Musashi-1 Research Articles

Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells

BackgroundCell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window.MethodsWe performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry.ResultsHuman somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes.ConclusionsHuman drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination.

MicroRNA-143/Musashi-2/KRAS cascade contributes positively to carcinogenesis in human bladder cancer.

It has been well established that microRNA (miR)‐143 is downregulated in human bladder cancer (BC). Recent precision medicine has shown that mutations in BC are frequently observed in FGFR3, RAS and PIK3CA genes, all of which correlate with RAS signaling networks. We have previously shown that miR‐143 suppresses cell growth by inhibiting RAS signaling networks in several cancers including BC. In the present study, we showed that synthetic miR‐143 negatively regulated the RNA‐binding protein Musashi‐2 (MSI2) in BC cell lines. MSI2 is an RNA‐binding protein that regulates the stability of certain mRNAs and their translation by binding to the target sequences of the mRNAs. Of note, the present study clarified that MSI2 positively regulated KRAS expression through directly binding to the target sequence of KRAS mRNA and promoting its translation, thus contributing to the maintenance of KRAS expression. Thus, miR‐143 silenced KRAS and MSI2, which further downregulated KRAS expression through perturbation of the MSI2/KRAS cascade.

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.

Knockdown of HMGA2 regulates the level of autophagy via interactions between MSI2 and Beclin1 to inhibit NF1-associated malignant peripheral nerve sheath tumour growth

BackgroundMalignant peripheral nerve sheath tumours (MPNSTs) are sarcomas of Schwann cell lineage origin that occur sporadically or in association with the inherited syndrome, neurofibromatosis type 1 (NF1). This study aimed to examine the function of High mobility group protein A2 (HMGA2) in NF1 MPNST progression and the underlying molecular mechanism.MethodsImmunohistochemistry (IHC) was used to detect HMGA2 expression in MPNST and neurofibroma patient samples. Cell Cycle Kit-8 (CCK-8) and 5-ethynyl-20-deoxyuridine (EdU) assays, terminal deoxynucleotidyl transferase-mediated nick end labelling, and transmission electron microscopy were performed to reveal HMGA2 functions in NF1 MPNST cells in vitro and in vivo. Chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) were used to detect HMGA2-modulated genes regulating autophagy and growth in NF1 MPNSTs in vitro and in vivo.ResultsNF1 MPNST samples exhibit higher HMGA2 positivity rates (13/16) than sporadic MPNST (16/41) and neurofibroma (0/7) patient samples. High HMGA2 expression is correlated with poor prognosis. Neurofibromin 1 (NF1)-deficient MPNST cells display elevated HMGA2 expression. Functional experiments revealed that HMGA2 knockdown inhibits NF1 MPNST cell growth in vitro and in vivo. In addition to promoting cell cycle arrest and apoptosis, HMGA2 knockdown inhibits autophagy, favouring cell death. RNA-Seq and ChIP-Seq revealed that HMGA2 directly activates the Musashi-2 (MSI2) promoter region, and MSI2 overexpression reverses autophagy and growth in shHMGA2-transfected cells. MSI2 interacts with Beclin1, and Beclin1 blockade inhibits autophagy, thereby inhibiting cell proliferation.ConclusionsHMGA2 knockdown regulates autophagy via MSI2-Beclin1 interactions to inhibit NF1 MPNST growth, revealing potential therapeutic targets for these untreatable tumours.

Expression of Musashi-1 During Osteogenic Differentiation of Oral MSC: An In Vitro Study.

Background: Musashi-1 (MSI1) is a negative regulator of mesenchymal stromal cell (MSC) differentiation which in turn favors cell proliferation. However, little is known about its expression by MSC from the oral cavity and in the context of osteogenic differentiation. Aim: The aim of this study was to analyze the expression of MSI1 in the context of osteogenic differentiation of MSC derived from the oral cavity. Material/methods: For this in vitro study, MSC were isolated from six different origins of the oral cavity. They were extensively characterized in terms of proliferative and clonogenicity potential, expression of stemness genes (MYC, NANOG, POU5F1, and SOX2), expression of surface markers (CD73, CD90, CD105, CD14, CD31, CD34, and CD45) and adipo-, chondro- and osteogenic differentiation potential. Then, osteogenic differentiation was induced and the expression of MSI1 mRNA and other relevant markers of osteogenic differentiation, including RUNX2 and Periostin, were also evaluated. Results: Cell populations from the alveolar bone (pristine or previously grafted with xenograft), dental follicle, dental germ, dental pulp, and periodontal ligament were obtained. The analysis of proliferative and clonogenicity potential, expression of the stemness genes, expression of surface markers, and differentiation potential showed similar characteristics to those of previously published MSC from the umbilical cord. Under osteogenic differentiation conditions, all MSC populations formed calcium deposits and expressed higher SPARC. Over time, the expression of MSI1 followed different patterns for the different MSC populations. It was not significantly different than the expression of RUNX2. In contrast, the expression of MSI1 and POSTN and RUNX2 were statistically different in most MSC populations. Conclusion: In the current study, a similar expression pattern of MSI1 and RUNX2 during in vitro osteogenic differentiation was identified.

