SHP2 promotes osteosarcoma via regulating STAT3/TET3/HOXB2 signaling

Abstract

To investigate the effect of SHP2 on the STAT3/TET3/HOXB2 signaling pathway in osteosarcoma, and its role in the proliferation, migration and invasion of osteosarcoma cells. First, bioinformatics analysis was used to identify relevant expressed genes. In vitro experiments, the expression levels of SHP2, p-STAT3, TET3, HOXB2, c-Myc, NANOG, NUSAP1 proteins in 143B cells and MG63 cells were detected by Western blot assay. The levels of TET3 and HOXB2 were detected by immunofluorescence double staining. Cell proliferation was detected by plate clone formation and CCK-8 assay. Cell migration was detected by scratch assay, and cell migration and invasion were detected by Transwell assay. Overexpression of SHP2 promotes osteosarcoma proliferation by upregulating STAT3, TET3 and HOXB2 proteins. VEGF activates RTK receptors and induces SHP2 autophosphorylation, which in turn activates STAT3 and enhances TET3 synthesis. TET3 then promotes HOXB2 transcription through demethylation. HOXB2 further upregulate the expression of c-Myc, NANOG and NUSAP1, ultimately driving the proliferation, migration and invasion of osteosarcoma and promoting tumor progression. This study confirmed that SHP2 promotes the proliferation, invasion and invasive ability of osteosarcoma cells by activating the STAT3/TET3/HOXB2 pathway, providing a new strategy for targeted treatment of osteosarcoma.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-35493-7.

Introduction

Osteosarcoma (OS), the most prevalent primary malignant bone tumor in adolescents, is characterized by high aggressiveness and early metastasis tendency. It has a poor prognosis and a high risk of disability and death due to its high aggressiveness, early metastasis, and medication resistance^1–3. Nowadays, the 5-year survival rate has increased to 60%–70% because to a comprehensive treatment plan that combines neoadjuvant chemotherapy with surgical resection. But throughout the last 30 years, this growth has come to a standstill. The dearth of effective therapeutic treatments has resulted in a very dismal prognosis, with the 5-year survival rate falling to around 20%, especially for patients with initial lung metastases or post-treatment recurrence^4,5. The molecular regulatory mechanisms of osteosarcoma have been the subject of more recent research, with the SHP2 signaling pathway emerging as a crucial field of study.

The PTPN11 gene encodes Shp2 (Src homology-2 domain-containing phosphatase 2), a member of the non-receptor protein tyrosine phosphatase (nRPTP) family. It is a widely distributed phosphatase in cells that regulates target protein phosphorylation in conjunction with protein kinases. It is essential for tissue development, cell division, apoptosis, and proliferation⁶. However, little is known about the exact role and underlying molecular processes of SHP2 in osteosarcoma, which limits the development of targeted therapeutics and prevents a thorough understanding of the disease’s molecular structure.

A crucial modulator of angiogenesis in both healthy and diseased states is vascular endothelial growth factor (VEGF). The majority of malignant tumors and the microenvironments that surround them, such as the stroma and tumor-infiltrating immune cells, often exhibit overexpression of VEGF⁷. Through its binding to receptor tyrosine kinases (RTKs), VEGF triggers downstream signaling pathways that increase the invasiveness, migration, and proliferation of tumor cells⁸. Studies have shown that RTK-driven cancer cell survival depends on SHP2⁹. Xu Z et al. conducted research indicating that SHP2 is activated in tumor endothelium cells. In several animal tumor models, SHP2 deletion or pharmacological suppression decreases tumor growth and microvessel density. Furthermore, tumor vascular normalization results from SHP2 lack¹⁰. As a downstream protein in nearly all RTK signaling pathways, SHP2 contributes to tumor proliferation, invasion, metastasis, and chemotherapy resistance in various RTK-driven cancers^11–14. Targeting SHP2, a key downstream protein in multiple RTKs, may represent a potential therapeutic strategy for tumor suppression¹⁵. Studies have shown that several RTK inhibitors used in clinical treatment suppress the phosphorylation of SHP2’s Tyr542 and Tyr580 residues^9,13, thereby inhibiting tumor cell growth^16,17. Therefore, inhibiting the VEGF-RTK pathway could effectively suppress the development and progression of osteosarcoma.

One important downstream effector of SHP2, signal transducer and activator of transcription 3 (STAT3), has several functions in carcinogenesis¹⁸. STAT3 not only regulates tumor cell proliferation and survival but also promotes immune escape by influencing immune cell functions in the tumor microenvironment¹⁹. Continuous activation of STAT3 results in the aberrant expression of multiple pro-tumor genes, such as c-Myc and NANOG, which are essential transcription factors for sustaining cancer stem cell characteristics and facilitating tumor advancement^20,21. According to recent research, STAT3 may affect gene expression patterns via epigenetic control mechanisms, offering fresh perspectives on the plasticity and heterogeneity of tumors. Nevertheless, little is known about the precise processes by which STAT3 contributes to the development of SHP2-mediated osteosarcoma. TET (ten-eleven translocation) methylcytosine dioxygenase 3 (TET3), a crucial enzyme in DNA demethylation, plays a key role in maintaining DNA methylation/demethylation balance, which promotes tumor development and abnormal biological differentiation. TET proteins bind to target genes via their N-terminal domains and catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) through their C-terminal CD domains, regulating gene expression, X chromosome inactivation, genomic imprinting, and cancer development²². Although TET3 is expressed in various cancers, its role in osteosarcoma remains controversial.

