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

Abstract

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.

Figure 1

Musashi-1 promotes CD44⁺ CRC traits. (A) Establishment of Musashi-1-overexpressing CRC cells (FLAG/FLAGMusashi-1). HT-29, HCT-116, and LoVo cells were transfected with 3× FLAG and 3× FLAGMusashi-1 expression vectors, yielding the stable clones of HT-29, HCT-116, and LoVo cells with FLAG/FLAGMusashi-1, respectively. Stably transfected cells were selected by G418 (4 mg/mL) in culture medium for 4 weeks. Total protein of selected stable cell lines was obtained by lysis in RIPA buffer with protease and phosphatase inhibitors. Samples were subjected to immunoblotting analysis with a monoclonal anti-FLAG antibody (left panel). Spheroid formation was determined by culturing FLAG (HT-29, HCT-116, and LoVo cells) and FLAGMusashi-1 (HT-29, HCT-116, and LoVo cells) cells in spheroid formation buffer for 2 weeks. Images were acquired with Olympus cellSens software v1.12 (right panel). (B) Immunoblotting analysis of CSC marker expression in HT-29, HCT-116, and LoVo cells /FLAG and HT-29, HCT-116, and LoVo cells/FLAGMusashi-1 cells. Total cell lysates of FLAG- or FLAGMusashi-1-overexpressing stable cell lines were collected in RIPA lysis buffer. Total proteins were subjected to immunoblotting analyses with Lgr5-, CD133-, CD44-, and CD44v6-specific antibodies. (C) Silencing of HT-29/FLAGMusashi-1. HT-29/FLAGMusashi-1 cells were transfected with Musashi-1-specific siRNAs using LipoMax (Invitrogen) for 72 h. Total cell lysates were collected and subjected to immunoblotting analyses using a monoclonal anti-FLAG antibody. Musashi-1 knockdown in HT-29/FLAGMusashi-1 cells (upper left panel). HT-29/FLAGMusashi-1 cells were transfected with pre-designed siRNAs against Musashi-1 by liposome-mediated nucleic acid delivery. Relative fold change is indicated in the bar chart (lower left panel). Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05. After 72 h, HT-29/FLAG and HT-29/FLAGMusashi-1 cells were subjected to CD44 expression analysis by flow cytometry (middle panels). Relative fold change is indicated in the bar chart (right panel). Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05.

Figure 2

Musashi-1 promotes cell migration. (A) Increased migration in Musashi-1-overexpressing HT-29 cells. HT-29/FLAG and HT-29/FLAGMusashi-1 stably expressing HT-29 cells were subjected to the migration assay in 8 μm transwell chambers. Cells were fixed and stained by crystal violet after 8 and 16 h incubations (left panel). Relative migration was quantified, and the results are shown as a bar chart. Error bars indicate the mean ± SD from three independent experiments. *p < 0.01, ^#p < 0.05 (right panel). (B) Cell migration is Musashi-1-dependent. HT-29/FLAGMusashi-1 cells were pretreated with Musashi-1-specific or scrambled siRNAs and then subjected to the transwell assay. Cells in the transwells were fixed and stained at the indicated time points (left panel). The results of the cell migration assay were quantified. Error bars indicate the mean ± SD from three independent experiments. *p < 0.01 (right panel). (C) Degradation of extracellular material by HT-29 cells overexpressing Musashi-1. HT-29/FLAGMusashi-1 and control HT-29/FLAG cells were plated on FITC-gelatin-coated coverslips. Cells were fixed at the indicated time points and images acquired using Olympus cellSens software v1.12 (left panel). Images were analysed for clear zone areas by ImageJ software. Clear zone areas were measured, normalized, and are presented as a bar chart. Error bars indicate the mean ± SD from three independent experiments. *p < 0.01, ^#p < 0.05 (right panel). (D) Increased expression of metastasis-associated genes. Total cell lysates of HT-29, HCT-116, and LoVo cells/FLAG and HT-29, HCT-116, and LoVo cells/FLAGMusashi-1 cells were collected in RIPA buffer with protease and phosphatase inhibitors. Expression of N-cadherin (N-Cad) and vimentin was analysed by immunoblotting with specific antibodies.

