Expression Of Musashi-1 Research Articles

Musashi-1 promotes a cancer stem cell lineage and chemoresistance in colorectal cancer cells

Colorectal cancers (CRCs) are a critical health issue worldwide. Cancer stem cell (CSC) lineages are associated with tumour transformation, progression, and malignant transformation. However, how lineages are transformed and how chemoresistance is acquired by CRCs remain largely unknown. In this report, we demonstrated that the RNA-binding protein Musashi-1 enhanced the development of CD44+ colorectal CSCs and triggered the formation of anti-apoptotic stress granules (SGs). Our results indicated that CD44+ CSC lineage-specific induction of tumour malignancies was controlled by Musashi-1. In addition, Musashi-1 formed SGs when CRC cell lines were treated with 5-fluorouracil. The C-terminal domain of Musashi-1 was critical for recruitment of Musashi-1 into SGs. Intracellular Musashi-1 SGs enhanced the chemoresistance of CRCs. Analysis of clinical CRC samples indicated that Musashi-1 expression was prominent in CRC stage IIA and IIB. In summary, we demonstrated that Musashi-1, a stemness gene, is a critical modulator that promotes the development of CD44+ colorectal CSCs and also enhances CRC chemoresistance via formation of SGs. Our findings elucidated a novel mechanism of CRC chemoresistance through increased anti-apoptotic effects via Musashi-1-associated SGs.

Overexpression of Musashi-1 protein is associated with progression and poor prognosis of gastric cancer.

Musashi-1, an evolutionally conserved RNA-binding protein, has been implicated in the promotion of pathological stem cell proliferation, including tumorigenesis. The objective of the present study was to evaluate the expression of Musashi-1 protein and its implications in the progression and prognosis of gastric cancer. The expression level of Musashi-1 protein in gastric cancer was determined by western blotting and immunohistochemistry, and compared with the clinicopathological parameters. The present study revealed that the expression level of Musashi-1 protein in gastric cancer was significantly upregulated and correlated with the tumor size, tumor-node-metastasis (TNM) stage, Lauren classification, depth of invasion, vessel invasion, lymph node metastasis and distant metastasis. The mean survival time for patients with low expression levels of Musashi-1 was significantly longer compared with patients with high expression levels of Musashi-1. For each TNM stage, the mean survival time for patients with a low Musashi-1 expression levels was also significantly longer compared with patients with a high Musashi-1 expression level. Notably, TNM stage II patients with a low Musashi-1 expression level demonstrated a longer mean survival time compared with TNM stage I patients with high Musashi-1 expression level (56.8 vs. 42.3 months; P=0.001), and TNM stage III patients with low Musashi-1 expression level exhibited a longer mean survival time compared with TNM stage II patients with a high Musashi-1 expression level (44.0 vs. 33.8 months; P=0.034). Multivariate Cox's regression test demonstrated that Musashi-1 protein expression level was an independent prognostic indicator for the survival rate of the patients with gastric cancer. The results of the present study highlighted an important role for Musashi-1 protein in the progression of gastric cancer. The detection of the Musashi-1 protein expression level alone or in combination with TNM staging may aid the prediction of the prognosis of patients with gastric cancer.

Musashi1, a potential prognostic marker in esophageal squamous cell carcinoma.

