RNA-binding Protein Musashi1 Research Articles

SAT-292 Musashi: A Novel Regulator of the Gonadotrope Transcriptome

Sufficient nutrition is critical for reproduction. We have previously shown that leptin, a circulating indicator of fat stores, signals to pituitary gonadotropes to maintain gonadotropin releasing hormone receptor (GnRHR) protein levels in female mice. We hypothesized that this process is post-transcriptional, happening primarily through regulation of the RNA-binding protein Musashi (MSI). We showed that MSI binds to Gnrhr and inhibits translation, and a gonadotrope-specific deletion of Msi1 and Msi2 (Gon-Msi1/2-null) leads to increased GnRHR protein levels. This culminates in dysregulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH). We have recently identified other gonadotrope and pituitary targets of MSI. We therefore suspected that MSI plays a role in both the maturation of gonadotropes and the normal cyclic regulation of gonadotropes. We hypothesized that the deletion of MSI would lead to downstream effects on (1) the composition of the gonadotrope population and (2) the molecular landscape of these cells. Using our adult, diestrous Gon-Msi1/2-null females, we performed single-cell RNA-sequencing on methanol-fixed dispersed pituitary cells. Libraries were made from two control pools and two mutant pools (n=3 pituitaries/pool) using 10x Genomics v3.1 Single-Cell Gene Expression technology and initially sequenced on an Illumina Next-seq mid-output flow-cell, yielding 5,000 reads/cell. Subsequent high-output sequencing obtained 25,000 reads/cell. We recovered single-cell mRNA transcript information from 18,206 control pituitary cells and 16,255 Gon-Msi1/2-null cells. Our analyses revealed that the Gon-Msi1/2-null pools had a higher % of cells expressing Fshb, as well as an expected significant drop in Msi2-expressing gonadotropes and no change in Lhb-expressing cells. We have recently identified Fshb as an MSI target in silico, and qRT-PCR of female pituitary lysate immunoprecipitated with anti-MSI1 shows a 7-fold enrichment in Fshb mRNA. We identified differentially expressed genes comparing the control and Gon-Msi1/2-null gonadotrope clusters. Using Gene Ontology analyses, the Gon-Msi1/2-null gonadotrope cluster appears to have aberrant expression of mRNAs involved in protein folding and cellular responses to nutrients. Our high-output sequencing has allowed us to achieve 25,000 reads/cell and will provide greater resolution of the role of Musashi in control of gonadotrope function. Taken together, our data indicate that Musashi influences the molecular landscape and subsequent physiology of the female gonadotrope. We have identified potential gonadotrope-specific MSI targets, including pathways that may underlie the dysregulated gonadotropin production and secretion seen in our Gon-Msi1/2-null females. Future studies will compare pubertal and adult females, as well as females from different estrous cycle stages.

RNA-binding protein Musashi2 stabilizing androgen receptor drives prostate cancer progression.

The androgen receptor (AR) pathway is critical for prostate cancer carcinogenesis and development; however, after 18‐24 months of AR blocking therapy, patients invariably progress to castration‐resistant prostate cancer (CRPC), which remains an urgent problem to be solved. Therefore, finding key molecules that interact with AR as novel strategies to treat prostate cancer and even CRPC is desperately needed. In the current study, we focused on the regulation of RNA‐binding proteins (RBPs) associated with AR and determined that the mRNA and protein levels of AR were highly correlated with Musashi2 (MSI2) levels. MSI2 was upregulated in prostate cancer specimens and significantly correlated with advanced tumor grades. Downregulation of MSI2 in both androgen sensitive and insensitive prostate cancer cells inhibited tumor formation in vivo and decreased cell growth in vitro, which could be reversed by AR overexpression. Mechanistically, MSI2 directly bound to the 3′‐untranslated region (UTR) of AR mRNA to increase its stability and, thus, enhanced its transcriptional activity. Our findings illustrate a previously unknown regulatory mechanism in prostate cancer cell proliferation regulated by the MSI2‐AR axis and provide novel evidence towards a strategy against prostate cancer.

Interplay between the RNA binding-protein Musashi and developmental signaling pathways.

