EID3 Promotes Cancer Stem Cell-Like Phenotypes in Osteosarcoma through the Activation of PI3K-AKT Signaling Pathway

The aim of this study is to elucidate molecular mechanism by which E1A-like inhibitor of differentiation 3 (EID3) promotes cancer stem cell-like phenotypes in osteosarcoma. Overexpression of EID3 in osteosarcoma cells generated more spherical clones, enhanced the expression of stemness-associated genes, and promoted chemoresistance, invasion, and metastasis. Furthermore, osteosarcoma cells overexpressing EID3 had increased ability to grow in suspension as osteospheres with high expression of Sox2 and stem cell marker CD133. In addition, knockdown of EID3 reduced sphere formation and inhibited osteosarcoma cell migration and invasion. RNA sequencing and bioinformatics analysis revealed that PI3K-Akt signaling pathway and MAPK pathwayrelated genes were enriched in osteosarcoma cells with high expression of EID3. Taken together, EID3 promotes osteosarcoma, and EID3–PI3K-Akt axis is a potential therapeutic target for osteosarcoma treatment.


Introduction
Osteosarcoma is the main cause of tumor death in children and adolescents [1]. With the development of modern medical technology, radical surgery and neoadjuvant chemotherapy has significantly increased the 5-year survival rate to 60% [2,3]. However, due to the resistance of tumor cells to chemotherapeutic drugs, the survival rate has reached a plateau [4]. The submachine system of chemotherapy resistance of osteosarcoma is not yet clear, and the existence of osteosarcoma stem cells (OSCs) is considered to be a major reason for chemotherapy resistance of osteosarcoma [5,6]. Although the role of osteosarcoma stem cells in chemotherapy resistance has not been fully elucidated, evidence shows that tumor stem cells can inhibit apoptosis through a variety of mechanisms, such as high expression of special drug transporters and effective DNA repair in osteosarcoma cells, which is related to the ability of tumor stem cells to maintain tumorigenicity through self-renewal and differentiation [5][6][7]. Therefore, to find the molecular mechanism related to the osteosarcoma stem cells and develop new targets to enhance drug sensitivity of osteosarcoma is an urgent problem to improve the treatment of osteosarcoma [8].
Molecular genetic analysis showed that the inactivation of tumor suppressor Rb and p53 played an important role in the occurrence and development of human osteosarcoma [9]. In vivo studies have also shown that osteosarcoma can be induced by mutations of genes such as (MSC) and/or bone progenitor cells such as p53 and RB or abnormal signal transduction of Hedgehog and NOTCH in mesenchymal stem cells [9,10]. Recent studies have shown that osteosarcoma contains OSCs responsible for tumorigenesis, growth, recurrence, and chemoresistance [11]. OSCs can maintain their stemness through self-renewal and differentiation [12]. The molecular mechanism of chemoresistance of osteosarcoma is not clear, but the existence of OSCs is considered to be a major reason for chemoresistance of osteosarcoma.
Based on the self-renewal characteristics of stem cells, several methods have been developed to identify and isolate OSCs [13]. Functional in vitro tests for the formation of tumor spheres under nonadhesive and serum-free conditions are usually used as an initial step in the enrichment of OSC-like cell populations. The in vivo verification of OSCs is to evaluate the tumorigenicity of low cell count by continuously transplanting isolated hypothetical OSC into immunocompromised mice [14]. At present, proteins that can be used as surface markers of osteosarcoma stem cells are CD133, CD117, and Stro1; especially, CD133 is widely used [15,16].
E1A-like inhibitor of differentiation 3 (EID3) is the third member of the EID family [17]. EID3 is homologous to a region of EID1, binds to p300/CBP, and acts as an inhibitor of p300/CBP-dependent transcription by direct interaction with nuclear receptors SHP and SRC1 [17]. EID is associated with a variety of tumors, and tumors expressing EID have strong invasiveness and poor prognosis. As a member of EID family, EID3 can inhibit histone acetyl transfer of CBP/p300 enzyme activity, and different from EID1, EID3 is specifically highly expressed in the testis [18]. In general, EID3 degrades rapidly through ubiquitin-dependent protein degradation pathway at the end of the cell cycle [19,20], but the inactivated pRb protein mutation will lead to the stability of EID3 protein, resulting in the inhibition of cell differentiation [17,21]. In fact, it has been reported that colon cancer cells with high expression of EID3 are more resistant to radiotherapy and chemotherapy and promote the formation of tumor stem cells [22]. In human umbilical cord blood mesenchymal stem cells, EID3 is highly expressed, and EID3 expression decreases during induced differentiation into neural stem cells [23]. However, the role of EID3 in osteosarcoma has not been reported.
In this study, we investigated the expression and biological function of EID3 in osteosarcoma cells. We demon-strated that the mRNA and protein levels of EID3 significantly increased in osteosarcoma cells. We found that osteosarcoma cells overexpressing EID3 generated more osteospheres and promoted cell invasion and had high expression of Sox2 and the stem cell marker CD133. Furthermore, RNA sequencing and bioinformatics analysis revealed that EID3 regulated stemness by interacting with PI3K-Akt signaling pathway.

