Msi1 Expression Research Articles

Characterization of E-Cadherin, SSEA-1, MSI-1, and SOX-2 Expression and Their Association with Pale Cells in Adenomyosis

Adenomyosis (AM) is a gynecological disease characterized by the invasion of endometrial glands and stroma within the myometrium. The etiology and pathogenesis of AM remain inadequately understood. Pale cells were identified as a novel cell type characterized by the absence of desmosomal contacts and light-colored cytoplasm. These cells were observed to migrate individually through ultra-micro ruptures in the basal membrane of the endometrial glands, translocating into the stroma and then further into the myometrium. Our study aimed to explore the possible stem cell properties of these pale cells. Forty hysterectomy specimens were analyzed using immunohistochemistry and immunofluorescence to assess negative E-cadherin expression and the positive expression of stem cell markers SSEA-1, MSI-1, and SOX-2. Immunohistochemical analysis revealed the presence of pale cells and occasionally rounded, enlarged E-cadherin-negative cells predominantly in the basal endometrial epithelium. The stem cell marker SSEA-1 was significantly elevated in the basalis epithelium, as well as in the ectopic epithelium. SSEA-1 positive cells were also identified in the stroma and myometrium. Sporadic colocalization of SSEA-1+/E-cadherin– cells was confirmed through immunofluorescence. The positive staining of pale cells for SSEA-1 and MSI-1 was also confirmed at the ultrastructural level by immunoelectron microscopy. These findings indicate that pale cells may possess stem cell characteristics, particularly a positive SSEA-1 profile, warranting further in vitro investigation into their role in the pathogenesis of adenomyosis.

Alternative splicing of ALDOA confers tamoxifen resistance in breast cancer.

The cancer-associated alternative splicing (AS) events generate cancer-related transcripts which are involved in uncontrolled cell proliferation and drug resistance. However, the key AS variants implicated in tamoxifen (TAM) resistance in breast cancer remain elusive. In the current study, we investigated the landscape of AS events in nine pairs of primary and relapse breast tumors from patients receiving TAM-based therapy. We unrevealed a notable association between the inclusion of exon 7.2 in the 5'untranslated region (5'UTR) of ALDOA mRNA and TAM resistance. Mechanistically, the inclusion of ALDOA exon 7.2 enhances the translation efficiency of the transcript, resulting in increased ALDOA protein expression, mTOR pathway activity, and the promotion of TAM resistance in breast cancer cells. Moreover, the inclusion of exon 7.2 in ALDOA mRNA is mediated by MSI1 via direct interaction. In addition, elevated inclusion of ALDOA exon 7.2 or expression of MSI1 is associated with an unfavorable prognosis in patients undergoing endocrine therapy. Notably, treatment with Aldometanib, an ALDOA inhibitor, effectively restrains the growth of TAM-resistant breast cancer cells in vitro and in vivo. The present study unveils the pivotal role of an AS event in ALDOA, under the regulation of MSI1, in driving TAM resistance in breast cancer. Therefore, this study provides a promising therapeutic avenue targeting ALDOA to combat TAM resistance.

Conditioned medium-enriched umbilical cord mesenchymal stem cells: a potential therapeutic strategy for spinal cord injury, unveiling transcriptomic and secretomic insights.

