IGF‑IR promotes clonal cell proliferation in myelodysplastic syndromes via inhibition of the MAPK pathway

Abstract

Type 1 insulin‑like growth factor receptor (IGF‑IR) signaling is considered to serve a key role in the development of cancer. However, the effects of IGF‑IR on the malignant characteristics of myelodysplastic syndrome (MDS) clonal cells remains to be determined. In the present study it was demonstrated that knockdown of IGF‑IR reduced the proliferation and increased the apoptosis of MDS/leukemia cells. Integrated analysis of gene expression profiles using bioinformatics identified the MAPK signaling pathway as a critical downstream factor of IGF‑IR, and this was confirmed in vitro using western blotting which revealed that IGF‑IR knockdown significantly increased the expression of activated MAPK. Furthermore, IGF‑IR signaling was inhibited to investigate the potential of IGF‑IR as a therapeutic target of MDS. The results revealed that the IGF‑IR inhibitor picropodophyllin (PPP) inhibited cell proliferation, promoted cell apoptosis and arrested the cell cycle at the G2/M phase in MDS/leukemia cells. Similar to the effects of IGF‑IR knockdown, PPP treatment also increased MAPK signaling in vitro. In conclusion, IGF‑IR may serve as a potential therapeutic target of MDS.

Keywords: myelodysplastic syndrome; IGF-IR; clonal cell; proliferation; MAPK.

Figure 1.

IGF-IR knockdown inhibits the proliferation and increases apoptosis of MDS/leukemia cell lines. (A) Transfection of IGF-IR-shRNA lentivirus resulted in a decrease in IGF-IR expression both in the SKM-1 and K562 cells. Colony formation assays revealed that SKM-1 and K562 cells with IGF-IR knockdown significantly reduced colony formation compared with the control cells. (B) Cell Counting Kit-8 assays revealed that IGF-IR knockdown inhibited cell growth compared with the control cells. (C) IGF-IR knockdown increased cell apoptosis in SKM-1 and K562 cell lines. ***P<0.001, ****P<0.0001 (ANOVA). shRNA, short hairpin RNA; MDS, myelodysplastic syndrome; IGF-IR, type 1 insulin-like growth factor receptor.

Figure 2.

Gene expression profile analysis of IGF-IR knockdown in SKM-1 cells. (A) Pathway analysis revealed that the pathways significantly affected by genes upregulated in the IGF-IR knockdown cells, included MAPK signaling, apoptosis, transcriptional dysregulation in cancer, p53 signaling and Notch signaling, and the pathways significantly affected by downregulated genes in the knockdown cells included metabolism, insulin signaling, PI3K-Akt signaling and Toll-like receptor signaling. (B) GO analysis revealed that the ontologies significantly affected by upregulated genes in the knockdown cells included DNA-dependent transcription, negative regulation of cell proliferation, apoptosis signaling pathway, positive regulation of NF-κB transcriptional factor activity and cell cycle arrest, and the ontologies significantly affected by downregulated genes included metabolic processes, innate immunity, cell adhesion, protein phosphorylation and inflammatory response. Gene expression profile analysis of IGF-IR knockdown in SKM-1 cells. (C) Further pathway-net analysis revealed that MAPK was a key node among genes downregulated as a result of IGF-IR knockdown, and the main upregulated genes were apoptosis-related pathway genes. IGF-IR, type 1 insulin-like growth factor receptor; GO, gene ontology.

Figure 2.

Gene expression profile analysis of IGF-IR knockdown in SKM-1 cells. (A) Pathway analysis revealed that the pathways significantly affected by genes upregulated in the IGF-IR knockdown cells, included MAPK signaling, apoptosis, transcriptional dysregulation in cancer, p53 signaling and Notch signaling, and the pathways significantly affected by downregulated genes in the knockdown cells included metabolism, insulin signaling, PI3K-Akt signaling and Toll-like receptor signaling. (B) GO analysis revealed that the ontologies significantly affected by upregulated genes in the knockdown cells included DNA-dependent transcription, negative regulation of cell proliferation, apoptosis signaling pathway, positive regulation of NF-κB transcriptional factor activity and cell cycle arrest, and the ontologies significantly affected by downregulated genes included metabolic processes, innate immunity, cell adhesion, protein phosphorylation and inflammatory response. Gene expression profile analysis of IGF-IR knockdown in SKM-1 cells. (C) Further pathway-net analysis revealed that MAPK was a key node among genes downregulated as a result of IGF-IR knockdown, and the main upregulated genes were apoptosis-related pathway genes. IGF-IR, type 1 insulin-like growth factor receptor; GO, gene ontology.

