Dat T. Vo

Genomic Analyses of Musashi1 Downstream Targets Show a Strong Association with Cancer-related Processes

Musashi1 (Msi1) is a highly conserved RNA-binding protein with pivotal functions in stem cell maintenance, nervous system development, and tumorigenesis. Despite its importance, only three direct mRNA targets have been characterized so far: m-numb, CDKN1A, and c-mos. Msi1 has been shown to affect their translation by binding to short elements located in the 3′-untranslated region. To better understand Msi1 functions, we initially performed an RIP-Chip analysis in HEK293T cells; this method consists of isolation of specific RNA-protein complexes followed by identification of the RNA component via microarrays. A group of 64 mRNAs was found to be enriched in the Msi1-associated population compared with controls. These genes belong to two main functional categories pertinent to tumorigenesis: 1) cell cycle, cell proliferation, cell differentiation, and apoptosis and 2) protein modification (including ubiquitination and ubiquitin cycle). To corroborate our findings, we examined the impact of Msi1 expression on both mRNA (transcriptomic) and protein (proteomic) expression levels. Genes whose mRNA levels were affected by Msi1 expression have a Gene Ontology distribution similar to RIP-Chip results, reinforcing Msi1 participation in cancer-related processes. The proteomics study revealed that Msi1 can have either positive or negative effects on gene expression of its direct targets. In summary, our results indicate that Msi1 affects a network of genes and could function as a master regulator during development and tumor formation.

The oncogenic RNA-binding protein Musashi1 is regulated by tumor suppressor miRNAs

Musashi1 (Msi1) is an evolutionarily conserved RNA-binding protein that has been implicated in processes like stem cell fate, nervous system development, and tumorigenesis via its activities as a specific regulator of translation. While Msi1 is barely detected in normal adult tissue, it has been observed to be highly expressed in numerous tumor types (e.g. breast, colon, medulloblastoma, glioblastoma, and et cetera). Unfortunately, the molecular cues that are responsible for Msi1 upregulation in cancer cells are largely unknown. Tumor suppressor microRNAs (miRNAs) are known for targeting genes with oncogenic properties like Msi1 and for being either downregulated or deleted in tumor tissue. We observed that Msi1 long 3’UTR region is potentially targeted by several tumor suppressor miRNAs (miR-34a, -101, -128, -137, and -138). Western blotting of endogenous Msi1 protein as well as luciferase assays confirmed Msi1 regulation by these tumor suppressor miRNAs. Furthermore, we observed when examining different cellular states that these miRNAs and Msi1 have opposite expression profiles. Cell proliferation inhibition induced by the tumor suppressor miRNAs was partially rescued by Msi1 transgenic expression. We conclude that tumor suppressor miRNAs are direct and influential regulators of Msi1, affecting its expression pattern during tumorigenesis of malignant nervous system tumors.

The RNA-Binding Protein Musashi1 Affects Medulloblastoma Growth via a Network of Cancer-Related Genes and Is an Indicator of Poor Prognosis

Musashi1 (Msi1) is a highly conserved RNA-binding protein that is required during the development of the nervous system. Msi1 has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation, and has also been implicated in tumorigenesis, being highly expressed in multiple tumor types. We analyzed Msi1 expression in a large cohort of medulloblastoma samples and found that Msi1 is highly expressed in tumor tissue compared with normal cerebellum. Notably, high Msi1 expression levels proved to be a sign of poor prognosis. Msi1 expression was determined to be particularly high in molecular subgroups 3 and 4 of medulloblastoma. We determined that Msi1 is required for tumorigenesis because inhibition of Msi1 expression by small-interfering RNAs reduced the growth of Daoy medulloblastoma cells in xenografts. To characterize the participation of Msi1 in medulloblastoma, we conducted different high-throughput analyses. Ribonucleoprotein immunoprecipitation followed by microarray analysis (RIP-chip) was used to identify mRNA species preferentially associated with Msi1 protein in Daoy cells. We also used cluster analysis to identify genes with similar or opposite expression patterns to Msi1 in our medulloblastoma cohort. A network study identified RAC1, CTGF, SDCBP, SRC, PRL, and SHC1 as major nodes of an Msi1-associated network. Our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target.

Musashi1: an RBP with versatile functions in normal and cancer stem cells.

Musashi1 (Msi1) is a highly conserved RNA binding protein that was initially identified in Drosophila by its ability to regulate sensory organ development and asymmetric cell division. Studies in mammalian cells reveal multiple functions for Musashi1 in normal and abnormal processes by mediating different post-transcriptional processes. According to our recent studies, Musashi1 very likely controls hundreds of targets, forming networks that regulate apoptosis, differentiation, proliferation and cell cycle. Musashi1 is a characteristic stem cell marker that regulates the balance between self-renewal and differentiation. Over-expression of Musashi1 has been associated with numerous tumor types and its function is required for tumor growth in breast, colon, medulloblastoma and glioblastoma. Musashi1 has also been implicated in neurogenesis and neurodegenerative diseases, and is emerging as a potential therapeutic target in both regenerative medicine and cancer.

