S100 calcium-binding protein A10 contributes to malignant traits in osteosarcoma cells by regulating glycolytic metabolism via the AKT/mTOR pathway.

As an aggressive musculoskeletal malignancy, osteosarcoma (OSa) is popular among young adults and teenagers worldwide. S100 calcium-binding protein A10 (S100A10) functioned as a novel tumor-promoting protein in several human cancers. However, its role in OSa remains obscure. In this study, gene and protein levels were respectively determined by RT-qPCR or Western blotting. OSa cell proliferation, apoptosis, and metastasis were evaluated via CCK-8, colony formation, flow cytometry, and Transwell assays. To assess the glycolysis level, glucose consumption and lactate production were detected. It was found S100A10 was highly expressed in OSa tissues and cell lines. Besides, S100A10 facilitated proliferation and metastasis, and inhibited apoptosis in OSa cells. In addition, S100A10 regulated OSa cell proliferation, metastasis and apoptosis via mediating the glycolysis process. Furthermore, S100A10-mediated AKT/mTOR signaling accelerated glycolysis, thereby promoting malignant behaviors in OSa cells. Taken together, our findings indicated that S100A10 might promote malignant phenotypes of OSa cells by accelerating glycolysis and activating the AKT/mTOR signaling, providing a promising target for OSa treatment.

Introduction

Osteosarcoma (OSa), a rare musculoskeletal malignancy originating from osteogenic mesenchymal cells [1], exhibits low incidence (3 per million) globally [2]. However, OSa is prevalent in children, adolescents, and young adults under 25 years old [3] and manifests high disability and mortality rates [4]. Despite great progress made in developing therapeutic methods for bone treatment [5,6], surgical excision combined with chemotherapy is still a primary therapeutic strategy for OSa patients in clinical treatment [7]. However, its clinical efficacy is quite limited due to rapid development and early metastasis of OSa [8,9]. The reality is that there is no substantial improvement in the overall survival of OSa patients in recent decades [10]. With few common features between OSa cases, its pathogenesis remains complex and elusive [11]. Therefore, identification of new biomarkers and molecular mechanisms for OSa is in urgent need.

As a well-supported metabolic process in cancer cells [12], glycolysis is a major approach for energy generation in cancer cells, with lactic acid as its product [13]. In cancer cells, glucose uptake and lactic acid production are dramatically increased with the fast cell growth and proliferation [14]. Previous studies also evidenced that glycolysis could promote cancer cell survival by facilitating proliferation and migration [15,16]. In addition, it was demonstrated by Sottnik et al. that 2-Deoxy-D-glucose (2-DG) could inhibit tumor growth in a postsurgical OSa model [17], suggesting the promoting role of glycolysis in OSa development and progression. Hence, glycolysis may be a promising target for OSa treatment.

S100 calcium-binding proteins are a type of low-molecular-weight proteins generally expressed in vertebrates [18]. The S100 family consists of more than twenty S100 proteins and each encoded by a specific gene [19]. As for their biological functions in intracellular and extracellular activities, S100 proteins act as key regulators in enzyme regulation, protein phosphorylation, energy metabolism, cell differentiation, proliferation, and apoptosis [20-22]. Through affecting cellular responses via the Ca2+ signal transduction pathway, the S100 family is deeply implicated in a number of human malignancies [23]. For example, Yang et al. uncovered that S100B contributed to the resistance of ovarian cancer stem cells to cisplatin via p53 inactivation [24]. Meng et al. found S100A11 facilitated cervical cancer cell proliferation and metastasis via activation of the Wnt/β-Catenin pathway [25]. In addition, Xiao et al. elucidated that S100A4, another member of the S100 family, could promote OSa growth and metastasis [26]. Of note, a study by Wang et al. identified S100 calcium-binding protein A10 (S100A10) as an upregulated gene in OSa [27]. However, the functional role of S100A10 in OSa remains largely unknown.

In the present work, we intended to probe the role and latent mechanism of S100A10 in OSa. Functional experiments demonstrated that S100A10 knockdown inhibited proliferation, migration, invasion, and induced apoptosis in OSa cells by modulating glycolysis via the AKT/mTOR pathway, indicating S100A10 might be a putative target for OSa treatment.

Materials and methods

Differential gene analysis

The gene expression profile datasets (GSE28424 and GSE36001) deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) database were used to investigate the differential expression of S100A10 in OS. Differential genetic analysis was performed using GEO online analytical tool GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/).