Musashi comprises an evolutionarily conserved family of RNA-binding proteins (RBP) that regulate cell fate decisions during embryonic development and play key roles in the maintenance of self-renewal and differentiation of stem cells and adult tissues. More recently, several studies have shown that any dysregulation of MSI1 and MSI2 can lead to cellular dysfunctions promoting tissue instability and tumorigenesis. Moreover, several reports have characterized many molecular interactions between members of the Musashi family with ligands and receptors of the signaling pathways responsible for controlling normal embryonic development: Notch, Transforming Growth Factor Beta (TGF-β), Wingless (Wnt) and Hedgehog Signaling (Hh); all of which, when altered, are strongly associated with cancer onset and progression, especially in pediatric tumors. In this context, the present review aims to compile possible cross-talks between Musashi proteins and members of the above cited molecular pathways for which dysregulation plays important roles during carcinogenesis and may be modulated by these RBP.

Musashi1 enhances chemotherapy resistance of pediatric glioblastoma cells in vitro.

Glioblastoma (GBM) is the most aggressive form of glioma in adults and children and is associated with very poor prognosis. Pediatric tumors are biologically distinct from adult GBM and differ in response to current GBM treatment protocols. Regarding pediatric GBM, new drug combinations and the molecular background of chemotherapy effects need to be investigated, in order to increase patient survival outcome. The expression of the RNA-binding protein Musashi1 (MSI1) in pediatric glioma samples of different WHO tumor grades was investigated on the protein (immunohistochemistry) and on the RNA level (publicly accessible RNA sequencing dataset). The impact of the chemotherapeutic temozolomide (TMZ) in combination with valproic acid (VPA) was tested in two pediatric glioblastoma-derived cell lines. The supportive effect of MSI1 expression against this treatment was investigated via transient knockdown and protein overexpression. MSI1 expression correlates with pediatric high-grade glioma (HGG). The combination of TMZ with VPA significantly increases the impact of drug treatment on cell viability in vitro. MSI1 was found to promote drug resistance to the combined treatment with TMZ and VPA. MSI1 expression is a potential marker for pediatric HGG and increases chemoresistance. Inhibition of MSI1 might lead to an improved patient outcome and therapy response.

Zika Virus as Oncolytic Therapy for Brain Cancer: Myth or Reality?

The ability of self-replicating oncolytic viruses (OVs) to preferentially infect and lyse cancer cells while stimulating anti-tumor immunity of the host strongly indicates its value as a new field of cancer therapeutics to be further explored. The emergence of Zika virus (ZIKV) as a global health threat due to its recent outbreak in Brazil has caught the attention of the scientific community and led to the discovery of its oncolytic potential for the treatment of glioblastoma multiforme (GBM), the most common and fatal brain tumor with poor prognosis. Herein, we evaluate the neurotropism of ZIKV relative to the receptor tyrosine kinase AXL and its ligand Gas6 in viral entry and the RNA-binding protein Musashi-1 (MSI1) in replication which are also overexpressed in GBM, suggesting its potential for specific targeting of the tumor. Additionally, this review discusses genetic modifications performed to enhance safety and efficacy of ZIKV as well as speculates future directions for the OV therapy.

Upregulation of microRNA-143-3p induces apoptosis and suppresses proliferation, invasion, and migration of papillary thyroid carcinoma cells by targeting MSI2

As a tumor-associated biological molecule, microRNA-143-3p (miR-143-3p) is implicated in the progression of papillary thyroid carcinoma (PTC). We conducted this study to elucidate the effects of miR-143-3p mediated by Musashi RNA binding protein 2 (MSI2) on the biological activities of PTC cells. The K1 cells with the lowest miR-143-3p expression were selected for the experiments. The targeting relationship between miR-143-3p and MSI2 was verified. The biological functions of miR-143-3p and MSI2 with respect to K1 cell proliferation, cycle distribution, apoptosis, invasion, migration, and tumorigenesis were studied using gain- and loss-of-function assays both in vitro and in vivo. MSI2 was verified to be a target gene of miR-143-3p. Cells treated with upregulation of miR-143-3p or silencing of MSI2 exhibited significantly decreased the expression of Bcl-2, PCNA, MCM2, Ki67, MSI2, MMP-2, and MMP-9. This was accompanied by inhibited cell proliferation, cell invasion, and migration, as well as a significant increase in Bax expression, cell cycle arrest, and cell apoptosis. More importantly, the tumor inhibitory effects of upregulated miR-143-3p were also confirmed in the tumor xenografts in nude mice. Our results indicate that upregulation of miR-143-3p suppresses the progression of PTC by impeding cell growth, invasion, and migration via downregulation of MSI2, highlighting the potential of miR-143-3p as a target for future PTC treatment.

