Yan Ru Qin

MicroRNA-375 inhibits tumour growth and metastasis in oesophageal squamous cell carcinoma through repressing insulin-like growth factor 1 receptor

Background To understand the involvement of micro-RNA (miRNA) in the development and progression of oesophageal squamous cell carcinoma (ESCC), miRNA profiles were compared between tumour and corresponding non-tumour tissues. Methods miRCURY LNA array was used to generate miRNA expressing profile. Real-time quantitative PCR was applied to detectthe expression of miR-375 in ESCC samples and its correlation with insulin-like growth factor 1 receptor (IGF1R). Methylation-specific PCR was used to study the methylation status in the promoter region of miR-375. The tumour-suppressive effect of miR-375 was determined by both in-vitro and in-vivo assays. Results The downregulation of miR-375 was frequently detected in primary ESCC, which was significantly correlated with advanced stage (p=0.003), distant metastasis (p<0.0001), poor overall survival (p=0.048) and disease-free survival (p=0.0006). Promoter methylation of miR-375 was detected in 26 of 45 (57.8%) ESCC specimens. Functional assays demonstrated that miR-375 could inhibit clonogenicity, cell motility, cell proliferation, tumour formation and metastasis in mice. Further study showed that miR-375 could interact with the 3′-untranslated region of IGF1R and downregulate its expression. In clinical specimens, the expression of IGF1R was also negatively correlated with miR-375 expression (p=0.008). Conclusions This study demonstrates that miR-375 has a strong tumour-suppressive effect through inhibiting the expression of IGF1R. The downregulation of miR-375, which is mainly caused by promoter methylation, is one of the molecular mechanisms involved in the development and progression of ESCC.

A CD90+ Tumor-Initiating Cell Population with an Aggressive Signature and Metastatic Capacity in Esophageal Cancer

Tumor-initiating cells (TIC), also known as cancer stem cells, are regarded widely as a specific subpopulation of cells needed for cancer initiation and progression. TICs have yet to be identified in esophageal tumors that have an increasing incidence in developed countries. Here, we report a CD90(+) cell population found in esophageal squamous cell carcinoma (ESCC), which is endowed with stem cell-like properties and high tumorigenic and metastatic potential. mRNA profiling of these cells suggested pathways through which they drive tumor growth and metastasis, with deregulation of an Ets-1/MMP signaling pathway and epithelial-mesenchymal transition figuring prominently. These cells possessed higher self-renewal activity and were sufficient for tumor growth, differentiation, metastasis, and chemotherapeutic resistance. CD90(+) TICs were isolated and characterized from ESCC clinical specimens as well as ESCC cell lines. In freshly resected clinical specimens, they represented a rare cell population, the levels of which correlated with strong family histories and lymph node metastasis. Our results prompt further study of this CD90(+) population of esophageal TICs as potential therapeutic targets.

Rab25 Is a Tumor Suppressor Gene with Antiangiogenic and Anti-Invasive Activities in Esophageal Squamous Cell Carcinoma

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

Adenosine-to-inosine RNA editing mediated by adars in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC), the major histologic form of esophageal cancer, is a heterogeneous tumor displaying a complex variety of genetic and epigenetic changes. Aberrant RNA editing of adenosine-to-inosine (A-to-I), as it is catalyzed by adenosine deaminases acting on RNA (ADAR), represents a common posttranscriptional modification in certain human diseases. In this study, we investigated the status and role of ADARs and altered A-to-I RNA editing in ESCC tumorigenesis. Among the three ADAR enzymes expressed in human cells, only ADAR1 was overexpressed in primary ESCC tumors. ADAR1 overexpression was due to gene amplification. Patients with ESCC with tumoral overexpression of ADAR1 displayed a poor prognosis. In vitro and in vivo functional assays established that ADAR1 functions as an oncogene during ESCC progression. Differential expression of ADAR1 resulted in altered gene-specific editing activities, as reflected by hyperediting of FLNB and AZIN1 messages in primary ESCC. Notably, the edited form of AZIN1 conferred a gain-of-function phenotype associated with aggressive tumor behavior. Our findings reveal that altered gene-specific A-to-I editing events mediated by ADAR1 drive the development of ESCC, with potential implications in diagnosis, prognosis, and treatment of this disease.