Musashi-1 Enhances Glioblastoma Migration by Promoting ICAM1 Translation

Glioblastoma multiforme (GBM) is a lethal brain tumor with a mean survival time of 1 year. One major reason for therapeutic failure is that GBM cells have an extraordinary capacity to invade normal brain tissue beyond the surgical margin, accounting for the lack of treatment efficacy. GBM cells that can infiltrate into the healthy brain possess tumor properties of stemness and invasion, and previous studies demonstrate that Musashi-1 (MSI1), a neural stem cell marker, plays an important role in the maintenance of stem cell status, cellular differentiation, and tumorigenesis in cancers. By analyzing neuronal progenitor cell markers and stemness genes, we predicted that MSI1 might be an important factor in GBM pathogenesis. Because inflammation aids in the proliferation and survival of malignant cells, the inflammatory microenvironment also promotes GBM invasion, and intercellular adhesion molecule-1 (ICAM1), a member of the immunoglobulin superfamily, is involved in inflammation. Our results indicate that the above phenomena are likely due to MSI1 upregulation, which occurred simultaneously with higher expression of ICAM1 in GBM cells. Indeed, MSI1 knockdown effectively suppressed ICAM1 expression and blocked GBM cell motility and invasion, whereas overexpressing ICAM1 reversed these effects. According to RNA immunoprecipitation assays, MSI1-mediated mRNA interactions promote ICAM1 translation. Finally, immunohistochemical analysis showed MSI1 and ICAM-1 to be coexpressed at high levels in GBM tissues. Thus, the MSI1/ICAM1 pathway plays an important role in oncogenic resistance, including increased tumor invasion, and MSI1/ICAM1 may be a target for GBM treatment.

The role of Arbovirus genome untranslated regions on neurotropism

Purpose: Arboviruses are a group of viruses transmitted by arthropods, mostly ticks, and mosquitoes. Among the members of this group are the families Togaviridae and Flaviviridae, (+)ssRNA viruses, such as Chikungunya, Dengue, and Zika. Multiple of these viruses have demonstrated their ability to cause neuropathology in humans. In Zika, a protein binding motif known as Musashi binding element (MBE) has been attributed to promoting replication, neurotropism, and pathology. Musashi-1 (MSI1) is an RNA-binding protein involved in the maintenance and self-renewal of stem cells and a translational regulator in many biological systems. MSI1 predominantly binds single-stranded UAG motifs in the 3’ untranslated region (UTR) of RNA. We have recently analyzed Musashi binding elements (MBEs) in the 3’UTR of flaviviruses (FV) in silico. In this study, we could show that MBEs in the 3’UTR of neurotropic viruses such as Zika, West Nile, and Powassan virus are highly accessible, and mostly occur in an unpaired structural context, which renders them optimal Musashi binding targets and corroborates previous experimental studies by a theoretical model. Methods & Materials: In order to expand to other related viruses, we systematically analyzed the properties of Musashi binding elements (MBEs) in the 3’UTR of Alphaviruses (AV) based on a thermodynamic model for RNA folding and correlated to the currently described pathogenicity in literature. Results: Our preliminary results indicate that MBEs in the 3’UTR of Chikungunya, Venezuelan Equine Encephalitis, and Eastern Equine Encephalitis viruses occur predominantly in paired, double-stranded structural context, suggesting that the mechanism to cause neurotropism in these AV must be a different one. Analyses in other AV are currently being performed to investigate the role of MBEs in a more broad dataset. Our initial analysis suggests that the MBE role in neuropathology may be an exclusive feature of FV. Conclusion: Our study addresses the broader question whether other emerging arboviruses can cause similar neurotropic effects. We expanded our study from a previous Flavivirus dataset to all (+)ssRNA arboviruses to evaluate the link between MBE and currently described neuropathology. Moreover, our thermodynamic model can be the initial indicative for in vivo studies to evaluate the potential neurotropic effect of MBEs on different viral families.

