Abstract
Background/Aim: Neuroblastoma remains a major cause of pediatric cancer mortality and although intensive multimodal treatment strategies have improved survival, they have also led to an increased risk of long-term treatment-related toxicities among survivors. This study aimed to evaluate the potential involvement of (Musashi) Msi1 in neuroblastoma oncogenesis and etoposide treatment response.
Materials and Methods: The expression of Msi1, an RNA-binding protein and stem cell marker, was examined in MYCN amplified and non-amplified cells, which were then quantified by densitometry. Immunoblotting was used to assess total protein levels in all cell lines. In addition, the impact of Msi1 silencing on etoposide sensitivity was also assessed.
Results: The study found that increased Msi1 expression is associated with MYCN-amplification and, in a publicly available clinical database, Msi1 upregulation correlates with decreased overall survival and is seen in older patients and those with more advanced disease. Furthermore, in vitro silencing of Msi1 was associated with decreased cell proliferation and colony formation, as well as increased sensitivity to etoposide treatment. These changes correlated with altered expression of several cell cycle, proliferation, and DNA damage repair genes that are known Msi1 targets in other malignancies.
Conclusion: These findings indicate that Msi1 could serve as a novel therapeutic target for high-risk, treatment-refractory neuroblastoma.
Introduction
Neuroblastoma is the most common extracranial solid tumor of childhood, accounting for approximately 10% of cancer-related mortality in children (1). While advances in treatment have led to long-term survival rates of greater than 90% in children with low- and intermediate-risk disease, children with high-risk neuroblastoma continue to face survival rates of less than 50% despite intensive, multimodal treatment regimens (2). These regimens frequently include chemotherapy, surgery, stem cell transplant, radiation therapy, and immunotherapy (3). Since the advent of these anti-cancer therapies, combined modality regimens have been shown to be highly effective in the pediatric population (4). Although this aggressive approach has improved survival rates, it has also led to higher rates of long-term complications related to treatment toxicity, including secondary malignancies, chronic health conditions, and psychosocial impairment (5, 6). As such, there remains a significant need for advances in the management of high-risk neuroblastoma that will decrease treatment-related toxicity without compromising survival.
Musashi1 (Msi1), an RNA-binding protein (RBP) and stem cell marker, exhibits upregulated expression in cancer (7-9). The Musashi family of RBPs, including Msi1, consists of a highly conserved group of proteins involved in the regulation of cell fate decisions during embryonic development, as well as stem cell self-renewal and differentiation in both fetal and adult tissues (10). The oncogenic role of Msi1 is characterized by the post-transcriptional regulation of targets involved in cell growth and division, maintenance of cancer stem cell phenotypes, and enhanced DNA damage repair, particularly double-strand break (DSB) repair (7, 10). Potential mechanisms have been identified through which Msi1 enhances DSB repair, and etoposide is a potent topoisomerase II inhibitor known to cause DSBs that lead to cell death if not repaired. Msi1 has been identified as a negative prognostic marker and chemo- and radio-resistance driver in multiple malignancies (11-13). However, its role in neuroblastoma has yet to be elucidated. Therefore, this study aimed to evaluate the potential involvement of Msi1 in neuroblastoma oncogenesis and etoposide treatment response.
Materials and Methods
Antibodies and reagents. The primary antibody against Musashi1 (Msi1) (14-9896-82) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The primary antibody against p27 (554069) was purchased from BD Bioscience (Franklin Lakes, NJ, USA). Primary antibodies p-AKT (cs-2971), PTEN (cs-9556s), Notch1 (cs-3608s), and RAD51 (cs-8875s) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against Numb (sc-136554) and DNA Protein Kinase catalytic subunit (DNA-PKcs) (sc-5282), as well as secondary anti-mouse (sc-516132), anti-rabbit (sc-2357), and anti-rat antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Dallas, TX, USA). Primary antibodies against β-actin (A1978) and Ponceau S solution were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Cell lines and culture. The human neuroblastoma cell lines, including the MYCN-amplified BE(2)-M17(CRL-2267), CHP-212 (CRL-2273), IMR-32 (CCL-127), JF (from Dr. Jason M. Shohet, Baylor College of Medicine, Houston, TX, USA) and LAN-1 (from Dr. Seeger, University of California, Los Angeles, CA, USA), SK-N-BE(2) (CRL-2271), BE(2)-C (CRL-2268), SK-N-DZ (CRL-2149), and the non-MYCN-amplified SHEP (subclone of SK-N-SH), SK-N-AS (CRL-2137), SK-N-SH (HTB-11), and SH-SY5Y (CRL-2266), were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Roswell Park Memorial Institute (RPMI) culture medium 1640 (#10040, Corning, Manassas, VA, USA) with 10% fetal bovine serum (FBS, #A31606-02, Life Technologies Corp., Grand Island, NY, USA) at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. Aseptic technique was exercised to ensure cell cultures were free from mycoplasma contamination. All cells in this study have been authenticated.