Esophageal cancer ranks as the sixth leading cause of cancer-related deaths worldwide. Cancer stemness is mainly considered to be the key factor for cancer recurrence particularly in esophageal cancer. It is important to identify cancer stem cell markers as targets in future therapies. The present study aimed to investigate the expression of putative cancer stem cell-related marker musashi1 (Msi1) and assess the correlation with clinicopathologcal status of esophageal squamous cell carcinoma (ESCC) cases. We then clarified the role of Msi1 in esophageal cancer cells during proliferation, apoptosis, sphere formation and migration. Finally, we investigated the relationship of Msi1 with the prognosis of ESCC patients. ESCC tissue samples from 93 patients and 20 paired histologically normal tissues were procured for immunohistochemical analysis. We analyzed the characteristics of Msi1, using sphere formation and anchorage independent growth. Moreover, using flow cytometry and Cell Counting Kit-8 (CCK-8) assay, we investigated the role of Msi1 in cancer cell proliferation and apoptosis. Furthermore, we clarified the role of Msi1 in the process of sphere formation and migration of ESCC cells through knockdown of Msi1 expression by siRNA in ESCC cell lines. The results revealed that there was a higher expression of Msi1 in ESCC specimens compared with normal tissues. In addition, Msi1 expression was significantly associated with clinical stage and lymph node metastasis. Most importantly, the increased immunocytochemical staining of Msi1 in spheroid cells revealed the stemness characteristics of Msi1 in ESCC. In addition, we found that silencing of Msi1 decreased cell proliferation, migration and induced apoptosis in TE-7 and KYSE70 cells. Furthermore, downregulation of Msi1 attenuated the sphere formation ability of ESCC cells. Patients with higher expression of Msi1 had a shorter survival. In conclusion, Msi1 acts as a stemness-associated gene in esophageal cancer cell lines and could serve as a prognostic marker in patients with ESCC.

Decellularized Matrix Derived from Neural Differentiation of Embryonic Stem Cells Enhances the Neurogenic Potential of Dental Follicle Stem Cells

IntroductionDental follicle stem cells (DFSCs) possess neurogenic potential because they originate from the embryonic neural crest. This study investigated whether neural differentiation of DFSCs can be enhanced by culture on decellularized matrix substrata (NSC-DECM) derived from neurogenesis of human embryonic stem cells (hESCs). MethodsThe hESCs were differentiated into neural stem cells (NSCs), and NSC-DECM was extracted from confluent monolayers of NSCs through treatment with deionized water. DFSCs seeded on NSC-DECM, Geltrex, and tissue culture polystyrene (TCPS) were subjected to neural induction during a period of 21 days. Expression of early/intermediate (Musashi1, PAX6, NSE, and βIII-tubulin) and mature/late (NGN2, NeuN, NFM, and MASH1) neural markers by DFSCs was analyzed at the 7-, 14-, and 21-day time points with quantitative real-time polymerase chain reaction. Immunocytochemistry for detection of βIII-tubulin, PAX6, and NGN2 expression by DFSCs on day 7 of neural induction was also carried out. ResultsQuantitative RT-PCR showed that expression of PAX6, Musashi1, βIII-tubulin, NSE, NGN2, and NFM by DFSCs was enhanced on NSC-DECM versus either the Geltrex or TCPS groups. Immunocytochemistry showed that DFSCs in the NSC-DECM group displayed more intense staining for βIII-tubulin, PAX6, and NGN2 expression, together with more neurite outgrowths and elongated morphology, as compared with either Geltrex or TCPS. ConclusionsDECM derived from neurogenesis of hESCs can enhance the neurogenic potential of DFSCs.

Expression profiles of cancer stem cell markers: CD133, CD44, Musashi-1 and EpCAM in the cardiac mucosa—Barrett’s esophagus—early esophageal adenocarcinoma—advanced esophageal adenocarcinoma sequence

IntroductionBarrett's esophagus (BE), which develops as a result of gastroesophageal reflux disease, is a preneoplastic condition for esophageal adenocarcinoma (EAC). A new hypothesis suggests that cancer is a disease of stem cells, however, their expression and pathways in BE − EAC sequence are not fully elucidated yet. AimsWe used a panel of putative cancer stem cells markers to identify stem cells in consecutive steps of BE-related cancer progression. MethodsImmunohistochemistry was performed on formalin-fixed, paraffin-embedded blocks from 58 patients with normal cardiac mucosa (n=5), BE (n=14), early EAC (pT1) from mucosal resection (n=17) and advanced EAC (pT1-T4) from postoperative specimens (n=22). Expression of the CD133, CD44, Musashi-1 and EpCAM was analyzed using respective monoclonal antibodies. ResultsAll markers showed a heterogeneous expression pattern, mainly at the base of the crypts of Barrett’s epithelium and EAC, with positive stromal cells in metaplastic and dysplastic lesions. Immuno-expression of EpCAM, CD44 and CD133 in cardiac mucosa was significantly lower (mean immunoreactivity score (IRS)=1.2; 0.0; 0.4; respectively) compared to their expression in Barrett’s metaplasia (mean IRS=4.3; 0.14; 0.7; respectively), in early adenocarcinoma (mean IRS=4.4; 0.29; 1.3; respectively) and in advanced adenocarcinoma (mean IRS=6.6; 0.7; 2.7; respectively) (p<0.05). On the contrary, Musashi-1 expression was higher in BE and early ADC compared to GM and advanced ADC (NS). ConclusionOur results suggest that the stem cells could be present in premalignant lesions. EpCAM, CD44 and CD133 expression could be candidate markers for BE progression, whereas Musashi-1 may be a marker of the small intestinal features of Barrett's mucosa.