Identification of alpha-actinin 4 and 67 kDa laminin receptor as stage-specific markers in esophageal cancer via proteomic approaches

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies in the world with a very poor prognosis. The majority of ESCC patients present with advanced metastatic disease upon diagnosis. Therefore, it is important to understand the molecular mechanism in the tumor invasion process and to find new biomarkers for early diagnosis and prognostic evaluation.

Identification of PTK6, via RNA Sequencing Analysis, as a Suppressor of Esophageal Squamous Cell Carcinoma

BACKGROUND & AIMS Esophageal squamous cell carcinoma (ESCC) is the most commonly observed histologic subtype of esophageal cancer. ESCC is believed to develop via accumulation of numerous genetic alterations, including inactivation of tumor suppressor genes and activation of oncogenes. We searched for transcripts that were altered in human ESCC samples compared with nontumor tissues. METHODS We performed integrative transcriptome sequencing (RNA-Seq) analysis using ESCC samples from 3 patients and adjacent nontumor tissues to identify transcripts that were altered in ESCC tissue. We performed molecular and functional studies of the transcripts identified and investigated the mechanisms of alteration. RESULTS We identified protein tyrosine kinase 6 (PTK6) as a transcript that was significantly down-regulated in ESCC tissues and cell lines compared with nontumor tissues or immortalized normal esophageal cell lines. The promoter of the PTK6 gene was inactivated in ESCC tissues at least in part via hypermethylation and histone deacetylation. Knockdown of PTK6 in KYSE30 ESCC cells using small hairpin RNAs increased their ability to form foci, migrate, and invade extracellular matrix in culture and form tumors in nude mice. Overexpression of PTK6 in these cells reduced their proliferation in culture and tumor formation in mice. PTK6 reduced phosphorylation of Akt and glycogen synthase kinase (GSK)3β, leading to activation of β-catenin. CONCLUSIONS PTK6 was identified as a transcript that is down-regulated in human ESCC tissues via epigenetic modification at the PTK6 locus. Its product appears to regulate cell proliferation by reducing phosphorylation of Akt and GSK3β, leading to activation of β-catenin. Reduced levels of PTK6 promote growth of xenograft tumors in mice; it might be developed as a marker of ESCC.

Characterization of Tumor-Suppressive Function of SOX6 in Human Esophageal Squamous Cell Carcinoma

Purpose: By using cDNA microarray analysis, we identified a transcriptional factor, SOX6, was frequently downregulated in esophageal squamous cell carcinoma (ESCC). The aim of this study is to investigate the role of SOX6 in human esophageal cancer development, and to examine the prevalence and clinical significance of SOX6 downregulation in ESCC. Experimental Design: Expressions of SOX6 mRNA in 50 ESCCs and SOX6 protein in 300 ESCCs were investigated by semiquantitative RT-PCR and immunohistochemistry, respectively. The tumor-suppressive function of SOX6 was characterized by cell growth, foci formation, wound-healing and cell invasive assays, and tumor xenograft experiment. Western blot analysis was applied to detect protein expression levels. Results: SOX6 was frequently downregulated in primary ESCCs in both mRNA level (29/50, 58%) and protein level (149/219, 68.0%), which was significantly associated with the poor differentiation (P = 0.029), lymph node metastases (P = 0.014), advanced TNM stage (P = 0.000), and disease-specific survival (P < 0.001). Multivariate analysis indicated that the downregulation of SOX6 (P = 0.000) was a significant independent prognostic factors for ESCC. Functional studies showed that SOX6 was able to suppress both in vitro and in vivo tumorigenic ability of ESCC cells. The tumor-suppressive mechanism of SOX6 was associated with its role in G1/S cell-cycle arrest by upregulating expressions of p53 and p21WAF1/CIP1 and downregulating expressions of cyclin D1/CDK4, cyclin A, and β-catenin. Conclusions: We provided the first evidence that SOX6 is a novel tumor-suppressor gene in ESCC development and is a potential prognostic marker in ESCC. Clin Cancer Res; 17(1); 46–55. ©2010 AACR.