Stability Over Time and Between Generations of Differential DNA Methylation Identified by Epigenome-Wide Association Study of Adolescent Adiposity

Background: Obesity predisposes to cardiovascular disease, itself influenced by perinatal factors. Epigenetics is hypothesized to link perinatal influences with later obesity. We aimed to identify, using an epigenome wide association study, differentially methylated CpGs measured at 17-years-old that associated with concurrent adiposity, then to ascertain whether the strongest of these associations were stable over time, between generations, and mediated the association between early life environment and later body composition. Methods: Genome-wide DNA methylation profiles were assayed using Infinium HumanMethylation 450K BeadChip arrays on blood samples from 1050 17-year-olds from The Raine Study (Generation 2). CpGs associated with adiposity measures (anthropometry, subcutaneous/visceral fat thickness by ultrasound) were pyrosequenced at 17 and 20-years-old (n=817), and in their mothers (n=406). Perinatal factors were examined as mediators of associations between CpG methylation and adiposity. Findings: Increased methylation of pyrosequenced CpGs within Musashi RNA Binding Protein 2 (MSI2), solute carrier family 25 member 10 (SLC25A10) and Ras Association (RalGDS/AF-6) Pleckstrin Homology Domains 1 (RAPH1) were associated with adiposity. Adjusting for perinatal factors, these CpGs independently predicted adolescent BMI accounting for 24% of the variance. MSI2 remained associated with BMI over time (β/SD, false discovery adjusted p-values: 17-years-old β=0.026, p=0.0025; 20-years-old β=0.027, p=0.0029), between generations (mother β=0.044, p=7.5e-04), with high sensitivity C-reactive protein (β=0.120, p=0.023) and leptin (β=0.149, p=0.0004). Interpretation: We report novel associations between DNA methylation within MSI2, SLC25A10 and RAPH1 with increased adolescent adiposity. For MSI2 these were stable over time, consistent across mother and child and, associated with inflammation. Funding Statement: The Raine Study Team, The NH&MRC, and The Telethon Kids Institute provided long term support of the Study. The University of Western Australia (UWA), Curtin University, The Telethon Kids Institute, Women and Infants Research Foundation, Edith Cowan University, Murdoch University, The University of Notre Dame Australia and Raine Medical Research Foundation provided funding for Core Management of the Raine Study. The DNA methylation work was supported by NHMRC grant 1059711. The 17 year follow-up was supported by National Health and Medical Research Council Program grant (ID353514) and Project grant (ID403981). RCH and TAM are supported by NHMRC Fellowships (grant number 1053384 and 1042255, respectively). S.R is supported by National Health and Medical Research Council EU grant (1142858) and the Department of Health, Western Australia FutureHealth fund in connection with the European Union's Horizon2020 grant 733206. This work was supported by resources provided by The Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia. KMG is supported by the UK Medical Research Council (MC_UU_12011/4), the National Institute for Health Research (NIHR Senior Investigator (NF-SI-0515-10042) and the NIHR Southampton Biomedical Research Centre) and the European Union (Erasmus+ Programme Early Nutrition eAcademy Southeast Asia-573651-EPP-1-2016-1-DE-EPPKA2-CBHE-JP). Declaration of Interests: KMG has received reimbursement for speaking at conferences sponsored by companies selling nutritional products, and is part of an academic consortium that has received research funding from Abbott Nutrition, Nestec and Danone. All other authors declare no conflicts of interest. Ethics Approval Statement: Institutional ethics approval has been obtained through the University of Western Australia Ethics Committee (RA/4/1/6613, 1214-EP, RA-4-1-2646). Informed consent was obtained from legal guardians or parents of those under the age of 17 years with assent of the child. Informed consent was obtained for those 17 years and older.

