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. 2001 Jun 19;98(13):7319-24.
doi: 10.1073/pnas.131568898. Epub 2001 Jun 12.

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis

T Bowman  1 , M A Broome, D Sinibaldi, W Wharton, W J Pledger, J M Sedivy, R Irby, T Yeatman, S A Courtneidge, R Jove
Affiliations

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis

T Bowman et al. Proc Natl Acad Sci U S A. .

Abstract

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3beta protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3beta protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF --> Src --> Stat3 --> Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

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Figures

Figure 1
Figure 1
Stat3-dependent c-Myc expression is required for Src transformation. (a) NIH 3T3 cells were transfected with the indicated expression vectors, and v-Src-induced focus formation in monolayer was quantified. (b) Northern blot analysis of c-myc expression in normal NIH 3T3 fibroblasts or these cells stably transformed by v-Src (NIH 3T3/v-Src). (c) Northern blot analysis of c-myc expression in FACS-selected NIH 3T3/v-Src cells transiently transfected with vectors encoding GFP alone (−) or together with Stat3β.
Figure 2
Figure 2
Fibroblasts lacking the c-myc gene are refractory to transformation by v-Src. (a) Parental rat-1 (TGR1) and c-myc knockout cells (HO15.19) were transfected with v-Src, and monolayer focus formation assays were performed. (Inset) Northern blot, demonstrating lack of c-myc mRNA expression in HO15.19 cells. (b) HO15.19 cells were transfected with v-Src vector alone or together with an expression vector encoding human c-Myc protein and subjected to focus formation assays.
Figure 3
Figure 3
Stat3 signaling contributes to Src oncogenesis by stimulating cell cycle progression and preventing apoptosis. (a) NIH 3T3/v-Src cells were mock-transfected or transfected with vectors encoding GFP alone or together with Stat3β followed by treatment with nocodazole. Cell cycle distribution of mock-transfected or GFP-positive cells with or without Stat3β expression was determined by staining with propidium iodide and FACS analysis. (b) NIH 3T3 or NIH 3T3/v-Src cells were mock-transfected or transfected with vectors encoding GFP alone or together with Stat3β. GFP-positive cells with or without Stat3β expression were harvested by FACS and analyzed for apoptosis with Annexin V staining.
Figure 4
Figure 4
PDGF-induced mitogenesis depends on Stat3 signaling and c-Myc expression. (a) Quiescent NIH 3T3 cells were microinjected with expression vectors encoding the indicated cDNAs and stimulated with PDGF or not stimulated, and DNA synthesis was measured by BrdUrd incorporation. (b) NIH 3T3 cells were treated with DMSO or the Src family kinase-selective inhibitor SU6656 (1 μM) for 1 h before stimulation with PDGF. Stat3 proteins were immunoprecipitated from cell lysates and analyzed by Western blot by using antibodies to activated phosphotyrosine-Stat3 or total Stat3 protein.
Figure 5
Figure 5
Model for normal mitogenic PDGF signal transduction through Stat3 that is subverted by Src oncogenesis. In this model, PDGF-induced receptor dimerization leads to activation of receptor-associated c-Src kinase activity, which, in turn, phosphorylates Stat3 monomers on tyrosine. Phosphorylated Stat3 forms activated dimers that translocate to the nucleus and induce c-myc gene expression that is required for normal PDGF-stimulated mitogenesis. In cells transformed by the Src oncoprotein, constitutive activation of Stat3 leads to continuous induction of c-myc expression, thereby short-circuiting PDGF receptor signaling and contributing to oncogenesis. It remains to be determined whether Stat3 regulates the c-myc promoter directly or indirectly in this model. In addition, other signaling pathways independent of Stat3 regulate c-myc expression and cell proliferation.
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