Nucleolar spindle associated protein 1 (NUSAP1) is a vascular binding protein with a molecular mass of 55kD. It is located in the chromosome arm and regulates spindle assembly and the maintenance of normal mitosis by binding to microtubule and DNA domains²³. NUSAP1 contributes to cell cycle progression and mitotic fidelity, potentially fostering tumorigenesis by compromising genomic stability, facilitating chromatin reorganization, and modulating signal transduction pathways. Based on this, we propose the core hypothesis that SHP2 promotes the proliferation, invasion and invasion ability of osteosarcoma cells by activating STAT3/TET3/HOXB2 pathway.

Materials and methods

Bioinformatics analysis

The osteosarcoma dataset GSE16088 (comprising 6 normal controls and 14 tumor samples) was obtained from the GEO database. After background correction, normalization, and probe merging using the Robust Multi-array Average (RMA) algorithm, the original data underwent batch effect correction via the Combat method. Differential expression genes were screened from the corrected data using the limma package with a threshold of |log2 Fold Change|> 2 and p-value < 0.05. The identified DEGs from both datasets were analyzed using Venny 2.1 to extract the intersection of up-regulated and down-regulated genes. Gene Ontology (GO) analysis (covering cell components CC, molecular functions MF, and biological processes BP) and KEGG pathway enrichment analysis were performed on the intersecting genes using the ClusterProfiler package to reveal key biological functions and signaling pathways. Spearman correlation analysis was employed to validate multi-group molecular associations. Finally, the ggplot2 package was used to construct visualizations such as volcano plots and heatmaps to systematically display the distribution characteristics of differentially expressed genes.

Animals and cells

Twenty-four male BALB/c-nu mice, aged 4–6 weeks and weighing 17–19 g, were purchased from Henan Sikebeisi Biological Company (Production License No.: SCXK (Yu) 2020-0007). The mice were housed in an SPF-grade animal facility at 20–25℃ with a relative humidity of 50%-60%, with free access to food and water. This study was approved by the Animal Experiment Ethics Committee. The human osteosarcoma cell line 143B and the human osteosarcoma cell line MG63 were purchased from Hunan Fenghui Biotechnology Co., Ltd.

Experimental materials

Fetal bovine serum (FBS) was purchased from Global Kang Qinhuangdao Biotechnology Co., Ltd.; DMEM basal medium, penicillin-streptomycin solution (double antibiotics), phosphate-buffered saline (PBS), and 0.05% trypsin were obtained from Wuhan Procell Life Science & Technology Co., Ltd.; Matrigel was sourced from Corning, USA; Transwell chambers, dimethyl sulfoxide (DMSO), and SDS-PAGE protein loading buffer were acquired from Shanghai Beyotime Biotechnology Co., Ltd.; High-efficiency RIPA lysis buffer and protease phosphatase inhibitors were purchased from Beijing Solarbio Science & Technology Co., Ltd.; The BCA protein concentration assay kit was obtained from GLPBIO, USA; The one-step PAGE quick preparation kit, SDS-PAGE rapid electrophoresis buffer, rapid transfer buffer, high-efficiency Western blocking buffer, and universal antibody diluent were procured from Shanghai Jifu Life Science Co., Ltd.; PVDF membrane was sourced from Merck, Germany; p-STAT3 antibody, nuclear TET3 antibody, nuclear HOXB2 antibody, c-Myc antibody, NANOG antibody, and NUSAP1 antibody were purchased from Jiangsu Qinke Biological Research Center Co., Ltd.; Goat anti-rabbit IgG (H + L) HRP antibody was obtained from Wuhan Sanying Biotechnology Co., Ltd.; SHP2-OE, TET3-KD, SHP2-KD, and HOXB2-OE lentiviruses were purchased from Shanghai Jiman Biological Company; STAT3 agonist Colivelin Stattic and TET inhibitor TETi76 were purchased from MCE.

Tumor formation in nude mice

143B and MG63 cells were grown in DMEM medium supplemented with 10% FBS and incubated at 37℃ with 5% CO2. The state of cell growth was observed, and the medium was routinely substituted. Upon achieving 70%-80% confluence, the cells were subjected to digestion with 0.25% trypsin and subsequently passaged at a ratio of 1:2 to 1:4. Logarithmically proliferating 143B cells were inoculated into 6-well plates at a concentration of 1 × 10^8 cells/L and incubated overnight at 37℃ with 5% CO2. Packaged SHP2-OE, TET3-KD, and HOXB2-OE lentiviruses were introduced for transfection in accordance with the manufacturer’s guidelines. Subsequent to mixing, the media was substituted with new DMEM supplemented with 10% FBS, and the cells were grown at 37℃ in an atmosphere of 5% CO2. Twenty-four BALB/c-nu nude mice were subcutaneously injected with 143B cells to create an osteosarcoma xenograft model. Transfected 143B cells were produced at a concentration of 1 × 10^7 cells/mL, and 0.1 mL of the cell suspension was administered into the right axilla of each mouse. Mice were weighed, and tumor volumes were assessed every five days. The tumor volume was determined using the formula 0.52×a×b^2, where ‘a’ represents the long diameter and ‘b’ denotes the short diameter. After 25 days, the mice were euthanized using CO2, and the tumor tissues were excised, washed with pre-chilled saline, and weighed. All procedures in this experiment adhered to applicable guidelines and regulations, complying with the 3R principles and ARRIVE recommendations.

