The 3'-untranslated region of p21WAF1 mRNA is a composite cis-acting sequence bound by RNA-binding proteins from breast cancer cells, including HuR and poly(C)-binding protein.
Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21(WAF1) is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21(WAF1) mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21(WAF1) 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21(WAF1) 3'-UTR constructs identified multiple cis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5' region of the p21(WAF1) 3'-UTR, whereas EGF induced reporter activity in constructs containing sequences 3' of the UVC-responsive region. These cis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21(WAF1) mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21(WAF1) protein expression or growth inhibition. However, binding of HuR to its target 3'-UTR cis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21(WAF1) mRNA via different mechanisms. We conclude that EGF-induced p21(WAF1) protein expression is mediated largely by stabilization of p21(WAF1) mRNA elicited via multiple 3'-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21(WAF1) expression or growth inhibition in this system. CP1 is a novel p21(WAF1) mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21(WAF1) mRNA stability.
Giles
Keith M
KM
Laboratory for Cancer Medicine and University Department of Medicine, Western Australian Institute for Medical Research and Centre for Medical Research, the University of Western Australia, Perth, Western Australia 6001, Australia.
Daly
John M
JM
Beveridge
Dianne J
DJ
Thomson
Andrew M
AM
Voon
Dominic C
DC
Furneaux
Henry M
HM
Jazayeri
Jalal A
JA
Leedman
Peter J
PJ
D020413:3' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D004268:DNA-Binding Proteins; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011066:Poly C / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C094604:PCBP1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011066:Poly C; D004815:Epidermal Growth Factor; D008156:Luciferases