USP10 regulates Musashi-2 stability via deubiquitination and promotes tumour proliferation in colon cancer.

Recent studies have demonstrated that ubiquitin-specific protease 10 (USP10) plays a catalytic role in tumour suppression mainly by deubiquitinating its target proteins to enhance their stabilities. However, we found that USP10 could interact with and regulate the expression of oncogenic factor Musashi-2 (MSI2). We investigated whether USP10 positively regulates the expression of MSI2 by deubiquitination and confirmed the type of polyubiquitin chain that is linked to MSI2. We also explored the role of USP10 in regulating the proliferation of colon cancer through different experiments. This study provides a completely new perspective in understanding the role of USP10 in deubiquitination. In the future, USP10 may serve as a target for colon cancer treatment.

Increased musashi 2 expression indicates a poor prognosis and promotes malignant phenotypes in gastric cancer.

Musashi 2 (MSI2), a marker of stem and progenitor cells, has been identified as an oncogene. Various investigations have revealed that MSI2 is differently expressed in several types of blood cancer and solid cancers. However, its expression and biological functions in gastric cancer (GC) remain unclear. In the present study, MSI2 mRNA and protein expression were assessed in GC tissue samples. The associations between MSI2 mRNA expression and the clinicopathological characteristics of patients with GC were analyzed, and the effect of MSI2 on the prognosis of patients with GC was verified. The biological functions of MSI2 in GC cells were assessed using gain-of-function assays in vitro. The results revealed that MSI2 was overexpressed in the majority of GC tissue samples, although this difference was not significant. MSI2 mRNA expression levels were associated with invasion depth, tumor-node-metastasis stage, degree of differentiation and tumor size (P<0.05), but were not associated with sex, age, tumor location or human epidermal growth factor receptor 2 expression. Increased MSI2 expression resulted in a poorer prognosis in patients with GC (χ2=4.221; P=0.040). In vitro assays revealed that MSI2 promoted MKN-28 cell proliferation, migration and invasion, and promoted tube formation in HUVECs. Although no significance of MSI2 expression was found, its oncogenic functions in the GC cell line indicated that MSI2 may be a potential oncogene that may serve as a biomarker for GC diagnosis and prognosis with verification from a larger sample and more GC cell lines.

Musashi 2 Is Overexpressed in Poor Outcome CLL Patients and Their Proliferative Fraction and Silencing This Gene Induces Apoptosis and Increases Cell Adhesion and Movement

