Shangbin Yang

β-Catenin/TCF pathway upregulates STAT3 expression in human esophageal squamous cell carcinoma

Precise roles of beta-catenin/TCF pathway involved in esophageal tumorigenesis remain elusive. Here we found STAT3 overexpression in esophageal cancer cells and tissues, and its overexpression in esophageal squamous cell carcinoma (ESCC) tissues correlated with beta-catenin cytoplasmic/nuclear accumulation. A functional TCF binding element was detected in STAT3 promoter which specifically bound to TCF4. Transfected beta-catenin induced STAT3 transcriptional activity dose-dependently, and also enhanced STAT3 mRNA and protein levels. These inductions were specifically abolished by dominant-negative TCF4. These results suggest that STAT3 is a target of beta-catenin/TCF pathway and might participate in esophageal tumorigenesis.

Identification of genes regulated by Wnt/β-catenin pathway and involved in apoptosis via microarray analysis

BackgroundWnt/β-catenin pathway has critical roles in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of this pathway, little is known regarding Wnt/β-catenin pathway modification of the cellular apoptosis.MethodsTo identify potential genes regulated by Wnt/β-catenin pathway and involved in apoptosis, we used a stably integrated, inducible RNA interference (RNAi) vector to specific inhibit the expression and the transcriptional activity of β-catenin in HeLa cells. Meanwhile, we designed an oligonucleotide microarray covering 1384 apoptosis-related genes. Using oligonucleotide microarrays, a series of differential expression of genes was identified and further confirmed by RT-PCR.ResultsStably integrated inducible RNAi vector could effectively suppress β-catenin expression and the transcriptional activity of β-catenin/TCF. Meanwhile, depletion of β-catenin in this manner made the cells more sensitive to apoptosis. 130 genes involved in some important cell-apoptotic pathways, such as PTEN-PI3K-AKT pathway, NF-κB pathway and p53 pathway, showed significant alteration in their expression level after the knockdown of β-catenin.ConclusionCoupling RNAi knockdown with microarray and RT-PCR analyses proves to be a versatile strategy for identifying genes regulated by Wnt/β-catenin pathway and for a better understanding the role of this pathway in apoptosis. Some of the identified β-catenin/TCF directed or indirected target genes may represent excellent targets to limit tumor growth.

Suppression of Aurora-A oncogenic potential by c-Myc downregulation.

The abnormality of serine/threonine kinase Aurora-A is seen in many types of cancers. Although in physiological context it has been shown to play a vital role in cellular mitosis, how this oncogene contributes to tumorigenesis remains unclear. Here we demonstrate that Aurora-A overexpression enhances both the expression level and transcriptional activity of c-Myc. The inhibition of c-Myc expression by RNA interference significantly impaired the oncogenic potential of Aurora-A, resulting in attenuated cellular proliferation and transformation rates as well as fewer centrosomal aberrations. Furthermore, downregulation of c-Myc effectively overcame Aurora-A-induced resistance to cisplatin in esophageal cancer cells. Taken together, our results suggest an important role for c-Myc in mediating the oncogenic activity of Aurora-A, which may in turn allow for future targeting of c-Myc as a potential therapeutic strategy for tumors with Aurora-A overexpression.

Aurora kinase A induces miR-17-92 cluster through regulation of E2F1 transcription factor

Aurora kinase A (AURKA) is an essential mitotic serine/threonine kinase and its abnormal expression is observed in many malignancies, yet the exact role for AURKA in tumorigenesis still remains elusive. Here, through a transcription factor array, we show that the transcription activity of E2F1 was increased by AURKA overexpression. Meanwhile, the E2F1 protein level was found to be upregulated and a correlation between AURKA and E2F1 expression was observed in cancer specimens. Further analysis revealed that AURKA increased E2F1 protein stability by inhibiting proteasome-dependent degradation of this protein. Additionally, a microRNA cluster, miR-17-92, was found to be upregulated upon AURKA overexpression, and this stimulation was largely repressed by E2F1 knockdown. Chromatin immunoprecipitation further demonstrated that AURKA enhanced E2F1 occupancy to the promoter of the miR-17-92 cluster. These data reveal a novel link between AURKA and microRNAs via the regulation of E2F1, providing new clues for understanding the role of AURKA in tumorigenesis.

Krüppel-like factor 4 represses transcription of the survivin gene in esophageal cancer cell lines

Abstract Aberrant expression of survivin has been shown to be regulated at the transcription level in cancer cells. In this study, we demonstrate that there are six putative binding sites of Krüppel-like factor 4 (KLF4) within the 2000-bp region upstream of the transcription start site of the human survivin gene. Luciferase reporter gene assays revealed that survivin promoter activity is repressed upon overexpression of KLF4 in EC9706 cells. A chromatin immunoprecipitation assay indicated that KLF4 indeed binds the survivin promoter in vivo. It specifically binds the site located at position -40 among the six binding sites as determined by electrophoretic mobility shift assay. Ectopic expression of KLF4 decreases the mRNA and protein levels of survivin. Furthermore, overexpression of survivin partially reverses KLF4-induced cell apoptosis. These results indicate that KLF4 is a transcriptional repressor of the human survivin gene in esophageal squamous cancer cells.