BE(2)-C and LAN-1 cells were stably transfected to silence Msi1 (sc-106836-SH) or control utilizing shRNA (sc-108060) plasmids per the manufacturer’s instructions (Santa Cruz Biotechnology, Dallas, TX, USA). Puromycin (Thermo Fisher Scientific, Waltham, MA, USA) was used as a selection antibiotic. The stably transfected cells BE(2)-C and LAN-1 cells were utilized for clonogenic assay and cell viability experiments. Stably transfected LAN-1 cells were utilized for etoposide sensitivity experiments. BE(2)-C and LAN-1 cells were transiently transfected to silence Msi1 or control using shRNA plasmids per the manufacturer’s instructions (Santa Cruz Biotechnology). The transiently transfected BE(2)-C and LAN-1 cells were utilized for cell viability and immunoblotting experiments. The sRNAs consisted of pools of three to five target-specific 19- to 25-nucleotide sequences in length.
Immunoblotting. Cells were collected using cell lysis buffer and protein samples were prepared for immunoblotting using our lab’s previously described method (14). Equal amounts of protein were loaded and separated using NuPAGE 4-12% Bis-Tris gels (Invitrogen by Thermo Fisher Scientific) and were then transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% nonfat milk in TBS-T for 1 h at room temperature. The membranes were then incubated with antibodies against the human target proteins by using rabbit, mouse, or rat anti-human antibodies (1:500-2,000 dilution) overnight at 4°C. Anti-rabbit, anti-mouse, or anti-rat secondary antibodies (1:10,000 dilution) conjugated with HRP were incubated for 1 h at room temperature and visualized using an enhanced chemiluminescence detection system (PerkinElmer, Waltham, MA, USA). Densitometry analysis was then utilized to assess quantitative protein expression using the ImageJ software (Image J.JS v0.6.0, National Institutes of Health, Bethesda, MD, USA, https://ij.imjoy.io/). Densitometry analysis of Msi1 protein expression was performed by calculating a ratio of the density of each band relative to the density of each housekeeping control band.
Cell viability assay. BE(2)-C and LAN-1 control and Msi1-KD cells were plated in triplicate in RPMI culture medium with 10% FBS in 96-well plates at 1,000 and 3,000 cells per well, respectively. Cell viability was measured at 24, 48, 72, and 96 h after plating. A second experiment evaluating the impact of Msi1 silencing on etoposide sensitivity was also performed and utilized the same cells and plating method described above. After plating, the cells were allowed to attach overnight before treatment with etoposide. The cells were treated in triplicate with etoposide doses ranging from 25-100,000 nM. Cell viability was then measured 72 h after treatment. All cell viability measurements were performed using the Cell Counting Kit-8 (CCK-8) colorimetric assay (CK04, Dojindo Molecular Technologies, Rockville, MD, USA) and counted using the Cytation 5 Cell Imaging Multi-Mode reader (OD 450 nm) (Agilent, Santa Clara, CA, USA). The coefficient of drug interaction (CDI) was calculated using the equation CDI=AB/(AxB), where AB was the percent cell viability for Msi1 silencing and etoposide treatment combined, A was etoposide treatment alone, and B was Msi1 silencing alone. Synergistic effect was defined as CDI <1, while CDI=1 was indicative of an additive effect, and CDI >1 an antagonistic effect.
Clonogenic assay. BE(2)-C and LAN-1 control and Msi1-KD cells were plated in 6-well plates at 1000 and 3000 cells per well, respectively. Cells were cultured for 14 days. Colonies were stained with 0.02% crystal violet dye, photographed and counted using the Bio-Rad Gel Doc XR+ Imager (Bio-Rad).
Patient database. R2 (http://r2.amc.nl), a publicly available genomics analysis and visualization platform, was used to analyze microarray and RNA-sequencing data of human neuroblastoma tumors from the Kocak neuroblastoma database, which contains gene expression profiles from 649 neuroblastoma tumors. This database was used to investigate possible correlations between Msi1 expression and International Neuroblastoma Staging System (INSS) stage, characteristics associated with high-risk disease (such as MYCN-amplification and age >18 months), and patient survival, as well as any associations between Msi1 expression and that of p27, PTEN, Numb, Notch1, RAD51, and DNA-PKcs.
Statistical and experimental analysis. Experiments were performed in triplicate. The scoring index and relative expression values were expressed as mean±standard error of mean (SEM). Statistical analyses were performed using Student’s t-tests, one-way ANOVA, and Mantel-Cox tests, using GraphPad Prism (version 10.2.3, GraphPad Software, San Diego, CA, USA). Hazard ratio (HR) was derived from Mantel-Cox tests. A p-value <0.05 was considered significant.