The growth of CLL cells stems from a small fraction of dividing CD5+B cells. The size and rate of growth of this proliferative fraction (PF) correlates directly with poor outcome prognostic markers and inversely with time-to-first-treatment. Furthermore, since dividing cells upregulate DNA mutators (AID and APOBEC), the PF can acquire new DNA abnormalities that can lead to more lethal disease. Hence, cells of the PF are important targets for therapy.By gene expression profile analysis, we found that Musashi 2 (MSI2) is highly expressed in the PF (CXCR4DimCD5Bright) compared with the resting fraction (RF) that expresses the reciprocal phenotype (CXCR4BrightCD5Dim). In normal cells, MSI2 binds to mRNA and blocks or enhances protein translation. MSI2 levels are higher in proliferating normal and malignant stem cells and during tumorigenesis. In CLL, high MSI2 mRNA levels associate with poor outcome. Nevertheless, nothing is known about the function of MSI2 in CLL cells. Therefore, we studied the biological role of MSI2 in CLL B cells.We found that CLL B cells express higher levels of MSI2 protein than those of healthy donors (HD). MSI2 levels were higher in U-CLL than M-CLL, and M-CLL B cells expressed more than HD cells, consistent with MSI2 associating with poor prognosis. Within a CLL clone, MSI2 was higher in PF than RF. Also, microenvironment signals that induce B cell proliferation also increased MSI2 protein levels. Consistent with these observations in patients, in vitro experiments showed that dividing cells contain more MSI2 protein than undivided cells.Next, we investigated the importance of MSI2 on CLL survival. In primary CLL cells, we knocked down MSI2 levels using MSI2 siRNA and co-cultured CLL cells with stromal cells (HS5) or stimulated them with CpG ODN + IL15, with or without HS5 cells. In the three conditions tested, knock-down of MSI2 resulted in a significant decrease in tumor-cell survival compared with the control (P = 0.0342, 0.0079, and 0.0274, respectively). In addition, down regulation of MSI2 led to an upregulation of cleaved caspase3 (1.3 fold change;P= 0.029), p27kip1 (1.10 fold change; P= 0.0026) and phospho p53 (1.16 fold change; P= 0.017) compared with siRNA control (siCTR). Collectively, the results highlight the importance of MSI2 in primary CLL-cell survival and suggest that these are mediated by caspase 3, p27kip1 and p53 signaling pathways.Reported comparisons of the RNAs that MSI2 binds in different cell types suggest that MSI2 regulates both common and unique mRNA targets in a cell type-specific manner. Hence to study the role of MSI2 in CLL, we analyzed the proteomes of MSI2 knockdown B cells from 12 CLL patients and controls. This showed that MSI2 knock down significantly increased the levels of 12 proteins (P ≤ 0.01 and fold change ≤ -1.2 ≥ 1.2). Since Ingenuity Pathway Analysis of these proteins suggested an increase cellular movement, we confirmed protein levels by flow cytometry of proteins involved in migration: Fer (non-tyrosine-protein kinase receptor), VAV1 (guanine nucleotide exchange factors) and its active form (phosphorY174 VAV, pVAV). Since Fer and VAV1 play a role in actin cytoskeleton regulation, we next knocked down MSI2 in B cells from the same 12 CLL patients and evaluated by microscopy cytoskeleton rearrangement of cells bound to fibronectin-coated slides. We used an established method to classify round cells as having a non-modified cytoskeleton and elongated cells as having undergone cytoskeleton rearrangement. Also, we counted the number of cells with an accumulation of polarized actin and others with multiple protrusions. MSI2 downregulation increased the number of elongated, polarized and cells with multi protrusions.Thus, MSI2 levels are higher in B cells from poor outcome patients and also in the dividing/divided cells of the PF before and after stimulation. Also, MSI's downregulation induces apoptosis of CLL cells and increases adhesion and migration. Therefore, we propose that MSI2 is a valuable target for therapeutic intervention since inhibiting its functions will likely abort clonal evolution and disease progression, making CLL an even more chronic and manageable condition. DisclosuresBarrientos:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kolitz:Magellan Health: Consultancy, Honoraria. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

Small-Molecule Targeting of Musashi RNA-Binding Activity in Acute Myeloid Leukemia