Cell culture

The lentiviral vector pLVX-Puro for SHP2-KD was constructed, and the SHP2-KD lentivirus (5’-AGTTACATTGCCACTCAAGGCTGC-3’) was designed by Shanghai Hanheng Biotechnology Co., Ltd. The 143B and MG63 cells (from Wuhan Pnose Life Science Co., Ltd.) were transfected using Lipofectamine 3000. After 48 h of infection, the culture supernatant was collected, cell debris was removed by centrifugation, and a viral concentration kit (Jiangsu Feitian Biotechnology Co., Ltd., product code: C2901M) was used. The 143B and MG63 cells were seeded in 6-well plates and cultured until achieving 60%-70% confluence. The cells were then infected with 40µL of viral titer and 400µL of medium containing 8 µg/ml Polybrene for 24 h. Finally, the cells were screened for 7 days using 2 µg/mL Puromycin.

Human osteosarcoma cell lines 143B and MG63 were categorized into the following groups after different treatments: NC group, SHP2-KD group, SHP2-KD + TFA group, SHP2-KD + TFA + TETi76 group, SHP2-KD + TFA + TETi76 + HOXB2-OE group, SHP2-OE group, SHP2-OE + Stattic group, SHP2-OE + Stattic + TET3-OE group, and SHP2-OE + Stattic + TET3-OE + HOXB2-KD group. The STAT3 agonist Colivelin TFA (TFA) was used at 50 µg/mL, the STAT3 inhibitor Stattic at 10 µM, and the TET inhibitor TETi76 at 20 µM.

Western blot

Protein was recovered from 143B and MG63 osteosarcoma cells in each group utilizing RIPA lysis buffer, which contained PMSF and phosphatase inhibitors. Following centrifugation at 4℃ and 12,000×g for 10 min, the supernatant was obtained, and protein content was assessed with the BCA kit. Samples were combined with 5×SDS-PAGE loading buffer and denatured at 95℃ for 10 min. Proteins were fractionated using 10% SDS-PAGE and subsequently transferred to a PVDF membrane. The membrane was treated with 5% skim milk at room temperature for 1 h and thereafter exposed to primary antibodies (p-STAT3, nuclear TET3, nuclear HOXB2, c-Myc, NANOG, and NUSAP1) at a dilution of 1:1000 at 4℃ overnight. Following a TBST wash (3 × 10 min), the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (1:5000) at ambient temperature for 1 h. Following washing, protein bands were identified via ECL and seen on a chemiluminescence imager. Gray analysis was conducted utilizing ImageJ software. GAPDH used as the internal reference protein for normalization purposes.

Colony formation assay

Logarithmically proliferating 143B and MG63 cells were produced as a cell suspension and inoculated into 6-well plates at a density of 1.5 × 10^3 cells/mL. Post-treatment, cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min, then stained with 0.1% crystal violet for 1 h. Following washing, colonies were enumerated under a microscope, and the colony formation rate was computed as (average colony count / number of implanted cells) x 100%.

CCK-8 assay

Logarithmically growing 143B cells and MG63 cells were seeded into 96-well plates at a density of 1 × 10^4 cells/mL and treated as described in Sect. “Cell culture ”. After washing with PBS, 100 µL of CCK-8 reagent (10% in medium) was added to each well, and cells were incubated at 37℃ in the dark for 1 h. Absorbance (A) was measured at 490 nm using a microplate reader, and cell proliferation was calculated.

Transwell invasion assay

Logarithmically proliferating 143B and MG63 cells were produced at a concentration of 1 × 10^5 cells/mL. Matrigel was diluted at a ratio of 1:5 with DMEM basal media, and 60 µL was dispensed into each Transwell chamber. Following a 30-minute incubation at 37℃, 200 µL of cell suspension was introduced into the upper chamber, while 600 µL of full DMEM media supplemented with serum was added to the lower chamber. After 48 h of cell culture, the upper chamber was removed and the cells were fixed with 4% paraformaldehyde for 30 min. The cells were stained with 0.1% crystal violet. Finally, the cells adhered in the upper chamber were wiped off with cotton swabs and the migration of cells was recorded by microscope.

Cellular scratch assay

143B and MG63 cells were uniformly seeded at 5 × 10⁵ cells per well in a 6-well plate. After 14 days of culture, the medium was removed and residual cells were washed with PBS. The cells were then fixed at room temperature with 4% paraformaldehyde for 15 min, followed by three PBS washes. A 0.1% crystal violet solution was added for 1-hour staining at room temperature. After washing away excess stain, the cells were dried and photographed.

Statistical evaluation

The data were analyzed by GraphPad Prism 8.0 software. The mean ± standard deviation was used to express the quantitative data. One-way ANOVA was used to compare the data between groups, and Bonferroni test was used to compare the data between groups. P < 0.05 was considered as statistically significant.

