Abstract 1509: Transcriptome analysis reveals significant effects of EGFR signaling on the secretome of mesenchymal stem cells.

Abstract

Abstract Mesenchymal stem cell (MSCs) are recruited by developing tumors, where MSCs release factors that enhance tumor cell proliferation and migration. Since different carcinomas synthesize epidermal growth factor (EGF)-like peptides and MSCs express the EGF receptor (EGFR), we hypothesize that EGFR signaling might mediate the interaction between MSCs and tumor cells. In order to comprehensively assess the mechanisms and factors that are regulated by EGFR signaling in MSCs and are involved in the interaction between MSCs and tumor cells, we analysed the transcriptome of MSCs cultured in absence or in presence of transforming growth factor α (TGFα), one of the main ligands of the EGFR, by using massively parallel sequencing. Libraries were prepared from poly-A RNA from not-stimulated (MSC-CTRL) or TGFα-stimulated (MSC-TGFα) MSCs. Sequencing was performed on the SOLiD 5500xl platform using the paired-end protocol (75bp + 35bp). The experiments were repeated in quadruplicate, and the average number of reads obtained in the 4 replicates resulted of about 37 million for MSC-CTRL, and 47 million for MSC-TGFα. The quality analysis of raw data showed that 75% of reads at the 75th position had quality values above 14. The mapping of the sequence reads to human hg19 reference genome evidenced a coverage ranging between 79.5% and 81.7% for MSC-CTRL, and between 76.3% and 79.0% for MSC-TGFα. The number of observed counts was normalized for the length of the element and the number of mapped reads, namely RPKM (Reads Per Kilobase per Million of mapped reads). The RPKM values obtained in the 4 replicates were compared by ANOVA test and the very high p-value (of about 1) demonstrated that the replicates were not statistically different. The differential gene expression in MSC-CTRL and MSC-TGFα was represented as fold induction (FI). When selecting genes for which the average count value of the 4 replicates was equal or higher than 1, 8946 genes resulted to have FI>1 and 10305 genes FI<1 according to RPKM values. Next, we focused the attention on 274 genes coding for secreted proteins, including interleukins, chemokines, angiogenesis factors, growth factors of the EGF, IGF and FGF families and their receptors. Using as cutoff a count value equal or higher than 1, 47 genes were considered not expressed, 126 genes resulted to have FI>1.0, 91 genes FI<1.0 and 10 genes FI=1.0. Among the secreted proteins investigated, a number of angiogenic factors were significantly modulated in MSCs by TGFα treatment. Analysis of secreted proteins, performed with XMAP based assays, confirmed that activation of EGFR signaling induces in MSCs the secretion of several pro-angiogenic factors, including VEGF, IL6, IL8, HGF and G-CSF. These findings strongly suggest that the EGFR-mediated interaction between MSCs and tumor cells induces in MSCs the secretion of factors that play an important role in tumor progression. Citation Format: Antonella De Luca, Cristin Roma, Francesca Fenizia, Francesca Bergantino, Marianna Gallo, Daniela Frezzetti, Susan Costantini, Nicola Normanno. Transcriptome analysis reveals significant effects of EGFR signaling on the secretome of mesenchymal stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1509. doi:10.1158/1538-7445.AM2013-1509