Abstract 1172: Expression of notch1, numb, and musashi1 in non-small cell lung cancer

Abstract Background: Deregulation of Notch pathway is associated with carcinogenesis, and Notch signaling is identified as tumor activator in non-small cell lung cancer(NSCLC). The function of Musashi1 has been found to activate Notch signaling through the translational repression of Numb, which represses an intracellular Notch signaling. In NSCLC, the association between Numb or Musashi1 expression and clinicopathological factors or prognosis has remained unclear. In this study, we evaluated the expression of Notch1, Numb, and Musashi1 in NSCLC. Methods: A total of 135 surgically resected NSCLCs were immunohistochemically assessed for Notch1, Numb, and Musashi1 expression. Only cytoplasm and nuclear staining was considered in the evaluation of activated Notch1 expression. The immunoscores were determined, and then its correlation with either the clinicopathological variables or the survival outcomes was analyzed. Results: Immunohistochemical reactivity for Numb, and Musashi1 was detected in the cytoplasm and nuclear of the tumor cells. Notch1 expression was associated with several clinicopathological factors, including pathological histology (p = 0.002), and differentiation of tumor (p = 0.024). Numb expression was also associated with several clinicopathological factors, including sex (p = 0.037), smoking habit (p = 0.033), pathological histology (p = 0.002), and differentiation of tumor (p = 0.015). No correlations were noted between Musashi1 expression and any of the variables. Analysis of cellular biological expression demonstrated a link between low Numb expression and high Notch expression. Multivariate Cox regression analysis showed that positive Numb expression was an independent favorable prognostic factor. Conclusions: We demonstrate that Numb expression was associated with better prognosis in NSCLC. Numb might have the function as tumor suppressor in NSCLC. Citation Format: Hajime Kikuchi, Jun Sakakibara-Konishi, Yasuyuki Ikezawa, Taichi Takashina, Hidenori Mizugaki, Eiki Kikuchi, Junko Kikuchi, Naofumi Shinagawa, Satoshi Oizumi, Yasuhiro Hida, Kichizo Kaga, Ichiro Kinoshita, Hirotoshi Dosaka-Akita, Masaharu Nishimura. Expression of notch1, numb, and musashi1 in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1172.

Immunolocalization of Musashi1 and Fibronectin Type III Domain Containing 3B in Water Buffalo Mammary Glands

Water buffaloes are the principle source of milk in south Asia and Africa. Mammary gland repeatedly undergoes the cycles of growth and regeneration during pregnancy, lactation and involution. It is assumed that buffalo mammary gland has mammary stem and progenitor cells that regulate gland growth and regeneration. In the present study the authors analyzed percentage of cellular composition, proliferation status and putative mammary stem/progenitor cell population. Identification of putative buffalo mammary stem/progenitor cells was attempted using immunohistochemical staining with Musashi1 (MSI1), an adult stem cell marker and fibronectin type III domain containing 3B (FNDC3B), a mammary stem and cancer cell marker. Immunolocalization of MSI1 and FNDC3B showed nuclear and cytoplasmic staining of alveolar and ductal mammary epithelial cells (MEC) and a few stromal cells. The percentage of MSI1-positive MEC in non-lactating (3.31 ± 1.11 %), lactating (2.73 ± 0.78 %) and mastitic glands (3.30 ± 0.97 %) were equivalent, indicating that the proportion of putative stem/progenitor cell population did not differ during various physiological stages. Likewise, the percentage of FNDC3B-positive MEC in non-lactating (12.40 ± 3.22 %) tended to be higher than lactating (8.19 ± 2.71 %) and mastitic glands (4.88 ± 2.37 %). In some cases, expression of MSI1 and FNDC3B was exceptionally high with high proliferative indices (37.6 ± 2.4 %)-an indication of tumor cells. This is the first report on expression of MSI1 and FNDC3B in buffalo mammary gland. Identification of buffalo mammary stem cells using MSI1 and FNDC3B requires further studies and functional validation.

