The 3'-Untranslated Region of p21 super(WAF1) mRNA Is a Composite cis-Acting Sequence Bound by RNA-binding Proteins from Breast Cancer Cells, Including HuR and Poly(C)-binding Protein

• Published in: The Journal of biological chemistry Vol. 278; no. 5; pp. 2937 - 2946
• Main Authors: Giles, K M, Daly, J M, Beveridge, D J, Thomson, A M, Voon, D C, Furneaux, H M, Jazayeri, JA, Leedman, P J
• Format: Journal Article
• Language: English
• Published: 31-01-2003
• ISSN: 0021-9258; 1083-351X

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 super(WAF1) is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21 super(WAF1) mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21 super(WAF1) 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21 super(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 super(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 super(WAF1) mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21 super(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 super(WAF1) mRNA via different mechanisms. We conclude that EGF-induced p21 super(WAF1) protein expression is mediated largely by stabilization of p21 super(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 super(WAF1) expression or growth inhibition in this system. CP1 is a novel p21 super(WAF1) mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21 super(WAF1) mRNA stability.