Results

Bioinformatics analysis results

Box plots were used to first evaluate the distribution of the raw data before the data were normalized. The expression profile data’s dimensionality was subsequently decreased using Principal Component Analysis (PCA). Significant sample heterogeneity was shown using scatter plots. 1,347 substantially differentially expressed genes (DEGs), including 409 downregulated and 938 upregulated genes, were found using the limma method. Multilevel functional annotation was done out utilizing the ClusterProfiler package: Cellular components like collagen-containing extracellular matrix, focal adhesion, and stress fibers; molecular functions like collagen binding, transmembrane receptor protein tyrosine kinase activity, and MAP kinase activity; and biological processes like epithelial cell proliferation, mesenchymal cell differentiation, and epithelial-to-mesenchymal transition were all significantly enriched in the differentially expressed genes, according to Gene Ontology analysis. KEGG pathway analysis revealed that DEGs were significantly enriched in the PI3K-Akt signaling pathway, MAPK signaling pathway, JAK-STAT signaling pathway, TGF-beta signaling pathway, and ECM-receptor interaction. The crucial role that these routes play in the development of osteosarcoma was highlighted by the integrated representation of bubble plots and bar charts. A framework of important molecular interactions was mapped out by Spearman correlation network analysis: the tyrosine phosphatase SHP2 showed a moderately positive correlation with the transcription factor STAT3 (r = 0.477), which was significantly associated with the epigenetic regulator TET3 (r = 0.539), and TET3 showed strong co-expression with the homeobox gene HOXB2 (r = 0.714). Additionally, HOXB2 and the nuclear proliferation marker NUSAP1 formed a radial regulatory network (r = 0.314). Downstream validation supported the biological significance of this network: c-Myc exhibited strong co-expression with the cell proliferation marker Ki-67 (r = 0.626) and NANOG was highly correlated with the EMT driver Snail (r = 0.67), indicating that HOXB2 may promote tumor progression by activating the stemness-EMT-cell cycle triad. Importantly, all significant correlations in this molecular interaction network were highly consistent in the independent validation cohort GSE87437, confirming the universality and reproducibility of this regulatory framework in osteosarcoma biology (Fig. 1).

Fig. 1.

Bioinformatics Analysis of SHP2 Promoting Osteosarcoma by Regulating STAT3/TET3/HOXB2 Signal Pathway. (A) Box plot of standardized sample expression data; (B) Scatter plot of sample clustering by principal component analysis; (C) Volcano plot for screening differentially expressed genes; (D) Heatmap/sorting map of clustering of differentially expressed gene profiles; (E) Bubble chart of multi-level functional enrichment analysis of gene ontology; (F) Bubble chart of KEGG signaling pathway enrichment analysis; (G) Lollipop chart of KEGG signaling pathway enrichment analysis; (H) Heatmap of molecular interaction network correlation in GSE16088; (I) Heatmap of independent cohort validation in GSE87437.

SHP2 inhibited the progression of osteosarcoma by suppressing the expression of STAT3, TET3 and HOXB2 proteins

To clarify the molecular mechanism by which SHP2 knockdown influences STAT3/TET3/HOXB2 proteins, we executed Western blot analysis to assess the protein expression levels of p-SHP2, p-STAT3, nuclear TET3, and nuclear HOXB2 in 143B and MG63 cells, and performed immunofluorescence staining to evaluate TET3 and HOXB2 protein expression. The Western blot results indicated that, in comparison to the NC group, the expression levels of SHP2, p-STAT3, nuclear TET3, and nuclear HOXB2 were diminished in the SHP2-KD group. In comparison to the SHP2-KD group, the SHP2-KD + TFA group exhibited no significant variation in SHP2 protein expression, whereas all other proteins, save SHP2, were markedly elevated. In comparison to the SHP2-KD + TFA group, the SHP2-KD + TFA + TETi76 group exhibited no significant variations in SHP2 and p-STAT3 protein expression levels; nevertheless, nuclear expressions of TET3 and HOXB2 were dramatically reduced. In comparison to the SHP2-KD + TFA + TETi76 group, the SHP2-KD + TFA + TETi76 + HOXB2-OE group exhibited no significant variations in the expression levels of SHP2, p-STAT3, and nuclear TET3 proteins; nevertheless, nuclear HOXB2 expression was markedly elevated (Fig. 2A).

Fig. 2.

SHP2-KD inhibits the progression of osteosarcoma by suppressing the expression of STAT3, TET3 and HOXB2 proteins. (A) Western blot analysis of SHP2, p-STAT3, nuclear TET3, and nuclear HOXB2 protein expression in each group. The blot results show the protein expression patterns in tumor tissues, and the statistical graphs display the relative expression levels. Groups: NC, SHP2-KD, SHP2-KD + TFA, SHP2-KD + TFA + TETi76, and SHP2-D + TFA + TETi76 + HOXB2-OE. (B) Immunofluorescence images and statistical graph of HOXB2 fluorescence intensity in each group. Groups: NC, SHP2-KD, SHP2-KD + TFA, SHP2-KD + TFA + TETi76, and SHP2-KD + TFA + TETi76 + HOXB2-OE. Data are presented as mean ± standard deviation. N = 7; P < 0.05 indicates a statistically significant difference; ^ns P > 0.05, *P < 0.05, **P < 0.01.

