Ras and TGF[beta] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways.

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras-transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor beta (TGFbeta) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFbeta receptor (TGFbeta-R) and oncogenic Ras signaling and is stabilized by autocrine TGFbeta production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFbeta alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFbeta-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFbeta-R and Raf/MAPK signaling.

Transferrin receptor hyperexpression in primary erythroblasts is lost on transformation by avian erythroblastosis virus.

In primary chicken erythroblasts (stem cell factor [SCF] erythroblasts), transferrin receptor (TfR) messenger RNA (mRNA) and protein were hyperexpressed as compared to nonerythroid chicken cell types. This erythroid-specific hyperexpression was abolished in transformed erythroblasts (HD3E22 cells) expressing the v-ErbA and v-ErbB oncogenes of avian erythroblastosis virus. TfR expression in HD3E22 cells could be modulated by changes in exogenous iron supply, whereas expression in SCF erythroblasts was not subject to iron regulation. Measurements of TfR mRNA half-life indicated that hyperexpression in SCF erythroblasts was due to a massive stabilization of transcripts even in the presence of high iron levels. Changes in mRNA binding activity of iron regulatory protein 1 (IRP1), the primary regulator of TfR mRNA stability in these cells, correlated well with TfR mRNA expression; IRP1 activity in HD3E22 cells and other nonerythroid cell types tested was iron dependent, whereas IRP1 activity in primary SCF erythroblasts could not be modulated by iron administration. Analysis of avian erythroblasts expressing v-ErbA alone indicated that v-ErbA was responsible for these transformation-specific alterations in the regulation of iron metabolism. In SCF erythroblasts high amounts of TfR were detected on the plasma membrane, but a large fraction was also located in early and late endosomal compartments, potentially concealing temporary iron stores from the IRP regulatory system. In contrast, TfR was almost exclusively located to the plasma membrane in HD3E22 cells. In summary, stabilization of TfR mRNA and redistribution of Fe-Tf/TfR complexes to late endosomal compartments may contribute to TfR hyperexpression in primary erythroblasts, effects that are lost on leukemic transformation.