Spinal cord injury (SCI) leads to significant destruction of nerve tissue, causing the degeneration of axons and the formation of cystic cavities. This study aimed to examine the characteristics of human umbilical cord-derived mesenchymal stem cells (HUCMSCs) cultured in a serum-free conditioned medium (CM) and assess their effectiveness in a well-established hemitransection SCI model. In this study, HUCMSCs cultured medium was collected and characterized by measuring IL-10 and identifying proteomics using mass spectroscopy. This collected serum-free CM was further used in the experiments to culture and characterize the HUMSCs. Later, neuronal cells derived from CM-enriched HUCMSC were tested sequentially using an injectable caffeic acid-bioconjugated gelatin (CBG), which was further transplanted in a hemitransection SCI model. In vitro, characterization of CM-enriched HUCMSCs and differentiated neuronal cells was performed using flow cytometry, immunofluorescence, electron microscopy, and post-transplant analysis using immunohistology analysis, qPCR, in vivo bioluminescence imaging, and behavioral analysis using an infrared actimeter. The cells that were cultured in the conditioned media produced a pro-inflammatory cytokine called IL-10. Upon examining the secretome of the conditioned media, the Kruppel-like family of KRAB and zinc-finger proteins (C2H2 and C4) were found to be activated. Transcriptome analysis also revealed an increased expression of ELK-1, HOXD8, OTX2, YY1, STAT1, ETV7, and PATZ1 in the conditioned media. Furthermore, the expression of Human Stem-101 confirmed proliferation during the first 3 weeks after transplantation, along with the migration of CBG-UCNSC cells within the transplanted area. The gene analysis showed increased expression of Nestin, NeuN, Calb-2, Msi1, and Msi2. The group that received CBG-UCNSC therapy showed a smooth recovery by the end of week 2, with most rats regaining their walking abilities similar to those before the spinal cord injury by week 5. In conclusion, the CBG-UCNSC method effectively preserved the integrity of the transplanted neuronal-like cells and improved locomotor function. Thus, CM-enriched cells can potentially reduce biosafety risks associated with animal content, making them a promising option for clinical applications in treating spinal cord injuries.

Expression of MSI1 and HER2 in mammary Paget's disease and their correlation with clinicopathological features and prognosis

Objective: To investigate the expression of MSI1 and HER2 in mammary Paget's disease, and the correlation between the expression levels of MSI1 and HER2 and the clinicopathologic characteristics and prognosis of the patients. Methods: Clinical data and paraffin-embedded specimens of 34 pairs of mammary Paget's disease and underlying breast cancer were collected at the Department of Pathology, Affiliated Lianyungang Oriental Hospital of Xuzhou Medical University from March 2011 to December 2019. Immunohistochemistry was used to detect the expression of MSI1 and HER2 in mammary Paget's disease and the accompanying breast cancer, and to analyze the correlation between the expression levels of MSI1 and HER2 and their clinicopathologic features, as well as their influence on prognosis. Results: In mammary Paget's disease, the positive rate of MSI1 was 91.2% (31/34) and the positive rate of HER2 was 88.2% (30/34); the expression of MSI1 and HER2 was positively correlated (P=0.001, r=0.530). The expression of MSI1 was positively correlated with menopausal status (r=0.372, P=0.030) and lymph node metastasis (r=0.450, P=0.008). HER2 expression was positively correlated with menopausal status (r=0.436, P=0.010), and negatively correlated with ER expression (r=-0.365, P=0.034). The co-expression of MSI1 and HER2 was positively correlated with age (r=0.347, P=0.044) and menopausal status (r=0.496, P=0.003), and negatively correlated with ER expression (r=-0.461, P=0.006). Conclusions: MSI1 and HER2 are highly expressed in mammary Paget's disease and their expression levels are positively correlated. The correlation analysis between clinicopathological features and prognosis suggests that both of them may be involved in the occurrence and development of mammary Paget's disease and are potential therapeutic targets for mammary Paget's disease.

1′-O-methyl-averantin isolated from the endolichenic fungus Jackrogersella sp. EL001672 suppresses colorectal cancer stemness via sonic Hedgehog and Notch signaling

