Date of Award
Doctor of Philosophy (PhD)
Molecular Therapeutics and Cell Signaling
Taosheng Chen, Ph.D.
Leonard Lothstein, Ph.D. Leta Nutt, Ph.D. Edwards A. Park, Ph.D. Gerard P. Zambetti, Ph.D.
nuclear receptor, NR4A2, miRNA, miR-34, p53, rhabdomyosarcoma
The nuclear receptor (NR) superfamily represents a structurally-conserved group of ligand-regulated transcription factors. These proteins have critical roles in various physiological and pathological processes, including cancer, and have been targets of drug therapy. The orphan NR subfamily 4A (NR4A), which includes the NR4A1 (Nur77), NR4A2 (Nurr1), and NR4A3 (Nor-1) genes, has been implicated in adult solid tumors and has been characterized as pro-tumorigenic mediator of cell proliferation, transformation, migration, and drug resistance. Alternatively, in leukemia, NR4A1 and NR4A3 have been described as tumor suppressors in hematologic malignancies. Members of the NR4A family are commonly overexpressed in cancer and this has been attributed to their regulation by other oncogenic signaling pathways.
Despite the understanding of signaling cascades that lead to overexpression of the NR4A members, little is known about their regulation by microRNAs (miRNAs). miRNAs are small, non-coding, endogenous RNAs that are transcribed, processed, and used to direct cellular proteins that destabilize or block translation of target mRNA. In this study, we first sought to determine the miRNAs that are responsible for regulating NR4A2. Using a 3ʹ UTR reporter assay, we identified miR-34 as a regulator of the NR4A2 through its 3ʹ UTR, which was confirmed using mutagenesis of the predicted binding region of the miR-34 seed region to its target site. We demonstrated that overexpression of exogenous or induction of endogenous miR-34 expression downstream of p53 activation by Nutlin-3a was associated with decreased endogenous NR4A2. Additionally, overexpression of NR4A2 was capable of suppressing the activation of p53 target genes, and was also able to attenuate the sensitivity of cells to the anti-proliferative effect of Nutlin-3a.
We further explored the roles of the NR4A family in pediatric cancer, an area that has not been fully investigated. We first determined that the members of the NR4A family are overexpressed in rhabdomyosarcoma (RMS) cell lines compared to normal muscle cells. Knockdown of NR4A1 or NR4A2 led to a reduction in cell proliferation and transformation, while knockdown of NR4A2 could also affect cell migration. Using a microarray approach, we sought to investigate the transcriptome-level changes in response to NR4A knockdown, and determined that knockdown of NR4A2 led to a unique gene signature, while NR4A1 and NR4A3 knockdown had large overlaps in expression changes. These unique gene expression changes in response to NR4A2 knockdown could explain the unique effects that NR4A2 has on migration.
Overall, this study has discovered miR-34 as a novel regulator of NR4A2, and places NR4A2 in a potential feedback mechanism involving p53, miR-34, and NR4A2. This could indicate that NR4A2 mediates at least some of its pro-oncogenic effects through the inhibition of p53, which is relieved by p53 itself upon activation. Alternatively, NR4A2, is shown to have other roles in cancer progression, potentially through novel downstream target genes. These data may be used in understanding the effects of miR-34 replacement therapy, as this method of treatment is progressing through clinical trials, allowing us to understand the diverse regulator cascades being modulated.