Date of Award

8-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Taosheng Chen, PhD

Committee

Meiyun Fan, PhD Mark Hatley, MD, PhD Ronald N. Laribee, PhD Erin Schuetz, PhD

Abstract

Nuclear receptors (NRs) constitute a major class of drug targets in the treatment of various cancer types. NRs respond to cellular signals and become activated upon ligand binding to transcriptionally modulate expression of target genes. NR4A1 (Nur77) is a member of the NR4A family of nuclear receptors and displays an oncogenic profile in many cancer models. It is often upregulated in adult solid malignancies and is known to promote cell proliferation and survival. Knockdown studies of NR4A1 in cancer cell lines results in decreased cell growth and angiogenesis and increased apoptosis, suggesting NR4A1 is an oncogenic protein. Due to the elevated levels of NR4A1 in cancer, it is important to determine the regulatory mechanisms behind this expression pattern.

One such mechanism is through microRNAs (miRNAs), which regulate gene expression by binding to the 3ʹUTR of target mRNA and effectively inhibit translation into protein. Prior to

this study, no miRNAs had been identified to directly target NR4A1. By using luciferase reporter assays, we identified miR-124, miR-15a, and miR-224 as potential NR4A1 regulators. The direct binding of these miRNAs to their potential seed regions within the 3ʹUTR of NR4A1 was confirmed by mutagenesis of their respective seed sequences. This abrogated the binding and thereby confirmed the direct targeting of these miRNAs to these particular sequences. To further study the relationship between NR4A1 and these miRNAs, we analyzed endogenous expression levels in several pediatric cancer cell lines. NR4A1 was upregulated in RD, Rh41, and Rh30 rhabdomyosarcoma cells and D341 and Daoy medulloblastoma cells as well as NB3 neuroblastoma cells. All three miRNAs were downregulated in Daoy cells. Considering that miR-124 is highly expressed in the brain and is a tumor suppressor, we decided to investigate the functional significance between NR4A1 and miR-124 in Daoy cells. We found that miR-124 could decrease NR4A1 mRNA and protein levels as well as the expression of several NR4A1 target genes. Overexpression of NR4A1 led to enhanced cell viability and proliferation while knockdown resulted in the opposite phenotype. Furthermore, stable expression of miR-124 in Daoy cells resulted in decreased proliferation and smaller spheroid formation. Lastly, we examined expression levels in granule neuron precursors (GNPs), which are the most common cell type in the cerebellum where medulloblastoma arises. Interestingly, there was an inverse expression pattern in which miR-124 was increased while Nr4a1 was decreased in the differentiated GNPs, suggesting a potential role for NR4A1 in neuronal development.

In addition to cancer cell proliferation, the role of NR4A1 in skeletal muscle differentiation was also explored. We found that NR4A1 increased during the differentiation of human LHCN myoblasts, and that knockdown of NR4A1 impairs differentiation and reduces expression of myogenic markers in LHCN as well as SkMC, and HSMM primary human skeletal muscle cells. This data agrees with previous studies performed in mouse models and mouse C2C12 cells showing increased Nr4a1 expression during differentiation as well as the ability of NR4A1 to enhance muscle mass and myofiber size.

Together, these two studies highlight two different and opposing functional roles of NR4A1 in medulloblastoma and skeletal muscle. The first study identified three miRNAs capable of directly targeting and suppressing NR4A1 and also provides a rationale for the use of miRNA mimics as a potential therapeutic in cancers with high NR4A1 expression. In the second study, we provided evidence that further confirms the pro-myogenic function of NR4A1 during skeletal muscle differentiation. It is important to understand this basic biology as it can help further understand and treat diseases related to muscle such as rhabdomyosarcoma and muscular dystrophy.

ORCID

http://orcid.org/0000-0002-8213-5253

DOI

10.21007/etd.cghs.2016.0411

Comments

Two year embargo expires August 2018.

Available for download on Wednesday, September 19, 2018

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