BACKGROUND: Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for 15-20% of all pediatric brain tumors. In patients with MB, prognosis depends heavily on the molecular makeup of the tumor. New genomic approaches over the last decade have enabled researchers to sub-classify MB based on differences in the transcriptome: WNT, Sonic hedgehog (SHH), Group 3 (MYC-amplified), and Group 4 (heterogeneous). SHH tumors represent a third of all MB cases, and small-molecule inhibitors have already been developed that target SHH signaling. Most notably, vismodegib has shown great promise in the treatment of MB and other SHH-driven cancers by targeting Smoothened (SMO), an upstream regulator of GLI activity. However, most patients who had initially responded to the drug quickly acquired point mutations in SMO that led to treatment resistance. In addition, patients who harbored mutations downstream of SMO had no response to treatment and were found to be intrinsically resistant. Although most patients with SHH-MB can be cured, current treatments often require broad base therapies, such as radiation and chemotherapy, which can have harmful and long-lasting side effects. These observations underscore the need for less toxic, more targeted therapies that act at the level of the GLI family of transcription factors themselves. However, as transcription factors are generally considered undruggable, Dr. Robbins’ group at the University of Miami Miller School of Medicine sought to address this need by using focused screens of siRNAs or small molecules that target epigenetic GLI regulators. They identified several candidates that act as readers, writers, and/or erasers of protein acetylation and methylation and showed that a subset of these candidates act downstream of SMO to attenuate GLI signaling (data not yet published). Bromodomain- and Plant Homeodomain-linked Zinc Finger-containing Protein 1 (BRPF1) was one of these candidates and further analysis revealed that its knockdown reduced Gli1 expression by more than 50%. Recent studies link BRPF1 to cerebellar development and tumor formation in SHH-MB and may be suggestive of its role as a negative regulator.
OBJECTIVES: We sought to compare basal levels of Brpf1 expression in normal versus MB in mice; to characterize Brpf1 knockdown versus overexpression in SHH cell lines; and to determine if BRPF1 merits further investigation as a candidate for future drug targeting therapies in MB and other SHH-driven cancers.
METHODS: We used RT-qPCR and immunoblotting analysis to look at Brpf1 expression in Ptch+/- and adult wild-type mice. cDNA and protein samples were donated by colleagues in the lab. We also grew and maintained SHH Light2 cells in culture and then used these cells to carry out siRNA and plasmid DNA transfections. RNA extraction, RT-PCR, and RT-qPCR were used to examine transfection efficiency and its effect on Gli1 expression.
RESULTS: Brpf1 levels were higher in SHH-MB compared to normal cerebellum. However, BRPF1 proteins were not detected in either normal or tumor samples. Brpf1 knockdown in Light2 cells correlated with an overall decrease in Gli1 expression while overexpression had no obvious affect on Gli1 expression.
CONCLUSIONS: Our findings suggest that BRPF1 may function as a positive regulator of GLI activity. Recent studies verify this claim at least partially stating that BRPF1 acts as both a positive and negative regulator of gene expression depending on the context. Thus, before we can draw any final conclusions, more research is needed to look at BRPF1 in the specific context of the SHH pathway and developing cerebellum.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/23795 |
| Date | 12 July 2017 |
| Creators | Drozdowicz, Kelly |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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