Immunofluorescence examination demonstrated that, in comparison to the NC group, the fluorescence intensities of TET3 and HOXB2 were diminished in the SHP2-KD group. In comparison to the SHP2-KD group, the SHP2-KD + TFA group exhibited elevated fluorescence intensities of TET3 and HOXB2. In comparison to the SHP2-KD + TFA group, the SHP2-KD + TFA + TETi76 group had reduced fluorescence intensities of TET3 and HOXB2. In contrast to the SHP2-KD + TFA + TETi76 group, the SHP2-KD + TFA + TETi76 + HOXB2-OE group exhibited elevated fluorescence intensities of TET3 and HOXB2 (Fig. 2B). The results indicated that SHP2 knockdown inhibited osteosarcoma growth by reducing the expression of STAT3, TET3, and HOXB2 proteins.

SHP2 downregulated the expression of c-Myc/NANOG/NUSAP1 proteins, thereby inhibiting osteosarcoma proliferation

To investigate the mechanism by which SHP2 knockdown regulates c-Myc/NANOG/NUSAP1 in the progression of osteosarcoma, we performed Western blot analysis to detect the protein expression levels of c-Myc, NANOG, and NUSAP1 in 143B and MG63 cells, and conducted CCK-8 assays to evaluate cell proliferation ability. Compared with the NC group, the protein expression levels of c-Myc, NANOG, and NUSAP1 were significantly decreased in the SHP2-KD group. Compared with the SHP2-KD group, the protein expression levels of c-Myc, NANOG, and NUSAP1 were significantly increased in the SHP2-KD + TFA group. Compared with the SHP2-KD + TFA group, the protein expression levels of c-Myc, NANOG, and NUSAP1 were significantly decreased in the SHP2-KD + TFA + TETi76 group. Compared with the SHP2-KD + TFA + TETi76 group, the protein expression levels of c-Myc, NANOG, and NUSAP1 were significantly increased in the SHP2-KD + TFA + TETi76 + HOXB2-OE group (Fig. 3A).

Fig. 3.

SHP2-KD the expression of c-Myc/NANOG/NUSAP1 proteins, thereby inhibiting osteosarcoma proliferation. (A) Western blot analysis of protein expression bands in 143B and MG63 cells, with statistical graphs showing relative protein expression levels. Groups: NC, SHP2-KD, SHP2-KD + TFA, SHP2-KD + TFA + TETi76, and SHP2-KD + TFA + TETi76 + HOXB2-OE. (B) Colony formation assay results assessing the proliferative capacity of 143B and MG63 cells, with statistical analysis graphs of colony numbers. (C) CCK-8 assay results evaluating the proliferative capacity of 143B and MG63 cells, with statistical analysis graphs of proliferating cell counts. Data are presented as mean ± standard deviation. n = 3; P < 0.05 was considered statistically significant; ^nsP>0.05, *P < 0.05, **P < 0.01.

The colony formation assay results showed that compared with the NC group, SHP2-KD significantly reduced proliferation ability. Compared with the SHP2-KD group, the SHP2-KD + TFA group exhibited increased proliferation ability. Compared with the SHP2-KD + TFA group, the SHP2-KD + TFA + TETi76 group showed reduced proliferation ability. Compared with the SHP2-KD + TFA + TETi76 group, the SHP2-KD + TFA + TETi76 + HOXB2-OE group significantly increased proliferation ability (Fig. 3B).

The CCK-8 assay results showed that compared with the NC group, SHP2-KD significantly reduced proliferation ability. Compared with the SHP2-KD group, the SHP2-KD + TFA group exhibited increased proliferation ability. Compared with the SHP2-KD + TFA group, the SHP2-KD + TFA + TETi76 group showed reduced proliferation ability. Compared with the SHP2-KD + TFA + TETi76 group, the SHP2-KD + TFA + TETi76 + HOXB2-OE group significantly increased proliferation ability (Fig. 3C). These results indicated that knockdown of SHP2 inhibited the proliferation of osteosarcoma by regulating the expression of c-Myc/NANOG/NUSAP1 proteins.

SHP2 inhibited the migration and invasion of osteosarcoma

To examine the mechanism by which SHP2 knockdown influences osteosarcoma progression, we performed scratch tests to evaluate cell migratory capacity and Transwell experiments to measure cell invasive potential. The scratch assay results indicated that, in comparison to the NC group, the scratch breadth in the SHP2-KD group was markedly diminished. In comparison to the SHP2-KD group, the scratch breadth in the SHP2-KD + TFA group was markedly enlarged. In comparison to the SHP2-KD + TFA group, the scratch width in the SHP2-KD + TFA + TETi76 group was markedly diminished. In comparison to the SHP2-KD + TFA + TETi76 group, the scratch width in the SHP2-KD + TFA + TETi76 + HOXB2-OE group was markedly augmented (Fig. 4A).

Fig. 4.

SHP2-KD can inhibit the migration and invasion of osteosarcoma. (A) Wound healing assay results evaluating the migration ability of 143B and MG63 cells, with statistical analysis graphs of the scratch width. (B) Transwell assay results assessing the migration and invasion abilities of 143B and MG63 cells, with statistical analysis graphs of the number of migrating and invading cells. Data are presented as mean ± standard deviation. N = 3; P < 0.05 was considered statistically significant; ^nsP > 0.05, *P < 0.05, **P < 0.01.