Clinical samples

OSa and adjacent normal tissue samples were collected from 43 OSa patients during surgery at Taizhou People’s Hospital from July 2019 to July 2021. All the tissue samples were immediately put in liquid nitrogen after collection and stored at −80°C till use. Each OSa patient enrolled signed the informed consent. None of these OSa patients had received any radiotherapy, chemotherapy, or immunotherapy before operation. The study protocol was permitted by the Hospital Ethics Committee.

Cell culture and transfection

Human osteoblast cell line (hFOB1.19) and Human OSa cell lines (HOS, U2OS, SAOS-2, LM7, and HOS-SL) were acquired from BeNa Culture Collection (Beijing, China) and cultivated in DMEM (10% FBS) in a humid atmosphere (37°C; 5% CO2).

Small interfering RNA (siRNA) against S100A10 (si-S100A10), siRNA negative control (si-NC), S100A10 pcDNA3.1 vector (S100A10-oe), and pcDNA3.1 empty vector (Vector) acquired from Genechem (Shanghai, China) were respectively transfected into HOS and LM7 cells via Lipofectamine 2000 (Invitrogen, USA). After 24 hours’ transfection, cells were harvested for further experiments.

RT-qPCR

Total RNA was isolated from tissues and cells via TRIZOL (Invitrogen) and reversely transcribed to cDNA with M-MLV (Invitrogen). Then, qPCR was performed with SYBR Premix Ex Taq reagent kit (Takara, Japan) on the ABI7900 real-time PCR system (Applied Biosystems, USA). Relative gene expression was normalized to GAPDH and calculated by 2‐ΔΔCt method. The primer sequences used were: S100A10 forward (F): 5′-AACAAAGGAGGACCTGAGAGTAC-3′ and reverse (R): 5′-CTTTGCCATCTCTACACTGGTCC-3′; GAPDH F: 5′-GTCTCCTCTGACTTCAACAGCG-3′ and R: 5′-ACCACCCTGTTGCTGTAGCCAA-3′.

Western blotting

Total protein was extracted from cells via lysis buffer (Beyotime, China), quantified for concentration via BCA Protein Assay Kit, separated by SDS-PAGE, and then transferred onto PVDF membranes. Next, PVDF membranes were blocked with 5% skim dry milk for 1 hour and cultivated with primary antibody (against S100A10, hexokinase 2 (HK2), pyruvate kinase isozymes M2 (PKM2), and glucose transporter 1 (GLUT1), phospho-Akt (p-Akt), Akt, phospho-mTOR (p-mTOR), mTOR, or GAPDH) and secondary antibody. Finally, the protein bands were visualized with an ECL system (Thermo Scientific, USA).

CCK-8

OSa cells (3 × 103 cells/well) were seeded onto 96-well plates and cultivated in DMEM with 10% FBS (Gibco) in a humid atmosphere (37°C; 5% CO2). After CCK-8 reagent was added (10 µl/well) at the indicated time, the cells were cultivated for another 2 hours. The absorbance was measured with a microplate-reader at 450 nm wavelength.

Colony formation

Transfected OSa cells were placed to six-well plates (500 cells/well) for 14 days’ cultivation. Then, the cell colonies were fixed with methanol and dyed with crystal violet. Afterward, the plates were rinsed three times to remove excess staining. Finally, the colonies (>100 cells) were counted under a microscope.

Flow cytometry

Apoptosis detection for OSa cells was performed with Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen). In brief, OSa cells were rinsed twice with PBS, centrifugated at 1500 rpm for 5 min, and resuspended in Binding Buffer. Then, the OSa cells were cultured with Annexin V-FITC and propidium iodide (PI) for 15 min in darkness. The apoptotic OSa cells were analyzed with a Flow Cytometer.

Transwell

24-well Transwell filters (8 μm pore size; Corning Costar, USA) with or without Matrigel coating on the membrane were used for cell invasion and migration assays. In brief, transfected OSa cells were seeded in the top chamber containing serum-free medium. DMEM medium (10% FBS) was added to the bottom chamber. After 24 hours’ cultivation, OSa cells above the membrane were removed. Then, OSa cells migrated or invaded through the membrane were fixed with paraformaldehyde, dyed with crystal violet, and finally counted under a light microscope.