Materials and Methods
2.1. Cell Culture. The human fetal osteoblast cell line hFOB and the human osteosarcoma cell lines MG-63 and U-2 OS were purchased from the American Type Cell Culture Collection (ATCC, USA) and cultured in DMEM-F12 (Gibco, USA), DMEM (Gibco, USA), and McCoy's 5a MeMo (Gibco, USA) plus 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS), respectively. The human osteosarcoma cell line MNNG/HOS cells were purchased from the Cell Bank of the China Science Academy (Shanghai, China) and cultured in RPMI 1640 plus 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% FBS. The hMSCs were kindly provided by Dr. Caixia Wang (Guangzhou First People's Hospital, Guangzhou, China) and cultured in conditioned medium composed of DMEM, 1 mmol/L L-glutamine (Gibco Laboratories, USA), 1% penicillin-streptomycin (Invitrogen, USA), and 10% FBS. All cells were maintained at 37°C with 5% CO 2 and 100% humidity except that the hFOB cells were maintained at 34°C.

Quantitative
Real-Time PCR. Total RNA was extracted using the RNeasy Plus Mini Kit, and the concentration and purity was determined using an ND-1000 spectrophotometer as previously reported [24]. Total RNA was prepared and detected. The primers are shown in Table 1.

Western
Blotting. Total cellular proteins were extracted with protein lysis buffer. Lysates were centrifuged at 10,000g at 4°C for 10 min, and supernatants were collected. The concentrations of proteins were detected by BCA Protein Assay Reagent Kit (Thermo, USA). Cell lysates containing 40 μg protein were separated on a 12% SDS-PAGE gel (Bio-rad, USA) and then transferred on polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h; incubated with primary antibodies for EID3, SOX2, and GAPDH; and then washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Finally, membranes were developed using an enhanced chemiluminescent (ECL) kit. Quantification of bands was performed using ImageJ Software. 2.7. Transwell Assay. Cell invasion was evaluated by the Matrigel invasion assay with a Corning Invasion Chamber (8 μm pore size) (Corning, USA) according to the manufacturer's instructions. 1 × 10 4 cells were seeded into the upper chamber of each well in serum-free medium, and the bottom chambers were filled with DMEM containing 10% FBS as chemoattractant. Cells were seeded in 10 mm diameter transwell plates with polycarbonate filters. After incubation for 24 h, the noninvading cells were gently removed with a cotton swab. Invasive cells were fixed for 30 min in 4% formaldehyde and stained for 15 min with crystal violet, air dried, and photographed. The number of invading cells was counted in five evenly spaced fields using an inverted phase-contrast microscope.
2.8. Wound Healing Assay. Cell migration was assessed by wound healing assay. In brief, cells were seeded in six-well plates in DMEM supplemented with 10% FBS. A scratch was created using a 200 μL tip and washed twice with serum-free medium. The migration was measured at 0 h and 24 h. Three images were taken per well, and data were analyzed using ImageJ software.