The Transwell assay results indicated that, in comparison to the NC group, the quantity of invading cells in the SHP2-KD group was markedly diminished. In comparison to the SHP2-KD group, the quantity of invading cells in the SHP2-KD + TFA group was markedly elevated. In comparison to the SHP2-KD + TFA group, the quantity of invading cells in the SHP2-KD + TFA + TETi76 group was markedly diminished. In comparison to the SHP2-KD + TFA + TETi76 group, the SHP2-KD + TFA + TETi76 + HOXB2-OE group exhibited a considerable increase in the number of invading cells (Fig. 4B). The results demonstrated that SHP2 knockdown impeded the migration and invasion of osteosarcoma.

SHP2 overexpression promoted osteosarcoma proliferation, migration, and invasion by regulating the STAT3/TET3/HOXB2 and c-Myc/NANOG/NUSAP1 signaling pathways

To elucidate the mechanism by which SHP2 modulates the STAT3/TET3/HOXB2 and c-Myc/NANOG/NUSAP1 pathways in osteosarcoma progression, we executed Western blot analysis to assess the protein expression levels of c-Myc, NANOG, and NUSAP1 in 143B and MG63 cells, and performed CCK-8 assays to evaluate cellular proliferation capacity. The Western blot results indicated that, in comparison to the NC group, the protein expression levels of SHP2, p-STAT3, HOXB2, TET3, c-Myc, NANOG, and NUSAP1 were markedly elevated in the SHP2-OE group. In comparison to the SHP2-OE group, the protein expression levels of p-STAT3, HOXB2, TET3, c-Myc, NANOG, and NUSAP1 were markedly reduced in the SHP2-OE + Stattic group, although the SHP2 protein level remained unchanged. In comparison to the SHP2-OE + Stattic group, the protein expression levels of TET3, HOXB2, c-Myc, NANOG, and NUSAP1 were markedly elevated in the SHP2-OE + Stattic + TET3-OE group, although the protein levels of SHP2 and p-STAT3 exhibited no significant alterations. While the protein levels of SHP2, p-STAT3, and TET3 did not significantly change, the protein expression levels of HOXB2, c-Myc, NANOG, and NUSAP1 were significantly reduced in the SHP2-OE + Stattic + TET3-OE + HOXB2-KD group compared to the SHP2-OE + Stattic + TET3-OE group (Fig. 5A).

Fig. 5.

SHP2 overexpression promotes osteosarcoma proliferation by regulating the STAT3/TET3/HOXB2 and c-Myc/NANOG/NUSAP1 signaling pathways. (A) Western blot analysis of protein expression bands in 143B and MG63 cells, with statistical graphs showing relative protein expression levels. Groups: NC, SHP2-OE, SHP2-OE + Stattic, SHP2-OE + Stattic + TET3-OE, and SHP2-OE + Stattic + TET3-OE + HOXB2-KD. (B) Colony formation assay results assessing the proliferative capacity of 143B and MG63 cells, with statistical analysis graphs of colony numbers. (C) CCK-8 assay results showing the proliferative capacity of 143B and MG63 cells, with statistical analysis of proliferating cell counts. Data are presented as mean ± standard deviation. N = 3; P < 0.05 was considered statistically significant; ^nsP > 0.05, *P < 0.05, **P < 0.01.

As shown by colony formation tests, SHP2-OE markedly enhanced proliferative capacity in comparison to the NC group. Proliferation was significantly lower in the SHP2-OE + Stattic group than in the SHP2-OE group. In contrast to the SHP2-OE + Stattic group, the SHP2-OE + Stattic + TET3-OE group demonstrated a rescue in proliferative capacity. However, this increase was greatly diminished in the SHP2-OE + Stattic + TET3-OE + HOXB2-KD group (Fig. 5B).

In a similar vein, the findings of the CCK-8 experiment showed that SHP2-OE significantly increased proliferation in comparison to the NC group. Treatment with Stattic decreased this effect, whereas TET3 overexpression restored it. Notably, HOXB2 knockdown later on resulted in a significant reduction in proliferative capacity once again (Fig. 5C). According to our results, SHP2 overexpression stimulates the growth of osteosarcoma via modifying the c-Myc/NANOG/NUSAP1 and STAT3/TET3/HOXB2 signaling axis.

Overexpression of SHP2 promoted migration and invasion in osteosarcoma

We used scratch assays to measure cellular migration and Transwell assays to measure invasive capacity in order to explore the mechanism by which SHP2 overexpression affects osteosarcoma progression. As illustrated in Fig. 6A, the results of the Transwell assay demonstrated that the SHP2-OE group had a higher number of invading cells than the NC group. In contrast to the SHP2-OE group, the SHP2-OE + Stattic group showed a significant reduction in cell invasion. In addition, invasive cells were significantly higher in the SHP2-OE + Stattic + TET3-OE group than in the SHP2-OE + Stattic condition. Lastly, compared to the SHP2-OE + Stattic + TET3-OE group, invasion was considerably reduced in the SHP2-OE + Stattic + TET3-OE + HOXB2-KD group.

Fig. 6.