Results
Increased Msi1 expression was associated with high-risk disease and inferior clinical outcomes in neuroblastoma. Increased expression of Msi1 has been associated with aggressive disease and poor outcomes in multiple malignancies (15-17). However, the role it plays in neuroblastoma has yet to be determined (18). As such, we utilized the publicly available Kocak database, accessible via the R2: Genomics Analysis and Visualization Platform (19), to determine whether Msi1 expression has any correlation with clinical outcomes in neuroblastoma. Higher Msi1 expression was seen in samples from patients with high-risk disease characteristics such as MYCN amplification (8.18 vs. 7.82, p<0.0001) (Figure 1A), age >18 months (mean 2log Msi1 expression 8.05 vs. 7.77, p<0.0001) (Figure 1B), and increased International Neuroblastoma Staging System (INSS) stage [F(3, 567)=21.03, p<0.001, η2=0.1] (Figure 1C). Furthermore, increased Msi1 expression was associated with lower 5-year survival. Those in the high Msi1 expression group experienced a 5.38 times higher risk of death at five years than those in the low expression group [HR=5.38, 95% confidence interval (CI)=2.52-11.47, p<0.0001] (Figure 1D).
Increased Msi1 expression is associated with MYCN-amplification and aggressive neuroblastoma phenotypes. (A) Increased Msi1 mRNA expression is associated with MYCN amplification in the publicly available Kocak R2 database, which contains samples from 649 neuroblastoma patients (p<0.0001, relative Msi1 mRNA expression 8.18 for MYCN-amplified tumors vs. 7.82 for non-amplified tumors). (B) In the Kocak database, increased Msi1 mRNA expression is seen in older patients (p<0.0001, relative Msi1 mRNA expression 7.77 for age <18 months vs. 8.05 for age >18 months). (C) Msi1 expression with INSS staging [F(3, 567)=21.03, p<0.001, η2=0.1]. (D) Kaplan-Meier survival analysis demonstrated increased Msi1 mRNA expression associated with decreased survival in the Kocak database (p<0.0001). (E) Msi1 protein expression is increased in MYCN-amplified neuroblastoma cell lines (molecular weight 39 kDa and 42 kDa for Msi1 and β-actin, respectively). Densitometry analysis of Msi1 protein expression (p=0.0049, MYCN-A: 15.47 vs. MYCN-NA: 1.0). Statistical analysis was performed using Student’s t-tests and a one-way ANOVA to evaluate differences in means between groups. Results are demonstrated as mean±standard error of the mean (SEM). *p<0.05, **p<0.01, and ****p<0.0001.
To determine whether a correlation between Msi1 expression and MYCN-amplification persisted at the protein level, immunoblotting was performed using 11 human neuroblastoma cell lines, including the MYCN-amplifying and non-amplifying cell lines. The selection of MYCN-amplifying and non-amplifying cell lines reflects genetic, not necessarily clinical, diversity. MYCN-amplifying cell lines included BE(2)-M17, CHP-212, IMR-32, JF, LAN-1, SK-N-BE(2)-C (a clonal subline of SK-N-BE(2), hereafter referred to as BE(2)-C), and SK-N-DZ. Non-MYCN-amplifying cell lines included SHEP, SK-N-AS, SK-N-SH, and SH-SY5Y. There was a pronounced increase in Msi1 protein expression among MYCN-amplifying cell lines as compared to non-amplifying lines (Figure 1E).
Silencing of Msi1 was associated with decreased colony formation. The BE(2)-C and LAN-1 cell lines were then both stably transfected to silence Msi1 to perform functional and mechanistic experiments. Successful transfection was confirmed via immunoblotting, which showed decreased Msi1 protein expression in the BE(2)-C and LAN-1 Msi1-KD cells (siMsi1 in Figure 2A and B), relative to their respective controls (iControl). Silencing of Msi1 resulted in decreased colony formation of both BE(2)-C cells (Control: 128.0±16.3 colonies vs Msi1-KD: 38.3±9.0 colonies, p=0.0085) and LAN-1 cells (Control: 78.3±6.5 colonies vs. Msi1-KD: 49.3±1.5 colonies, p=0.0121) (Figure 2C). These findings correlate with a 70.1% and 37.0% decrease in colony formation among Msi1-silenced cells compared to control cells, in BE(2)-C and LAN-1 cell lines, respectively.