Expression and clinical significance of RNA-binding protein Musashi1 in colorectal cancer

Musashi1 (Msi1) is an evolutionary conservative RNA-binding protein (RBP), and it is a stem marker in a variety of organizations, including intestinal, neural system. Msi1 maintains the balance between self-renewal and differentiation. Recently, many researchers report that Msi1 is overexpressed in many types of tumors, especially in colorectal neoplasms, participating in the regulation of cell cycle, proliferation, apoptosis and so on. Msi1 becomes a key regulator of many cancers, which is expected to turn into a new target for cancer therapy. Key words: Colorectal neoplasms; RNA-binding proteins; Musashi1

Putative stem cell markers in cervical squamous cell carcinoma are correlated with poor clinical outcome.

BackgroundThe aim of this study was to elucidate the value of putative cancer stem cell markers Musashi-1, ALDH1, Sox2, and CD49f in predicting the prognosis in cervical squamous cell carcinoma (CSCC).MethodsReal-time PCR and immunohistochemistry staining was performed to examine Musashi-1, ALDH1, Sox2, and CD49f expression in archived specimens of CSCC patients with postoperative chemotherapy. Kaplan–Meier analysis and Cox proportional hazards model were used to assess the prognostic impact of CSC markers for overall survival (OS) and recurrent-free survival (RFS).ResultsThe Real-time PCR data showed that the expression of all markers were increased in CSCC tissues compared with in paired normal cervical tissues (P < 0.05). The IHC result showed that high expression of Msi1, ALDH1, Sox2, and CD49f was found in 25.7 %, 43.0 %, 62.0 % and 29.0 % CSCC samples, respectively. Moreover, high expression of Msi1 (P = 0.033 and P = 0.003, respectively), ALDH1 (P = 0.015 and P = 0.002, respectively), and Sox2 (P = 0.005 and P = 0.003, respectively), and low expression of CD49f (P = 0.027 and P = 0.025, respectively) were correlated with poor OS and PFS in CSCC patients. Interestingly, tumors with Msi1high/CD49flow expression had the poorest prognosis according to Msi1/CD49f stratification. In multivariate Cox regression analysis, Sox2 expression (P = 0.047 and P = 0.018, respectively), ALDH1 expression (P = 0.013 and P = 0.003, respectively), and CD49f expression (P = 0.008 and P = 0.003, respectively) were independent prognostic markers for both OS and RFS.ConclusionsOur results suggest that cancer stem cell markers are linked with poor prognosis of CSCC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1826-4) contains supplementary material, which is available to authorized users.

A Mouse Model of Targeted Musashi1 Expression in Whole Intestinal Epithelium Suggests Regulatory Roles in Cell Cycle and Stemness.

The intestinal epithelium is very peculiar for its continuous cell renewal, fuelled by multipotent stem cells localized within the crypts of Lieberkühn. Several lines of evidence have established the evolutionary conserved RNA-binding protein Musashi1 as a marker of adult stem cells, including those of the intestinal epithelium, and revealed its roles in stem cell self-renewal and cell fate determination. Previous studies from our laboratories have shown that Musashi1 controls stem cell-like features in medulloblastoma, glioblastoma, and breast cancer cells, and has pro-proliferative and pro-tumorigenic properties in intestinal epithelial progenitor cells in vitro. To undertake a detailed study of Musashi1's function in the intestinal epithelium in vivo, we have generated a mouse model, referred to as v-Msi, overexpressing Musashi1 specifically in the entire intestinal epithelium. Compared with wild type litters, v-Msi1 mice exhibited increased intestinal crypt size accompanied by enhanced proliferation. Comparative transcriptomics by RNA-seq revealed Musashi1's association with gut stem cell signature, cell cycle, DNA replication, and drug metabolism. Finally, we identified and validated three novel mRNA targets that are stabilized by Musashi1, Ccnd1 (Cyclin D1), Cdk6, and Sox4. In conclusion, the targeted expression of Musashi1 in the intestinal epithelium in vivo increases the cell proliferation rate and strongly suggests its action on stem cells activity. This is due to the modulation of a complex network of gene functions and pathways including drug metabolism, cell cycle, and DNA synthesis and repair.