Overexpression of SHP2 promotes the migration and invasion of osteosarcoma. (A) Wound healing assay results demonstrating the migration ability of 143B and MG63 cells, with statistical analysis of scratch width. (B) Transwell assay results evaluating the migration and invasion abilities of 143B and MG63 cells, with statistical analysis of the number of migrating and invading cells. Data are presented as mean ± standard deviation. N = 3; P < 0.05 was considered statistically significant; ^nsP > 0.05, *P < 0.05, **P < 0.01.

The SHP2-OE group’s wound breadth was significantly greater than the NC group’s in the scratch test (Fig. 6B). The SHP2-OE + Stattic group, on the other hand, demonstrated a significant decrease in scratch breadth as compared to the SHP2-OE condition. When TET3 overexpression was added further (SHP2-OE + Stattic + TET3-OE), the scratch breadth was once again noticeably wider than in the SHP2-OE + Stattic group. In contrast to the SHP2-OE + Stattic + TET3-OE group, the following HOXB2 knockdown (SHP2-OE + Stattic + TET3-OE + HOXB2-KD) caused a significant narrowing of the scratch width. The findings showed that osteosarcoma migration and invasion were aided by SHP2 overexpression.

SHP2 overexpression promoted osteosarcoma progression by upregulating STAT3/TET3/HOXB2 protein expression

We performed subcutaneous tumor transplantation in nude mice to track tumor development in order to examine the impact of SHP2 overexpression on osteosarcoma and its molecular pathways. The protein expression of p-SHP2, p-STAT3, nuclear TET3, and nuclear HOXB2 in tumor tissues was evaluated by Western blot analysis. The results showed that the SHP2-OE group had significantly higher tumor weight and volume than the NC group. Tumor weight and volume were significantly lower in the SHP2-OE + TET3-KD group than in the SHP2-OE group. On the other hand, the tumor weight and volume significantly increased in the SHP2-OE + TET3-KD + HOXB2-OE group (Fig. 7A).

Fig. 7.

SHP2-OE promotes osteosarcoma progression by upregulating STAT3, TET3, and HOXB2 proteins. (A) Experimental results of nude mouse subcutaneous tumor transplantation, including statistical analysis charts for tumor weight and volume. (B) Western blot analysis of protein expression bands in tumor tissues, with statistical comparison charts for relative protein expression levels. Mouse groups: NC group, SHP2-OE group, SHP2-OE + TET3-KD group, SHP2-OE + TET3-KD + HOXB2-OE group. (C) SHP2 promotes malignant progression of osteosarcoma through the STAT3/TET3/HOXB2 signaling pathway. Data are presented as mean ± SD. N = 6, P < 0.05 indicates statistically significant difference, ^nsP> 0.05, *P < 0.05, **P < 0.01.

Western blot findings also showed that the SHP2-OE group expressed more p-SHP2, p-STAT3, nuclear TET3, and nuclear HOXB2 than the NC group did. The SHP2-OE + TET3-KD group exhibited reduced nuclear TET3 and nuclear HOXB2 expression along with a significant increase in p-SHP2 and p-STAT3 levels when compared to the SHP2-OE group. While nuclear TET3 expression was decreased, the SHP2-OE + TET3-KD + HOXB2-OE group showed elevated levels of p-SHP2, p-STAT3, and nuclear HOXB2 (Fig. 7B).

These findings implied that SHP2 overexpression promoted the growth of osteosarcoma by upregulating the proteins HOXB2, TET3, and STAT3. The development of osteosarcoma was significantly influenced by the SHP2/STAT3/TET3/HOXB2 signaling axis. By activating the RTK receptor, VEGF caused SHP2 autophosphorylation, which in turn activated STAT3 and increased the production of TET3. TET3 then used demethylation to stimulate HOXB2 transcription. The expression of c-Myc, NANOG, and NUSAP1 was further upregulated by HOXB2, creating a self-reinforcing loop that promoted tumor invasion, dissemination, and epithelial-mesenchymal transition (EMT) (Fig. 7C). The original membranes for this study were supplemented in Supplementary file 1. The original data for this study are were in Supplementary file 2.

Discussion

The most common primary malignant bone tumor in children and adolescents is osteosarcoma²⁴. According to studies, between 20,000 and 30,000 people worldwide get a diagnosis of this illness each year²⁵. The crucial function of SHP2 as a central signaling hub in the osteosarcoma microenvironment is methodically explained by this work. Through the regulation of the STAT3/TET3/HOXB2 pathway, SHP2 stimulates the growth and invasion of tumors. The results demonstrate the pivotal role of SHP2 in the regulatory network, offering a fresh theoretical perspective on the malignant development of osteosarcoma and perhaps paving the way for targeted treatments.

Tumor development, infiltration of adjacent tissues, and distant metastasis are all dependent on tumor angiogenesis. Vascular endothelial cell activation is essential to this process²⁶. In osteosarcoma, vascular endothelial growth factor (VEGF), which promotes angiogenesis, is markedly overexpressed. VEGF promotes tumor cell invasion, migration, and proliferation by binding to receptor tyrosine kinases (RTKs) and activating downstream signaling pathways¹³. SHP2 is one of the downstream signaling molecules that are recruited and activated by the VEGF-RTK interaction, which also causes receptor dimerization and autophosphorylation.

