Ras-mediated Loss of the Pro-apoptotic Response Protein Par-4 Is Mediated by DNA Hypermethylation through Raf-independent and Raf-dependent Signaling Cascades in Epithelial Cells

Channing J Der,Karen Frantz,Vanessa M Muniz-Medina,Aylin S Ülkü,Vivek M Rangnekar,Kevin Pruitt,Rafael J Rojas,Janiel M Shields

doi:10.1074/jbc.m503083200

Abstract

The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.

Highlights

Proper development and tissue homeostasis are regulated in part through a complex balance between cell proliferation and cell death
Emerging complexities of Ras signaling include the involvement of Raf-independent effectors in mediating Ras transformation, cell type differences in Ras signaling, and the complexity of genes deregulated in Ras-transformed cells [11]. par-4 has been identified as a gene down-regulated by activation of the Raf/ERK MAPK cascade in Ras-transformed fibroblasts (6 – 8)
In light of differences in the signaling pathways involved in Ras transformation of fibroblasts versus epithelial cells, we evaluated the mechanism and role of Par-4 down-regulation in Ras transformation of epithelial cells

Summary

Introduction

Proper development and tissue homeostasis are regulated in part through a complex balance between cell proliferation and cell death. A consequence of constitutive Ras activation is downstream changes in gene expression, which in turn act to modulate a variety of processes, including proliferation, differentiation, angiogenesis, and apoptosis [11]. Ras is known to cause a shift in the growth/apoptosis balance to favor growth over death and in doing so promotes cell proliferation [12]. Oncogenic Ras protects cells from apoptosis by modulation of a variety of apoptosisrelated proteins, including down-regulation of Par-4 expression [6, 7]. Consistent with this possibility, transient Par-4 expression causes an apoptotic response in Ras-transformed NIH 3T3 cells [7]. Ras binds to and activates Raf, which in turn phosphorylates and activates MEK1 1 and

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Conclusion