Role of 3’UTR Elements in the Regulation of the Cyclin D1 Proto-oncogene

Deshpande, Anagha (2009): Role of 3’UTR Elements in the Regulation of the Cyclin D1 Proto-oncogene. Dissertation, LMU München: Medizinische Fakultät

Abstract

Deregulation of the cell cycle regulator cyclin D1 in a wide variety of tumors has highlighted the role of cell cycle alterations in cancer. Genomic amplifications, mutations or balanced chromosomal translocations involving this gene are believed to lead to its aberrant overexpression in tumors. Somatic mutations in the 3’UTR of cyclin D1 gene have been reported in breast cancer, neuroblastoma and mantle cell lymphoma patients although their contribution to the cyclin D1 deregulation is unclear. In our study, we confirmed a regulatory role of the 3’UTR in cyclin D1 expression. Our results demonstrated that deletion of the cyclin D1 3’UTR significantly alters cyclin D1 protein expression and function. Similarly, the introduction of mutations observed in MCL patients in the cyclin D1 3’UTR significantly increased the expression of the cyclin D1 protein. These results underline that in malignancies such as MCL, truncation of the 3’UTR due to genomic deletions or somatic mutations is a likely cause of cyclin D1 overexpression. In order to ascertain whether the deletion of the cyclin D1 3’UTR could impart proliferative properties to cells, thereby contributing to transformation, we assessed the phenotype of fibroblasts retrovirally transduced with cyclin D1 with or without the 3’UTR. Interestingly our results demonstrated marked changes in cyclin D1 function upon deletion of the cyclin D1 3’UTR. Cells expressing cyclin D1 without the 3’UTR proliferated significantly more than those expressing the full length cyclin D1. Similar results were observed in rat ileum epithelial cells which lack the endogenous cyclin D1. Thus our data confirm that the deletion of the 3’UTR confers a proliferative advantage to cells. Furthermore, in this dissertation, we focused on the different potential regulatory elements of the cyclin D1 3’UTR to assess their role in controlling cyclin D1 expression. We reasoned that elements in the 3’UTR that are responsible for the controlled expression of the cyclin D1 protein are lost in 3’UTR deleted tumors. Therefore, it would be interesting to specifically pinpoint the role of these elements and highlight their contribution to cyclin D1 protein expression. It is assumed that since AU-rich elements (AREs) in the 3’UTR of cyclin D1 could have a potential destabilizing effect on the cyclin D1 mRNA, their loss could contribute to the observed overexpression of cyclin D1. Importantly, using highly sensitive reporter assays, we showed that the targeted loss of AREs from an otherwise intact 3’UTR leads to a decrease in reporter expression. These results demonstrate that the loss of these cis-acting elements in 3’UTR deleted tumors cannot account for cyclin D1 overexpression and there must be additional factors involved. Using bioinformatic analysis, we identified putative binding sites for microRNAs, small regulatory non-coding RNAs that have been shown to have important roles in cancer. Our study confirmed that microRNAs of the miR-15/16 family and the miR-17-92 cluster directly target the cyclin D1 gene through post-transcriptional regulation. These microRNAs have been shown to be involved in a cell cycle regulation and in a number of malignancies, especially in B-cell lymphoma. The various forms of cyclin D1 generated by deletions or mutations in the 3”UTR of cyclin D1 in tumors exclude these microRNA binding sites. Taken together, our results demonstrate a regulatory role for the 3’UTR in cyclin D1 expression and function. We show that the deletion of the cyclin D1 3’UTR leads to cyclin D1 overexpression and confers a proliferative advantage to cells. Finally, our results characterize the regulators functions of the different cis and trans-acting elements of the cyclin D1 3’UTR and identify this region as a bona fide target of cell cycle regulatory microRNAs. Extending these findings to other oncogenes, it is conceivable that the escape of 3’UTR mediated regulation by the acquisition of additional mutations of this region is an under-appreciated mechanism in the pathogenesis of cancer.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Cyclin D1, 3'UTR, MicroRNA, mantle cell lymphoma, AU rich elements
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 15. Juli 2009
• 1. Bericht­erstatter:in: Dreyling, Martin