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.
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.
Pruitt
Kevin
K
Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, 27599-7295, USA.
Ulkü
Aylin S
AS
Frantz
Karen
K
Rojas
Rafael J
RJ
Muniz-Medina
Vanessa M
VM
Rangnekar
Vivek M
VM
Der
Channing J
CJ
Shields
Janiel M
JM
D000362:Agar / Q000737:chemistry; D000483:Alleles; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D018076:DNA, Complementary / Q000378:metabolism; D000077209:Decitabine; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D009570:Nitriles / Q000494:pharmacology; D010053:Ovary / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D015854:Up-Regulation; D048490:raf Kinases / Q000502:physiology*
D000362:Agar; D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine; D002070:Butadienes; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D018076:DNA, Complementary; D000077209:Decitabine; D004791:Enzyme Inhibitors; D005419:Flavonoids; D047908:Intracellular Signaling Peptides and Proteins; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D012313:RNA; D012333:RNA, Messenger; D048490:raf Kinases; C000717967:DNMT3A protein, human; C113580:U 0126; C101296:prostate apoptosis response-4 protein; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse; C000619382:Dnmt1 protein, rat; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one