Abstract 3682: Therapeutic targeting of stem cell self-renewal in childhood medulloblastoma: Strategies for blocking recurrence

Abstract Introduction: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Group 3 MB patients face the highest incidence of metastasis and poor overall patient survival. The early onset and highly aggressive nature of MB suggest a stem cell origin, where a highly self-renewing transformed cell of the postnatal cerebellum drives MB tumorigenesis. In this work, we explore how WNT signaling and other essential drivers of self-renewal, BMI1 and MSI1, promote MB progression. We subsequently generate new strategies to therapeutically target mechanisms of MB stem cell self-renewal that drive treatment resistance and relapse in Group 3 MB. Experimental procedures: We apply stem cell assays, patient-derived human-mouse xenograft (PDX) models, and genomic and bioinformatic profiling of recurrent patient-derived MB. Our established brain tumor initiating cell (BTIC) model provides an excellent tool for the examination of developmental pathways implicated in MB. New Unpublished Data: A small molecule Bmi1 inhibitor, PTC-028, induced a remarkable decrease in self-renewal as well as reduction of local and spinal metastatic disease in recurrent MB, which is striking as no prior drug has shown efficacy against recurrent Group 3 MB. Although mouse and human neural stem cells (NSCs) express Bmi1 and are mildly sensitive to Bmi1 inhibitors, no significant toxicity was observed in either mouse or human NSCs upon PTC-028 treatment, at doses that induced efficacious killing of MB cells. Another novel therapeutic paradigm includes activating Wnt signaling in otherwise non-Wnt MB, which abrogates self-renewal and tumorigenicity of these highly aggressive tumors. For safe and non-toxic activation of Wnt in preclinical models, we identified L807mts, a novel inhibitor that functions through a substrate-to-inhibitor conversion mechanism within the catalytic site of GSK. A final therapeutic strategy to target self-renewal lies in the discovery of the targetable MB-specific interactome of the RNA binding protein (RBP) Musashi1, another key regulator of stem cell self-renewal. Msi1 is overexpressed in Group 3 MB compared to normal cerebellum, and is associated with poor patient prognosis. shRNA knockdown of Msi1 decreased the self-renewal capacity of MB stem cells and significantly decreased tumor burden and increased survival in our PDX model. Finally, comparative eCLIP (enhanced cross-linking and immunoprecipitation) of MB stem cells and normal NSCs, combined with mass spectrometry and RNA-sequencing of shMsi1 MB cells, has elucidated novel therapeutic targets in the RBP interactome of Msi1 Conclusion: Characterization and therapeutic targeting of self-renewal mechanisms unique to MB BTICs may provide an opportunity to limit treatment-resistant stem cell populations from driving patient relapse in recurrent Group 3 MB, a disease currently lacking any targeted therapies. Note: This abstract was not presented at the meeting. Citation Format: David Bakhshinyan, Michelle Kameda-Smith, Branavan Manoranjan, Ashley Adile, Chitra Venugopal, Sheila Kumari Singh. Therapeutic targeting of stem cell self-renewal in childhood medulloblastoma: Strategies for blocking recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3682.

Abstract 3657: Defining the role of the RNA-binding protein MSI2 in neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor of childhood with a 5-year survival rate approaching 50% in high-risk patients and like other pediatric cancers, lacks many targetable mutations. Thus, novel therapeutic approaches are needed. Previous research has shown that the RNA-binding protein (RBP), LIN28B, enhances MYCN expression and induces NB formation. Additionally, we previously demonstrated that LIN28B-RAN-AURKA signaling drives NB oncogenesis. These results highlight the role of RBPs in modulating NB tumorigenesis. The Musashi family includes RBPs that have been previously shown to be associated with worse prognosis in adult malignancies, including colon cancer and pancreatic cancer, among others. Thus, we speculated that the Musashi family might influence aggressive phenotypes in pediatric tumors, such as neuroblastoma. MSI2 is robustly expressed in neuroblastoma tumors, and, indeed, increased levels of Musashi-2 (MSI2) correlate with worse NB prognosis. In order to begin to elucidate its role in NB tumorigenesis, we knocked-down MSI2 in human NB cell lines using multiple independent short hairpin RNAs (shRNA) and small interfering RNAs (siRNA) and examined the impact on proliferation, survival, and downstream target genes relative to scrambled control RNA-treated cells. From these experiments, we observed decreased colony formation in clonogenic assays, increased apoptosis and decreased proliferation. To gain an initial understanding of the downstream targets that MSI2 influences, we performed Nanostring analysis in 2 human NB cell lines, Kelly and SK-N-DZ and found that knocking down MSI2 led to a downregulation of many cancer-related genes including IDH2 and other metabolic targets. IDH2 is a mitochondrial enzyme and its mutant is known to be involved in gliomagenesis. Changes in IDH2 expression have also been linked to epigenetic alterations. Finally, wild-type IDH2is upregulated in lung cancer and contributes to tumor growth. Therefore, we are currently examining the functional relationship between MSI2 and IDH2 in NB. Citation Format: Adeiye Pilgrim, Selma Cuya, Dongdong Chen, Robert Schnepp. Defining the role of the RNA-binding protein MSI2 in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3657.