RNA-binding proteins (RBPs) play critical roles in cell homeostasis by controlling gene expression post-transcriptionally, contributing to mRNA processing events (splicing, polyadenylation, localization, stability, export and translation). The involvement of RBPs to tumorigenesis, through genetic perturbation or epigenetic dysregulation, has been found in a variety of human cancers. The RBP MUSASHI-2 (MSI2) contributes to the pathogenesis of a spectrum of solid tumors and hematologic malignancies and predicts a worse clinical outcome in patients with myeloid and acute lymphoblastic leukemia (MDS, AML and ALL). Thus, MSI2 has been proposed as a putative biomarker for diagnosis as well as a potential therapeutic target for AML. However, there are currently no specific inhibitors for MSI.Previous work from our lab reported a Fluorescent Polarization (FP) screen with 6,208 compounds identifying small-molecules with MSI RNA-binding inhibition activity. Here, we characterize Ro 08-2750 (Ro), best FP screen hit, as a MSI RNA-competitive inhibitor. Electrophoresis Mobility Shift Assays (EMSA) demonstrated Ro inhibition of MSI2-RNA complexes formation. MicroScale Thermophoresis (MST) interaction studies showed that the compound interacts with MSI2 full-length and RNA-Recognition Motif 1 (RRM1) with μM affinity and with nearly 20-fold lower KD to an RBP control (SYNCRIP). We obtained the crystal structure of MSI2 RRM1 at 1.7Å and docking and mutagenesis validation confirmed K22, F66, F97 and R100 as crucial binding residues in the RNA-binding pocket. To further prove structure activity relationship, we used two chemical analogs: Ro-OH, an alcohol derivative of the Ro's aldehyde, showed 10-fold reduced activity and Ro-NGF, containing the Ro isoalloxazine scaffold, showed no binding or activity in vitro. Of note, in proliferation assays Ro EC50 was 2.6±0.1 μM in MLL-AF9 bone marrow cells and an average of 8.4±1.1 μM in MOLM13 and K562 human AML cells, whereas RoOH and RoNGF showed 10-fold or >50 μM EC50, respectively. Ro significantly reduced binding of MSI2 to its mRNA targets (such as cMYC, CDKN1A or SMAD3) in an RNA-IP and a direct effect in their protein translation in human leukemia cells. RNA-sequencing of 4h Ro treated MOLM13 and K562 AML cells resulted in gene expression changes that enriched for the gene expression profiling after shRNA mediated depletion of MSI2 in CML-BC and AML cell lines. Ro demonstrated a significant therapeutic index abolishing MLL-AF9+ BM colony formation at concentrations that did not affect the plating efficiency of normal Lin-Sca+cKit+ (LSK) cells. Similarly, Ro demonstrated differential sensitivity in three AML patient samples colony formation compared to normal human CD34+ cord blood cells. Finally, we sought to determine Ro in vivo activity by using an aggressive murine MLL-AF9 murine leukemia model. Acute treatment (4h and 12hr) with 13.75 mg/kg Ro in DMSO reduced c-KIT protein abundance and intracellular c-MYC. Administration of the same Ro dose every 3 days was well tolerated and showed a significant reduction in spleen weights, white blood cell counts and c-MYC levels compared to the controls. These data provide the feasibility that targeting MSI in vivo could have therapeutic efficacy in AML.This study identifies and characterizes Ro 08-2750 as a compound selectively inhibiting the oncogenic RNA-binding activity of MSI in myeloid leukemia. Ro targeting an RRM motif to block RNA activity represents a valuable proof of concept for the general inhibition of these class of RNA regulators. Overall, we provide a framework to identify and test novel RBP inhibitors thus validating this class of proteins as chemically “druggable” novel therapeutic targets in cancer. DisclosuresChodera:Schrödinger: Consultancy, Membership on an entity's Board of Directors or advisory committees.

MSI1 associates glioblastoma radioresistance via homologous recombination repair, tumor invasion and cancer stem-like cell properties

IntroductionGlioblastoma multiforme (GBM) is the most common brain malignancy in adults, and currently available GBM treatments present several unique challenges. It is known that GBM involves cancer stem-like cells (CSCs) and tumor cells that aggressively invade normal brain tissues, and both cell types may cause resistance to radiotherapy (RT) and are thus responsible for therapeutic failure. The radioresistance of GBM cells relies on the efficient activation of the DNA damage response (DDR), but the mechanisms linking this response with stem-cell status and tumor invasion remain unclear. Materials and methodsWe used irradiation to treat patient-derived GBM (Par) cells and then purified radioresistant GBM (R2M2) cells through two rounds of irradiation and an invasion assay. Musashi-1 (MSI1) is a neural stem-cell marker and key oncogenic factor of GBM. We identified MSI1 expression to predict radioresistance through silencing an MSI1-high-expressing R2M2 cell line or inducing overexpression in a Par cell line with low/no MSI1 expression and assessing the subsequent DDR. ResultMSI1 enhances tumor invasion via VCAM1 and modulates GBM radioresistance via the hyperactivation of the DDR through increasing homologous recombination repair and evading apoptosis. MSI1 knockdown induces DNA damage accumulation in irradiated GBM cells and promotes their depletion in vitro; MSI1 knockdown also inhibits the formation of GBMs generated by irradiated xeno-transplanted cells. MSI1 inhibition may radiosensitize tumors, prevent CSC-positive selection induced by RT, and reduce tumor invasion. ConclusionMSI1 may involve in regulating GBM radioresistance, invasion, and recurrence and could be a novel target for GBM treatment.

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.

WNT and NOTCH signaling pathways as activators for epidermal growth factor receptor in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is the most common histological type of esophageal cancer, with a poor prognosis. Deregulation of WNT and NOTCH signaling pathways is important in ESCC progression, which can be due to either malfunction of their components or crosstalk with other pathways. Therefore, identification of new crosstalk between such pathways may be effective to introduce new strategies for targeted therapy of cancer. A correlation study was performed to assess the probable interaction between growth factor receptors and WNT/NOTCH pathways via the epidermal growth factor receptor (EGFR) and Musashi1 (MSI1), respectively.MethodsLevels of MSI1/EGFR mRNA expression in tumor tissues from 48 ESCC patients were compared to their corresponding normal tissues using real-time polymerase chain reaction.ResultsThere was a significant correlation between EGFR and MSI1 expression (p = 0.05). Moreover, there was a significant correlation between EGFR/MSI1 expression and grade of tumor differentiation (p = 0.02).ConclusionThis study confirms a direct correlation between MSI1 and EGFR and may support the important role of MSI1 in activation of EGFR through NOTCH/WNT pathways in ESCC.