Musashi-1 Expression is a Prognostic Factor in Ovarian Adenocarcinoma and Correlates with ALDH-1 Expression.

The presence of cancer stem-like cells (CSCs) has been demonstrated to be associated with tumor metastasis, chemoresistance, and rapid recurrence of various tumors. The impact of CSC-related markers in the metastasis and prognosis of ovarian cancer has not been well established. In this study, the protein expression of musashi-1 and ALDH1 was measured using immunohistochemistry. Results demonstrated that the percentage of positive musashi-1 and ALDH1 expression were significantly higher in ovarian serous adenocarcinomas, mucinous adenocarcinomas and clear cell adenocarcinomas than in cystadenomas and normal tissues. The percentage of positive musashi-1 and ALDH1 expression were significantly lower in patients identified with clinical stage I or II ovarian adenocarcinomas without lymph node metastasis compared to patients with clinical stage III or IV tumors and lymph node metastasis. The expression of musashi-1 and ALDH1 was found to be highly consistent in ovarian adenocarcinomas. Univariate Kaplan-Meier analysis showed a negative correlation between musashi-1 or ALDH1 expression and overall survival. Multivariate Cox regression analysis showed that positive expression of musashi-1 or ALDH1 in ovarian adenocarcinoma was an independent predictor of poor prognosis. Our study suggested that musashi-1 and ALDH1 expression are closely related to metastasis of ovarian adenocarcinoma. The positive expression of musashi-1 and ALDH1 might be a poor-prognostic factor of ovarian adenocarcinoma.

Correlation of Musashi-1, Lgr5, and pEGFR expressions in human small intestinal adenocarcinomas.

Recent studies have revealed that Musashi-1 and Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) were putative stem cell genes. The epidermal growth factor receptor (EGFR) has also been extensively studied; it was known as an oncogenic driver in cancers. Overexpressions of Musashi-1, EGFR, and Lgr5 have been reported in some tumor tissues and cell lines. In this study, we used immunohistochemical analysis to investigate the expression pattern of Musashi-1, Lgr5, and pEGFR in 38 small intestinal adenocarcinomas (SIAs) resection specimens, 20 matched normal specimens and tried to analyze the correlations among them. The positive rate of Musashi-1, Lgr5, and pEGFR in SIAs, respectively, was 71 % (27/38), 55 % (21/38), and 45 % (17/38). Compared with the adjacent normal small intestinal mucosa, Musashi-1, Lgr5, and pEGFR protein were overexpressed in SIAs (P< 0.05). Furthermore, Musashi-1 and Lgr5 expressions were significantly correlated with the depth of wall invasion (P = 0.0011, P = 0.0017, respectively). Musashi-1 expression was closely correlated with Lgr5 (P = 0.015, r = 0.392). However, pEGFR expression was not associated with age, gender, tumor size, differentiation, depth of invasion, lymphatic metastasis, TNM stage, and pEGFR expression was not correlated with Musashi-1 or Lgr5 (P > 0.05, r = 0.064; P > 0.05, r = 0.307, respectively). Thus, we suggest that Musashi-1, Lgr5, and pEGFR are overexpressed in human SIAs and may play roles in human SIA carcinogenesis and progression.

Comparison of the effects of human dental pulp stem cells and human bone marrow-derived mesenchymal stem cells on ischemic human astrocytes in vitro.