Figure 3.

IGF-IR knockdown activates the MAPK signaling pathway in MDS/leukemia cell lines. Western blot analysis revealed that MAPK signaling characterized by p-p38 MAPK and p-p44/42 MAPK was activated by knockdown of IGF-IR in (A) SKM-1 and (B) K562 cells. p21 and MYC are considered critical target genes of the MAPK signaling pathway. IGF-IR knockdown increased the mRNA expression levels of (C) p21 whereas the expression of (D) MYC was decreased. **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA). IGF-IR, type 1 insulin-like growth factor receptor; p-, phosphorylated; MDS, myelodysplastic syndrome; shRNA, short hairpin RNA; KD, knockdown.

Figure 4.

PPP inhibits proliferation, promotes apoptosis and induces cell cycle arrest at the G2/M phase in MDS/leukemia cell lines. (A) PPP inhibited proliferation of both SKM-1 and K562 cell lines. The mean cell viability of SKM-1 cells was reduced from 97.2 to 16.6%, and the mean cell viability of K562 cell reduced from 97.1 to 70.3%. (B) PPP promoted apoptosis. The mean apoptotic rate of the SKM-1 cell line increased from 1.7 to 6.2%, and the mean apoptotic rate of the K562 cell line increased from 1.0 to 7.2%. *P<0.05, **P<0.01 (unpaired t-test). (C) PPP significantly induced G2/M phase block and decreased the percentage of cells in the S phase in SKM-1 and K562 cells. M1 represents G0/G1 phase cells, M2 represents S phase cells, M3 represents G2/M phase cells, and M1-M3 was flanked by fragments and clumps of cells, thus, the sum of the three was <100%. **P<0.01, ***P<0.001 (Chi-square test). PPP, picropodophyllin; MDS, myelodysplastic syndrome.

Figure 5.

PPP inhibits proliferation, promotes apoptosis and induces cell cycle arrest in G2/M in CD34⁺ cells of patients with MDS. (A) Following PPP treatment, cell viability of CD34⁺ cells of 8 patients with MDS was also significantly reduced. Collectively, the mean cell viability was reduced from 84.2 to 67.1%. The decrease in proliferation was the most significant, decreasing to 60.1% after 48 h of treatment, after which proliferation increased to varying degrees in the different groups of cells obtained from patients. Representative graphs for two patients (RCMD and RAEB2) are presented. (B) PPP increased apoptosis in the CD34⁺ cells of 6 MDS patients, among which the differences of 2 patients were statistically significant. *P<0.05 (unpaired t-test). (C) The cell cycles of the CD34⁺ cells of 7 MDS patients were arrested in the G2/M phase when treated with PPP. The percentage of cells in the S-phase in 5 patients was reduced, among which the reduction in patients no. 7 and 8 were the most significant. M1 represents G0/G1 phase cells, M2 represents S phase cells, M3 represents G2/M phase cells, and M1-M3 was flanked by fragments and clumps of cells, thus, the sum of the three was <100%. *P<0.05, **P<0.01 vs. the G2/M phase; ^#P<0.05, ^##P<0.01 vs. the S phase (Chi-square test). PPP, picropodophyllin; MDS, myelodysplastic syndrome.

Figure 6.

PPP activates the MAPK signaling pathway in MDS/leukemia cell lines. (A) Western blot analysis revealed that treatment with PPP upregulated the expression of MAPK signaling-related proteins including p-p38 MAPK and p-p44/42 MAPK in SKM-1 cells. (B) PPP increased the expression of p-p44/42 MAPK whereas the expression of p-p38 MAPK in K562 cells was not altered. (C and D) PPP increased the mRNA expression levels of p21 and inhibited the expression of MYC in SKM-1 and K562 cells. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (ANOVA). PPP, picropodophyllin; MDS, myelodysplastic syndrome; p-, phosphorylated; IGF-IR, type 1 insulin-like growth factor receptor.