Increased Expression of Musashi-1 Evidences Mesenchymal Repair in Maxillary Sinus Floor Elevation

This study aimed to analyze the expression of Musashi-1 (MSI1) in maxillary native bone and grafted bone after maxillary sinus floor elevation. To do so, fifty-seven bone biopsies from 45 participants were studied. Eighteen samples were collected from native bone while 39 were obtained 6 months after maxillary sinus grafting procedures. Musashi-1 was analyzed by immunohistochemistry and RT-PCR. MSI1 was detected in osteoblasts and osteocytes in 97.4% (38/39) of grafted areas. In native bone, MSI1 was detected in only 66.6% (12/18) of the biopsies, mainly in osteocytes. Detection of MSI1 was significantly higher in osteoprogenitor mesenchymal cells of grafted biopsies (p < 0.001) but minor in smooth muscle and endothelial cells; no expression was detected in adipocytes. The mesenchymal cells of the non-mineralized tissue of native bone showed very low nuclear expression of MSI1, in comparison to fusiform cells in grafted areas (0.28(0.13) vs. 2.10(0.14), respectively; p < 0.001). Additionally, the detection of MSI1 mRNA was significantly higher in biopsies from grafted areas than those from native bone (1.00(0.51) vs. 60.34(35.2), respectively; p = 0.029). Thus, our results regardig the significantly higher detection of Musashi-1 in grafted sites than in native bone reflects its importance in the remodeling/repair events that occur after maxillary sinus floor elevation in humans.

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.

Abstract 2863: Dissecting the structural basis for inhibitors of RNA-binding proteins

Abstract RNA-binding proteins (RBPs) are key regulators of cellular functions, especially in post-transcriptional regulations. Dysregulation of RBPs is implicated in many diseases including cancer. One of the RBPs that is overexpressed in a variety of human cancer is Musashi-2 (MSI2). Elevated MSI2 expression is associated with ectopic oncogenic pathways, including but not limited to NUMB/Notch, PTEN/mTOR, TGF-β/SMAD3, making MSI2 a promising therapeutic target for cancer. In the case of NUMB, MSI2 binds to and negatively regulates translation of NUMB, a negative regulator of Notch signaling. Protein structure is critical for drug discovery and structure-based rational design. However, so far there is no structure available for MSI2 protein. Recently, using nuclear magnetic resonance (NMR) we solved the first solution structure of MSI2 RNA-recognition Motif 1 (MSI2-RRM1) that forms the major long narrow RNA-binding pocket. Using a fluorescence polarization (FP) chemical libraries screening, we identified several groups of hit compounds that disrupt the binding of MSI2-NUMB RNA potently. These compounds induced apoptosis, inhibited cancer cell proliferation, invasion and metastasis in multiple tumor models tested in vitro and in vivo. They also interfered with cancer stem cell functions with reduced tumorsphere formation. Several lead compounds showed promising efficacy in animal tumor models of human breast, prostate and colon cancer. To investigate the specificity of the compounds towards other RBPs, the compounds were tested in biophysical binding assays such as FP, time resolved Fluorescence Resonance Energy Transfer assay (TR-FRET), NMR spectroscopy and cell-based assays. We compared the affinity of these MSI2 inhibitors towards other RBPs such as MSI1 and HuR. Using docking and molecular dynamics simulation, we analyzed the structure-activity relationship of these inhibitors towards different RBP targets. This work adds significant information to the structure of MSI2-RRM1 and the structural basis for designing more potent and specific inhibitors of RBPs. Citation Format: Lan Lan, Minli Xing, Xiaoqing Wu, Philip Gao, Justin T. Douglas, Yu Zhan, Gulhumay Gardashova, Jiajun Liu, Robert P. Hanzlik, Jeffrey Aubé, Kristi L. Neufeld, Berl R. Oakley, Roberto N. De Guzman, Liang Xu. Dissecting the structural basis for inhibitors of RNA-binding proteins [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 2863.

