Abstract
Metastasis is the most lethal hallmark of esophageal squamous cell carcinoma (ESCC). The aim of the study is to identify key signaling pathways that control metastasis in ESCC. Highly invasive ESCC sublines (designated I3 cells) were established through three rounds of selection of cancer cells invading through matrigel-coated chambers. Gene expression profile of one of the I3 sublines was compared with that of its parental cell line using cDNA microarray analysis. Gene ontology and pathway analyses of the differentially expressed genes (both upregulated and downregulated) indicated that genes associated with cellular movement and the AKT pathway were associated with increased cancer cell invasiveness. Western blot analysis confirmed increased phosphorylated AKT (p-AKT), N-cadherin and decreased E-cadherin expression in the I3 cells. Immunohistochemistry was used to evaluate the clinical significance of p-AKT expression in ESCC, and the results showed higher p-AKT nuclear expression in lymph node metastases when compared with primary carcinoma. Inactivation of the PI3K/AKT pathway with specific inhibitors, or with PTEN overexpression, resulted in reversed cadherin switching and inhibited cancer cell motility. Inhibition of the pathway by treatment with wortmannin markedly suppressed experimental metastasis in nude mice. Our data demonstrated the importance of the PI3K/AKT signaling pathway in ESCC metastasis and support PI3K/AKT as a valid therapeutic target in treatment of metastatic ESCC.
Highlights
There was no activation of the Src pathway, which is another important regulatory pathway in cancer cell invasion and metastasis [9, 10], as indicated by the comparable protein expression level of p-Src and Src between the I3 sublines and corresponding parental cells (Figure 2D), even though the ingenuity pathway analysis (IPA) analysis suggested the dysregulation of the Src pathway in KYSE410-I3 cells
Wortmannin suppresses metastasis of esophageal cancer cells in nude mice Since our results showed that PI3K/AKT inhibition reduced the migration and invasive potential of esophageal cancer cells in vitro (Figure 3), we evaluated the efficacy of PI3K/AKT blockade in inhibiting metastasis of esophageal cancer cells in vivo
The highly invasive esophageal squamous cell carcinoma (ESCC) sublines that were established in this study confirms the significance of the PI3K/ AKT pathway in ESCC metastasis, but will be valuable tools for evaluating novel therapeutic agents which can curb tumor metastasis
Summary
Esophageal cancer is associated with poor prognosis because over 50% of patients with esophageal cancer present with metastasis [1]. Cancer metastasis is a multistage process which involves enhanced cell motility, intravasation, transit in lymphatic and blood vessels, extravasation and growth at a new location. Each of these events is attributed, at least in part, to the acquisition www.impactjournals.com/oncotarget of genetic alterations in the tumor cells [2]. Since new evidence supports that the clinical application of wortmannin may be revived through nanoparticle drug delivery [8], wortmannin was used in this study to investigate the effects of PI3K/AKT blockade on in vivo metastasis of human ESCC cells in mice. Because increased invasiveness may be conferred by EMT during which epithelial markers are usually downregulated while mesenchymal markers are upregulated, we examined the expression levels of EMT markers including E-cadherin and N-cadherin in ESCC cells (including the I3 cells), and determined whether PI3K/AKT inhibition by LY294002 and wortmannin could reverse the EMT program
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