Epidermal Growth Factor-mediated T-cell Factor/Lymphoid Enhancer Factor Transcriptional Activity Is Essential but Not Sufficient for Cell Cycle Progression in Nontransformed Mammary Epithelial Cells

Abstract

Because ␤-catenin target genes such as cyclin D1 are involved in cell cycle progression, we examined whether ␤-catenin has a more pervasive role in normal cell proliferation, even upon stimulation by non-Wnt ligands. Here, we demonstrate that epidermal growth factor (EGF) stimulates T-cell factor/lymphoid enhancer factor (Tcf/Lef) transcriptional activity in nontransformed mammary epithelial cells (MCF-10A) and that its transcriptional activity is essential for EGF-mediated progression through G 1 /S phase. Thus, expression of dominant-negative Tcf4 blocks EGF-mediated Tcf/Lef transcriptional activity and bromodeoxyuridine uptake. In fact, the importance of EGF-mediated Tcf/Lef transcriptional activity for cell cycle progression may lie further upstream at the G 1 /S phase transition. We demonstrate that dominant-negative Tcf4 inhibits a reporter of cyclin D1 promoter activity in a dose-dependent manner. Importantly, dominant-negative Tcf4 suppresses EGF-mediated cell cycle activity specifically by thwarting EGF-mediated Tcf/Lef transcriptional activity, not by broader effects on EGF signaling. Thus, although expression of dominant-negative Tcf4 blocks EGF-mediated TOPFLASH activation, it has no effect on either EGF receptor or ERK phosphorylation, further underscoring the fact that Tcf/Lef-mediated transcription is essential for cell cycle progression, even when other pro-mitogenic signals are at normal levels. Yet, despite its essential role, Tcf/Lef transcriptional activity alone is not sufficient for cell cycle progression. Serum also stimulates Tcf/Lef transcriptional activation in MCF-10A cells but is unable to promote DNA synthesis. Taken together, our data support a model wherein EGF promotes Tcf/Lef transcriptional activity, and this signal is essential but not sufficient for cell cycle activity.

Tunable interplay between epidermal growth factor and cell–cell contact governs the spatial dynamics of epithelial growth

“…20. For experiments, cells were plated on either glass coverslips (VWR) or 2-chambered coverslides (Lab-Tek) in growth medium for 24 h. For G 0 synchronization, cells were maintained in serum free medium for 24 h (20). The following antibodies were used: anti-actin (Santa Cruz), anti-BrdU (Roche Applied Science), anti-E-cadherin (BD Transduction Laboratories), anti-HA (Covance), anti-phospho-Thr202/Tyr204-Erk 1/2 (Cell Signaling Technology), anti-phospho-serine 473-Akt (Cell Signaling Technology), HECD-1 (Zymed), mouse IgG (Sigma-Aldrich), and Alexa dye-labeled secondary antibodies (Invitrogen).…”

“…Retrovirus was produced by triple transfection of HEK 293T cells and used to infect MCF-10A cells as described in ref. 20.…”

“…MCF-10A cells were cultured in growth medium as described in ref. 20. For experiments, cells were plated on either glass coverslips (VWR) or 2-chambered coverslides (Lab-Tek) in growth medium for 24 h. For G 0 synchronization, cells were maintained in serum free medium for 24 h (20).…”

Wnt signalling and the mechanistic basis of tumour development

“…The β-catenin protein thus liberated can be recycled to the cytoplasmic pool, which is then followed by increased expression of β-catenin target genes. Thus, surface receptors, such as c-RON, epidermal growth factor receptor (EGFR) and c-ErbB2, can stimulate canonical Wnt signalling [82][83][84] as well as activating the signalling pathways with which they are more usually associated.…”

Activator Protein 2α Associates with Adenomatous Polyposis Coli/β-Catenin and Inhibits β-Catenin/T-cell Factor Transcriptional Activity in Colorectal Cancer Cells

“…Inhibits ␤-Catenin/TCF/LEF-dependent Transcriptional Activity in Colorectal Cancer Cells-To investigate whether AP-2␣ modulates ␤-catenin/TCF/LEF-dependent transcriptional activity, experiments were performed with the reporter TOPflash (1,20,(22)(23)(24)(25) or with the corresponding negative control FOPflash, and pSV-␤-galactosidase was included to normalize for transfection efficiency. Initially, wild type ␤-catenin was overexpressed by transient transfection in HEK293 cells (Fig.…”