Search

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3' untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

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.

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.

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.

A gene critical to esophageal cancer has been identified. Functional studies using microcell-mediated chromosome transfer of intact and truncated donor chromosomes 3 into an esophageal cancer cell line and nude mouse tumorigenicity assays were used to identify a 1.61 Mb tumor suppressive critical region (CR) mapping to chromosome 3p14.2. This CR is bounded by D3S1600 and D3S1285 microsatellite markers. One candidate tumor suppressor gene, ADAMTS9, maps to this CR. Further studies showed normal expression levels of this gene in tumor-suppressed microcell hybrids, levels that were much higher than observed in the recipient cells. Complete loss or downregulation of ADAMTS9 gene expression was found in 15 out of 16 esophageal carcinoma cell lines. Promoter hypermethylation was detected in the cell lines that do not express this gene. Re-expression of ADAMTS9 was observed after demethylation drug treatment, confirming that hypermethylation is involved in gene downregulation. Downregulation of ADAMTS9 was also found in 43.5 and 47.6% of primary esophageal tumor tissues from Hong Kong and from the high-risk region of Henan, respectively. Thus, this study identifies and provides functional evidence for a CR associated with tumor suppression on 3p14.2 and provides the first evidence that ADAMTS9, mapping to this region, may contribute to esophageal cancer development.

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.

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.

E-cadherin, a cell adhesion molecule, is regarded as an invasion-suppressor molecule and a prognostic marker in many types of human cancers. Downregulation of E-cadherin is common in esophageal carcinoma and is associated with an increase in invasive and metastatic potential. To study the mechanisms responsible for inactivation of this gene in esophageal squamous cell carcinoma (ESCC), we investigated the methylation status around the 5' promoter region of E-cadherin gene of six ESCC cell lines by methylation-specific polymerase chain reaction, and compared it with E-cadherin protein and mRNA expression. We also studied the methylation status of 20 ESCC clinical specimens. Methylation was noted in four of the six cell lines (one fully methylated and three partially methylated). The completely methylated cell line lacked E-cadherin protein expression and mRNA transcription. E-cadherin expression and transcription were reduced in a partially methylated cell line but preserved in the other partially methylated cell lines. Treatment of E-cadherin-negative carcinoma cells with the demethylating agent, 5-aza-2'-deoxycytidine, induced re-expression of the gene. A high frequency of methylation (16/20, 80%) was also noted in the 20 ESCC clinical samples. Our results indicate that 5' CpG island methylation is common in esophageal carcinoma and may play an important role in downregulation of E-cadherin.

Human papillomavirus (HPV), in particular types 16 and 18, is positively associated with anogenital cancers and may be an important etiologic factor in their pathogenesis. The goal of our study was to investigate the role of HPV infection in the pathogenesis of esophageal squamous cell carcinoma (ESCC) and its relationship with the p53 mutation. We have examined ESCC collected from Sichuan, China, for the presence of HPV infection and p53 mutation. The presence of HPV DNA was detected by PCR-Southern analysis while the p53 mutation was analyzed by PCR-SSCP. High-risk HPV (types 16 and 18) DNA was detected in 32 out of 152 cases of ESCC examined. In contrast, HPV DNA was not detected in normal esophageal tissues excised from the distant end (tumor free) of resected ESCC. Mutation of the p53 gene was detected in 22 out of 55 cases of ESCC. The distribution of the 22 p53 mutation was: 5 in exon 5, 1 in exon 6, 5 in exon 7, 10 in exon 8 and 1 in exon 10. The p53 mutation was detected at a significantly lower rate in ESCC with HPV infection. Our results support a role of HPV infection in the pathogenesis of ESCC from a high-incidence area.

