Patricia C. Sanchez-Diaz

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.

Post-transcription meets post-genomic: the saga of RNA binding proteins in a new era.

Gene expression is regulated by a complex series of events that take place both at the transcriptional and post-transcriptional levels. The mechanisms and factors involved in transcriptional regulation are relatively well understood, whereas post-transcriptional regulation, in comparison, is still a poorly appreciated process. RNA binding proteins (RBPs) are the key regulators of all post-transcriptional events (RNA splicing, stability, transport and translation). Essentially, in order to improve our knowledge in post-transcriptional regulation, we need to elucidate the mechanisms employed by RBPs to control gene expression. The combination of genomic tools with traditional biochemical approaches generated novel technologies, like ribonomics. The application of these novel technologies not only had a profound impact in the study of RBPs but also created the ground work necessary for the identification of post-transcriptional gene networks; a selected group of mRNAs associated with a particular function or biological pathway/process that is regulated by the same RBP. In this review article, we explore the latest achievements and discuss future challenges that lie ahead in the RBP world.

Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy.

BackgroundMusashi1 (Msi1) is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer.MethodsWe used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells.ResultsWe observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.ConclusionOur data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

Structural and Functional Analysis of SmeT, the Repressor of the Stenotrophomonas maltophilia Multidrug Efflux Pump SmeDEF

Stenotrophomonas maltophilia is an opportunistic pathogen characterized for its intrinsic low susceptibility to several antibiotics. Part of this low susceptibility relies on the expression of chromosomally encoded multidrug efflux pumps, with SmeDEF being the most relevant antibiotic resistance efflux pump so far studied in this bacterial species. Expression of smeDEF is down-regulated by the SmeT repressor, encoded upstream smeDEF, in its complementary DNA strand. In the present article we present the crystal structure of SmeT and analyze its interactions with its cognate operator. Like other members of the TetR family of transcriptional repressors, SmeT behaves as a dimer and presents some common structural features with other TetR proteins like TtgR, QacR, and TetR. Differing from other TetR proteins for which the structure is available, SmeT turned out to have two extensions at the N and C termini that might be relevant for its function. Besides, SmeT presents the smallest binding pocket so far described in the TetR family of transcriptional repressors, which may correlate with a specific type and range of effectors. In vitro studies revealed that SmeT binds to a 28-bp pseudopalindromic region, forming two complexes. This operator region was found to overlap the promoters of smeT and smeDEF. This finding is consistent with a role for SmeT simultaneously down-regulating smeT and smeDEF transcription, likely by steric hindrance on RNA polymerase binding to DNA.

De-Regulated MicroRNAs in Pediatric Cancer Stem Cells Target Pathways Involved in Cell Proliferation, Cell Cycle and Development

Background microRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years, the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs. Methodology/Results Using a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG, Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle, cell proliferation, p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p, hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays). Conclusion Our findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers.

Ubiquitin carboxyl-Terminal esterase L1 (UCHL1) is associated with stem-like cancer cell functions in pediatric high-grade glioma

Pediatric high-grade gliomas represent 8–12% of all primary tumors of the nervous system in children. Five-year survival for these pediatric aggressive tumors is poor (15–35%) indicating the need to develop better treatments for pediatric high-grade gliomas. In this work we used SF188 and SJ-GBM2 cell lines to study the function of the ubiquitin carboxyl-terminal esterase L1 (UCHL1), a deubiquitinase de-regulated in several cancers, in pediatric high-grade gliomas. UCHL1 depletion in SF188 and SJ-GBM2 glioma cells was associated with decreased cell proliferation and invasion, along with a reduced ability to grow in soft agar and to form spheres (i.e. self-renewal measure). A 70% reduction in Wnt signaling was also observed in the SF188 and SJ-GBM2 UCHL1 knockdowns (KDs) using a TCF-dependent TOPflash reporter assay. Transcriptome comparisons of UCHL1 KDs versus vector control identified a list of 306 differentially expressed genes (at least 2-fold change; p <0.05) which included genes known to be involved in cancer like ACTA2, POSTN, LIF, FBXL7, FBXW11, GDF15, HEY2, but also potential novel genes such us IGLL5, ABCA4, AQP3, AQP4, CALB1, and ALK. Bioinformatics gene ontology (GO) analysis of these 306 genes revealed significant enrichment in “signal peptides”, “extracellular matrix”and “secreted proteins” GO Terms. “Angiogenesis and blood vessel development”, “neuron differentiation/development”, cell adhesion”, and “cell migration” also showed significant enrichment in our GO analysis. Top canonical pathways identified by Ingenuity Pathway Analysis (IPA) included “Clathrin-mediated Endocytosis Signaling” (p = 5.14x10-4), “Virus Entry via Endocytic Pathways” (p = 6.15x 10−4), and “High Mobility Group-Box 1 (HMGB1) Signaling” (p = 6.15x10-4). While FGF2, IL1B, TNF and PDGFB were predicted as top upstream regulators (p < 2x10-16) of the UCHL1 KD-associated transcriptome. Aberrant expression of UCHL1 in pediatric high-grade gliomas may promote cell invasion, transformation, and self-renewal properties, at least in part, by modulating Wnt/Beta catenin activity. UCHL1 might act as an oncogene in glioma within the gene network that imparts stem-like characteristics to these cancer cells.

