Transcriptional profile of Rous Sarcoma Virus transformed chicken embryo fibroblasts reveals new signaling targets of viral-src

Abstract

Transformation of chicken fibroblasts in vitro by Rous Sarcoma Virus represents a model of cancer in which a single oncogene, viral src, uniformly and rapidly transforms primary cells in culture. We experimentally surveyed the transcriptional program affected by Rous Sarcoma Virus (RSV) in primary culture of chicken embryo fibroblasts. As a control, we used cells infected with non-transforming RSV mutant td106, in which the src gene was deleted. Using Affymetrix GeneChip Chicken Genome Arrays, we report 811 genes that were modulated more than 2.5 fold in the virus transformed cells. Among these, 409 genes were induced and 402 genes were repressed by viral src. From the repertoire of modulated genes, we selected 20 genes that were robustly changed. We then validated and quantified the transcriptional changes of most of the 20 selected genes by real-time PCR. The set of strongly induced genes contains vasoactive intestinal polypeptide, MAP kinase phosphatase 2 and follistatin, among others. The set of strongly repressed genes contains TGF beta 3, TGF beta-induced gene, and deiodinase. The function of several robustly modulated genes sheds new light on the molecular mechanism of oncogenic transformation.

FIGURE 1. Functional network of robustly induced (red) and repressed (blue) transcripts and their interacting partners (black) in primary chicken cells transformed by Rous Sarcoma Virus

By surveying the literature on the function of each of the 20 gene products that were most dramatically changed in RSV transformed CEFs, we propose a model of their concerted action of promoting cellular transformation and tumor progression. Detailed study of individual genes rather than computer-aided annotation of the genes and their networks was employed to reveal functional outputs of changed transcripts, assuming that changes in transcription corresponded to changes in the protein level. Out of the 20 genes modulated by v-src, 10 could be linked to promoting a cancer phenotype based on their functional characteristics. These include: Follistatin (FST) that contributes to cell growth stimulation by titrating out activin(s) (ACT) and keeping TGF beta receptor (TGFBR) signaling in “low gear”. The low activity of TGFBR is also kept in check by decreased levels of TGF beta 3 (TGFB3). As a consequence, one of the TGF beta-induced genes (TGFBI) that encodes an adhesion protein, is down regulated, contributing to the anchorage-independent growth of transformed cells. Reduced levels of tissue factor pathway inhibitor, (TFPI) together with high levels of urokinase-type plasminogen activator (PLAU), play a role in tumor metastasis. Upregulated arachidonate 5-lipoxygenase (ALOX5) should contribute to tumor development and progression by promoting inflammation. Downregulated type 2 deiodinase (DIO2) would decrease the levels of endogenous tri-iodothyronine and indirectly activate AP-1 transcription factor contributing to signals that enhance cell growth, inflammation and metastasis. The role of four and half LIM domain protein 1 (FHL1) in cell adhesion has been documented and the repression of FHL1, which is regulated by src-phosphorylated CAS, promotes anchorage-independent growth. Tumor suppressive action of nephroblastoma overexpressed gene (NOV) is linked to the control of cell adhesion. The reduced levels of NOV promote anchorage-independent growth. Induction of AHNAK, a gene that encodes a large phosphoprotein, may also contribute to the loss of contact inhibition. See the text for more detailed description of the scheme plus relevant references.

FIGURE 2. Immunoblot analysis of VIP in normal and RSV-SR transformed CEFs

Upper three-partite panel, top panel (lanes A–G): Western blot analysis of cell lysates from CEFs (lane A), CEFs infected with RSV-SR td106 mutant (lanes B, C, D), and CEFs transformed by RSV-SR (lanes E, F, G). The blot was probed with a monoclonal antibody raised against human VIP. Lysates from cells derived from three independent experiments (infection with RSV-SR td106 mutant and transformation with RSV-SR) were used. Arrow points to VIP of 52 kDa molecular weight. Middle panel, the same Western blot was probed with src phospho-Tyr 416-specific antibody. Arrow points to c-src of 60 kDa molecular weight. Lower panel, Coomassie blue staining of the parallel SDS-polyacrylamide gel analyzed in the upper panels. Lower two panels, top panel (lanes H–O): Western blot analysis of conditioned media from normal and RSV-SR transformed CEFs; lane H, lysate from normal, uninfected CEFs that serves as a positive control for the presence of VIP; lanes I–K, supernatant from CEFs infected with RSV-SR td 106 mutant; and lanes M–O, supernatant from CEFs transformed with RSV-SR. Arrow indicates VIP migrating at the apparent 52 kDa molecular weight. Lower panel, Coomassie blue staining of the parallel SDS-polyacrylamide gel analyzed in the upper panel.