MSI2-TGF-β/TGF-β R1/SMAD3 positive feedback regulation in glioblastoma.

Glioblastoma is the most malignant glioma tumors with inevitable relapse and resistance to chemotherapy; however, the mechanisms driving chemoresistance remain to be fully elucidated. This study is to explore the molecular and cellular mechanisms involving in the chemoresistance of glioblastoma. The expression of musashi (MSI) RNA-binding protein in the tumor tissues and cells of glioblastoma was measured. The effects of MSI2 in epithelial-to-mesenchymal transition (EMT), resistance to temozolomide (TMZ), tumor cell invasion, migration, and proliferation and associated signaling were evaluated. High MSI2 expression was observed in the glioblastoma tissues. Silencing or overexpression of MSI2 significantly affected tumor cells invasion, migration, and proliferation. Silencing of MSI2 expression significantly inhibited O6-methylguanine-DNA methyltransferase (MGMT) expression and tumor growth, and reversed resistance to TMZ in xenograft tumor models. MSI2 expression regulated EMT through activating the transcription factors Snail and the TGFβ R1/SMAD3 signaling. Our study demonstrated a positive feedback loop of MSI2-TGFβ/SMAD3 signaling which activates the EMT and MGMT which may contribute to chemoresistance in glioblastoma. This study also highlights that MSI2 could be a new target for the therapy of glioblastoma.

Effect of RNA-binding protein MSI2 deficiency on progression in triple-negative breast cancer via stabilizing TP53INP1.

e12555 Background: The RNA-binding protein Musashi-2 (MSI2) has been implicated in the tumorigenesis and tumor progression of human cancers. This study investigated the clinical significance of MSI2 and the potential molecular mechanisms involved in triple-negative breast cancer (TNBC) progression. Methods: The Illumina sequencing platform was used to analyze the mRNA transcriptome in TNBC vs. normal tissues, while quantitative reverse transcription–polymerase chain reaction and immunohistochemistry validated MSI2 expression in breast cancer tissues and the effects of MSI2 on TNBC cells were assayed in vitro and in viv o. RNA immunoprecipitation (RIP) DNA and RNA sequencing was performed to identify the potential MSI2-targeted mRNAs. RIP and luciferase analyses were used to confirm the mRNA targets of MSI2. Results: MSI2 expression was significantly downregulated in TNBC vs. normal tissues and was associated with a poor overall survival of patients. MSI2 overexpression in vitro and in vivo inhibited TNBC cell proliferation and invasion as well as the extracellular signal-regulated kinase 1/2 (ERK1/2) activity. Molecularly, MSI2 expression promoted the stability of TP53INP1 mRNA by its interaction with the TP53INP1 mRNA 3ʹ-untranslated region. Furthermore, knockdown of TP53INP1 expression was able to reverse MSI2-induced suppression of TNBC cell invasion, whereas ectopic expression of TP53INP1 and inhibition of the ERK1/2 activity blocked MSI2 knockdown-induced TNBC cell invasion. Conclusions: The current study demonstrated that downregulated MSI2 expression was associated with TNBC progression and a poor prognosis and that MSI2 expression inhibited TNBC proliferation and invasion by induction of TP53INP1 stability and inhibition of ERK1/2 activity.

Musashi binding elements in Zika and related Flavivirus 3\u2032UTRs: A comparative study in silico

Zika virus (ZIKV) belongs to a class of neurotropic viruses that have the ability to cause congenital infection, which can result in microcephaly or fetal demise. Recently, the RNA-binding protein Musashi-1 (Msi1), which mediates the maintenance and self-renewal of stem cells and acts as a translational regulator, has been associated with promoting ZIKV replication, neurotropism, and pathology. Msi1 predominantly binds to single-stranded motifs in the 3′ untranslated region (UTR) of RNA that contain a UAG trinucleotide in their core. We systematically analyzed the properties of Musashi binding elements (MBEs) in the 3′UTR of flaviviruses with a thermodynamic model for RNA folding. Our results indicate that MBEs in ZIKV 3′UTRs occur predominantly in unpaired, single-stranded structural context, thus corroborating experimental observations by a biophysical model of RNA structure formation. Statistical analysis and comparison with related viruses show that ZIKV MBEs are maximally accessible among mosquito-borne flaviviruses. Our study addresses the broader question of whether other emerging arboviruses can cause similar neurotropic effects through the same mechanism in the developing fetus by establishing a link between the biophysical properties of viral RNA and teratogenicity. Moreover, our thermodynamic model can explain recent experimental findings and predict the Msi1-related neurotropic potential of other viruses.