A protein tyrosine phosphatase of the non-receptor type, SHP2 is essential to many cellular signaling pathways. In order to further control the activity of downstream signaling molecules, SHP2’s N-terminal SH2 domain self-phosphorylates after binding to phosphorylated tyrosine residues upon recruitment by RTKs. SHP2 is a proto-oncogene that is important for tumor invasion, growth, metastasis, and death in a number of malignancies²⁷. According to studies, tumor cell growth is decreased when SHP2 activity is inhibited²⁸. Experiments on subcutaneous tumor growth in naked mice showed that overexpression of SHP2 significantly enhanced the volume of tumors. SHP2 overexpression improves the proliferative ability of 143B and MG63 cells, suggesting its function in promoting osteosarcoma development, according to monoclonal proliferation studies and CCK-8 assays. Cell scratch tests and Transwell studies in this work showed that SHP2 overexpression increases 143B and MG63 cells’ capacity to migrate and invade, validating its function in promoting osteosarcoma metastasis.

One of the main routes linked to cytokine activation is the STAT3 signaling pathway, which is a proto-oncogene. By either directly interacting with STAT3 or dephosphorylating JAK2, SHP2 stimulates STAT3 phosphorylation. Changes in the angiogenesis and proliferation capacities of cancer cells are essential for their development and metastasis, and the main cause of these alterations is persistent STAT3 activation. STAT3 transcriptional activity has been demonstrated to be positively linked with tumor aggressiveness in a number of human cancers, including ovarian, lung, esophageal, breast, and prostate cancers^29–33. Due to its high expression in clinical osteosarcoma tissues, STAT3 may be used as a molecular therapeutic target and biomarker for early detection. In vitro and in vivo, downregulation of the STAT3 signaling pathway may cause tumor cells to undergo apoptosis, prevent proliferation, and lower the incidence of metastases. Therefore, one of the most important strategies for slowing the malignant evolution of osteosarcoma is to reduce STAT3 expression levels and downstream transcriptional activation.

One important enzyme in DNA demethylation is TET (ten-eleven translocation) methylcytosine dioxygenase 3 (TET3). It facilitates DNA demethylation and controls gene expression by catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine^34,35.

Chromosome 17 contains the HOXB2 gene (Homeobox B2), which is a member of the HOX family. A transcription factor that is essential for controlling cellular morphogenesis and differentiation is encoded by HOXB2³⁶. t is essential for both physiological and pathological processes, including carcinogenesis, cell differentiation, and embryonic development^37,38. Through demethylation, TET3 stimulates the transcription of HOXB2. Research has shown that increasing HOXB2 expression increases tumor invasion and metastasis in osteosarcoma³⁹.

Research shows that via upregulating c-Myc, NANOG, and NUSAP1, HOXB2 stimulates tumor cell motility, invasion, and proliferation⁴⁰. Up to 70% of human tumors, especially those that are very aggressive and resistant to therapy, have aberrant overexpression of c-Myc, which is extensively expressed in a variety of tissues and organs⁴¹. The c-Myc protein is a key player in promoting cell division and is a hallmark of unrestricted cell proliferation. Numerous investigations have shown that c-Myc stimulates osteosarcoma migration and proliferation^42–44. The proto-oncogene NANOG is intimately linked to stem cell properties, differentiation, and cell division. Many malignant tumors have aberrant activation of the STAT3/NANOG signaling pathway, which is highly associated with a poor prognosis^45,46. The epithelial-mesenchymal transition (EMT) is facilitated by high NANOG expression, which increases tumor invasion and metastatic potential. One essential mitotic regulator is nucleolar and spindle-associated protein 1 (NUSAP1). Numerous cellular processes, including spindle assembly and cytokinesis, depend on NUSAP1 activation⁴⁷. Malignant tumor formation is also significantly influenced by aberrant mitotic processes.

NUSAP1 is intimately linked to the formation of malignant tumors, according to recent research⁴⁸. Increasing NUSAP1 expression has been shown in many studies to improve osteosarcoma migration and invasion^49,50. SHP2 enhances HOXB2 transcription, which in turn raises the expression of c-Myc, NANOG, and NUSAP1 by promoting STAT3 phosphorylation and upregulating TET3 expression, according to in vitro and in vivo Western blot and immunofluorescence investigations.

In summary, our work clarifies how SHP2 controls the STAT3/TET3/HOXB2 signaling pathway to facilitate the growth of osteosarcoma. Clarifying this process improves our knowledge of the pathophysiology of hepatocellular carcinoma and offers a strong theoretical basis and translational promise for creating combination treatments that target important nodes, thereby improving the prognosis for patients with osteosarcoma. Future studies will concentrate on improving combination treatment approaches, clinical validation, and better clarifying the mechanism.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Hua Yang: Conceptualization, Methodology, Data Curation, Writing - Original Draft Preparation, Writing - Review & Editing. Supervision. Jiangfeng Ji: Investigation, Visualization, Validation, Writing - Review & Editing. All authors read and approved the final manuscript.

Funding

The 2024 Annual Plan for Medical Science Research Projects in Hebei Province, NO:20240585.

Data availability

The data that support the findings of this study are available on request from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study has been approved by the Medical Ethics Committee of Xingtai general Hospital.(Approval number:2023023).