There is no scientific study that has investigated the association between human papilloma virus (HPV) and p53 mutation in Hong Kong Chinese patients with esophageal cancers. The aim of this survey is to evaluate in details the prevalence and relationship of HPV and p53 mutation in these patients with esophageal squamous cell carcinomas. Fresh tissues from the resected specimens of 70 Chinese patients (59 men, 11 women) with primary esophageal squamous cell carcinomas (20 well-differentiated, 36 moderately differentiated, and 14 poorly differentiated squamous cell carcinomas) were tested for the presence of HPV and p53 mutation using the polymerase chain reaction (PCR), single-strand conformational polymorphism (SSCP) analysis, and DNA sequencing. No HPV type 18 was detected, whereas HPV type 16 was identified in 8.6% (6 of 75) of the cases. p53 mutation was found in 44% (31 of 70) of the tumors. The mean ages of HPV-positive and HPV-negative groups of patients were 55 and 64 years, respectively (P = .046, t-test). There was no correlation between the prevalence of HPV and p53 mutation in these tumors. The presence of HPV and p53 also had no relation to the sex of the patients or to the grade of the carcinomas. It is concluded that the overall low prevalence of HPV in esophageal carcinomas may suggest that the virus may not play an important role in the pathogenesis of these tumors in Hong Kong Chinese patients. Also, p53 mutation and integrated HPV DNA are not mutually exclusive in esophageal cancer.

The tissues from 70 Chinese patients with oesophageal squamous cell carcinoma were prospectively collected to study for the pattern of p53 mutations and its relationship with clinico-pathological features and prognosis using immunohistochemistry, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. p53 over-expression and p53 mutations were detected in 73% and 44% of the patients. These p53 aberrations had no relationship with the patient age, sex, smoking/drinking habits and tumor site, size or stage. The p53 over-expression was more intense in moderately/poorly-differentiated squamous cell carcinomas. Thirty-three p53 mutations were noted in 31 patients; 18.2% in exon 5, 15.2% in exon 6, 33.3% in exon 7 and 33.3% in exon 8. Mutations were primarily point mutations and common in codons 248, 273 and 285. There were 46% transversions, 36% transitions and 18% frameshift. The survival of the patients depended mainly on the extent of resection. In patients with stage III oesophageal cancer, the median survival of those with p53 mutations was 6.8 months whereas those without was 12.5 months. The results were of clinical importance although the value did not reach statistical significance. Thus, there was a definite role of p53 mutations in the pathogenesis of oesophageal squamous cell carcinomas. p53 mutations were not synonymous with p53 over-expression. The distribution of p53 mutations in oesophageal cancers suggested that the etiologic contribution might be complex and probably involve different exogenous and endogenous exposures. p53 mutations also appear to play a role in predicting the survival of patients with stage III oesophageal squamous cell carcinomas.

Human papillomavirus (HPV) infection, especially high risk types HPV 16 and 18, have been studied widely in cervical cancer. However, HPV infection in esophageal cancer has not been well defined. In the present study, immunohistochemistry, PCR and Southern blot hybridization methods were used to detect HPV infection in 127 cases of esophageal squamous cell carcinoma. Immunohistochemistry results indicated that the virus was detected frequently in well differentiated carcinoma. The positive rates for BPV and HPV E6 protein were 60.6% (77/127) and 43% (54/127) respectively. Meanwhile, PCR and Southern hybridization showed that 35.9% (37/103) of esophageal squamous cell carcinomas have HPV DNA, which included 20.4% (21/103) HPV 16 and 7.8% (8/103) HPV 18. Of the 103 cases, only 1 had both HPV 16 and HPV 18 DNA. Our results suggest that HPV infection is present in esophageal squamous cell carcinoma and may play a role in its pathogenesis.

Terminal differentiation is usually achieved in normal as well as transformed squamous epithelial cells when cultured. On the other hand, tumor cells at various differentiation stages and with different biological characteristics comprise the heterogeneous properties of tumors which have been one of the barriers to effective treatments. Recently, a surface membrane protein has been reported in squamous cell carcinomas of the head and neck, which is recognized by a murine monoclonal antibody, SQM1. This glycoprotein was further suggested to be related to squamous cell differentiation and intercellular adhesion. In a recent study, the esophageal carcinoma cells of EC/CUHK2 cell line were induced to various differentiation stages as evidenced by the increasing amount of intracellular desmosomes and tonofilaments and greater binding ratios of cytokeratin and involucrin antibodies than in those cells that maintained lower calcium ion concentrations. The expression of SQM1 antigen was found to increase in intensity when the tumor cells were cultured in moderate to high calcium ion levels for 10 to 15 hours when the differentiation patterns were beginning to appear. The intensity declined gradually thereafter. Thus SQM1 protein might be related to the stage when the cells started committing with squamous differentiation.