Msi1 is associated with decreased colony formation and cell proliferation in neuroblastoma. (A1-A2) Msi1 (39kDa) silencing at the protein level was confirmed using Western Blot in BE(2)-C (left) and LAN-1 (right) cells following transfection with sRNA to produce stable Msi1 silencing (siControl/siMsi1) and siRNA to produce transient Msi1 silencing (shControl/shMsi1). (B1-B2) Densitometry analysis of Msi1 protein expression was performed by calculating a ratio of the density of each band relative to the density of each housekeeping control band [BE(2)-C, left side, Msi1 protein expression relative to control 0.24 for shMsi1, p=0.0005, and 0.19 for siMsi1, p<0.0001] (LAN-1, right side, Msi1 protein expression relative to control 0.58 for shMsi1, p<0.0001, and 0.57 for siMsi1, p=0.0285). (C1-C2) Decreased colony formation by clonogenic assay in Msi1-silenced cells in both BE(2)-C cells (left side, Control: 128.0±16.3 colonies vs. Msi1-KD: 38.3±9.0 colonies, p=0.0085) and LAN-1 cells (right side, Control: 78.3±6.5 colonies vs. Msi1-KD: 49.3±1.5 colonies, p=0.0121). Representative images of colony formation of BE(2)-C (left) and LAN-1 (right) control cells vs. Msi1-KD cells. (D1-D2) On CCK-8 proliferation and cytotoxicity assays performed at 24, 48, 72, and 96 h after plating, BE(2)-C cells (left) showed significantly decreased proliferation among Msi1-KD cells at 96 h (30.8% vs. 24.6%, p=0.0027) and LAN-1 cells (right) showed decreased proliferation among Msi1-KD cells at 72 h (37.4% vs. 25.0%, p=0.0068) and 96 h (48.5% vs. 42.0%, p=0.0407). Statistical analysis was performed using Student’s t-tests to evaluate differences in means between groups. Results are demonstrated as mean±standard error of the mean (SEM). *p<0.05.
Silencing of Msi1 was associated with decreased proliferation of neuroblastoma cells. Due to a delay between experiments, the persistence of Msi1 silencing in both cell lines was evaluated using immunoblotting prior to beginning cell viability testing. This revealed stable Msi1 silencing in the LAN-1 cell line, but diminished silencing in the BE(2)-C cell line. To allow for continued analysis of the role of Msi1 in both cell lines, both lines were transiently transfected to silence Msi1. Successful transfection was confirmed via immunoblotting, which showed decreased Msi1 protein expression in the BE(2)-C and LAN-1 Msi1-KD cells (siMsi1 in Figure 2A and B), relative to their respective controls (siControl). We then proceeded with cell viability experiments using transiently silenced BE(2)-C cells and stably silenced LAN-1 cells. We found that silencing of Msi1 was associated with decreased proliferation in both cell lines. In BE(2)-C cells, the decrease in proliferation became significant at 96 h (30.8% vs. 24.6%, p=0.0027), while LAN-1 Msi1-KD cells showed decreased proliferation at both 72 h (37.4% vs. 25.0%, p=0.0068) and 96 h (48.5% vs. 42.0%, p=0.0407) (Figure 2D).
Treatment with etoposide resulted in an increase in Msi1 expression. Msi1 expression has been found to increase in response to drug and radiation treatment in multiple malignancies, and elevated Msi1 has been implicated in increased chemo- and radio-resistance (20-22). For instance, silencing Msi1 in human gastric cancer cells was shown to decrease drug resistance when compared to chemotherapy alone (14). Msi1 expression also correlated with pediatric glioblastoma, the most aggressive form of glioma, and was found to increase chemo- and radioresistance (23, 24). To determine whether Msi1 plays a role in treatment response in neuroblastoma, stably silenced LAN-1 cells were treated with etoposide, a drug commonly utilized in treating neuroblastoma. After 72 h, the treatment with 1,000 nM etoposide increased Msi1 mRNA expression in treated control and Msi1-KD cells compared to their untreated counterparts (Figure 3A). Immunoblotting confirmed increased Msi1 expression at the protein level in control cells following etoposide treatment (untreated control: 0.37, treated control: 0.39, p=0.0081), while, as anticipated, Msi1 protein expression did not increase in Msi1-KD cells despite etoposide treatment (Figure 3B).