This study assesses the cytoprotective effects of human dental pulp stem cells (hDPSCs) and conditioned medium from hDPSCs (CM-hDPSCs) on ischemic human astrocytes (hAs) in vitro compared with human bone marrow-derived mesenchymal stem cells (hMSCs). Ischemia of hAs was induced by oxygen-glucose deprivation (OGD). CM-hDPSCs and hMSCs were collected after 48 hr of culture. Cell death was determined by 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide and cellular ATP assays. The expression of glial fibrillary acidic protein (GFAP) and musashi-1 as markers of reactive astrogliosis was examined with immunochemical staining. mRNA expression and reactive oxygen species (ROS) were analyzed by RT-PCR and flow cytometry, respectively. OGD increased cytotoxicity in a time-dependent manner and decreased cellular ATP content concomitantly in hAs. Pretreatment and posttreatment with hDPSCs were associated with greater recovery from OGD-induced cytotoxicity in hAs compared with hMSCs. Similarly, CM-hDPSCs had a greater effect on OGD-induced cytotoxicity in a dose-dependent manner. Pre- and posttreatment with CM-hDPSCs or CM-hMSCs attenuated OGD-induced GFAP, nestin, and musashi-1 expression in hAs. Furthermore, treatment of cells with CM-hDPSCs and hMSCs blocked OGD-induced ROS production and interleukin-1ß upregulation. This study demonstrates for the first time that hDPSCs and CM-hDPSCs confer superior cytoprotection against cell death in an in vitro OGD model compared with hMSCs as shown by cell viability assay. Reactive gliosis, ROS production, and inflammatory mediators might contribute to this protective effect. Therefore, hDPSCs could represent an alternative source of cell therapy for ischemic stroke.

Association of hepatocyte-derived growth factor receptor/caudal type homeobox 2 co-expression with mucosal regeneration in active ulcerative colitis.

To characterize the regeneration-associated stem cell-related phenotype of hepatocyte-derived growth factor receptor (HGFR)-expressing cells in active ulcerative colitis (UC). On the whole 38 peripheral blood samples and 38 colonic biopsy samples from 18 patients with histologically proven active UC and 20 healthy control subjects were collected. After preparing tissue microarrays and blood smears HGFR, caudal type homeobox 2 (CDX2), prominin-1 (CD133) and Musashi-1 conventional and double fluorescent immunolabelings were performed. Immunostained samples were digitalized using high-resolution Mirax Desk instrument, and analyzed with the Mirax TMA Module software. For semiquantitative counting of immunopositive lamina propria (LP) cells 5 fields of view were counted at magnification × 200 in each sample core, then mean ± SD were determined. In case of peripheral blood smears, 30 fields of view with 100 μm diameter were evaluated in every sample and the number of immunopositive cells (mean ± SD) was determined. Using 337 nm UVA Laser MicroDissection system at least 5000 subepithelial cells from the lamina propria were collected. Gene expression analysis of HGFR, CDX2, CD133, leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), Musashi-1 and cytokeratin 20 (CK20) were performed in both laser-microdisscted samples and blood samples by using real time reverse transcription polymerase chain reaction (RT-PCR). By performing conventional and double fluorescent immunolabelings confirmed by RT-PCR, higher number of HGFR (blood: 6.7 ± 1.22 vs 38.5 ± 3.18; LP: 2.25 ± 0.85 vs 9.22 ± 0.65; P < 0.05), CDX2 (blood: 0 vs 0.94 ± 0.64; LP: 0.75 ± 0.55 vs 2.11 ± 0.75; P < 0.05), CD133 (blood: 1.1 ± 0.72 vs 8.3 ± 1.08; LP: 11.1 ± 0.85 vs 26.28 ± 1.71; P < 0.05) and Musashi-1 (blood and LP: 0 vs scattered) positive cells were detected in blood and lamina propria of UC samples as compared to controls. HGFR/CDX2 (blood: 0 vs 1 ± 0.59; LP: 0.8 ± 0.69 vs 2.06 ± 0.72, P < 0.05) and Musashi-1/CDX2 (blood and LP: 0 vs scattered) co-expressions were found in blood and lamina propria of UC samples. HGFR/CD133 and CD133/CDX2 co-expressions appeared only in UC lamina propria samples. CDX2, Lgr5 and Musashi-1 expressions in UC blood samples were not accompanied by CK20 mRNA expression. In active UC, a portion of circulating HGFR-expressing cells are committed to the epithelial lineage, and may participate in mucosal regeneration by undergoing mesenchymal-to-epithelial transition.