Endolichenic fungi are host organisms that live on lichens and produce a wide variety of secondary metabolites. Colorectal cancer stem cells are capable of self-renewal and differentiation into cancer cells, which makes cancers difficult to eradicate. New alternative therapeutics are needed to inhibit the growth of tumor stem cells. This study examined the ability of an extract of Jackrogersella sp. EL001672 (derived from the lichen Cetraria sp.) and the isolated compound 1′-O-methyl-averantin to inhibit development of cancer stemness. The endolichenic fungus Jackrogersella sp. EL001672 (KACC 83021BP), derived from Cetraria sp., was grown in culture medium. The culture broth was extracted with acetone to obtain a crude extract. Column chromatography and reverse-phase HPLC were used to isolate an active compound. The anticancer activity of the extract and the isolated compound was evaluated by qRT-PCR and western blotting, and in cell viability, spheroid formation, and reporter assays. The acetone extract of EL001672 did not affect cell viability. However, 1′-O-methyl-averantin showed cytotoxic effects against cancer cell lines at 50 μg/mL and 25 μg/mL. Both the crude extract and 1′-O-methyl-averantin suppressed spheroid formation in CRC cell lines, and downregulated expression of stemness markers ALDH1, CD44, CD133, Lgr-5, Msi-1, and EphB1. To further characterize the mechanism underlying anti-stemness activity, we examined sonic Hedgehog and Notch signaling. The results showed that the crude extract and the 1′-O-methyl-averantin inhibited Gli1, Gli2, SMO, Bmi-1, Notch-1, Hes-1, and the CSL complex. Consequently, an acetone extract and 1′-O-methyl-averantin isolated from EL001672 suppresses colorectal cancer stemness by regulating the sonic Hedgehog and Notch signaling pathways.

Effect of 3D Spheroid Culturing on NF-κB Signaling Pathway and Neurogenic Potential in Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (hAFSCs) are known for their advantageous properties when compared to somatic stem cells from other sources. Recently hAFSCs have gained attention for their neurogenic potential and secretory profile. However, hAFSCs in three-dimensional (3D) cultures remain poorly investigated. Therefore, we aimed to evaluate cellular properties, neural differentiation, and gene and protein expression in 3D spheroid cultures of hAFSCs in comparison to traditional two-dimensional (2D) monolayer cultures. For this purpose, hAFSCs were obtained from amniotic fluid of healthy pregnancies and cultivated in vitro, either in 2D, or 3D under untreated or neuro-differentiated conditions. We observed upregulated expression of pluripotency genes OCT4, NANOG, and MSI1 as well as augmentation in gene expression of NF-κB-TNFα pathway genes (NFKB2, RELA and TNFR2), associated miRNAs (miR103a-5p, miR199a-3p and miR223-3p), and NF-κB p65 protein levels in untreated hAFSC 3D cultures. Additionally, MS analysis of the 3D hAFSCs secretome revealed protein upregulation of IGFs signaling the cascade and downregulation of extracellular matrix proteins, whereas neural differentiation of hAFSC spheroids increased the expression of SOX2, miR223-3p, and MSI1. Summarizing, our study provides novel insights into how 3D culture affects neurogenic potential and signaling pathways of hAFSCs, especially NF-κB, although further studies are needed to elucidate the benefits of 3D cultures more thoroughly.

Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

Active intestinal stem cells are prone to injury by ionizing radiation. We previously showed that upon radiation-induced injury, normally quiescent reserve intestinal stem cells (rISCs) (marked by BMI1) are activated by Musashi-1 (MSI1) and exit from the quiescent state to regenerate the intestinal epithelium. This study aims to further establish the mechanism that regulates activation of Bmi1-CreER;Rosa26eYFP (Bmi1-CreER) rISCs following γ radiation-induced injury. Bmi1-CreER mice were treated with tamoxifen to initiate lineage tracing of BMI1 (eYFP+) cells and exposed to 12 Gy of total body γ irradiation or sham. Intestinal tissues were collected and analyzed by immunofluorescence, Western blot, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and chromatin immunoprecipitation real-time polymerase chain reaction. After irradiation, increased expression of Msi1 in eYFP+ cells was accompanied by increased expression of Axin2, a WNT marker. Promoter studies of the Msi1 gene indicated that Msi1 is a WNT target gene. Coculture of stromal cells isolated from irradiated mice stimulated Bmi1-CreER-derived organoid regeneration more effectively than those from sham mice. Expression of WNT ligands, including Wnt2b, Wnt4, Wnt5a, and Rspo3, was increased in irradiated stromal cells compared with sham-treated stromal cells. Moreover, expression of the Sonic hedgehog (SHH) effector Gli1 was increased in stromal cells from irradiated mice. This was correlated with an increased expression of SHH in epithelial cells postirradiation, indicating epithelial-stromal interaction. Finally, preinjury treatment with SHH inhibitor cyclopamine significantly reduced intestinal epithelial regeneration and Msi1 expression postirradiation. Upon ionizing radiation-induced injury, intestinal epithelial cells increase SHH secretion, stimulating stromal cells to secrete WNT ligands. WNT activators induce Msi1 expression in the Bmi1-CreER cells. This stromal-epithelial interaction leads to Bmi1-CreER rISCs induction and epithelial regeneration.