Msi1 expression increases with etoposide and Msi1 silencing decreases the expression of DNA damage repair genes and improves sensitivity to etoposide treatment. (A) Msi1 mRNA expression in treated control and Msi1-KD cells increased compared to their untreated control cells after 72 h of treatment with 1,000 nM etoposide. (B1-B2) Western Blot analysis of Msi1 protein expression (39 kDa) in LAN-1 Control and Msi1-KD cells collected 72 h after treatment with 1,000 nM etoposide showed increased expression of Msi1 in treated control cells compared to untreated control cells and decreased Msi1 protein expression in treated Msi1-KD cells compared to untreated Msi1-KD cells. (C) Densitometry analysis of Msi1 protein expression was performed by calculating a ratio of the density of each band relative to the density of each housekeeping control band (untreated control vs. treated control: p=0.0081, 0.37 vs. 0.39; untreated Msi1-KD vs. treated Msi1-KD: p<0.0001, 0.21 vs. 0.08). 72 h after treatment with etoposide (doses ranging from 25-100,000 nM), cell viability was measured using CCK-8 assays. Silencing of Msi1 decreased the IC50 dose of etoposide 3.1-fold compared to control (p=0.0002, IC50 Control: 785.6 nM vs. Msi1-KD: 250.4 nM). (D) DNA-PKcs and RAD51 protein expression was increased in LAN-1 cells 72 h after treatment with 1,000 nM etoposide and that increase was mitigated by Msi1-silencing (molecular weight was 460 kDa and 37 kDa for DNA-PKcs and RAD51, respectively). (E) Densitometry analysis of DNA-PKcs protein expression was performed by calculating a ratio of the density of each band relative to the density of each housekeeping control band; it showed a significant increase in DNA-PKcs expression in etoposide-treated control cells vs untreated control (1.15 vs. 1.08, p=0.0005) and a significant decrease in DNA-PKcs expression in treated Msi1-KD cells vs treated control cells (1.03 vs. 1.15, p=0.0002). (F) Densitometry analysis of RAD51 protein expression showed a similar increase in RAD51 expression in etoposide-treated control cells vs untreated control (1.38 vs. 1.08, p<0.0001) and a significant decrease in RAD51 expression in treated Msi1-KD cells vs treated control cells (1.25 vs. 1.38, p<0.0001). Statistical analysis was performed using Student’s t-tests to evaluate differences in means between groups. Results are demonstrated as mean±standard error of the mean (SEM). *p<0.05.
Msi1 silencing with etoposide treatment synergistically decreased neuroblastoma cell proliferation. Considering the known toxicity of the aggressive drug regimens used in the treatment of high-risk neuroblastoma and the frequency and morbidity of long-term toxicity-related complications experienced by neuroblastoma survivors, it is desirable to minimize the administered doses of these toxic agents as much as possible. As such, we sought to determine whether blockade of Msi1 activity might improve etoposide treatment response in neuroblastoma. We measured cell viability using CCK-8 assays 72 h after treating stably-transfected LAN-1 control and Msi1-KD cells with etoposide. Silencing of Msi1 decreased the IC50 dose of etoposide 3.1-fold compared to control (IC50 Control: 785.6 nM vs. Msi1-KD: 250.4 nM, p=0.0002) (Figure 3C). To better evaluate the interaction between Msi1 silencing and etoposide treatment, the coefficient of drug interaction (CDI) was determined for each etoposide dose administered. A synergistic effect was defined as CDI <1. The combination of Msi1 silencing and etoposide treatment synergistically decreased cell viability, with an average CDI of 0.76 (Table I).
Msi1 knockdown combined with etoposide treatment synergistically decreases cell proliferation in neuroblastoma.
Silencing of Msi1 was associated with decreased expression of DNA damage repair genes after etoposide treatment. Regulation of key proteins involved in DNA damage repair, particularly double-strand break (DSB) repair, has been theorized as a mechanism by which Msi1 increases treatment resistance. The upregulation of DNA-Protein Kinase Catalytic Subunit (DNA-PKcs) and RAD51 protein expression by Msi1 in response to DSBs induced by radiation therapy has been identified as a potential mechanism through which Msi1 enhances DSB repair by increasing non-homologous end-joining and homologous recombination repair capabilities, respectively (23, 25, 26). Etoposide is a potent topoisomerase II inhibitor known to cause DSBs that lead to cell death if not repaired. We sought to assess the impact of etoposide treatment and Msi1-silencing on DNA-PKcs and RAD51 expression to determine whether regulation of the expression of these proteins by Msi1 may contribute to the improved response to etoposide seen with Msi1-silencing. Protein samples were collected 72 h after treatment of LAN-1 control and Msi1-KD cells with 1,000 nM etoposide (Figure 3D). Compared to untreated control cells, etoposide-treated control cells showed a significant increase in the protein expression of both DNA-PKcs (1.08 vs. 1.15, p=0.0005) and RAD51 (1.08 vs. 1.38, p<0.0001), suggesting that expression of these proteins is upregulated in response to the DNA damage caused by etoposide treatment (Figure 3E and F). Furthermore, compared to etoposide-treated control cells, treated Msi1-KD cells showed significantly decreased protein expression of DNA-PKcs (1.03 vs. 1.15, p=0.0002) and RAD51 (1.25 vs. 1.38, p<0.0001). In combination, these findings suggest that Msi1 increases chemoresistance by enhancing the DNA damage repair capabilities of neuroblastoma cells through the upregulation of DNA-PKcs and RAD51 protein expression.
