and JS-2, which are located in chromosome 5p15.2 and are 5' upstream to delta catenin for their roles in molecular pathogenesis of ESCC. Eleven cell lines, 27 primary ESCC cases and multiple human tissue cDNA panels (MTC) of digestive system were studied for the expression level of JS-1 and JS-2 by RT-PCR. The full-length cDNA sequences of JS-1 and JS-2 were determined from a non-tumor esophageal epithelial cell line by 3' and 5' rapid amplification of cDNA ends (RACE). The transforming capacity of JS-1 and JS-2 was also investigated by transfecting NIH 3T3 cells with the expression vector pcDNA3.1(-) cloned with the full coding sequences and it was followed by the study of foci formation of the transfected cells under confluence growth and the anchorage-independent growth in soft agar. Forty-five percent (5/11) and 18% (2/11) of the ESCC cell lines showed overexpression of JS-1 and JS-2 respectively, while 55% (15/27) and 14% (3/22) primary ESCC cases showed overexpression of JS-1 and JS-2 respectively. JS-1 overexpression was most common in patients with stage II ESCC (6/27; 22%) whereas JS-2 was only overexpressed in a dysplastic lesion (1/22; 4%) and stage III tumors (2/22; 9%). The expression levels of JS-1 and JS-2 are both low in normal esophageal tissues. Overexpression of JS-1 in NIH 3T3 cells caused foci formation in confluence growth and colony formation in soft agar but not for JS-2. A high grade sarcoma was formed in the athymic nude mice when NIH 3T3 cells overexpressing JS-1 were injected subcutaneously. Our results thus indicate that the frequent overexpression of JS-1 in ESCC and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC. The present study also forms the ground work for further identification of novel mechanisms of molecular carcinogenesis in ESCC and other cancers.

GAEC1 and colorectal cancer: a study of the relationships between a novel oncogene and clinicopathologic features.

GAEC1 is a novel gene located at 7q22.1 that was detected in our previous work in esophageal cancer. The aims of the present study are to identify the copy number of GAEC1 in different colorectal tissues including carcinomas, adenomas, and nonneoplastic tissues and characterize any links to pathologic factors. The copy number of GAEC1 was studied by evaluating the quantitative amplification of GAEC1 DNA in 259 colorectal tissues (144 adenocarcinomas, 31 adenomas, and 84 nonneoplastic tissues) using real-time polymerase chain reaction. Copy number of GAEC1 DNA in colorectal adenocarcinomas was higher in comparison with nonneoplastic colorectum. Seventy-nine percent of the colorectal adenocarcinomas showed amplification and 15% showed deletion of GAEC1 (P < .0001). Of the adenomas, 90% showed deletion of GAEC1, with the remaining 10% showing normal copy number. The differences in GAEC1 copy number between colorectal adenocarcinoma, colorectal adenoma, and nonneoplastic colorectal tissue are significant (P < .0001). GAEC1 copy number was significantly higher in adenocarcinomas located in distal colorectum compared with proximal colon (P = .03). In conclusion, GAEC1 copy number was significantly different between colorectal adenocarcinomas, adenomas, and nonneoplastic colorectal tissues. The copy number was also related to the site of the cancer. These findings along with previous work in esophageal cancer imply that GAEC1 is commonly involved in the pathogenesis of colorectal adenocarcinoma.

Functional evidence of decreased tumorigenicity associated with monochromosome transfer of chromosome 14 in esophageal cancer and the mapping of tumor-suppressive regions to 14q32.

Despite the abundant evidence of high allelic loss of chromosome arm 14q in human cancers, tumor-suppressor genes mapped to this chromosome have yet to be identified. To narrow the search for candidate genes, we performed monochromosome transfer of chromosome 14 into an esophageal carcinoma cell line, SLMT-1 S1. Statistically significant suppression of the tumorigenic potential of microcell hybrids containing the transferred chromosome 14 provided functional evidence that tumor-suppressive regions of chromosome 14 are essential for esophageal cancer. Tumor segregants emerging in nude mice during the tumorigenicity assay were analyzed by detailed PCR-microsatellite typing to identify critical nonrandomly eliminated regions (CRs). A 680-kb CR mapped to 14q32.13 and an approximately 2.2-Mb CR mapped to 14q32.33 were delineated. Dual-color BAC FISH analysis of microcell hybrids and tumor segregants verified the selective loss of the 14q32.13 region. In contrast, similar transfers of an intact chromosome 11 into SLMT-1 S1 did not significantly suppress tumor formation. These functional complementation studies showing the correlation of tumorigenic potential with critical regions of chromosome 14 validated the importance of the 14q32 region in tumor suppression in esophageal cancer. The present study also paved the path for further identification of novel tumor-suppressor genes that are relevant to the molecular pathogenesis of esophageal cancer.