Resveratrol impairs cellular mechanisms associated with the pathogenesis of endometriosis

Research questionDoes resveratrol exert a potent inhibitory effect on the development of endometriosis by interfering with some pivotal processes? DesignIn-vitro cultures of primary endometriotic stromal cells, immortalized endometrial stromal (St-T1b) and endometriotic epithelial (12Z) cells were used to assess the effects of resveratrol on endometrial cell mechanisms. The effects of resveratrol on 12Z and St-T1b cell viability were assessed by MTT assay, apoptosis by FITC Annexin V assay and cleaved caspase-3 levels and cell migration by wound healing assay. The effect of resveratrol on the expression of genes related to cell migration, angiogenesis and cell stemness was evaluated by qRT-PCR. ResultsResveratrol significantly decreased cell viability (P= 0.0065 to P = 0.0180), cell migration (P < 0.001 to P = 0.0225) and increased the number of apoptotic cells (P = 0.0031 to P = 0.0432) in both cell lines. In cell lines and primary culture, the treatment reduced MMP-2/TIMP-1 (P < 0.001 to P = 0.0180), VEGF (P = 0.0052 to P = 0.0243) and Ang-1 mRNA (P < 0.001 to P = 0.0382) expression. Among the stem cell phenotype markers, resveratrol 100 µM increased mRNA expression levels of Notch-1 (P < 0.001 to P = 0.0018), KLF-4 (P = 0.0011 to P = 0.0137), SOX-2 (P < 0.001 to P = 0.0070) and TERT (P < 0.001 to P = 0.0193) in both cell lines and primary cultures. The mRNA expression level of Snail-1 increased in the cell lines (P < 0.001 to P = 0.0087), whereas OCT-4 mRNA expression increased in St-T1b (P = 0.0396) and primary cultures (P = 0.0148). Vimentin mRNA expression showed a significant upregulation in primary cultures (P < 0.001). The expression of Msi-1 (P = 0.0145) and NANOG (P = 0.0080) decreased only in St-T1b cells. ConclusionResveratrol showed inhibitory effects on cell behaviour related to the development of endometriosis by differentially affecting growth, apoptosis, migration and stem cell phenotype of endometrial and endometriotic cells in vitro.

The transcript ENST00000444125 of lncRNA LINC01503 promotes cancer stem cell properties of glioblastoma cells via reducing FBXW1 mediated GLI2 degradation

LINC010503 is a novel oncogenic lncRNA in multiple cancers. In this study, we further explored the expression of LINC010503 transcripts and their regulations on the glioblastoma (GBM) stem cell (GSC) properties. LINC01503 transcription patterns in GBM and normal brain tissues were compared using RNA-seq data from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA)-GBM. GBM cell lines (U251 and U87) were used as in vitro cell models for cellular and molecular studies. The results showed that ENST00000444125 was the dominant transcript of LINC01503 in both normal and tumor tissues. Its expression was significantly elevated in the tumor group and associated with poor survival outcomes. LINC01503 had both cytoplasmic and nuclear distribution. It positively modulated the expression of multiple GSC markers, including CD133, SOX2, NESTIN, ALDH1A1, and MSI1, and tumorsphere formation in U251 and U87 cells. RNA pull-down and RIP-qPCR assay confirmed an interaction between ENST00000444125 and GLI2. ENST00000444125 positively regulated the half-life of the GLI2 protein in GBM cells. ENST00000444125 overexpression reduced GLI2 ubiquitination and partially attenuated FBXW1 overexpression induced GLI2 ubiquitination. ENST00000444125 overexpression could activate Wnt/β-catenin signaling in GBM cells. However, these activating effects were remarkedly hampered when GLI2 was knocked down. In conclusion, this study revealed that LINC01503 might have isoform-specific dysregulation in GBM. Among the two major transcripts expressed in GBM cells, ENST00000444125 might be the major functional transcript. Its upregulation might enhance the GSC properties of GBM cells via reducing FBXW1-mediated proteasomal degradation of GLI2.