Silencing of Msi1 altered the expression of several proteins involved in cell cycle progression, cell proliferation, and differentiation of neuroblastoma cells. In this study, we found that silencing of Msi1 led to decreased colony formation and diminished proliferation of neuroblastoma cells. In other malignancies, these Msi1-mediated changes in cell behavior have been correlated with altered expression of p27, a key regulator of cell cycle progression, as well as changes in Notch and PI3K/AKT signaling through the translational regulation of Numb and PTEN expression, respectively (27, 28). As such, we sought to determine whether the regulation of these proteins may also be responsible for the inhibitory effect of Msi1-silencing on neuroblastoma cell growth. We queried the R2 Kocak database to look for associations between Msi1 mRNA expression and that of p27 (CDKN1b), PTEN, Numb, and Notch1. We found a negative correlation between Msi1 expression and expression of p27 (p<0.001, r=−0.324, R2=0.105) (Figure 4A), PTEN (p<0.001, r=−0.321, R2=0.103) (Figure 4B), and no correlation between Numb (p=0.0085, r=−0.104, R2=0.011). Notch1 and Msi1 expression were positively correlated (p<0.0001, r=0.271, R2=0.073) (Figure 4C). Because Msi1 is known to post-transcriptionally regulate the expression of its target proteins, we then used immunoblotting to evaluate for changes in protein expression related to Msi1-silencing. We identified a significant increase in p27 protein expression in transiently silenced Msi1-KD cells from both BE(2)-C (p=0.016, 87.72 vs. 144.1) and LAN-1 (p<0.0001, 17.89 vs. 61.48) cell lines (Figure 4A). Increased PTEN expression was noted in Msi1-KD cells from both cell lines [BE(2)-C: p=0.0073, 7.56 vs. 10.6; LAN-1: p<0.0001, 17.13 vs. 37.47] (Figure 4B), as was increased expression of Numb [BE(2)-C: p<0.0001, 3.3 vs. 87.33; LAN-1: p=0.0136, 58.41 vs. 64.18] (Figure 4C). Interestingly, no change in Phospho-Akt or cleaved-Notch1 protein expression was identified. We questioned whether this might be related to the relatively short time interval between transfection and protein collection in the transiently-silenced cells, so we then assessed Phospho-Akt and cleaved-Notch1 protein levels in stably-transfected BE(2)-C and LAN-1 cells. This revealed associations in both cell lines between Msi1-silencing and decreased levels of Phospho-Akt [BE(2)-C: p=0.0055, 14.65 vs. 10.66; LAN-1: p<0.0001, 126.4 vs. 65.5] (Figure 4B) and cleaved-Notch1 [BE(2)-C: p<0.0001, 4.63 vs. 0.42; LAN-1: p=0.0032, 12.25 vs. 3.26] (Figure 4C). Together, these findings suggest that Msi1 acts as an activator of both the PI3K/Akt and Notch signaling pathways by regulating the expression of PTEN and Numb, respectively.
Msi1 silencing alters the expression of cell cycle, proliferation, and differentiation genes. (A1-A2) Querying of the R2 Kocak database to assess for associations between Msi1 and p27 (CDKN1b) mRNA expression revealed a negative association between the expression of Msi1 and p27 (p<0.0001, r=−0.324, R2=0.105). Protein expression of p27 (27 kDa) is upregulated following Msi1 knockdown in both BE(2)-C (p=0.016, control: 87.72 vs. Msi1-KD: 144.1) and LAN-1 (p<0.0001, control: 17.89 vs. Msi1-KD: 61.48) cells. Densitometry analysis of protein expression of each target gene was performed by calculating a ratio of the density of each band relative to the density of each housekeeping control band. (B1-B3) Expression of Msi1 and PTEN mRNA was negatively correlated in the Kocak database (p<0.0001, r=−0.321, R2=0.103). Protein expression of PTEN (54 kDa) is upregulated following Msi1 knockdown in both BE(2)-C (p=0.0073, control: 7.56 vs. Msi1-KD: 10.6) and LAN-1 (p<0.0001, control: 17.13 vs. Msi1-KD: 37.47) cells. Levels of phosphorylated Akt protein (60 kDa) were decreased in stably silenced (shMsi1) BE(2)-C Msi1-KD cells (p=0.0055, control: 14.65 vs. Msi-KD: 10.66) and stably silenced LAN-1 Msi1-KD cells (p<0.0001, control: 126.4 vs. Msi1-KD: 65.5), but no difference in phospho-Akt levels was seen in transiently silenced Msi1-KD cells (siMsi1) from either cell line. (C1-C4) In the Kocak database, mRNA expression of Msi1 and Numb was negatively correlated (p=0.0085, r=−0.104, R2=0.011), while expression of Msi1 and Notch1 was positively correlated (p<0.0001, r=0.271, R2=0.073). Numb (71 kDa) protein expression is upregulated following Msi1 knockdown in both BE(2)-C cells (p<0.0001, control: 53.3 vs. Msi1-KD: 87.33) and LAN-1 cells (p=0.0136, control: 58.41 vs. Msi1-KD: 64.18). Protein expression of Notch1 (120 kDa) is downregulated (p=0.0327, control: 5.04 vs. Msi1-KD: 3.87) in stably silenced (shMsi1) BE(2)-C Msi1-KD cells (p<0.0001, control: 4.63 vs. Msi1-KD: 0.42), but not transiently silenced (siMsi1) BE(2)-C cells. In LAN-1 Msi1-KD cells protein expression of Notch1 is downregulated following transient Msi1-silencing (siMsi1) (p=0.0327, control: 5.04 vs. Msi1-KD: 3.87) and stable Msi1-silencing (shMsi1) (p=0.0032, control: 12.25 vs. Msi1-KD: 3.26). Statistical analysis was performed using t-tests to evaluate differences in means between groups. Results are demonstrated as mean±standard error of the mean (SEM). *p<0.05.