β-Catenin/TCF pathway plays a vital role in selenium induced-growth inhibition and apoptosis in esophageal squamous cell carcinoma (ESCC) cells

Epidemiological and experimental studies have indicated selenium could reduce the risk of some cancers. In our present study, growth inhibition and apoptosis were detected upon methylseleninic acid (MSA) treatment in human esophageal squamous cell carcinoma cell lines EC9706 and KYSE150. MSA reduced beta-catenin protein levels, while there was no significant change observed on transcriptional levels. Moreover, we found MSA accelerated the degradation of beta-catenin and activated glycogen synthase kinase 3beta (GSK-3beta). Some targets of beta-catenin/TCF pathway and apoptosis-related genes altered after MSA treatment. Notably, utilizing the inducible 293-TR/beta-catenin cell line, we found the apoptotic phenotypes induced by MSA were partially reversed by the overexpression of beta-catenin. Overall, our data indicate the effects induced by MSA in ESCC cells may act on the inhibition of beta-catenin/TCF pathway.

BAG2 is a target of the c-Myc gene and is involved in cellular senescence via the p21CIP1 pathway

Suppression of c-Myc is likely to induce cellular senescence in many tumors with unclear mechanisms. A proteomics survey indicated that high levels of BCL2-associated athanogene 2 (BAG2) were found in response to c-Myc repression in TRE293 cells. This observation led to the investigation into the role of BAG2 in c-Myc-induced senescence. The association of the c-Myc/SP1 complex with the BAG2 promoter verified the role of c-Myc/SP1 in regulating BAG2 transcription. Furthermore, high levels of BAG2 were found to induce p21(CIP1)-dependent senescence and subsequent carcinogenetic arrest, suggesting its possible role as an indirect activator of the p21(CIP1) pathway.

P21-Activated Kinase 7 Mediates Cisplatin-Resistance of Esophageal Squamous Carcinoma Cells with Aurora-A Overexpression

Aurora-A overexpression is common in various types of cancers and has been shown to be involved in tumorigenesis through different signaling pathways, yet how the deregulation affects cancer therapeutics remains elusive. Here we showed that overexpression of Aurora-A rendered esophageal cancer cells resistance to cisplatin (CDDP) by inhibiting apoptosis. By using an apoptosis array, we identified a downstream gene, p21-activated kinase 7 (PAK7). PAK7 was upregulated by Aurora-A overexpression at both mRNA and protein levels. Importantly, the expression levels of Aurora-A and PAK7 were correlated in ESCC primary samples. Chromatin immunoprecipitation (ChIP) assay revealed that binding of E2F1 to the promoter of PAK7 was significantly enhanced upon Aurora-A activation, and knockdown of transcription factor E2F1 decreased PAK7 expression, suggesting that Aurora-A regulated PAK7 through E2F1. Furthermore, we demonstrated that PAK7 knockdown led to increased apoptosis, and Aurora-A-induced resistance to CDDP was reversed by downregulation of PAK7, suggesting PAK7 was a downstream player of Aurora-A that mediated chemoresistance of ESCC cells to CDDP. Our data suggest that PAK7 may serve as an attractive candidate for therapeutics in ESCC patients with Aurora-A abnormality.

Abstract 2943: Aurora kinase A induces miR-17-92 cluster through regulation of E2F1 transcription factor

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aurora Kinase A (AURKA) encodes an evolutionally conserved serine/threonine kinase that plays a pivotal role in centrosome cycling during mitosis. Abnormal expression of AURKA has been observed in many malignancies, yet the exact role for AURKA in tumorigenesis still remains elusive. Here through a transcription factor array, we show that the transcription activity of E2F1 was increased by AURKA overexpression. Meanwhile, E2F1 protein level was found to be upregulated upon AURKA induction and the correlation between AURKA and E2F1 expression was also observed in breast cancer specimens. Further analysis revealed that AURKA increased E2F1 protein stability by inhibiting proteasome-dependent degradation of this protein. Additionally, a microRNA cluster, miR-17-92 was found to be upregulated upon AURKA overexpression, and this stimulation was largely repressed by small interfering RNA (siRNA) against E2F1. Chromatin immunoprecipitation further demonstrates that AURKA enhanced the in vivo occupancy of E2F1 to the promoter of the miR-17-92 cluster. These data reveal a novel link between AURKA and microRNAs via the regulation of E2F1, providing new clues for understanding the role of AURKA in tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2943.