The RNA-Binding Protein Musashi1 Regulates a Network of Cell Cycle Genes in Group 4 Medulloblastoma.

Medulloblastoma is the most common malignant brain tumor in children. Treatment with surgery, irradiation, and chemotherapy has improved survival in recent years, but patients are frequently left with devastating neurocognitive and other sequelae. Patients in molecular subgroups 3 and 4 still experience a high mortality rate. To identify new pathways contributing to medulloblastoma development and create new routes for therapy, we have been studying oncogenic RNA-binding proteins. We defined Musashi1 (Msi1) as one of the main drivers of medulloblastoma development. The high expression of Msi1 is prevalent in Group 4 and correlates with poor prognosis while its knockdown disrupted cancer-relevant phenotypes. Genomic analyses (RNA-seq and RIP-seq) indicated that cell cycle and division are the main biological categories regulated by Msi1 in Group 4 medulloblastoma. The most prominent Msi1 targets include CDK2, CDK6, CCND1, CDKN2A, and CCNA1. The inhibition of Msi1 with luteolin affected the growth of CHLA-01 and CHLA-01R Group 4 medulloblastoma cells and a synergistic effect was observed when luteolin and the mitosis inhibitor, vincristine, were combined. These findings indicate that a combined therapeutic strategy (Msi1 + cell cycle/division inhibitors) could work as an alternative to treat Group 4 medulloblastoma.

Assessment of MUSASHI 1 and MUSASHI 2 expression in spermatozoa and testicular tissue.

MUSASHI (MSI) family plays the main role in the spermatogenesis process. The purpose of this study was the assessment of sperm MSI1 and MSI2, and sperm functional tests in infertile men (n=30) with varicocele and fertile men (n=30). Furthermore, MSI1 and MSI2 proteins were assessed in testicular tissue of azoospermic men (n=9) as well as epididymal spermatozoa and testis of mice. Expression of MSI1 and MSI2 was assessed at RNA and protein levels in human spermatozoa. Sperm concentration and motility were significantly lower, while abnormal sperm morphology, lipid peroxidation, DNA fragmentation and protamine deficiency were significantly higher in men with varicocele compared to fertile individuals. Any significant difference was not observed in the expression of MSI1 and MSI2 mRNA between the two groups. Unlike MSI1 protein that was not detectable in humans, the relative expression of MSI2 protein was similar in varicocele and fertile individuals. The expression level of both Msi1 and Msi2 proteins was also observable in mouse spermatozoa. No significant relationship was observed between sperm functional parameters with expression of these genes. The data of this study demonstrated that although MSI1 and MSI2 play important roles during spermatogenesis, their relative expression in spermatozoa was not affected by varicocele.

The pyriproxyfen metabolite, 4′–OH–PPF, disrupts thyroid hormone signaling in neural stem cells, modifying neurodevelopmental genes affected by ZIKA virus infection.

North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by the Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. Thyroid hormone (TH) represses MSI1. PPF is a suspected TH disruptor. We hypothesized that co-exposure to the main metabolite of PPF, 4′–OH–PPF, could exacerbate ZIKV effects through increased MSI1 expression. Exposing an in vivo reporter model, Xenopus laevis, to 4′–OH–PPF decreased TH signaling and increased msi1 mRNA and protein, confirming TH-antagonistic properties. Next, we investigated the metabolite's effects on mouse subventricular zone-derived neural stem cells (NSCs). Exposure to 4′–OH–PPF dose-dependently reduced neuroprogenitor proliferation and dysregulated genes implicated in neurogliogenesis. The highest dose induced Msi1 mRNA and protein, increasing cell apoptosis and the ratio of neurons to glial cells. Given these effects of the metabolite alone, we considered if combined infection with ZIKV worsened neurogenic events. Only at the fourth and last day of incubation did co-exposure of 4′–OH–PPF and ZIKV decrease viral replication, but viral RNA copies stayed within the same order of magnitude. Intracellular RNA content of NSCs was decreased in the combined presence of 4′–OH–PPF and ZIKV, suggesting a synergistic block of transcriptional machinery. Seven out of 12 tested key genes in TH signaling and neuroglial commitment were dysregulated by co-exposure, of which four were unaltered when exposed to 4′–OH–PPF alone. We conclude that 4′–OH–PPF is an active TH-antagonist, altering NSC processes known to underlie correct cortical development. A combination of the TH-disrupting metabolite and ZIKV could aggravate the microcephaly phenotype.