Musashi-2 and related stem cell proteins in the mouse suprachiasmatic nucleus and their potential role in circadian rhythms

BackgroundThe suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains the master circadian clock of the body and an unusually large number of cells expressing stem cell-related proteins. These seemingly undifferentiated cells may serve in entrainment of the SCN circadian clock to light cycles or allow it to undergo neural plasticity important for modifying its rhythmic output signals. These cells may also proliferate and differentiate into neurons or glia in response to episodic stimuli or developmental events requiring alterations in the SCN’s control of physiology and behavior. ProblemTo characterize expression of stem cell related proteins in the SCN and the effects of stem-like cells on circadian rhythms. MethodsExplant cultures of mouse SCN were maintained in medium designed to promote survival and growth of stem cells but not neuronal cells. Several stem cell marker proteins including SRY-box containing gene 2 (SOX2), nestin, vimentin, octamer-binding protein 4 (OCT4), and Musashi RNA-binding protein 2 (MSI2) were identified by immunocytochemistry in histological sections from adult mouse SCN and in cultures of microdissected SCN. A bioinformatics analysis located potential SCN targets of MSI2 and related RNA-binding proteins. ResultsCells expressing stem cell markers proliferated in culture. Immunostained brain sections and bioinformatics supported the view that MSI2 regulates immature properties of SCN neurons, potentially providing flexibility in SCN neural circuits. Explant cultures had ongoing mitotic activity, indicated by proliferating-cell nuclear antigen, and extensive cell loss shown by propidium iodide staining. Cells positive for vasoactive intestinal polypeptide (VIP) that are highly enriched in the SCN were diminished in explant cultures. Despite neuronal cell loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging of explants prepared from SCN of Per1::luc transgenic mice. The circadian rhythm in SCN gene expression persisted in brain slice cultures in stem cell medium. Prominent, widespread expression of RNA-binding protein MSI2 supported the importance of posttranscriptional regulation in SCN functions and provided further evidence of stem-like cells. ConclusionThe results show that the SCN retains properties of immature neurons and these properties persist in culture conditions suitable for stem cells, where the SCN stem-like cells also proliferate. These properties may allow adaptive circadian rhythm adjustments. Further exploration should examine stem-like cells of the SCN in vivo, how they may affect circadian rhythms, and whether MSI2 serves as a master regulator of SCN stem-like properties.

774 – Role of Cell Cycle Inhibitor P21WAF1/CIP1, RNA Binding Protein Musashi-1, and KrüPPEL-Like Factor 4 in Regulation of Mouse Bmi1+ Reserve Intestinal Stem Cells Function After γ Radiation-Induced Injury In Vivo

774 – Role of Cell Cycle Inhibitor P21WAF1/CIP1, RNA Binding Protein Musashi-1, and KrüPPEL-Like Factor 4 in Regulation of Mouse Bmi1+ Reserve Intestinal Stem Cells Function After γ Radiation-Induced Injury In Vivo

Antagonism between the RNA-binding protein Musashi1 and miR-137 and its potential impact on neurogenesis and glioblastoma development.

RNA-binding proteins (RBPs) and miRNAs are critical gene expression regulators that interact with one another in cooperative and antagonistic fashions. We identified Musashi1 (Msi1) and miR-137 as regulators of a molecular switch between self-renewal and differentiation. Msi1 and miR-137 have opposite expression patterns and functions, and Msi1 is repressed by miR-137. Msi1 is a stem–cell protein implicated in self-renewal while miR-137 functions as a proneuronal differentiation miRNA. In gliomas, miR-137 functions as a tumor suppressor while Msi1 is a prooncogenic factor. We suggest that the balance between Msi1 and miR-137 is a key determinant in cell fate decisions and disruption of this balance could contribute to neurodegenerative diseases and glioma development. Genomic analyses revealed that Msi1 and miR-137 share 141 target genes associated with differentiation, development, and morphogenesis. Initial results pointed out that these two regulators have an opposite impact on the expression of their target genes. Therefore, we propose an antagonistic model in which this network of shared targets could be either repressed by miR-137 or activated by Msi1, leading to different outcomes (self-renewal, proliferation, tumorigenesis).

