Musashi1 Contribution to Glioblastoma Development via Regulation of a Network of DNA Replication, Cell Cycle and Division Genes

Abstract

RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in their levels are often observed in tumors with numerous oncogenic RBPs identified in recent years. Musashi1 (Msi1) is an RBP and stem cell gene that controls the balance between self-renewal and differentiation. High Msi1 levels have been observed in multiple tumors including glioblastoma and are often associated with poor patient outcomes and tumor growth. A comprehensive genomic analysis identified a network of cell cycle/division and DNA replication genes and established these processes as Msi1's core regulatory functions in glioblastoma. Msi1 controls this gene network via two mechanisms: direct interaction and indirect regulation mediated by the transcription factors E2F2 and E2F8. Moreover, glioblastoma lines with Msi1 knockout (KO) displayed increased sensitivity to cell cycle and DNA replication inhibitors. Our results suggest that a drug combination strategy (Msi1 + cell cycle/DNA replication inhibitors) could be a viable route to treat glioblastoma.

Keywords: DNA replication; E2F2; E2F8; Musashi1; cell cycle; cell division; glioblastoma.

Figure 1

Musashi1 knockout (KO) affects a network of genes implicated in cell cycle/division and DNA replication/repair. (A) The diagram shows the Gene Ontology terms (biological processes) enriched among genes downregulated in both U251 and U343 Msi1 KO lines in comparison to respective controls, according to ShinyGo [27]. (B) Tables show downregulated genes in both U251 and U343 Msi1 KO lines and their functions. Gray boxes indicate the genes from this group that showed increased expression in a transgenic mouse line displaying Msi1 overexpression (OE) [21]. Msi1 targets previously identified by CLIP (Crosslinking and Immuno-precipitation) or RIP (Ribonuclear particle immuno-precipitation) [3,9,23] are labeled in red. (C) Western analysis of cell cycle and DNA replication genes affected by Msi1 KO in both glioblastoma (GBM) cell lines U251 and U343. The uncropped Western blots have been shown in Figure S4. (D) qRT-PCR analysis shows that transgenic expression of Msi1 in astrocytes increased the expression of cell cycle and DNA replication genes. Statistical significance calculated by multiple t-test. Data shown as means ±s.d. *** p < 0.001).

Figure 2

Msi1 impact on cell cycle and division. (A) Network shows cell cycle and/or division genes downregulated in Msi1 KO GBM cells in comparison to controls. The network was built using String [28] considering interaction (experimental evidence), text mining and co-occurrence. Different colors were used to indicate clusters of strongly related genes; Msi1 targets previously identified by CLIP or RIP [3,9,23] are labeled in red. (B) The impact of Msi1 KO on cell cycle is shown using KEGG’s cell cycle pathway. Downregulated genes in U251 and U343 Msi1 KO cells in comparison to respective controls are in blue boxes and Msi1 targets previously identified by CLIP or RIP [3,9,23] are labeled in red.

Figure 3

Musashi1 in cell cycle, cell division and DNA replication. (A) Cell cycle genes regulated by Msi1 according to different genomic analyses. (B) Important genes associated with the centromere that are regulated by Msi1 according to different genomic analyses. Centromere-associated network (CCAN). (C) Core DNA replication genes regulated by Msi1 according to different genomic analyses in the pre-initiation, activation of pre-replicative complex and initiation phases of DNA replication. Down-regulated genes in Msi1 KO cells in reference to controls are identified in purple, genes determined to be up-regulated in transgenic tissue with Msi1 OE [21] are identified in blue and Msi1 targets previously identified by CLIP or RIP [3,9,23] are labeled in red.

Figure 4

Musashi1 regulates DNA replication and cell cycle and division. (A) Graphs display the % of forks that are in restart, new orientation and stalled in Msi1 KO vs. control GBM cells. Statistics are shown for only stalled. The comparisons between no treatment (NT) and HU are shown in black. The comparisons between controls and Msi KO clones after HU treatment are shown in red. Statistical significance was calculated by Chi-square with Yate’s correction and Fisher’s exact test (ns: not significant). (B) The graph shows replication fork (RF) speed. The comparisons between controls and Msi1 KO cells after HU treatment are shown in red. Statistical significance calculated by Mann-Whitney test. The mean values are represented as horizontal red lines. RF speed of Msi1 KO clones showed a slowdown. (C) U251 and U343 Msi1 KO cells displayed changes in cell cycle progression (accumulation of cells in G1) in comparison to respective controls. (D) The aspect of U251 and U343 Msi1 KO and control cells exposed to Paclitaxel. On the left, staining with anti-α-tubulin; on the right, staining with DAPI shows an increased number of multinucleated cells in Msi1 KO cells. Scale bar = 10 µm. (E) The graph shows the number of multinucleated cells in U251 and U343 control and Msi1 KO cells after treatment with Paclitaxel (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

GBM cells with Msi1 KO are more sensitive to cell cycle and DNA replication inhibitors. Graphs show the impact of different cell cycle and DNA replication inhibitors on proliferation (72 h) of U251 and U343 Msi1 KO cells in reference to their respective controls (DMSO treated). Statistical significance was calculated by one-way ANOVA and t-test. Data shown as means ± s.d. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Synergy between “Msi1 KO” and cell cycle and DNA replication inhibitors was determined by the statistical response additivity approach [41], with # indicating the drug concentration in which we observed the statistically most significant synergy (# < 0.05, ## < 0.01, ### < 0.001).

Figure 6

E2F2 and E2F8 are mediators of Msi1 impact on cell cycle and DNA replication genes. (A) qRT-PCR data showing that E2F2 and E2F8 decreased expression upon Msi1 knockdown in U251 and U343 cells. Statistical significance calculated by multiple t-test (*** p < 0.001). (B) Expression correlation (R values) between Msi1, E2F2 and E2F8 in TCGA GBM samples (*** p < 0.001). (C) The table shows genes affected by Msi1 KO in U251 and U343 cells that also display high expression correlation with E2F2 and E2F8 in GBM samples. Pearson correlation, all genes *** p < 0.001, R > 0.3. (D) E2F2 and E2F8 mRNA expression levels in normal brain vs. glioblastoma according to GEPIA [54]. (E) E2F2 and E2F8 mRNA expression in gliomas grades II, II and IV (TCGA samples). Data shown in B, C and E was generated with Gliovis [55]. (F) qRT-PCR data shows the impact of Msi1, E2F2 and E2F8 knockdown on the expression of the cell cycle, cell division and DNA replication genes in U251 cells. (G) Model showing that Msi1 regulates the expression E2F2 and E2F8 via CDK6, p21 and p27. (H) qRT-PCR data shows the impact of CDK6, p21 and p27 knockdown on the expression of E2F2 and E2F8. Left panel, CDK6 KD was performed in U251 cells. Right panel, p21 and p27 double KD were performed in U251 Msi1 KO cells. Statistical significance calculated by multiple t-test. Data shown as means ± s.d. (* p < 0.05, *** p < 0.001).