Prognostic significance of neural stem cell markers, Nestin and Musashi-1, in oral squamous cell carcinoma: expression pattern of Nestin in the precancerous stages of oral squamous epithelium.

Besides the tissue-specific stem cell markers, neural and hematopoietic stem cell markers were found to play an important role in carcinogenesis. Based on this background, we have investigated the expression pattern and prognostic significance of neural stem cell markers, Nestin and Musashi-1, in oral cancer. We used immunohistochemistry and immunofluorescence analyses to study the expression pattern and correlation between Nestin and Musashi-1 in oral squamous cell carcinoma. The Kaplan-Meier method was used to construct overall and disease-free survival curves, and the differences were calculated using log-rank test. Nestin expression was gradually increased in the transformation stages of oral cancer. Both Nestin and Musashi-1 expressions were associated with higher stage and poorly differentiated status of oral carcinoma. Interestingly, Nestin and Musashi-1 double positive cases showed statistically highly significant correlation with poorer survival of oral carcinoma patients. Expression of Nestin in the preneoplastic lesions indicates its role in the transformation of oral squamous epithelium. Clinicopathological and survival analyses suggest that Nestin and Musashi-1 might be associated with invasion, differentiation and poorer survival in oral squamous cell carcinoma. In addition to their role as independent prognostic indicators, Nestin and Musashi-1 double positivity can be used to select high-risk cases for effective therapy and this is the novel finding of this study. Nestin and Musashi-1 are found to be independent prognostic markers of oral cancer, and they might be used as molecular targets for effective therapy.

Survival, proliferation and differentiation enhancement of neural stem cells cultured in three-dimensional polyethylene glycol-RGD hydrogel with tenascin.

Polyethylene glycol hydrogel (PEG) conjugated with arginyl glycyl aspartic acid (RGD) (PEG-RGD) has been considered to be a scaffold in three-dimensional (3D) culture that improves neurite outgrowth; on the other hand, tenascin C controls neural growth and differentiation. In this study, the effect of a combined RGD and tenascin C mixture in 3D culture (3D-PEG-RGD-TnC) on the survival, growth and differentiation of neural stem cells. The viability of the culture has been evaluated by live/dead assay and the results show that the viability of NSCs in 3D-PEG-RGD-TnC is significantly higher than its value in 3D-PEG-RGD. The proliferation was evaluated by MTS test and was found to be slightly improved but statistically not significant. Accordingly, the differentiation was evaluated by immunoreactivity to nestin, neurofilament 68, neurofilament 160, neurofilament 200 and GFAP; and the expression of nestin, neuro D, musashi1, β-tubulin III, GFAP, MBP and Oct4 was studied using RT-PCR. The results showed enhancement of the differentiation of NSCs into the neuronal phenotype in 3D-PEG-RGD-TnC. The morphology of NSCs cultured in 3D-PEG-RGD-TnC showed neurite outgrowths and increase in the contact between the differentiated cells' extensions. The conclusion of this study was that NSC survival, proliferation and differentiation are enhanced when the cells are cultured in 3D-PEG-RGD-TnC.

Mir-23a and mir-125b regulate neural stem/progenitor cell proliferation by targeting Musashi1

Musashi1 is an RNA binding protein that controls the neural cell fate, being involved in maintaining neural progenitors in their proliferative state. In particular, its downregulation is needed for triggering early neural differentiation programs. In this study, we profiled microRNA expression during the transition from neural progenitors to differentiated astrocytes and underscored 2 upregulated microRNAs, miR-23a and miR-125b, that sinergically act to restrain Musashi1 expression, thus creating a regulatory module controlling neural progenitor proliferation.

