ABSTRACT
Colorectal cancers (CRC) with microsatellite instability (MSI) have clinical, pathologic, genetic, and epigenetic features distinct from microsatellite-stable CRC. Examination of epidermal growth factor receptor (EGFR) mRNA and protein expression levels in a panel of colon cancer cell lines identified strong expression of EGFR in multiple cell lines with MSI. Although no relationship between EGFR overexpression and the length of a CA dinucleotide repeat in intron 1 was observed, a variant A13/A14 repeat sequence within the 3'-untranslated region (3'-UTR) of the EGFR gene was identified, which was mutated by either mononucleotide or dinucleotide adenosine deletions in 64% of MSI cell lines and 69% of MSI colon tumors. Using a Tet-Off system, we show that this mutation increases EGFR mRNA stability in colon cancer cells, providing a mechanistic basis for EGFR overexpression in MSI colon cancer cell lines. To determine whether this mutation is a driver or a bystander event in MSI colon cancer, we examined the effect of pharmacologic and molecular inhibition of EGFR in EGFR 3'-UTR mutant MSI cell lines. Cell lines with an EGFR 3'-UTR mutation and that were wild-type (WT) for downstream signaling mediators in the Ras/BRAF and PIK3CA/PTEN pathways were sensitive to EGFR inhibition, whereas those harboring mutations in these signaling mediators were not. Furthermore, in cell lines WT for downstream signaling mediators, those with EGFR 3'-UTR mutations were more sensitive to EGFR inhibition than EGFR 3'-UTR WT cells, suggesting that this mutation provides a growth advantage to this subset of MSI colon tumors.
Subject(s)
Colonic Neoplasms/genetics , ErbB Receptors/biosynthesis , Genes, erbB-1 , Mutation , Poly A/genetics , Repetitive Sequences, Nucleic Acid , 3' Untranslated Regions , Animals , Cell Line, Tumor , Colonic Neoplasms/enzymology , ErbB Receptors/genetics , Gene Amplification , Humans , Mice , Mice, SCID , Microsatellite Instability , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Ribonuclease III/antagonists & inhibitors , Ribonuclease III/genetics , Sequence DeletionABSTRACT
BACKGROUND: We have previously published results indicating that decreased expression of CDK2-AP1 in MSI human colorectal cancer is associated with deletion mutations in the poly (T) 8 repeat sequence within the 3'-UTR of the CDK2-AP1 gene. In this study, we test the hypothesis that the del T mutation results in decreased CDK2-AP1 expression by causing reduced mRNA stability. METHODS: We introduced wild-type and mutant 3'-UTR sequences fused to a green fluorescent protein (GFP) gene separately into human CRC cell lines and quantified the expression of the GFP gene. Native CDK2-AP1 mRNA stability was measured in human CRC cell lines, using an actinomycin D assay and the mRNA structure folding software mfold 3.2. RESULTS: Mutant GFP-3'-UTR samples demonstrated significantly reduced GFP expression compared with wild-type GFP-3'-UTR as measured by both FACS and real-time PCR. Both the actinomycin D assay and mfold software demonstrated significantly reduced mRNA stability for the del T poly (T) 8 transcript compared with the wild type. CONCLUSIONS: In summary, these novel results support our hypotheses that the del T poly (T) 8 observed in the 3'-UTR of the CDK2-AP1 gene in human MSI CRC is functionally significant and results in decreased CDK2-AP1 expression. The results also indicate the mechanism of this decreased expression is caused at least in part by decreased mRNA stability.