Increased expression of MUSASHI1 in epithelial breast cancer cells is due to down regulation of miR-125b

BackgroundMusashi1 (MSI1) is an oncogenic protein with a crucial role in the proliferation and characteristics of the epithelial cells in breast cancer. The change in expression of MSI1 has a role in solid tumor progression. There are different factors that regulate MSI1 expression in various cancer tissues including microRNAs which are considered as one of the most important of these factors. The aim of our study is identification of the molecular cause of maximal expression of MSI1 in epithelial breast cancer cell lines.ResultsAmong predicted microRNAs, miR-125b, miR-637 and miR-802 were able to significantly reduce the luciferase activity. In addition, the relative expression of these three miRNAs were measured in the cancerous cell lines that results showed a significant reduction in expression of all microRNAs. On the other hand, only the overexpression of miR-125b caused a change in the expression pattern of MSI1 in breast epithelial cancer cell lines. Accordingly, our results demonstrated that the exogenous expression of miR-125b decreased not only the MSI1 protein but also expression of epithelial markers in breast cancer cells.ConclusionsThe results of luciferase reporter assay showed that MSI1 is a direct target for miR-125b in epithelial breast cancer cells. Moreover, higher amount of MSI1 in those cell lines seems due to the reduced amount of miR-125b, which is responsible for epithelial features of those kinds of cancer cells. Therefore, the modulation of miR-125b may be a potential approach to help to combat against epithelial breast tumors.

Msi1 inhibits cervical cancer cell apoptosis by downregulating BAK through AKT signaling.

Musashi-1 (Msi1) is an RNA binding protein that functions as a regulator in multiple carcinomas. Our previous study demonstrated that Msi1 could promote the proliferation of cervical cancer cells by targeting the cell cycle proteins P21, P27 and P53. However, the mechanisms by which Msi1 affects the survival of cervical cancer cells, such as apoptosis, are still unclear. In this study, we found that the expression of Msi1 inhibited cervical cancer cell apoptosis in vitro and in vivo. Furthermore, the expression of Msi1 downregulated the expression of PTEN, while AKT signaling was activated, which resulted in a reduction in the proapoptotic protein BAK. In addition, rescue the expression of BAK in Msi1 expressing cervical cancer cells induced the increase of apoptosis cells. These findings indicate that Msi1 regulates cervical cancer cell apoptosis by inhibiting PTEN and activating AKT signaling, which leads to the downregulation of BAK.

Musashi1 expression is negatively correlated with numb expression in brain metastases.

The expression of tumor stem cell markers musashi1 (msi1) and numb in brain metastases were detected to explore their roles in the development of brain metastases.A total of 51 cases of brain metastasis, 29 cases of primary tumor and 15 cases of normal brain tissue were selected. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to detect msi1 and numb expression at the protein and mRNA levels. Correlation between msi1 and numb in brain metastases were evaluated.Immunohistochemistry and RT-PCR showed that no significant difference in the expression of msi1 and numb between brain metastases and primary tumors was observed (P > .05); the expression of msi1 and numb in brain metastases was significantly higher than that in normal brain tissues (P < .05); and the expression of msi1 and numb in primary tumors was significantly higher than that in normal brain tissues (P < .05). In general, the expression of msi1 gene was negatively correlated with the expression of numb at mRNA level by Pearson correlation analysis (r = −0.345, P < .05). Additionally, the expression of msi1 and numb in brain metastases was not related to gender, age, and tissue origin (P > .05).Msi1 is highly expressed in brain metastases and primary tumors, while numb is lowly expressed in brain metastases and primary tumors; msi1 and numb are negatively correlated in brain metastases, suggesting that msi1 and numb may have regulatory mechanisms in the development of brain metastases.

