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

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.