High expression of ID1 facilitates metastasis in human osteosarcoma by regulating the sensitivity of anoikis via PI3K/AKT depended suppression of the intrinsic apoptotic signaling pathway

Abstract

A lack of understanding of the molecular basis underlying the regulation of metastatic disease and its effective therapy are the primary causes of high mortality in osteosarcoma. Thus, new insights into metastases and novel effective targets for metastatic osteosarcoma are urgently required. Anoikis resistance is considered a hallmark of cancer cells with metastatic ability. However, the molecular mechanism of anoikis is poorly understood in osteosarcoma. We applied immunohistochemistry to investigate the correlation between inhibitor of differentiation or DNA binding 1 (ID1) and clinicopathological features, and investigated the correlation between ID1 and the metastatic behavior of osteosarcoma cells, in vitro and in vivo. The results revealed that ID1 is overexpressed in human osteosarcoma tissues, is positively associated with lung metastases, and is a potential biomarker of poor prognosis. Overexpression of ID1 could increase anoikis insensitivity of osteosarcoma cells to facilitate metastasis through the PI3K/AKT-dependent mitochondrial apoptosis pathway. Knockdown of ID1 partly reversed the high potential of metastasis in anoikis-resistant osteosarcoma cells. Our findings revealed, that ID1 is a candidate molecular target for metastatic potential osteosarcoma by highlighting the role of anoikis resistance. In addition ID1 might be a potential predictor of poor prognosis in patients with osteosarcoma.

Keywords: Osteosarcoma; anoikis; biomarker; inhibitor of differentiation or DNA binding 1; metastasis.

Figure 1

ID1 is overexpressed in osteosarcoma, positively associated with lung metastasis, and predicts a poor prognosis. A, B. Representative images (200 × and 400 ×) of IHC staining and the quantitative comparison of IHC scores for ID1 expression in fibrous dysplasia, osteoblastoma, and osteosarcoma. ID1 expression was notably and significantly higher in osteosarcoma than in fibrous dysplasia and osteoblastoma. C. Higher IHC scores of ID1, which symbolized protein expression intensity, were found in patients with metastasis compared with those in patients without metastasis. D. A comparison of ID1 expression between low-grade osteosarcoma and high-grade osteosarcoma: The IHC scores of ID1 were significantly higher in Enneking stage III patients. **P < 0.01, ***P < 0.001, Scale bars: 50 µm.

Figure 2

High expression of ID1 predicts a poor prognosis in patients with osteosarcoma and overexpressed or knocked down ID1 in osteosarcoma cells. A. Representative images (200 ×) of IHC for high or low expression staining of ID1 in human osteosarcoma tissues. IHC scores ≥ 5 were defined as high expression of ID1 (average score was 4.97). B. Kaplan-Meier curve showing that high expression of ID1 is significantly associated with poor prognosis and shorter overall survival (61 of 67 patients with exact follow-up data were included). Scale bars: 100 µm. ID1, inhibitor of differentiation or DNA binding 1; IHC, immunohistochemistry. C, D. Overexpressed ID1 expression using lentivirus transfection and knocked down ID1 expression using siRNAs in 143B and M/HOS osteosarcoma cell lines were confirmed by qPCR and Western blot analysis.

Figure 3

ID1 has no significant effect on osteosarcoma cell migration and invasion. A. Wound healing assays were conducted for cells transfected with control (Lv-Ctrl) and ID1-overexpression (Lv-ID1) lentiviruses in 143B or M/HOS cells for 20 h. B. Transwell invasion assay indicating that overexpression of ID1 does not affect 143B or M/HOS osteosarcoma cells invasion in vitro. C, D. ID1 knockdown by siRNAs also does not affect osteosarcoma cell migration and invasion significantly. E. Pearson correlation analysis between ID1 and common MMP genes mRNA expression in human osteosarcoma tissues by analyzing the R2 gene expression database (http://hgserver1.amc.nl). F. Representative images of western blotting analysis of ID1 and MMP2 in 143B and M/HOS cells. Cells were treated with or without ID1-overexpression. NS, no significant difference; Scale bars: 200 µm.