Mastication markedly affects mandibular condylar cartilage growth, gene expression, and morphology

Mandibular growth is believed to be strongly related to mastication. Furthermore, mandibular condylar cartilage is known to be derived from neural crest cells. We examined whether the degree of chewing affects condylar cartilage growth of the mandible. Mice were fed diets with varying hardness. Genes specific to neural crest-derived cells were measured by real-time polymerase chain reaction to compare the expression changes between the mandibular and tibia cartilages. The mandibular condylar cartilage was then evaluated histologically, and proliferation was evaluated using proliferating cell nuclear antigen. Immunostaining was conducted for osteopontin, type X collagen, and Musashi1, and real-time polymerase chain reaction was used to assess the expression levels of osteopontin and type X collagen. Markers including P75, Wnt-1, Musashi1, and Nestin were upregulated in the mandibular condylar cartilage as compared with the tibial cartilage. Histologic assessment of the mandibular cartilage showed that the hypertrophic chondrocyte zone was statistically significantly thicker in mice fed a hard diet. Chondrocyte proliferation and Musashi1 expression were lower in mice fed a hard diet. After 4weeks, numerous osteopontin and type X collagen-positive cells were observed in mice fed a mixed diet. Mastication affects the balance between differentiation and proliferation in the mandibular condylar cartilage. This phenomenon might be attributed to the presence of neural crest-derived cells.

Developmental Expression of Musashi-1 and Musashi-2 RNA-Binding Proteins During Spermatogenesis: Analysis of the Deleterious Effects of Dysregulated Expression1

Spermatogenesis is a complex developmental process whereby diploid spermatogenic stem cells become haploid and undergo a series of morphological changes to produce physically mature spermatozoa. Crucial to this process are a number of RNA-binding proteins, responsible for the posttranscriptional control of essential mRNAs and particularly pertinent to the two periods of inactive transcription that occur in spermatogenesis. One such group of RNA-binding proteins is the Musashi family, specifically Musashi-1 (MSI1) and Musashi-2 (MSI2), which act as key translational regulators in various stem cell populations and have been linked with the induction of tumorigenesis. In the present study, we examined the differential expression of mammalian MSI1 and MSI2 during germ cell development in the mouse testis. MSI1 was found to be predominately localized in mitotic gonocytes and spermatogonia, whereas MSI2 was detected in meiotic spermatocytes and differentiating spermatids. Extensive examination of the function of Musashi in spermatogenesis was achieved through the use of two transgenic mouse models with germ cell-specific overexpression of full-length isoforms of Msi1 or Msi2. These models demonstrated that aberrant expression of either Msi1 or Msi2 has deleterious effects on normal spermatogenesis, with Msi2 overexpression resulting in male sterility. Studies undertaken on human testicular seminoma tumors provide further insights into the relevance of MSI1 and MSI2 overexpression as diagnostic markers to human stem cell cancers. Overall this study provides further evidence for the unique functions that RNA-binding protein isoforms occupy within spermatogenesis, and introduces the potential manipulation of the Musashi family proteins to elucidate the mechanisms of posttranscriptional gene expression during germ cell development.

NOTCH3 Signaling Regulates MUSASHI-1 Expression in Metastatic Colorectal Cancer Cells

MUSASHI-1 (MSI-1) is a well-established stem cell marker in both normal and malignant colon cells and it acts by positively regulating the NOTCH pathway through inactivation of NUMB, a NOTCH signaling repressor. To date, the mechanisms of regulation of MSI-1 levels remain largely unknown. Here, we investigated the regulation of MSI-1 by NOTCH signaling in colorectal cancer cell lines and in primary cultures of colorectal cancer metastases. Stimulation by the NOTCH ligand DLL4 was associated with an increase of MSI-1 mRNA and protein levels, and this phenomenon was prevented by the addition of an antibody neutralizing NOTCH2/3 but not NOTCH1. Moreover, forced expression of activated NOTCH3 increased MSI-1 levels, whereas silencing of NOTCH3 by short hairpin RNA reduced MSI-1 levels in both colorectal cancer cells and CRC tumor xenografts. Consistent with these findings, enforced NOTCH3 expression or stimulation by DLL4 increased levels of activated NOTCH1 in colorectal cell lines. Finally, treatment of colorectal cancer cells with anti-NOTCH2/3 antibody increased NUMB protein while significantly reducing formation of tumor cell spheroids. This novel feed-forward circuit involving DLL4, NOTCH3, MSI-1, NUMB, and NOTCH1 may be relevant for regulation of NOTCH signaling in physiologic processes as well as in tumor development. With regard to therapeutic implications, NOTCH3-specific drugs could represent a valuable strategy to limit NOTCH signaling in the context of colorectal cancers overexpressing this receptor.