Genomic Analyses Reveal Broad Impact of miR-137 on Genes Associated with Malignant Transformation and Neuronal Differentiation in Glioblastoma Cells

miR-137 plays critical roles in the nervous system and tumor development; an increase in its expression is required for neuronal differentiation while its reduction is implicated in gliomagenesis. To evaluate the potential of miR-137 in glioblastoma therapy, we conducted genome-wide target mapping in glioblastoma cells by measuring the level of association between PABP and mRNAs in cells transfected with miR-137 mimics vs. controls via RIPSeq. Impact on mRNA levels was also measured by RNASeq. By combining the results of both experimental approaches, 1468 genes were found to be negatively impacted by miR-137 – among them, 595 (40%) contain miR-137 predicted sites. The most relevant targets include oncogenic proteins and key players in neurogenesis like c-KIT, YBX1, AKT2, CDC42, CDK6 and TGFβ2. Interestingly, we observed that several identified miR-137 targets are also predicted to be regulated by miR-124, miR-128 and miR-7, which are equally implicated in neuronal differentiation and gliomagenesis. We suggest that the concomitant increase of these four miRNAs in neuronal stem cells or their repression in tumor cells could produce a robust regulatory effect with major consequences to neuronal differentiation and tumorigenesis.

Computational challenges, tools, and resources for analyzing co- and post-transcriptional events in high throughput

Co‐ and post‐transcriptional regulation of gene expression is complex and multifaceted, spanning the complete RNA lifecycle from genesis to decay. High‐throughput profiling of the constituent events and processes is achieved through a range of technologies that continue to expand and evolve. Fully leveraging the resulting data is nontrivial, and requires the use of computational methods and tools carefully crafted for specific data sources and often intended to probe particular biological processes. Drawing upon databases of information pre‐compiled by other researchers can further elevate analyses. Within this review, we describe the major co‐ and post‐transcriptional events in the RNA lifecycle that are amenable to high‐throughput profiling. We place specific emphasis on the analysis of the resulting data, in particular the computational tools and resources available, as well as looking toward future challenges that remain to be addressed. WIREs RNA 2015, 6:291–310. doi: 10.1002/wrna.1274

RNA-Binding Protein Musashi1 Is a Central Regulator of Adhesion Pathways in Glioblastoma.

ABSTRACT The conserved RNA-binding protein Musashi1 (MSI1) has emerged as a key oncogenic factor in numerous solid tumors, including glioblastoma. However, its mechanism of action has not yet been established comprehensively. To identify its target genes comprehensively and determine the main routes by which it influences glioblastoma phenotypes, we conducted individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) experiments. We confirmed that MSI1 has a preference for UAG sequences contained in a particular structural context, especially in 3′ untranslated regions. Although numerous binding sites were also identified in intronic sequences, our RNA transcriptome sequencing analysis does not favor the idea that MSI1 is a major regulator of splicing in glioblastoma cells. MSI1 target mRNAs encode proteins that function in multiple pathways of cell proliferation and cell adhesion. Since these associations indicate potentially new roles for MSI1, we investigated its impact on glioblastoma cell adhesion, morphology, migration, and invasion. These processes are known to underpin the spread and relapse of glioblastoma, in contrast to other tumors where metastasis is the main driver of recurrence and progression.

Musashi1 Impacts Radio-Resistance in Glioblastoma by Controlling DNA-Protein Kinase Catalytic Subunit.

The conserved RNA-binding protein Musashi1 (MSI1) has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation and as a key oncogenic factor in numerous solid tumors, including glioblastoma. To explore the potential use of MSI1 targeting in therapy, we studied MSI1 in the context of radiation sensitivity. Knockdown of MSI1 led to a decrease in cell survival and an increase in DNA damage compared to control in cells treated with ionizing radiation. We subsequently examined mechanisms of double-strand break repair and found that loss of MSI1 reduces the frequency of nonhomologous end-joining. This phenomenon could be attributed to the decreased expression of DNA-protein kinase catalytic subunit, which we have previously identified as a target of MSI1. Collectively, our results suggest a role for MSI1 in double-strand break repair and that its inhibition may enhance the effect of radiotherapy.

RNA-binding protein HuR regulates the expression of ABCA1

ABCA1 is a major regulator of cellular cholesterol efflux and plasma HDL biogenesis. Even though the transcriptional activation of ABCA1 is well established, the posttranscriptional regulation of ABCA1 expression is poorly understood. Here, we investigate the potential contribution of the RNA binding protein (RBP) human antigen R (HuR) on the posttranscriptional regulation of ABCA1 expression. RNA immunoprecipitation assays demonstrate a direct interaction between HuR and ABCA1 mRNA. We found that HuR binds to the 3′ untranslated region of ABCA1 and increases ABCA1 translation, while HuR silencing reduces ABCA1 expression and cholesterol efflux to ApoA1 in human hepatic (Huh-7) and monocytic (THP-1) cells. Interestingly, cellular cholesterol levels regulate the expression, intracellular localization, and interaction between HuR and ABCA1 mRNA. Finally, we found that HuR expression was significantly increased in macrophages from human atherosclerotic plaques, suggesting an important role for this RBP in controlling macrophage cholesterol metabolism in vivo. In summary, we have identified HuR as a novel posttranscriptional regulator of ABCA1 expression and cellular cholesterol homeostasis, thereby opening new avenues for increasing cholesterol efflux from atherosclerotic foam macrophages and raising circulating HDL cholesterol levels.

The Musashi1 RNA-Binding Protein: A Critical Regulator in Glioblastoma

Musashi1 (Msi1) is an evolutionary conserved RNA-binding protein that plays important roles in neural stem cell maintenance, nervous system development, and tumorigenesis. In glioblastoma, Msi1 is found to be highly expressed and to control a network of cancerrelated genes. In this chapter, we will review the participation of RNA-binding proteins in tumorigenesis and the role of Msi1 in stem cells and in glioblastoma. Furthermore, we will discuss the results of a study done with The Cancer Genome Atlas (TCGA) dataset to map genes highly correlated in expression with Msi1 as an avenue to understand its function in gliomagenesis.