Figure 3

Overexpression of Musashi-1 enhances drug resistance in CRC cells. (A) FLAG- or FLAGMusashi-1-overexpressing HCT-116 were pretreated with increasing concentrations of 5-FU. Cells were then subcultured and seeded into new 6-well culture plates, fixed, and stained. Images were acquired using a Canon digital camera (upper panel). The relative cell survival curves were calculated and normalized. Square and circle indicate the mean ± SD from three independent experiments. *p < 0.01 (lower panel). (B) FLAG- or FLAGMusashi-1-overexpressing HT-29 cells were pretreated with increasing concentrations of 5-FU. Cells were then subcultured and seeded into new 6-well culture plates, fixed, and stained. Images were acquired using a Canon digital camera (upper panel). The relative cell survival curves were calculated and normalized. Square and circle indicate the mean ± SD from three independent experiments. ^#p < 0.05 (lower panel).

Figure 4

Musashi-1 forms SGs following treatment with arsenite. FLAGMusashi-1 transfected HT-29 cells were plated on coverslips and treated with 150 μM arsenite for 30 min. Cells were then fixed in 4% paraformaldehyde for 15 min at room temperature. After permeabilisation with 0.1% Triton X-100/PBS, FLAGMusashi-1-, PABP-, G3BP-, and eIF4E-specific antibodies were added to hybridization buffer at 4 °C overnight. The signals were amplified by Alexa488- or Alexa555-conjugated secondary antibodies. Images were acquired with a multiphoton confocal microscope. (A) Left panel: Co-localization of Musashi-1 and G3BP. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05. (B) Left panel: Co-localization of Musashi-1 and PABP1. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05. (C) Left panel: Co-localization of Musashi-1 and eIF4E. Arrow indicates Musashi-1 granules. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments.

Figure 5

The Musashi-1 C-terminal domain is required for granule formation. HCT-116 cells were transfected with various Musashi-1 domain swap constructs. Forty-eight hours after transfections, cells were stimulated with arsenite (150 μM) for 30 min. Cells were then fixed in 4% paraformaldehyde at room temperature and subjected to immunostaining. (A) Schematic diagram of Musashi-1 domain swap constructs. (B) Left panel: Co-localization of FLAGMusashi-1 and PABP1. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05. (C) Left panel: Co-localization of ΔNFLAGMusashi-1 and PABP1. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05. (D) Left panel: Co-localization of ΔR1FLAGMusashi-1 and PABP1. (E) Left panel: Co-localization of ΔR2FLAGMusashi-1 and PABP1. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05. (F) Left panel: Co-localization of ΔCFLAGMusashi-1 and PABP1. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments.

Figure 6

5-FU induces Musashi-1 granule formation. HCT-116 cells were transfected with FLAGMusashi-1 or ΔCFLAGMusashi-1 domain swap constructs. Forty-eight hours after transfections, cells were treated with 5-FU (400 μM) for 24 h. Cells were then fixed in 4% paraformaldehyde at room temperature and subjected to immunostaining as described in the Methods. (A) Left panel: Co-localization of FLAGMusashi-1 and G3BP. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05. (B) Left panel: Co-localization of ΔR1FLAGMusashi-1 and G3BP. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05. (C) Left panel: Co-localization of ΔCFLAGMusashi-1 and G3BP. Right panel: Statistical results of percentages of SG formation. Error bars indicate the mean ± SD from three independent experiments. Arrow indicates Musashi-1 granules.

Figure 7

Musashi-1 inhibits 5-FU-induced apoptosis in HCT-116 cells. (A) Upper panel: FLAG, FLAGMusashi-1, ΔR1FLAGMusashi-1, and ΔCFLAGMusashi-1 HCT-116 stable clones were seeded on 22 mm × 22 mm coverslips and treated with 5-FU (400 μM) for 24 h. Cells were fixed in 4% paraformaldehyde and subjected to the TUNEL assay as described in the Methods. Lower panel: Statistical results of percentages of TUNEL positive cells. Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05. (B) Upper panel: FLAG, FLAGMusashi-1, ΔR1FLAGMusashi-1, and ΔCFLAGMusashi-1 HCT-116 stable clones were treated with or without 5-FU (400 μM) for 24 h. Total cellular proteins were isolated and subjected to immunoblotting with antibodies specific to PARP. Increased cleaved PARP (c-PARP) signals were observed in ΔCFLAGMusashi-1 HCT-116 stable clones. Tubulin indicates Tubulin protein as an internal control. Lower panel: Statistical results of percentages of relative intensity of c-PARP. Error bars indicate the mean ± SD from three independent experiments. ^#p < 0.05.

Figure 8

Increased Musashi-1 expression in CRC cells. Paired clinical samples (one stage IIA and two stage IIB samples) of normal and CRC tissues were subjected to Musashi-1 immunohistochemical staining as described in the Methods. Relative Musashi-1 expression levels were detected by a Musashi-1-specific antibody and fluorescence dye-conjugated secondary antibodies.