Figure 4

ID1 regulates sensitivity of anoikis in osteosarcoma cells through mitochondrial apoptosis pathway suppression in vitro. A. Annexin V-PE/7-ADD assay indicating that after 48 hours of culture, the anoikis rate of cells with ID1 overexpression is much lower than that of the control cells. B. Knockdown of ID1 expression by siRNAs increased the anoikis rate of 143B and M/HOS cells significantly. C. Representative images and quantification of mitochondrial membrane potential (JC-1). Cells with or without ID1 silencing were cultured for 48 hours and then stained with JC-1 solution for flow cytometry analysis. D. Representative images of the western blotting analysis of ID1, BCL2, BAX, cleaved-caspase 3, and Cytochrome versions in 143B and M/HOS cells. Cells were subjected to ID1 overexpression or silencing. **P < 0.01, ***P < 0.001, compared with the 143B Ctrl group, ##P < 0.01, ###P < 0.01, compared with the M/HOS Ctrl group.

Figure 5

Crucial involvement of ID1 in osteosarcoma cells to acquire anoikis-resistance. A and F. Morphology of 143B and M/HOS clusters cultured in ultra low attachment 6-well plates at the indicated times captured under an inverted light microscope, scale bars: 100 µm. B. Annexin V-PE/7-ADD assay indicating that after culture for 48 hours, the apoptotic rate of 143B re-ad cells (143B-AR) was much lower than that of 143B cells, **P < 0.01. C. ID1 mRNA and protein expression was significantly higher in 143B-AR cells compared with that in 143B cells, **P < 0.01. D. Anoikis resistance in 143B-AR cells was partly reversed by siRNA-mediated knockdown of ID1, *P < 0.05, compared with the scrambled group. E. Representative images of western blotting analysis of ID1, BCL2, BAX, Cytochrome c, and cleaved-Caspase3 in 143B-AR cells treated as indicated. G. Annexin V-PE/7-ADD assay indicates there is no significant change of the apoptotic rate after suspended for 48 hours between M/HOS re-ad cells and M/HOS cells. H. ID1 mRNA and protein expression was found no significant change in M/HOS re-ad cells compared to M/HOS cells (Although there is a somehow significant increased mRNA expression of ID1 in M/HOS re-ad cells (*P < 0.05), but it is always thought to be no practical significance when the relative fold change of the mRNA expression is less than 1.5 times).

Figure 6

ID1 promotes osteosarcoma cells lung metastasis and ID1 silencing partly reverses the increased metastatic ability of anoikis-resistance cells in vivo. M/HOS and 143B cells were transfected as indicated and injected into the tail vein of nude mice to establish an in vivo model of lung metastasis. A and F. Representative macroscopic and microscopic images (H&E staining) of mouse lungs as indicated. Red dotted line sections were enlarged and red arrows indicate confirmed metastatic lesions. B and G. Percentage of mice bearing lung metastases in each group (n = 5). C and H. Numbers of lung metastatic nodules quantified on H&E-stained lung sections. D and E. ID1 was successfully silenced in 143B-AR cells. *P < 0.05, **P < 0.01, scale bars: 100 µm.

Figure 7

ID1 induces anoikis-resistance in osteosarcoma cells via activating the PI3K/AKT pathway to suppress mitochondrial apoptotic signals. A. Representative images of the western blotting analysis of ID1, ERK, p-ERK, AKT, and p-AKT levels in 143B and 143B-AR cells after 48 hours suspension. B. Representative images of the western blotting (WB) analysis of ID1, AKT, ERK, and their phosphorylated versions in 143B and M/HOS cells. Cells were treated with ID1-overexpression or knockdown. C. The ID1-induced-insensitive to anoikis were altered by PI3K/AKT inhibitors (LY294002). *P < 0.05, **P < 0.01. D. Representative images of the WB analysis of ID1, BCL2, BAX, cleaved-caspase 3, Cytochrome c, AKT and the phosphorylated versions in 143B and M/HOS cells.