Gianluca Occhi 1 , * , Daniela Regazzo 1 , Giampaolo Trivellin 1 , 2 , Francesca Boaretto 3 , Denis Ciato 1 , Sara Bobisse 3 , Sergio Ferasin 1 , Filomena Cetani 4 , Elena Pardi 4 , Márta Korbonits 2 , Natalia S. Pellegata 5 , Viktoryia Sidarovich 6 , Alessandro Quattrone 6 , Giuseppe Opocher 3 , 7 , Franco Mantero 1 , Carla Scaroni 1
21 March 2013
The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27 KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27 KIP1 expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5′UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF–encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27 KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27 KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27 KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27 KIP1 expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.
Gene expression can be modulated at different steps on the way from DNA to protein including control of transcription, translation, and post-translational modifications. An abnormality in the regulation of mRNA and protein expression is a hallmark of many human diseases, including cancer. In some eukaryotic genes translation can be influenced by small DNA sequences termed upstream open reading frames (uORFs). These elements located upstream to the gene start codon may either negatively influence the ability of the translational machinery to reinitiate translation of the main protein or, much less frequently, stimulate protein translation by enabling the ribosomes to bypass cis-acting inhibitory elements. CDKN1B, which encodes the cell cycle inhibitor p27 KIP1, includes an uORF in its 5′UTR sequence. p27 KIP1 expression is often reduced in cancer, and germline mutations have been identified in CDKN1B in patients affected with a syndrome (MEN4) characterized by varying combinations of tumors in endocrine glands. Here we show that a small deletion in the uORF upstream to CDKN1B reduces translation reinitiation efficiency, leading to underexpression of p27 KIP1 and coinciding with tumorigenesis. This study describes a novel mechanism by which p27 KIP1 could be underexpressed in human tumors. In addition, our data provide a new insight to the unique pathogenic potential of uORFs in human diseases.