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. 2023 Jan 2;67(1):3413.
doi: 10.4081/ejh.2023.3413.

DPY30 promotes the growth and survival of osteosarcoma cell by regulating the PI3K/AKT signal pathway

Gong Cheng  1 , Fengmin An  2 , Zhilin Cao  3 , Mingdi Zheng  4 , Zhongyuan Zhao  5 , Hao Wu  6
Affiliations

DPY30 promotes the growth and survival of osteosarcoma cell by regulating the PI3K/AKT signal pathway

Gong Cheng et al. Eur J Histochem. .

Abstract

Osteosarcoma (OS) is characterized by aggressive features including invasiveness and high incidence of metastasis. OS patients with metastases are difficult to treat and suffer from a poor prognosis. DPY30 (protein dpy-30 homolog) is a key component of SET1/MLL family of H3K4 methyltransferases, which is implicated in the progression of multiple cancers. However, the potential functional engagement of DPY30 in OS remains to be unveiled. The objective of this study is to investigate the potential roles of DPY30 in the regulation of malignant phenotypes of OS cells. We examined DPY30 expression from a published dataset (GSE28424) as well as in OS tissues and adjacent normal tissues from OS patients. The association of DPY30 expression level and clinicopathologic parameters was assessed by Chi-square test. The role of DPY30 in regulating the malignant phenotype of OS cells and tumorigenesis was examined by in vitro functional assays and xenograft mouse model. We reported an upregulation of DPY30 in OS tumor tissues in both published dataset and clinical samples. A high level of DPY30 expression was associated with larger tumor size and more metastasis in OS patients, as well as poor overall survival. DPY30 knockdown in OS cells significantly impairs proliferation, migration and invasion, but induced cellular apoptosis. We further demonstrated that the agonist of PI3K/AKT pathway can rescue the inhibitory effects of DPY30 knockdown in OS cells. Together, our data indicate that DPY30 functions as an oncogene to promote the malignancy of OS cells possibly through PI3K/AKT pathway. The dependency of OS cells on DPY30 overexpression is a targetable vulnerability in OS cells.

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Figures

Figure 1.
Figure 1.
The expression of DPY30 in clinical OS samples and cell lines. A) The expression of DPY30 from the GEO dataset (GSE28424; including 19 osteosarcoma cell lines and 4 normal bone cell lines). B)The expression of DPY30 in the OS tumor tissues and para-carcinoma normal tissues (n=44) were assessed by RT-qPCR.(C) Immunohistochemical (IHC) staining demonstrated the overexpression of DPY30 in OS tumor tissues in comparison to para-carcinoma normal tissues. D) DPY30 protein expression was determined by Western blot in 5 pairs of OS tumor tissues and para-carcinoma normal tissues. E) DPY30 protein expression was determined by Western blot in 4 different OS cells lines (U2OS, HOS, Saos2 and MG63) and human osteoblast cell line (hFOB). F) Kaplan Meier curve and log-rank test were used to compare the overall survival in 44 OS patients. G) Receiver operative characteristics (ROC) curve analysis of DPY30 in the prognosis of OS patients. ***p<0.001.
Figure 2.
Figure 2.
DPY30 knockdown suppresses the malignancy in OS cells. A) The knockdown efficiencies of three sh-RNAs targeting DPY30 were determined by Western blot in U2OS and HOS cells. B) CCK-8 proliferation assay in U2OS and HOS cells upon DPY30 knockdown. C) EdU incorporation assay in U2OS and HOS cells upon DPY30 knockdown. D) Flow cytometry analysis of apoptotic events in U2OS and HOS cells upon DPY30 knockdown. **p<0.01; ***p<0.001.
Figure 3.
Figure 3.
DPY30 knockdown suppresses the invasion and migration capacities of OS cells. In vitro migration (A) and invasion (B) assay in U2OS and HOS cells upon DPY30 knockdown. (C) Protein levels of E-cadherin and N-cadherin were determined by Western blot in U2OS and HOS cells upon DPY30 knockdown. **p<0.01; ***p<0.001.
Figure 4.
Figure 4.
PI3K/AKT signaling pathway underlies the effect of DPY30 in OS cells. A) The phosphorylation levels of PI3K and AKT in U2OS and HOS cells upon DPY30 knockdown. B) The phosphorylation levels of PI3K and AKT in U2OS and HOS cells upon DPY30 knockdown with or without the treatment of PI3K agonist (740 Y-P). C) CCk-8 proliferation assay, cellular apoptosis (D), migration (E) and invasion (F) assay in U2OS and HOS cells upon DPY30 knockdown with or without the treatment of PI3K agonist (740 Y-P). **p<0.01; ***p<0.001; ^^p<0.01; ^^^p<0.001; *compared to sh-NC; ^compared to sh-DPY30.
Figure 5.
Figure 5.
DPY30 silencing suppresses tumorigenesis in mouse model. HOS cells with sh-NC and sh-DPY30 were injected into nude mice. The tumor volume (A) and weight (B) were determined at day 28 between the two groups (n=6 in each group). C) Immunohistochemical (IHC) staining of DPY30 and Ki-67 in the xenograft tumor tissues from sh-NC and sh-DPY30 groups (n=6 in each group, 5 sections were analyzed from each xenograft tumor tissue). **p<0.01; ***p<0.001.
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Grants and funding

Funding: This work was supported by the Shandong Province Medical and Health Technology Development Plan Project (202104070255); the Shandong Province Key Research and Development Program (2018GSF118186); the Yantai Key Research and DevelopmentPlan Project (2017WS114), the General program of Shandong Natural Science Foundation (ZR2022MH199).