Экспрессия РНК-связывающего белка Musashi-2, как негативный прогностический маркер при метастатическом колоректальном раке.

Экспрессия РНК-связывающего белка Musashi-2, как негативный прогностический маркер при метастатическом колоректальном раке.

Inhibition of RNA-Binding Protein Musashi-1 Suppresses Malignant Properties and Reverses Paclitaxel Resistance in Ovarian Carcinoma.

Background and Aims: Ovarian carcinoma (OC) is one of the most lethal malignant tumors with a high reoccurrence and chemoresistance. The key mechanism relationship with chemoresistance in ovarian carcinoma is still unclear. The existence of cancer stem cells involves in chemoresistance and reoccurrence in OC. The objective of this study was to investigate the expression, suppression of malignant properties and reversal of paclitaxel resistance inhibiting RNA-binding protein Musashi-1 with siRNA in ovarian cancer cells.Methods: The expression of MSI-1 was analyzed in 39 normal ovarian epithelia tissues, 92 serous cystadenomas, 68 borderline serous cystadenomas, and 97 serous cystadenocarcinomas by immunohistochemistry. pLKO.1-MSI-1-siRNA expression vector was transfected into ovarian carcinoma cell line A2780 and its paclitaxel-resistant cell subline A2780/Taxol. The roles of MSI-1 in proliferation, apoptosis, migration and invasion were explored by cell proliferation analysis, Caspase 3 activity assay, wound healing assay, migration and matrigel invasion assay, respectively. Western Blotting and Real-time quantitative PCR were conducted to detect the expression of MSI-1 and the ERK signaling pathway. Reversal of paclitaxel resistance assay was used to evaluate the role of MSI-1 in paclitaxel resistance of OC cells. Finally, therapeutic effects of MSI-1 inhibition were investigated the xenogratfs of SCID mice in vivo of the paclitacel-resistant.Results: MSI-1 is overexpressed and associated with an unfavorable prognosis in OC patients. Knockdown of MSI-1 by small interfering RNA (siRNA) inhibits proliferation, promotes apoptosis, and reduces migration and invasion of cancer cells. Moreover, MSI-1 expression inhibition reverses paclitaxel-resistance in OC cells. We further display that MSI-1 effectively protects OC cells from paclitaxel-induced apoptosis by increasing the expression of p-Bcl-2 through ERK signaling pathway activation. In vivo, MSI-1 siRNA clearly showed a strong effect on tumor growth inhibition and paclitaxel-resistance reversal.Conclusions: These findings suggest that MSI-1 overexpression is associated with the prognosis of OC patients, and knockdown of MSI-1 can suppress malignant properties and reverse paclitaxel-resistance in OC cells. MSI-1 maybe act as a potential prognostic indicator and a therapeutic target in OC.

Long non-coding RNA DANCR promotes malignant phenotypes of bladder cancer cells by modulating the miR-149/MSI2 axis as a ceRNA

BackgroundAccumulating evidences have indicated that long non-coding RNAs (lncRNAs) are potential biomarkers that play key roles in tumor development and progression. Differentiation antagonizing non-protein noding RNA (DANCR) is a novel lncRNA that acts as a potential biomarker and is involved in the development of cancers. However, the clinical significance and molecular mechanism of DANCR in bladder cancer is still unknown.MethodsThe relative expression level of DANCR was determined by Real-Time qPCR in a total of 106 patients with urothelial bladder cancer and in different bladder cancer cell lines. Loss-of-function experiments were performed to investigate the biological roles of DANCR on bladder cancer cell proliferation, migration, invasion and tumorigenicity. Comprehensive transcriptional analysis, RNA-FISH, dual-luciferase reporter assay and western blot were performed to explore the molecular mechanisms underlying the functions of DANCR.ResultsIn this study, we found that DANCR was significantly up-regulated in bladder cancer. Moreover, increased DANCR expression was positively correlated with higher histological grade and advanced TNM stage. Further experiments demonstrated that knockdown of DANCR inhibited malignant phenotypes and epithelial-mesenchymal transition (EMT) of bladder cancer cells. Mechanistically, we found that DANCR was distributed mostly in the cytoplasm and DANCR functioned as a miRNA sponge to positively regulate the expression of musashi RNA binding protein 2 (MSI2) through sponging miR-149 and subsequently promoted malignant phenotypes of bladder cancer cells, thus playing an oncogenic role in bladder cancer pathogenesis.ConclusionThis study is the first to demonstrate that DANCR plays a critical regulatory role in bladder cancer cell and DANCR may serve as a potential diagnostic biomarker and therapeutic target of bladder cancer.

