The goal of this proposal is to determine if a recently discovered RNA-binding protein is expressed specifically in human cancer tissue. The protein binds to a segment of the c-myc mRNA coding region that causes the mRNA to be unstable. This segment is called the Coding Region instability Determinant or CRD. The protein that binds to the CRD is the CRD-Binding Protein or CRD-BP. We discovered the CRD-BP using a cell-free mRNA decay system. We observed that polysome-associated c-myc mRNA was specifically and rapidly degraded by an endonuclease when competitor RNA corresponding to the c-myc CRD was added to the reactions. Cleavage occurred within the CRD of the mRNA. We reasoned that a protein might be shielding the CRD from endonuclease attack and that competitor CRD RNA was titrating the protein off of the mRNA. This protein, the CRD-BP, was later purified, sequenced, and cloned in our laboratory. Four subsequent observations from our lab revealed strong links between the CRD-BP and human cancer. Two findings suggest that the CRD-BP is an oncofetal protein: (i) The CRD-BP is expressed abundantly in fetuses but not in adults. (ii) The CRD-BP is expressed in most transformed human tissue culture cells. Two other findings suggest that the CRD-BP is a potential human tumor marker: (iii) The CRD-BP gene is amplified in approximately one-third of human breast cancer cases. (iv) The CRD-BP is expressed in some cases of human colon cancer but not in normal colon. Our working hypotheses are that (i) the CRD-BP is overexpressed in some or many human carcinomas and (ii) the CRD-BP might therefore be a new cancer marker. The following aims/questions explore these hypotheses:
Aim I. The CRD-BP is overexpressed in colon cancer. Is it also overexpressed in lung, breast, and prostate cancers? The CRD-BP gene is amplified in breast cancer. Is it also amplified in colon, lung, and prostate cancers? If so, does CRD-BP gene amplification correlate with CRD-BP overexpression? Aim II. Do patients with colon, lung, breast, and prostate cancer make antibodies to the CRD-BP? Aim III. Is the CRD-BP itself detectable in serum from patients with colon, lung, breast, and prostate cancer? Aim IV. Does CRD-BP expression in these cancers correlate with cancer grade or with a poorer prognosis? These studies could provide clinicians with a novel tumor marker. They might also stimulate future studies to evaluate the CRD-BP as a therapeutic target.
| Mao, Chengjian; Flavin, Kathryn Goolsby; Wang, Stanley et al. (2006) Analysis of RNA-protein interactions by a microplate-based fluorescence anisotropy assay. Anal Biochem 350:222-32 |