Discussion
Children with high-risk neuroblastoma continue to have unacceptably high mortality rates despite aggressive, multimodal therapy, and survivors often face life-long complications related to the toxicity of the treatments they receive. As such, in order to develop treatments that will optimize survival and minimize toxicity, it is necessary to identify the biologic features of high-risk tumors that may be contributing to treatment resistance. In this study, we found that, in neuroblastoma, increased Msi1 expression is associated with decreased 5-year survival, as well as characteristics of high-risk disease, such as MYCN-amplification and patient age >18 months. Furthermore, by silencing Msi1 in MYCN-amplified neuroblastoma cells, we determined that loss of Msi1 function is associated with decreased proliferation and colony formation, as well as improved response to etoposide treatment. These findings suggest that Msi1 may be a novel therapeutic target in neuroblastoma. While all experiments were conducted in triplicate to verify results, limitations of this study include the limited number of cell lines studied. In addition, these experiments were not replicated in in vivo models. Patient-derived xenograft models, most commonly immunodeficient mice, would aid in understanding aspects of disease progression and metastasis.
Overexpression of Msi1 has been identified in numerous malignancies, including breast, gynecologic, urologic, gastrointestinal, lung, and brain tumors (29-31). In these cancers, the role of Msi1 in the promotion of cell growth, proliferation, and survival has been well-defined. In breast cancer, Msi1 regulates cell proliferation, contributes to the maintenance of a cancer stem cell phenotype, and is a negative prognostic marker (8). Similar results were also demonstrated in gastric cancer, as increased Msi1 expression correlated with poor prognosis (20). Msi1 was also identified as a sensitive and specific diagnostic marker and potential therapeutic target in lung cancer (31). In hepatocellular carcinoma, Msi1 overexpression is associated with enhanced cell growth and promotion of cell cycle progression (16). Furthermore, knockdown of Msi1 has been associated with inhibited growth in endometrial cancer (27), tumor regression in colon adenocarcinoma (28), and enhanced apoptosis in breast cancer (32). In this study, we found that silencing of Msi1 in two MYCN-amplified neuroblastoma cell lines led to a decrease in cell proliferation and colony formation. Our findings, in combination with these previous reports, suggest that upregulation of Msi1 expression in cancer cells increases cell proliferation and survival. Additionally, the decrease in cell viability noted in our study following Msi1-silencing, along with prior studies documenting tumor regression induced by Msi1-knockdown, supports the consideration of Msi1 as a potential therapeutic target in neuroblastoma.
The mechanisms by which Msi1 contributes to oncogenesis and tumor growth remain incompletely understood, however they have been studied in various other neoplasms. Translational regulation of Numb, an inhibitor of the Notch signaling pathway, was one of the earliest identified functions of Msi1 (33). Msi1 has since been found to regulate the expression of hundreds of proteins, most commonly those involved in cell cycle regulation, such as its suppression of p21, p27, and p53 expression (34), and in cell survival pathways, such as the activation of PI3K-AKT signaling via the suppression of PTEN expression (35). In medulloblastoma, Msi1 promotes cell proliferation through the translational regulation of key Notch, Hedgehog, and Wnt pathway proteins (36), regulates the expression of multiple cell cycle genes. Increased expression of Msi1 in medulloblastoma correlates with higher grade tumors and worse prognosis (37). Meanwhile, activation of the Notch and PI3K-Akt signaling pathways by Msi1 has been implicated in the enhancement of glioma cell growth (38). Activation of the Wnt and Notch pathways by Msi1 is associated with tumorigenesis in intestinal cancers (39). Furthermore, increased Msi1 expression correlates with decreased Numb expression and resultant increased Notch pathway activation in brain metastases from multiple types of primary tumors (40). In this study, we found that the decreased proliferation and colony formation seen with Msi1-silencing was likely related to decreased Notch signaling (as evidenced by increased Numb protein expression and decreased levels of cleaved Notch1), decreased PI3K-Akt signaling (as evidenced by increased PTEN expression along with decreased Phospho-Akt levels), and decreased cell cycle progression via increased p27 expression.