Single-nucleotide polymorphism-mass array reveals commonly deleted regions at 3p22 and 3p14.2 associate with poor clinical outcome in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common solid tumors in the world with poor prognosis. Deletion of chromosome 3p is one of the most frequent chromosomal alterations in ESCC, suggesting the existence of one or more tumor suppressor genes (TSGs) at this region. In the present study, a recently developed high‐throughput and high‐resolution technology, single‐nucleotide polymorphism (SNP)‐mass array, was applied to investigate loss of heterozygosity on 3p in 100 primary ESCC cases with 386 SNP markers. Four commonly deleted regions (CDRs) at 3p26.3, 3p22, 3p21.3 and 3p14.2 were identified. Absent and down‐regulated expression of several candidate TSGs, including CHL1, PCAF, RBMS3, PLCD1 and CACNA2D3, were detected in primary ESCC tumors and ESCC cell lines. Moreover, deletions of CDRs 2 and 4 were correlated with advanced tumor stage and deletion of CDR2 was associated with tumor metastasis in ESCC. Our findings provided evidence that minimal deleted regions at 3p26.3, 3p22, 3p21.3 and 3p14.2 containing potential TSGs may contribute to the pathogenesis of esophageal cancer.

Downregulation of the novel tumor suppressor DIRAS1 predicts poor prognosis in esophageal squamous cell carcinoma.

Loss of chromosome 19p is one of the most frequent allelic imbalances in esophageal squamous cell carcinoma (ESCC), suggesting the existence of one or more tumor suppressor genes within this region. In this study, we investigated a role in ESCCs for a candidate tumor suppressor gene located at 19p13.3, the Ras-like small GTPase DIRAS1. Downregulation of DIRAS1 occurred in approximately 50% of primary ESCCs where it was associated significantly with advanced clinical stage, lymph node metastasis, and poor overall survival. LOH and promoter methylation analyses suggested that loss of DIRAS1 expression was mediated by epigenetic mechanisms. Functional studies established that ectopic re-expression of DIRAS1 in ESCC cells inhibited cell proliferation, clonogenicity, cell motility, and tumor formation. Mechanistic investigations suggested that DIRAS1 acted through extracellular signal-regulated kinase (ERK1/2; MAPK3/1) and p38 mitogen-activated protein kinase (MAPK; MAPK14) signaling to trigger BAD Ser112 dephosphorylation and matrix metalloproteinase (MMP)2/9 transcriptional inactivation to promote apoptosis and inhibit metastasis, respectively. Taken together, our results revealed that DIRAS1 has a pivotal function in ESCC pathogenesis, with possible use as a biomarker and intervention point for new therapeutic strategies.

Characterization of a candidate tumor suppressor gene uroplakin 1A in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is increasing in incidence, but the knowledge of the genetic underpinnings of this disease remains limited. In this study, we identified the tetraspanin cell surface receptor uroplakin 1A (UPK1A) as a candidate tumor suppressor gene (TSG), and we investigated its function and mechanism in ESCC cells. UPK1A downregulation occurred in 68% of primary ESCCs examined, where it was correlated significantly with promoter hypermethylation (P < 0.05). Ectopic expression of UPK1A in ESCC cells inhibited cell proliferation, clonogenicity, cell motility, and tumor formation in nude mice. Mechanistic investigations suggested that these effects may be mediated by inhibiting nuclear translocation of β-catenin and inactivation of its downstream targets, including cyclin-D1, c-jun, c-myc, and matrix metalloproteinase 7 (MMP7). Cell cycle arrest elicited by UPK1A at the G(1)-S checkpoint was associated with downregulation of cyclin D1 and cyclin-dependent kinase 4, whereas metastasis suppression was associated with reduction of MMP7. These findings were consistent with evidence derived from clinical samples, where UPK1A downregulation was correlated with lymph node metastasis (P = 0.009), stage (P = 0.015), and overall survival (P < 0.0001). Indeed, multivariate cyclooxygenase regression analysis showed that UPK1A was an independent prognostic factor for overall survival. Taken together, our findings define a function for UPK1A as an important TSG in ESCC development.