Activating Transcription Factor 4 Promotes Esophageal Squamous Cell Carcinoma Invasion and Metastasis in Mice and Is Associated with Poor Prognosis in Human Patients

<div><p>Background</p><p>Activating transcription factor 4 (ATF4) is a stress response gene that is involved in homeostasis and cellular protection. However, its expression and function in esophageal squamous cell carcinoma (ESCC) remains unknown. In this study, we aimed to determine the clinicopathologic significance of ATF4 in ESCC and its potential role in ESCC invasion and metastasis.</p><p>Methodology/Principal Findings</p><p>We demonstrated that ATF4 overexpression is correlated with multiple malignant characteristics and indicates poor prognosis in ESCC patients. ATF4 expression was an independent factor that affected the overall survival of patients with ESCC after surgical resection. ATF4 promoted cell invasion and metastasis by promoting matrix metalloproteinase (MMP)-2 and MMP-7 expression, while its silencing significantly attenuated these activities both <i>in vitro</i> and <i>in vivo</i>.</p><p>Conclusions/Significance</p><p>We report that ATF4 is a potential biomarker for ESCC prognosis and that its dysregulation may play a key role in the regulation of invasion and metastasis in ESCC cells. The targeting of ATF4 may provide a new strategy for blocking ESCC metastasis.</p></div

Overexpression of ATF4 promotes tumor cell invasion and metastasis.

<p><i>In vitro</i> migration and invasion assays in TE-1LM (A) and Eca-109 (B) cells that were stably transfected with LV-vector or LV-ATF4. Inset, relative ATF4 protein expression levels as determined by Western blot. (C) Incidence of metastasis in mice implanted with TE-1LM-vector or TE-1LM-ATF4 cells. (D) Representative hematoxylin and eosin staining of the lungs and livers of mice.</p

Profiles of Patients with ATF4-Positive or ATF4-Negative ESCCs.

<p>Profiles of Patients with ATF4-Positive or ATF4-Negative ESCCs.</p

ATF4 does not affect the proliferation or colony formation of ESCC cells.

<p>(A) MTT assay of the proliferation of TE-1LM non-treated control (NC), TE-1LM-vector (Vector), and TE-1LM-ATF4 (ATF4) cells. The data represent the means ± S.D. of three independent experiments. (B) The effects of ATF4 on the proliferation of the cells as described above were also examined using a colony formation assay. The media was changed every 3 d. Cells were plated in triplicate, and the experiment was repeated three times. Representative wells are shown.</p

Representative IHC of ATF4 in matched ESCC and adjacent non-cancerous tissues.

<p>ESCC tissues with strong (A), moderate (B), weak (C), and negative (D) ATF4 staining. Adjacent non-cancerous tissues with weak (E) and negative (F) ATF4 staining. Magnification, 200×.</p

Histogram of COG classification.

<p>Histogram of COG classification.</p

The general structure of lanostane type and seco-lanostane type terpenoids produced by <i>W. cocos</i>.

<p>The general structure of lanostane type and seco-lanostane type terpenoids produced by <i>W. cocos</i>.</p

Overview of the sequencing and assembly.

<p>Overview of the sequencing and assembly.</p

Distribution of the homology search of expressed sequence tags against the nr database.

<p>Distribution of the homology search of expressed sequence tags against the nr database.</p

<i>De Novo</i> Sequencing and Transcriptome Analysis of <i>Wolfiporia cocos</i> to Reveal Genes Related to Biosynthesis of Triterpenoids

<div><p><i>Wolfiporia cocos</i> Ryvarden <i>et</i> Gilbertson is a saprophytic fungus in the Basidiomycetes. Its dried sclerotium is widely used as a traditional crude drug in East Asia. Especially in China, the dried sclerotium is regarded as the silver of the Chinese traditional drugs, not only for its white color, but also its medicinal value. Furthermore, triterpenoids from <i>W. cocos</i> are the main active compounds with antitumor and anti-inflammatory activity. Biosynthesis of the triterpenoids has rarely been researched. In this study, the <i>de novo</i> sequencing of the mycelia and sclerotia of <i>W. cocos</i> were carried out by Illumina HiSeq 2000. A total of 3,484,996,740 bp from 38,722,186 sequence reads of mycelia, and 3,573,921,960 bp from 39,710,244 high quality sequence reads of sclerotium were obtained. These raw data were assembled into 60,354 contigs and 40,939 singletons, and 56,938 contigs and 37,220 singletons for mycelia and sclerotia, respectively. The transcriptomic data clearly showed that terpenoid biosynthesis was only via the MVA pathwayin <i>W. cocos</i>. The production of total triterpenoids and pachymic acid was examined in the dry mycelia and sclerotia. The content of total triterpenoids was 5.36% and 1.43% in mycelia and sclerotia, respectively, and the content of pachymic acid was 0.458% and 0.174%. Some genes involved in the triterpenoid biosynthetic pathway were chosen to be verified by qRT-PCR. The unigenes encoding diphosphomevalonate decarboxylase (Unigene 20430), farnesyl diphosphate synthase (Unigene 14106 and 21656), hydroxymethylglutaryl-CoA reductase (NADPH) (Unigene 6395_All) and lanosterol synthase (Unigene28001_All) were upregulated in the mycelia stage. It is likely that expression of these genes influences the biosynthesis of triterpenoids in the mycelia stage.</p></div