In addition to its role in tumor growth and cell proliferation, Msi1 has been implicated in enhancing chemo- and radio-resistance in multiple malignancies. Increased expression of Musashi proteins in response to treatment with cisplatin and paclitaxel has been correlated with chemoresistance in inflammatory breast cancer (41), while in Group 3/4 medulloblastoma, Msi1 has been shown to decrease cell apoptosis, contributing to increased cisplatin resistance (21). In some studies, this function of Msi1 has been attributed to its role in DNA damage repair. In glioblastoma, it has been reported to increase radioresistance by promoting double-strand break homologous recombination repair via increased expression of RAD51 (24), and in both glioblastoma and endometrial cancer to promote DSB repair through non-homologous end joining mediated by increased expression of DNA-PKcs (23, 27). In the current study, we identified increased expression of Msi1 following etoposide treatment and Msi1-silencing was associated with improved sensitivity to etoposide, as evidenced by a 3.1-fold decrease in the etoposide IC50 dose. Additionally, we found that silencing of Msi1 was associated with decreased protein expression of both DNA-PKcs and RAD51 following etoposide treatment.
These results have immense potential for translational relevance. Further studies in xenograft models are necessary to validate these findings. Xenograft models have been used in other cancer studies. For example, a mouse model was used to demonstrate that volatile anesthetics promote breast cancer metastasis to the lung (41). Previous studies have reported that increased expression of DNA-PKcs and RAD51 in neuroblastoma is associated with radioresistance and chemoresistance, respectively (42, 43). Our findings, in combination with these previous reports, suggest that Msi1 may contribute to treatment resistance in neuroblastoma by enhancing DSB repair through the promotion of DNA-PKcs and RAD51 protein expression. Furthermore, knockdown of Msi1 has been reported to act synergistically with radiation therapy and chemotherapy in multiple malignancies, including glioblastoma (24) and medulloblastoma (21). The translational potential of these findings is evidenced by the disruption of oncogenic phenotypes associated with Msi1-inhibition by luteolin or gossypol in glioblastoma (43) and colon cancer (44), respectively.
Conclusion
High-risk neuroblastoma remains a significant cause of cancer-related morbidity and mortality in children. Treatment-refractory disease is not uncommon, despite aggressive, multimodal regimens that frequently lead to life-long toxicity-related complications. As such, the identification of biologic factors associated with treatment resistance and high-risk disease characteristics like MYCN-amplification and increased patient age is of great importance if we hope to maximize survival while minimizing treatment toxicity. This study suggests that RNA-binding protein Musashi1 may play an oncogenic role in neuroblastoma through the regulation of genes involved in cell cycle progression and cell proliferation and may promote chemo-resistance through enhanced DNA damage repair (Figure 5). Despite the preclinical nature of this study, our findings suggest that Msi1 could be a promising target for developing novel therapies for high-risk neuroblastoma.
Schematic of the proposed function of Musashi1 in neuroblastoma. Image created with BioRender.com.
Acknowledgements
Not applicable.
Footnotes
Authors’ Contributions
The Authors confirm contribution to the paper as follows: study conception and design: Elizabeth D. Cochran, Jingbo Qiao, Jillian C. Jacobson, Dai H. Chung; data collection: Elizabeth D. Cochran, Jingbo Qiao, Sullivan McCreery; analysis and interpretation of results: Elizabeth D. Cochran, Jingbo Qiao; draft manuscript preparation: Elizabeth D. Cochran, Jingbo Qiao, Arti Machchhar. All Authors reviewed the results and approved the final version of the manuscript.
Availability of Data and Materials
The data that support the findings of this study are available from the Corresponding Author, DC, upon reasonable request.
Conflicts of Interest
The Authors have no conflicts of interest to declare.
Funding
This work was supported by grants from the National Institutes of Health (R01 DK61470, DHC) and research funding from Children’s Health.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received October 27, 2025.
- Revision received February 26, 2026.
- Accepted March 3, 2026.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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