MSI2 knockdown represses extrahepatic cholangiocarcinoma growth and invasion by inhibiting epithelial-mesenchymal transition.

PurposeTo investigate the expression and functional role of Musashi2 (MSI2), an RNA-binding protein, in extrahepatic cholangiocarcinoma (eCCA).Patients and methodsWe measured MSI2 expression in human specimens and cell lines using Western blot and quantitative real-time polymerase chain reaction, and we analyzed its association with clinicopathologic features in eCCA patients. Univariate and multivariate analyses were performed to identify risk factors correlated with overall survival and disease-free survival. Functional experiments were used to study the mechanisms of MSI2 in regulating eCCA cell growth, migration, and invasion.ResultsMSI2 expression was upregulated significantly in both human specimens and cell lines, and high MSI2 expression was associated with lymph node metastasis, advanced TNM stage, and poor prognosis in eCCA patients. Additionally, MSI2 overexpression promoted eCCA cell growth, migration, and invasion, while MSI2 knockdown repressed eCCA cell migration and invasion by inhibiting epithelial–mesenchymal transition.ConclusionMSI2 is an independent prognostic factor for eCCA patients, and MSI2 downregulation inhibits eCCA cell growth and metastasis. MSI2 may be a potential therapeutic target for eCCA patients.

Functional Polymorphism in the MSI1 Gene Promoter Confers a Decreased Risk of Lung Cancer in Chinese by Reducing MSI1 Expression.

Background: Musashi1 (MSI1) is a characteristic stem cell marker that regulates the balance between cell self-renewal and differentiation. Evidence has identified MSI1 as a pivotal oncogenic regulator in diverse malignancies. However, little evidence uncovers the role of genetic variations of MSI1 gene in cancer etiology.Objective: The aim of this study was to investigate the association between genetic variants in the MSI1 gene and lung cancer risk.Methods: Based on a two-stage retrospective study with a total of 1559 patients with lung cancer and 1667 healthy controls, we evaluated the relevance between three putative functional SNPs in the MSI1 promoter (i.e., -2696T>C[rs7959801], -2297T>C[rs3742038] and -1081C>T[rs34570155]) and lung cancer risk.Results: We found that the SNP rs7959801T>C was significantly associated with lung cancer susceptibility. Compared to those with rs7959801TT wild-genotype, individuals with CT/CC variant genotypes exerted consistently beneficial roles in lung cancer risk in the discovery set (adjusted odd ratios [OR] = 0.67; 95% confidence interval [CI] = 0.57-0.80), and in the validation set (OR=0.69; 95%CI=0.54-0.88). Functional assays indicated that the allele transformation from T to C in rs7959801 of MSI1 gene arrestingly decreased its transcription activity in vitro. Furthermore, the expression levels of MSI1 were significantly lower in the patients with CT/CC variants than in those who were with TT genotype.Conclusion: Our findings suggested that the rs7959801T>C polymorphism in the MSI1 promoter conferred a decreased risk to lung cancer by reducing the expression of MSI1 and it may be a promising indicator for lung cancer predisposition.

Musashi-1 promotes chemoresistant granule formation by PKR/eIF2α signalling cascade in refractory glioblastoma

Musashi-1 (MSI1), one of the RNA-binding proteins, is abundantly found not only in neural stem cells but also in several cancer tissues and has been reported to act as a positive regulator of cancer progression. Growing evidence indicates that PKR and eIF2α play pivotal roles in the stimulation of stress granule formation as well as in the subsequent translation modulation in response to stressful conditions; however, little is known about whether MSI1 is involved in this PKR/eIF2α cancer stem cell-enhancing machinery. In this study, we demonstrated that MSI1 promotes human glioblastoma multiforme (GBM) stem cells and enhances chemoresistance when exposed to sublethal stress. The overexpression of MSI1 leads to a protective effect in mitigating drug-induced cell death, thus facilitating the formation of chemoresistant stress granules (SGs) in response to arsenic trioxide (ATO) treatment. SG components, such as PKR and eIF2α, were dominantly activated and assembled, while ATO was engaged. The activated PKR and eIF2α contribute to the downstream enhancement of stem cell genes, thereby promoting the progression of GBM. The silencing of MSI1 or PKR both obviously withdrew the phenomena. Taken together, our findings indicate that MSI1 plays a leading role in stress granule formation that grants cancer stem cell properties and chemoresistant stress granules in GBM, in response to stressful conditions via the PKR/eIF2α signalling cascade.