Glucose consumption and lactate production determination

To measure glucose consumption, OSa cells were seeded into 6-well plates and incubated in complete medium for 48 h. Then, glucose concentration in the medium was measured with Glucose Assay Kit (Sigma-Aldrich, USA). Glucose consumption is the glucose concentration in fresh complete medium minus that of the culture medium collected. To measure lactate production, the culture supernatant was collected to evaluate lactate production with Lactate Assay Kit (Sigma-Aldrich, USA).

Statistical analyses

In this study, GraphPad Prism 6.0 was employed for all statistical analyses, with Student’s t-test or one-way ANOVA for difference comparison. P < 0.05 was deemed significant. Each experiment was repeated more than 3 times. All data obtained were expressed as mean ± standard deviation.

Results

In this study, it was speculated that S100A10 could promote malignant traits and glycolysis in OSa cells. Functional experiments demonstrated that S100A10 promoted tumorigenesis and glycolysis in OSa cells by inhibiting the AKT/mTOR signaling pathway. Our findings demonstrated the oncogenic role of S100A10 in OSa, suggesting S100A10 might be a potential target for OSa treatment.

S100A10 is elevated in OSa

GEO datasets (GSE36001 and GSE28424) were downloaded to analyze the expression pattern of S100A10 in OSa, and the results indicated that S100A10 was significantly upregulated in OSa compared with normal samples (Figure 1(a,b)). For further confirmation, we detected S100A10 expression in OSa tissue samples (n = 43) and normal tissue samples (n = 43) by RT-qPCR. As shown in Figure 1(c), S100A10 expression was upregulated in OSa tissues, compared with normal tissues. Based on the mean level of S100A10 expression in OSa tissue samples, the OSa patients (n = 43) were divided into S100A10 low expression (n = 20) and S100A10 high expression (n = 23) groups. Next, the correlation between S100A10 expression level and clinicopathological features of these OSa patients was assessed. According to Table 1, high S100A10 expression was significantly correlated with large tumor size, advanced Enneking stage, and positive tumor metastasis. Similarly, RT-qPCR and Western blotting indicated that S100A10 level was dramatically elevated in human OSa cell lines (HOS, U2OS, SAOS-2, LM7, and HOS-SL), relative to that in human osteoblast cell line (hFOB1.19) (Figure 1(d,e)). As S100A10 expression was relatively higher in HOS and LM7 cell lines than in other OSa cell lines, they were chosen for the following experiments. All these results suggested that S100A10 was substantially upregulated in OSa and tended to assume a carcinogenic role in OSa.

Figure 1.

S100A10 is elevated in OSa. (a and b) The expression of S100A10 in OSa was analyzed using GEO database (GSE36001 and GSE28424). (c) RT-qPCR for determination of S100A10 mRNA expression in OSa tissue samples (n = 43) and normal tissue samples (n = 43). (d and e) RT-qPCR and Western blotting for determination of S100A10 mRNA expression in human osteoblast cell line (hFOB1.19) and human OSa cell lines (HOS, U2OS, SAOS-2, LM7, and HOS-SL). *P < 0.05; **P < 0.01; ***P < 0.001.

Table 1.

Association between S100A10 expression and OSa clinical features

	Number ofPatients (n = 43)	S100A10 mRNA expression		P-value
		Low (n = 20)	High (n = 23)
Age				0.778
<20	31	15	16
≥20	12	5	7
Gender				0.869
Male	24	11	13
Female	19	9	10
Anatomical location				0.923
Femur/tibia	33	16	17
Humerus	6	3	3
Elsewhere	4	1	3
Tumor size (cm)				0.007*
<8	24	17	7
≥8	19	4	15
Histological type				0.775
Osteoblastic	17	9	8
Chondroblastic	6	2	4
Fibroblastic	8	3	5
Others	12	6	6
Enneking stage				0.021*
I	16	12	4
II	14	6	8
III	13	2	11
Tumor metastasis				<0.001*
Absent	19	16	3
Present	24	4	20

S100A10 knockdown inhibits proliferation, migration, and invasion but induces apoptosis in OSa cells

To investigate the regulatory effect of S100A10 on the biological functions of OSa cells, S100A10 was firstly knocked down in HOS and LM7 cells, with transfection efficiency detected by RT-qPCR and Western blotting (Figure 2(a,b)). CCK-8 assay exhibited that S100A10 silencing remarkably suppressed the viability of HOS and LM7 cells (Figure 2(c)). According to the results of colony formation assays, the S100A10-knockdown group showed a significant decrease in the number of cell colonies (Figure 2(d)). In addition, flow cytometry assay indicated that S100A10 depletion led to increased apoptosis in HOS and LM7 cells, in contrast with the control group (Figure 2(e)). Moreover, transwell assays demonstrated that S100A10 inhibition remarkably attenuated the migrative and invasive capabilities of HOS and LM7 cells (Figure 2(f,g)). To sum up, the above results demonstrated that S100A10 exerted tumor-promoting effects on OSa cells.