The Stemness Gene Mex3A Is a Key Regulator of Neuroblast Proliferation During Neurogenesis.

Mex3A is an RNA binding protein that can also act as an E3 ubiquitin ligase to control gene expression at the post-transcriptional level. In intestinal adult stem cells, MEX3A is required for cell self-renewal and when overexpressed, MEX3A can contribute to support the proliferation of different cancer cell types. In a completely different context, we found mex3A among the genes expressed in neurogenic niches of the embryonic and adult fish brain and, notably, its expression was downregulated during brain aging. The role of mex3A during embryonic and adult neurogenesis in tetrapods is still unknown. Here, we showed that mex3A is expressed in the proliferative region of the developing brain in both Xenopus and mouse embryos. Using gain and loss of gene function approaches, we showed that, in Xenopus embryos, mex3A is required for neuroblast proliferation and its depletion reduced the neuroblast pool, leading to microcephaly. The tissue-specific overexpression of mex3A in the developing neural plate enhanced the expression of sox2 and msi-1 keeping neuroblasts into a proliferative state. It is now clear that the stemness property of mex3A, already demonstrated in adult intestinal stem cells and cancer cells, is a key feature of mex3a also in developing brain, opening new lines of investigation to better understand its role during brain aging and brain cancer development.

RNA-binding proteins Musashi and tau soluble aggregates initiate nuclear dysfunction

Oligomeric assemblies of tau and the RNA-binding proteins (RBPs) Musashi (MSI) are reported in Alzheimer’s disease (AD). However, the role of MSI and tau interaction in their aggregation process and its effects are nor clearly known in neurodegenerative diseases. Here, we investigated the expression and cellular localization of MSI1 and MSI2 in the brains tissues of Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) as well as in the wild-type mice and tau knock-out and P301L tau mouse models. We observed that formation of pathologically relevant protein inclusions was driven by the aberrant interactions between MSI and tau in the nuclei associated with age-dependent extracellular depositions of tau/MSI complexes. Furthermore, tau and MSI interactions induced impairment of nuclear/cytoplasm transport, chromatin remodeling and nuclear lamina formation. Our findings provide mechanistic insight for pathological accumulation of MSI/tau aggregates providing a potential basis for therapeutic interventions in neurodegenerative proteinopathies.

Cancer stem cell generation during epithelial-mesenchymal transition is temporally gated by intrinsic circadian clocks.

Epithelial-mesenchymal transition (EMT) is a key event preceding tumor cell metastasis that increases cell invasiveness and cancer stem cell (CSC)populations. Studies suggest that genes used in generating circadian rhythms also serve in regulating EMT. To test the role of circadian clocks in cellular EMT events two cancer cell lines were compared, one that has a well-established circadian clock, C6 from rat glioma, and one that does not, MCF-7 from human breast tumor. MCF-7 tumorsphere cultures were tested for evidence of circadian rhythms because of previously reported circadian rhythm enhancement in C6 tumorspheres shown by elevated rhythm amplitude and increased expression of circadian clock gene Per2. Bioluminescence imaging of Per2 gene expression in MCF-7 tumorspheres revealed a previously unconfirmed circadian clock in this important cancer research model. Inducing CSCgeneration through EMT in C6 and MCF-7 monolayer cultures revealed circadian oscillations in the size of the post-EMT CSCpopulation, confirming that circadian rhythms are additional processes controlling this stage of cancer progression. EMT was verified by distinct cellularmorphological changes and expression of stem cell proteins OCT4, nestin, MSI1, and CD133 along with EMT-related proteins ZEB1, vimentin, and TWIST. Quantifying single-cell events and behaviors through time-lapse imaging indicated the post-EMT population size was determined largely by circadian rhythms in epithelial-like cancer cells undergoing EMT. We then identified a specific phase of the circadian rhythm in Per2 gene activation as a potential target for therapeutic treatments that may suppress EMT, minimize CSCs, and limit metastasis.