EID3 Is Highly Expressed in Osteosarcoma Cells.
We examined EID3 expression in three human osteosarcoma cell lines and found that EID3 protein was overexpressed in all three osteosarcoma cell lines compared to primary human osteoblasts cell line hFOB. As shown in Figure 1(a), we observed that the expression levels of EID3 in osteosarcoma MG-63 cells was higher than that of other osteosarcoma cells, including U2OS and HOS cells. We further determined the expression levels of EID3 in spherecultured and monolayer-cultured MG-63 cells [25,26]. We found that EID3 expression was higher in the sarcospheres than in adherent cells (Figures 1(b) and 1(c)). In addition, 7 Oxidative Medicine and Cellular Longevity EID3 expression was higher in bone mesenchymal stem cells (BMSCs) than in other osteosarcoma cells (Figure 1(d)).

Discussion
Osteosarcoma is the most commonly diagnosed primary malignant bone tumor, with a peak in incidence occurring in the second decade of life. OSCs play important role in osteosarcoma. It is well recognized that EID3 represses transcription and inhibits cell differentiation [1]. In human umbilical cord blood mesenchymal stem cells, EID3 is highly expressed, and EID3 expression decreases during the induced differentiation into neural stem cells [23]. Recent studies have focused on the role of EID3 in tumorigenesis. It has been reported that colon cancer cells with high expression of EID3 participate in the inhibition of differentiation of colon cancer cells and the formation of tumor stem cells [22]. Our results showed that EID3 was highly expressed 14 Oxidative Medicine and Cellular Longevity cells were identified in three osteosarcoma cell lines (Saos2, MG63, and U2OS). These results suggest that EID3 may be related to the stemness of osteosarcoma cells. Further in vivo studies based on animal models are needed to confirm the role of EID3 in the maintenance of the stemness of osteosarcoma cells.
To explore the role of EID3 in osteosarcoma, we overexpressed or deleted the expression of EID3 in osteosarcoma cells and proved that EID3 played an important role in maintaining the stemness of osteosarcoma cells based on sphere-forming assay, chemoresistance, and cell migration and invasion assay. EID3 overexpression not only improved stem cell phenotype but also enhanced the enrichment of CD133 + cells and the expression of stem cell-related markers OCT3/4, ABCG2, and NANOG in osteosarcoma cells [14].
Next, we used transcriptome sequencing to explore the mechanism by which EID3 regulates the stemness of osteosarcoma cells. The results showed that DEGs were mainly involved in PI3K-Akt signaling pathway, MAPK signaling pathway, cytokine-cytokine receptor interaction, and focal adhesion. Moreover, we found that overexpression of EID3 can lead to high expression of GRB2, PDGFRA, MYC, VEGFA, and VEGFC gene. EID3 interacts with CBP and p300 to inhibit gene transcription and cell differentiation in part via the inhibition of histone acetyltransferase (HAT) activity of p300. Whether EID3 maintains the stemness of osteosarcoma cells by upregulating the expression of GRB2 and activating PI3K-AKT pathway remains to be further explored.

Conclusions
In conclusion, our study demonstrates high expression of EID3 in osteosarcoma cells, especially in sphere-cultured osteosarcoma cells. EID3-overexpressing MG-63 cells exhibited significantly higher sphere-forming activity and higher levels of GRB2, PDGFRA, MYC, and VEGFA. These findings reveal the mechanism by which EID3 promotes the stemness of osteosarcoma cells and chemoresistance and provides new approach for targeted therapy for osteosarcoma patients.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
All the authors declare that there is no conflict of interest regarding the publication of this paper.

Authors' Contributions
Yan Wang, Shiyong Luo, and Yuxuan Wang contributed equally to this work.