Dominant negative Ras enhances lactogenic hormone‐induced differentiation by blocking activation of the Raf–Mek–Erk signal transduction pathway

Abstract

EGF and Ras mitogenic signal transduction pathways are frequently activated in breast carcinoma and inhibit mammary differentiation and apoptosis. HC11 mouse mammary epithelial cells, which differentiate and synthesize β-casein following growth to confluency and stimulation with lactogenic hormones, were used to study EGF-dependent signaling during differentiation. Blocking Mek-Erk or phosphotidylinositol-3-kinase (PI-3 kinase) signaling with specific chemical inhibitors enhanced β-casein promotor-driven luciferase activity. Because EGF stimulation of HC11 cells resulted in the activation of Ras, the effect of activated Ras (RasV12) or dominant negative (DNRasN17) on lactogen induced differentiation was examined. HC11 cell lines expressing RasV12 or DNRasN17 under the control of a tetracycline(tet)-responsive promotor were constructed. Activated RasV12 expression resulted in reduced tyrosine phosphorylation of Stat5 and a delay in β-casein expression in response to prolactin. However, the expression of tet-regulated DNRasN17 and adenovirusencoded DNRasN17 enhanced Stat5 tyrosine phosphorylation, Stat5 DNA binding and β-casein transcription. The expression of DNRasN17 blocked the activation of the Mek-Erk pathway by EGF but did not prevent the phosphorylation of AKT, a measure of activation of the PI-3-kinase pathway. Moreover, the expression of DNRasN17 prevented the block to lactogenic differentiation induced by EGF. Stimulation of HC11 cells with prolactin resulted in the association of the SHP2 phosphatase with Stat5, and this association was prevented by DNRasN17 expression. These results demonstrate that in HC11 cells DNRas inhibits the Mek-Erk pathway and enhances lactogenic hormone-induced differentiation. This occurs, in part, by inhibiting the association of the SHP2 phosphatase with Stat5.

Tsg101 is upregulated in a subset of invasive human breast cancers and its targeted overexpression in transgenic mice reveals weak oncogenic properties for mammary cancer initiation

“…Functional differentiation of mammary epithelial cells is normally associated with reduced activation of downstream signals, such as MAP kinases, that mediate a proliferative response (Cerrito et al, 2004). Concurrent with this notion, phosphorylated Erk was expressed during pregnancy but barely detectable at day 4 and day 10 of lactation in the mammary gland of wild-type mice Tsg101 overexpression and mammary cancer KB Oh et al (Figure 5a and b).…”

EMT-Induced Stemness and Tumorigenicity Are Fueled by the EGFR/Ras Pathway

“…Matthias Drosten and Mariano Barbacid (CNIO; Madrid, Spain) [44]. pRev- and pBABE-based transfer vectors were co-transfected with the ecotropic helper retrovirus plasmid pCL-Eco (a gift from Motomi Osato) into HEK 293T cells using FuGENE HD (Roche, Basel, Switzerland).…”

A functional study of proximal goat β-casein promoter and intron 1 in immortalized goat mammary epithelial cells

“…An SRE, which binds a nuclear protein with a serum response factor (SRF), mediates activation of the c-fos gene in response to a growth factor (Treisman, 1987). The Raf/MEK/ERK signaling cascade was activated after accepting the growth factor signal, and this negatively regulated expression of the CSN2 gene by binding the SRE (Johansen and Prywes, 1994;Cerrito et al, 2004). The SRE motif (TCATATTTAG) in goat CSN2 is more similar to the consensus CCATATTAGG motif than is bovine (GCATATTTAG), and the pig motif is different again (GCATGTC-AG; Johansen and Prywes, 1994).…”