The effects of a mixture of Lactobacillus species on colorectal tumor cells activity through modulation of Hes1 pathway

BackgroundIn this study, the action mechanisms of a live potential probiotic Lactobacillus cocktail (L.C) on tumors growth and genes expression of Notch1/Hes1 pathway were investigated. MethodsThe anti-proliferative and apoptotic effects of thelactobacilli cocktail (L.C) on CT-26 cells investigated using MTT and Flow cytometry tests. Quantitative real-time polymerase chain reaction (qPCR) method used for identifying the expression levels of hes1and its associated genes. The effect of L.C was confirmed in a murine model of colorectal cancer. ResultsA live L.C inhibited the viability of the cell and induced late apoptosis of CT-26 cells by upregulating CASP3 and CASP9 (P < 0.05). Moreover, the expression of notch1, msi1, CTNNB1, gli1, and id1 (except for numb), which are required for the regulation of hes1, was decreased in L.C-treated mice in a time- and dose-dependent manner (P < 0.05). Also, data from the mouse model showed the reduction of pathological grade and stage as well as the modulation of hes1 regulatory genes and apoptosis in L.C-treated mice. ConclusionsThe results showed that the modulation of the gut microbiota with probiotics inhibiting hes1 and its regulatory genes expression led to apoptosis induction, and progression prevention in cancer cells.

Abstract 3669: Defining an ELAVL1-Musashi 2 signaling cascade in neuroblastoma tumorigenesis

Abstract Background: Patients with high-risk neuroblastoma endure an extremely intense, multidrug treatment regimen and yet, approximately half of them die, thus mandating a better approach to this cancer. Our laboratory is investigating the overarching hypothesis that RNA binding proteins (RBPs), which play key roles in balancing self-renewal, differentiation, and cell proliferation, are deregulated in neuroblastoma, and drive oncogenic signaling networks. Therefore, RBPs may constitute novel targets for therapeutic development. In this work, we focus on Musashi 2 (MSI2), an RBP that has been shown to play a critical role in the maintenance of stem cell populations and in the formation of aggressive tumors, notably in acute myeloid leukemia and in colon cancer. The role of MSI2 in neuroblastoma, however, has not been assessed and is the focus of this investigation. Methods: To test this hypothesis, we used in silico analysis of neuroblastoma gene expression datasets annotated with clinical parameters. Additionally, we used shRNAs to manipulate transcripts of interest in neuroblastoma cells and measured effects on cell proliferation, cell cycle status, and apoptosis. Results: Gene expression profiling revealed that MSI2 is robustly expressed in neuroblastoma cell lines, tumors, and patient derived xenografts, within both the MYCN and non-MYCN amplified context. Immunoblotting in a representative panel of lines confirmed the expression of MSI2 at the level of protein. High MSI2 expression was associated with more advanced stages of neuroblastoma across multiple independent datasets and was correlated with worse survival. Depletion of MSI2 using four independent shRNAs led to 2 to 3-fold decreased proliferation across four cell lines that was due in part to increased apoptosis. Another RBP, ELAVL1, is also expressed within neural lineages, and has been shown to regulate MSI1 expression. We hypothesized that ELAVL1 might similarly promote MSI2 expression and we demonstrated that, ELAVL1 depletion led to decreased MSI2 expression. Conclusions: MSI2 is robustly expressed in multiple models of neuroblastoma and appears to drive increased proliferation and survival. Currently, we are investigating the functional interplay between ELAVL1 and MSI2 providing a foundation for designing strategies to target the ELAVL1-MSI2 interaction for therapeutic effect. Citation Format: Selma M. Cuya, Adeiye Pilgrim, Komal Rathi, DongDong Chen, Robert Schnepp. Defining an ELAVL1-Musashi 2 signaling cascade in neuroblastoma tumorigenesis [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 3669.