Advances in Brief p14 Silencing by Promoter Hypermethylation Mediates Abnormal Intracellular Localization of MDM2

The INK4a/ARF locus encodes two distinct tumor suppressors, p16 and p14. Although the contribution of p16 to human tumorigenesis through point mutation, deletion, and hypermethylation has been widely documented, little is known about specific p14 lesions and their consequences. Recent data indicate that p14 suffers inactivation by promoter hypermethylation in colorectal cancer cells. Because it is known that p14 prevents MDM2 nucleocytoplasmic shuttling and thus stabilizes p53 by attenuating MDM2-mediated degradation, we studied the relationship of p14 epigenetic silencing to the expression and localization of MDM2 and p53. Cancer cell lines with an unmethylated p14 promoter showed strong nuclear expression of MDM2, whereas in a colorectal cell line with p14 hypermethylation-associated inactivation, MDM2 protein was also seen in the cytosol. Treatment with the demethylating agent 5-aza-2*-deoxycytidine was able to reinternalize MDM2 to the nucleus, and p53 expression was restored. No apparent changes in retinoblastoma localization were observed. We also studied the profile of p14 promoter hypermethylation in an extensive collection of 559 human primary tumors of different cell types, observing that in colorectal, gastric, renal, esophageal, and endometrial neoplasms and gliomas, aberrant methylation of p14 was a relatively common epigenetic event. MDM2 expression patterns revealed that lack of p14 promoter hypermethylation was associated with tumors showing exclusive nuclear MDM2 staining, whereas MDM2 cytosolic staining was frequently observed in neoplasms with aberrant p14 methylation. Taken together, these data support that epigenetic silencing of p14 by promoter hypermethylation is a key mechanism in the disturbance of the MDM2 nuclear localization in human cancer.