Myo1b promotes tumor progression and angiogenesis by inhibiting autophagic degradation of HIF-1α in colorectal cancer

Figures

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  Fig. 1. Myo1b is upregulated in colorectal cancer and associated with poor prognosis. A Venn diagram depicting the overlap of differential expressed genes (DEGs) between 68 pairs CRC with adjacent mucosa tissues in the GEO dataset (GSE1065862) and myosin family genes. B Heatmap enumerates the differential expressed genes encoding Myosins between adjacent mucosa tissues and colorectal cancer samples in the GEO dataset (GSE106582). C Scatter diagram represents quantification analysis of Myo1b expression in adjacent mucosa tissues (n = 68) and colorectal cancer (n = 68) from the GEO dataset (GSE106582). D Scatter diagram represents Myo1b mRNA expression level of adjacent mucosa tissues (n = 31) and colorectal cancer tissues (n = 31) from Nanfang Hospital. The data are normalized to GAPDH. E Western blot was conducted to examine the protein levels of Myo1b in 8 cases of CRC tumor (T) and matched adjacent mucosa (N) tissues. The scatter diagram (right) shows the relative expression of Myo1b in human CRC tumors and matched adjacent mucosa tissues (normalized to β-actin). F Representative immunohistochemical staining of Myo1b protein in normal colonic mucosae, colon adenocarcinoma and paired…

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  Fig. 2. Myo1b promotes CRC angiogenesis by enhancing VEGF secretion of CRC cells. A Gene set enrichment analysis (GSEA) indicated that VEGF pathway was positively enriched in patients harboring high Myo1b expression from GEO datasets (GSE14333). B RT-qPCR confirmed the efficiency of si-Myo1b as well as OE-Myo1b in CRC cells and detected the effects of Myo1b on the expression of multiple angiogenesis-related factors in HCT-116 and HT29 cells. The conditioned medium (CM) of CRC cells (HCT-116 and HT29) with different Myo1b expression levels affected cell tube formation (C), migration (D & E) and proliferation (F) of HUVECs. G Blood vessels formed in representative images of the CAM assay after CM treatment. H The concentration of VEGF in the conditioned medium (CM) of HCT-116 and HT29 cells with different Myo1b expression levels was detected by ELISA assays. Each bar represented the mean ± SD (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 3. Myo1b mediates autophagy process by blocking autophagosome-lysosome fusion. A GSEA based on GEO dataset (GSE106582) indicated autophagy-related pathway was enriched in adjacent mucosa tissues when compared with colorectal cancer. B, C Western blot analyses of Myo1b, p-mTOR, P62 and LC3 in the CRC cells silencing or overexpressing Myo1b were performed in the presence or absence of Chloroquine (50 μM, 6 h). D Intensity of MAP1LC3 punctate in HCT-8 cells was analyzed after transfected with Myo1b over-expression vector or cultured with RAPA (500 nM, 24 h) and CQ (50 μM, 6 h).

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  Fig. 4. Myo1b inhibited autophagic degradation of HIF-1α in CRC. A Western blot was conducted to detect the effects of Myo1b expression and 50 μM CQ treatment for 6 h on HIF-1α protein expression in CRC cells. B RT-qPCR detects the effects of Myo1b on HIF-1α mRNA expression in HCT-116 and HT29 cells. Immunoblot assessment of HIF-1α stability in HCT-116 cells transfected with si-Myo1b (C) and HT29 cells transfected with over-expression vector (D), si-P62 (E) or treated by 50 μM CQ for 6 h (F). G Immunoprecipitation assay and western blot conducted to confirm the interaction between P62 and HIF-1α. Each bar represented the mean ± SD (n ≥ 3). ns, p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 5. HIF-1α participated in the angiogenesis function of Myo1b in CRC. A Effects of si-P62 and si-HIF-1α on Myo1b mediated angiogenesis was analyzed through HUVEC tube formation assay. Wound healing assay (B) and transwell migration assay (C) were performed to explore the effects of si-P62 and si-HIF-1α on Myo1b mediated HUVEC migration. D Effects of CM treatment collected from indicated cells on HUVEC proliferation was evaluated by Cell Counting Kit-8 assays. E Effects of si-P62 and si-HIF-1α on Myo1b mediated VEGF secretion of HCT-116 and HT29 cells were measured using ELISA assay. Each bar represented the mean ± SD (n ≥ 3). *, **, ***: NC-CM VS OE-Myo1b-CM; #, ##, ###: OE-Myo1b-CM VS OE-Myo1b+si-HIF-1α-CM. *, #, p < 0.05; **, ##, p < 0.01; ***, ###, p < 0.001.

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  Fig. 6. Myo1b promoted CRC xenograft tumors growth and angiogenesis in vivo. A Western blot was conducted to confirm the efficiency of sh-Myo1b in HCT-116 and DLD1 cells. B Images of CRC orthotopic tumors from BALB/c nude mice after the orthotopic injection of stable Myo1b-knockdown HCT-116 cells (sh-Myo1b) or control HCT-116 cells (NC). C Kaplan–Meier survival analysis of mice bearing orthotopic xenografts. n = 6 for each group. D Images of xenograft tumors from groups of BALB/c nude mice 24 days after the subcutaneous injection of stable Myo1b-knockdown HCT-116 cells (sh-Myo1b) or control HCT-116 cells (NC). E Subcutaneous tumor size was monitored every 6 days after injection. F Weights of subcutaneous xenograft tumors were measured after sacrificed at day 24. G Representative images of HE and IHC staining with antibody targeting human CD34, VEGF, Ki67, P62 and LC3 in subcutaneous xenograft tumors. Original magnification, ×200 or ×400; Scale bar, 100 μm or 40 μm. Each bar represented the mean ± SD (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 7. Schematic diagram of the mechanism on Myo1b mediated autophagy and angiogenesis in CRC. The high-expression of Myo1b in CRC cells disrupts the fusion between autophagosomes and lysosomes, which blocks the selective degradation of HIF-1α. The accumulated HIF-1α further upregulates the proangiogenic related genes and consequently promotes angiogenesis in CRC.