Figure 2.

S100A10 knockdown inhibits proliferation, migration, and invasion but induces apoptosis in OSa cells. (a and b) S100A10 expression in HOS and LM7 cells transfected by si-NC or si-S100A10 was detected by RT-qPCR and Western blotting. (c) CCK-8 assay for HOS and LM7 cell viability evaluation. (d) Colony formation assay for colony-forming capability evaluation. (e) Flow cytometry for apoptosis evaluation. (f and g) Transwell assay for cell migration and invasion evaluation. *P < 0.05; **P < 0.01.

S100A10 accelerates glycolysis to promote malignant phenotypes in OSa cells

To explore whether S100A10 could regulate glycolysis in OSa cells, glucose consumption and lactate production were firstly measured. As shown in Figure 3(a,b), S100A10 knockdown caused a significant decrease in glucose consumption and lactate production in HOS and LM7 cells. Consistently, Western blotting results manifested that S100A10 depletion inhibited glycolysis-related proteins, such as HK2, PKM2, and GLUT1 (Figure 3(c–e)). Hence, it could be concluded that S100A10 promoted glycolysis in OSa cells.

Figure 3.

S100A10 accelerates glycolysis to promote malignant phenotypes in OSa cells. (a and b) Glucose consumption and lactate production were measured in HOS and LM7 cells transfected with si-NC or si-S100A10. (c-e) Western blotting for determination of HK2, PKM2, and GLUT1 levels in each group. (f and g) RT-qPCR and Western blotting for determination of S100A10 expression in HOS and LM7 cells transfected by Vector or S100A10-oe. (h-l) HOS and LM7 cells were transfected with Vector, S100A10-oe, or S100A10-oe +2-DG (10 mM). CCK-8, colony formation, flow cytometry, and Transwell assays were performed to evaluate cell viability, proliferation, apoptosis, migration, and invasion in each group. *P < 0.05; **P < 0.01.

To further investigate whether glycolysis participated in S100A10-mediated regulation on biological functions of OSa cells, the glycolysis inhibitor, 2-DG, was applied. Firstly, S100A10 was overexpressed in HOS and LM7 cells (figure 3(f,g)). According to functional assays, S100A10 upregulation led to increased proliferation, migration, invasion, and decreased apoptosis in OSa cells, which was partly abolished by 2-DG (Figure 3(h–l)). These data indicated that S100A10 promoted malignant traits in OSa cells via regulating glycolysis.

S100A10 activates the AKT/mTOR signaling to expedite malignant biological behaviors and glycolysis of OSa cells

The AKT/mTOR pathway is involved in a multiplicity of cellular processes, including cell proliferation, migration, apoptosis, and glycolysis [28]. The active engagement of the AKT/mTOR signaling pathway in OSa development and progression has been well documented by former studies [29]. Also, it has been demonstrated that S100A10 could accelerate glycolysis and malignant growth in gastric cancer by activating the AKT/mTOR signaling [30]. Therefore, it was hypothesized that the AKT/mTOR pathway might participate in S100A10-regulated malignant biological behaviors and glycolysis in OSa cells. To analyze the activity of the AKT/mTOR signaling, Western blotting was applied to measure relevant protein (p-AKT, AKT, p-mTOR, and mTOR) levels. As shown in Figure 4(a), S100A10 silencing almost made no difference to the total AKT and mTOR levels but significantly reduced the p-AKT and p-mTOR levels in HOS and LM7 cells, suggesting that S100A10 activated the AKT/mTOR pathway in OSa cells. Moreover, an AKT agonist, SC-79, markedly abated the suppressive effect of S100A10 deletion on OSa glycolysis, cell proliferation, migration, invasion, and apoptosis (Figure 4(b–j)). Given these results, it could be concluded that S100A10 could accelerate the malignant progression of OSa cells and glycolysis via activating the AKT/mTOR signaling.

Figure 4.