Transcriptome-wide analysis uncovers the targets of the RNA-binding protein MSI2 and effects of MSI2's RNA-binding activity on IL-6 signaling

The RNA-binding protein Musashi 2 (MSI2) has emerged as an important regulator in cancer initiation, progression, and drug resistance. Translocations and deregulation of the MSI2 gene are diagnostic of certain cancers, including chronic myeloid leukemia (CML) with translocation t(7;17), acute myeloid leukemia (AML) with translocation t(10;17), and some cases of B-precursor acute lymphoblastic leukemia (pB-ALL). To better understand the function of MSI2 in leukemia, the mRNA targets that are bound and regulated by MSI2 and their MSI2-binding motifs need to be identified. To this end, using photoactivatable ribonucleoside cross-linking and immunoprecipitation (PAR-CLIP) and the multiple EM for motif elicitation (MEME) analysis tool, here we identified MSI2's mRNA targets and the consensus RNA-recognition element (RRE) motif recognized by MSI2 (UUAG). Of note, MSI2 knockdown altered the expression of several genes with roles in eukaryotic initiation factor 2 (eIF2), hepatocyte growth factor (HGF), and epidermal growth factor (EGF) signaling pathways. We also show that MSI2 regulates classic interleukin-6 (IL-6) signaling by promoting the degradation of the mRNA of IL-6 signal transducer (IL6ST or GP130), which, in turn, affected the phosphorylation statuses of signal transducer and activator of transcription 3 (STAT3) and the mitogen-activated protein kinase ERK. In summary, we have identified multiple MSI2-regulated mRNAs and provided evidence that MSI2 controls IL6ST activity that control oncogenic signaling networks. Our findings may help inform strategies for unraveling the role of MSI2 in leukemia to pave the way for the development of targeted therapies.

Abstract LB-079: The interaction of MSI1 and AGO2 promotes glioblastoma multiforme progression under environmental stress

Abstract Glioblastoma multiforme (GBM) is the high-grade astrocytoma; it is the most common and aggressive malignant brain tumor. GBM is largely resistant to therapy and experiences a corresponding poor prognosis. Therefore, GBM progression and recurrence are the main therapeutic challenges frequently faced by patients with refractory tumors. Nevertheless, the underlined molecular mechanism remains unknown. The RNA-binding protein musashi1 (MSI1) is expressed in stem cell compartments and in the aggressive tumor, only little is known about its function and mechanism in fact decision of target mRNA and post-transcription control. In this study, we describe a mechanism by which stress induces tumor progression via elevated cytoplasmic translocation of MSI1 in malignant neoplasms. MSI1 forms ribonucleoprotein (RNP) complexes with its downstream mRNAs at the 3'-untranslated region (3'UTR) or coding domain sequence (CDS) in the nucleus. Upon stress conditions, the MSI1/mRNA complex translocates to the cytosol where Argonaute2 (AGO2) is recruited and directly interact with MSI1. The MSI1/AGO2 complex modulates mRNA stability depending on their binding locations on the mRNA targets. Blocking of MSI1 shuttling by either a nuclear export inhibitor (Leptomycin B, LMB) or NES/NLS mutant attenuates GBM tumor growth in the mouse model. Importantly, the MSI1-AGO2 complex enhances GBM cells proliferation and survival under stress stimulation. MSI1 directly binds to AGO2 in the cytosol through its C-terminus. Orthotopic delivery of MSI1-C-terminus decoy peptides, which disrupt the MSI1/AGO2 interaction, effectively reduces stress-induced tumor progression. These results provide new insights into the function of MSI1 and suggest a novel peptide-based therapeutic strategy against refractory malignancies. Citation Format: Hsiao-yun Chen, Shih-Hwa Chiou. The interaction of MSI1 and AGO2 promotes glioblastoma multiforme progression under environmental stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-079.