Gene sets enrichment analysis of miRNA expression profile

Alteration in expression levels of genes as well as their regulatory components, such as those levels of miRNAs in cancer cells will likely reflect in the enrichment of unique cellular functions and biological processes in their corresponding cell systems. By employing systems biology approaches, and instead of examining individual gene, we will directly and unambiguously identify the regulatory pathways and biological processes that are distinctive to specific disease states or responses to therapeutic treatment. In this study, we employ gene set enrichment analysis inspired method to explore the functional regulation of miRNAs. The analysis integrated a mathematic model that associates miRNAs to gene sets through miRNA target genes with a statistic test of enrichment to study the regulation change at a specific pathway. Different from conventional individual miRNA processing procedures, our method enables researchers to construct regulation hypothesis at pathway and functional levels.

Abstract 5144: Ubiquitin carboxy-terminal esterase L1 (UCHL1) in stem-like cancer cells of embryonal neural tumors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Emerging evidence suggest that most cancers have “stem cells” and that these stem-like cells are the drivers of tumor growth and metastases. Therefore targeting of these cancer stem cells will lead to advances in treatment of the disease. Accordingly, a rational option is to interfere with the function and or expression of genes or proteins that are essential for the self-renewal and expansion of the cancer stem cell fraction. We performed proteomic analysis to compare and identify proteins, the level of which is differentially altered between the neuropsheres (enriched cancer stem cell fraction) and the adherent cells, of human medulloblastoma cell line Daoy and human neuroblastoma cell line SK-N-BE(2). Several proteins important in cell cycle control, signal transduction, and cell differentiation were found to be over expressed in the cancer stem cell enriched neuropsheres. Intriguingly, we identified a deubiquitinating enzyme UCHL1 (ubiquitin carboxyl-terminal esterase L1) to be over-expressed in the neurospheres. UCHL1 is a component of the ubiquitin-proteasome system, the major machinery responsible for intracellular protein degradation and regulation of many key biological processes. The present study was undertaken to determine the role of UCHL1 in cancer stem cell functions. The differential level of UCHL1 was validated using western blot analyses in SK-N-BE(2), SH-SY5Y and Daoy cell lines. The levels of UCHL1 in the neurosphere culture of SKNBE(2), Daoy and SH-SY5Y were ∼1.6, 1.7, and 2.0 fold respectively, higher, than the adherent culture, thus confirming our proteomic results. The mRNA level of UCHL1 was determined using quantitative RT-PCR. There was no significant difference in expression of mRNA levels of UCHL1 between the adherent cultures and neurospheres cultures therefore suggesting the possibility of a post-transcriptional regulation of UCHL1. Blockade of the hydrolase activity of UCHL1 was performed using small molecule inhibitor LND-5744. IC50 was determined in Daoy and SK-N-BE(2) neurospheres and monolayer adherent cells. Interestingly, both Daoy and SK-N-BE(2) neurospheres were more sensitive to LND-5744 than the adherent cells (Daoy neurosphere IC50 ∼100µM vs Daoy adherent cells ∼125µM and SK-N-BE(2) neurosphere IC50 ∼10-25µM, vs SK-N-BE(2) adherent cells ∼100µM). Thus, its loss seems to have a detrimental effect in the ability of cancer stem cell self-renewal, readout as impairment in neurosphere formation in the presence of LND-5744. Additional functional validation using small interfering RNA silencing of UCHL1 to confirm that its elevated expression may contribute to cancer stem cell properties are ongoing. The findings from this study suggested that UCHL1 might serve as prospective molecular target for neural embryonic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5144.