IGF1/IGF1R signaling promotes the expansion of liver CSCs and serves as a potential therapeutic target in HCC

Abstract

The presence of cancer stem cells (CSCs) play important roles in hepatocellular carcinoma (HCC) relapse, metastasis, drug resistance. The IGF1/IGF-1R signaling pathway has been implicated in the development and progression of various cancers, and plays an important role in maintaining the stemness of various types of CSCs, but its role in liver CSCs remains unclear. Here, we report that IGF1R is highly expressed in HCC tumors and positively correlated with stemness markers. To further verify the role of IGF1R in liver CSCs, the positive correlation of IGF1R expression with liver CSCs was also validated in mRNA level using the TCGA database. After pretreatment with IGF1 or overexpression of IGF1R, we observed a expansion of CD133 + and CD90 + populations and concomitantly a increased expression of CSC-associated genes, and increased sphere formation. Furthermore, IGF1R inhibition by inhibitor effectively eliminated liver CSCs and inhibited the growth of tumors and metastasis in vivo. These findings uncover the important role of IGF1/IGF1R signaling in liver CSCs, and also provide a promising diagnostic marker as well as therapeutic intervention for HCC.

Keywords: HCC; IGF1; IGF1R; Liver cancer stem cell; Picropodophyllin.

Fig. 1

IGF1R is highly expressed in HCC tumors and positively correlated with stemness markers. A IHC staining IGF1R images from two matched pretumor and HCC clinical samples. B Kaplan-Meier survival analysis comparing the overall survival (n = 89) of HCC patients with different IGF1R expression levels. C Correlation of IGF1R and CD133 expression in 64 HCC clinical samples. r = Pearson correlation coefficient. D Correlation of IGF1R and CD90 expression in 64 HCC clinical samples. r = Pearson correlation coefficient. All experiments were performed in triplicate, and the results are shown as mean ± standard deviation. *P < 0.05

Fig. 2

Correlation between IGF1R and liver CSCs. A Correlation between IGF1R and CD90. B Correlation between IGF1R and CD133. C The correlation between IGF1R and stemness markers in HCC. D Heatmap of coexpression analysis of IGF1R and liver CSCs markers. E GSEA plot showing IGF1R expression level was positively correlated with activated stemness related gene pathways

Fig. 3

IGF1 triggers the expansion of liver CSCs. A and B Expression levels of stemness genes in Huh7 and MHCC97H cells treated with IGF1 or Control detected by Western blot. C and D The proportion of CD133 + cells in Huh7 and MHCC97H cells treated with IGF1 (200ng/ml) or Control evaluated by flow cytometric assay. E and F Spheroid formation assay of Huh7 and MHCC97H cells pretreated with IGF1 (200ng/ml) or Control

Fig. 4

IGF1R upregulation enhances the expansion of liver CSCs. A The protein expression levels of stemness genes after IGF1R upregulation in Huh7 and MHCC97H cells. B and C The proportion of CD133 + and CD90 + cells in IGF1R upregulation Huh7 cells or vector cells was evaluated by flow cytometric assay. D Spheroid formation assay of IGF1R upregulation Huh7 cells or vector cells. All experiments were performed in triplicate, and the results are shown as mean ± standard deviation. Scale bars,50 μm. *P < 0.05

Fig. 5

PPP treatment inhibits in vivo tumor grouth. A Xenograft tumors derived from subcutaneous injections of Huh7 cells treated with vehicle or PPP (20 mg/kg). B Tumor growth curves and weights curves of mice bearing Huh7 cells treated with PPP or vehicle (control) for 15 days. Tumor size was measured by a caliper. C IHC staining of CD133 in the xenografted tumors treated with vehicle or PPP. *P < 0.05

Fig. 6

PPP treatment suppresses HCC metastasis. A and B Representative images and quantification of the bioluminescence in pulmonary metastatic lesions. C Representative images of HE images of lungs resected from tail vein injection metastasis mouse models. *P < 0.05