S100A10 activates the AKT/mTOR signaling to expedite malignant biological behaviors and glycolysis of OSa cells. (a) Western blotting for determination of p-AKT, AKT, p-mTOR, and mTOR levels in HOS and LM7 cells transfected by si-NC or si-S100A10. (b and c) Western blotting for determination of HK2, PKM2, and GLUT1 levels in HOS and LM7 cells were transfected by si-NC, si-S100A10, or si-S100A10+ SC-79. (d and e) Glucose consumption and lactate production were measured in HOS and LM7 cells transfected by si-NC, si-S100A10, or si-S100A10+ SC-79. (f-j) CCK-8, colony formation, flow cytometry, and Transwell assays were performed to evaluate cell viability, proliferation, apoptosis, migration, and invasion in each group. *P < 0.05; **P < 0.01.

Discussion

OSa is characterized by rapid development and distant metastasis [9]. According to previous studies, OSa usually occurs in teenagers and young adults [3] and has high mortality [4]. Due to the limitations of current therapies, many survivors even suffer from permanent disability [31]. As a result of inadequate understanding of OSa pathogenesis, there are still no definitive diagnostic methods or curative therapies for OSa patients [32]. Therefore, it is imperative to explore possible mechanisms implicated in OSa development and determine specific targets for OSa diagnosis and treatment.

As one of the S100 proteins, S100A10 is widely expressed in cells, tissues, and tumors [33-35]. Accumulating evidence has identified S100A10 as an active regulator in a number of cellular functions, such as proliferation, migration, invasion, and apoptosis [36,37]. In addition, it has been demonstrated that S100A10 is upregulated and plays a tumor-promoting role in multiple human cancers. To cite an instance, Wang et al. found that succinylation of S100A10 promoted cell proliferation in gastric cancer [38]. Shan et al. disclosed that S100A10 showed high expression in hepatocellular carcinoma; also, miR-590-5p mediated S100A10 inhibition repressed HepG2 cell proliferation [39]. In addition, Wang et al. uncovered that S100A10 promoted proliferation, migration, invasion, and chemoresistance to carboplatin in ovarian cancer cells [40]. In our study, high S100A10 level was observed in OSa tissues and cell lines. Also, high S100A10 level was closely associated with large tumor size, advanced Enneking stage, and positive tumor metastasis of OSa patients, suggesting the cancerogenic role of S100A10 in OSa. Subsequent functional experiments demonstrated that S100A10 inhibition repressed the proliferative, migrative, and invasive capabilities of OSa cells, and accelerated OSa cell apoptosis, which further confirmed that S100A10 promoted the malignant progression of OSa in vitro.

Glycolysis is a vital metabolic process in OSa cells and participates in the regulation of several cellular functions [41]. For instance, Shen et al. disclosed that KCNQ1OT1 promoted OSa growth by accelerating glycolytic metabolism via the miR-34c-5p/ALDOA axis [42]. As elucidated by Yu et al., STC2 could enhance glycolysis in OSa cells to promote cell proliferation and metastasis [43]. In addition, a former study demonstrated that S100A10 could promote glycolysis in gastric cancer [30]. In this work, S100A10-silenced OSa cells exhibited lower glucose consumption and lactate production, as well as lower HK2, PKM2, and GLUT1 levels, implying that S100A10 expedited glycolysis in OSa cells. Furthermore, a glycolysis inhibitor, 2-DG, abated the increase in OSa cell proliferation, migration, and invasion as well as the decrease in OSa cell apoptosis caused by S100A10 overexpression. Taken together, S100A10 aggravated malignant behaviors of OSa cells via glycolysis regulation.

Interestingly, S100A10 is also a prospective activator of the AKT/mTOR signaling [30]. The AKT/mTOR pathway is deeply involved in the regulation of proliferation, migration, invasion, and apoptosis in tumor cells [44]. Besides, the AKT/mTOR pathway also participates in glycolytic regulation in OSa [45]. For example, Wang et al. revealed that Arbutin treatment inhibited OSa cell proliferation and metastasis by downregulating MTHFD1L via miR-338-3p and inactivating the AKT/mTOR signaling [46]. Huang et al. discovered that ARHGAP25-mediated inactivation of AKT/mTOR signaling impaired tumor growth and glycolysis in pancreatic adenocarcinoma [47]. Wu et al. uncovered that Deoxyshikonin inactivated the AKT/mTOR signaling to inhibit cell viability and glycolysis in acute myeloid leukemia [48]. Herein, S100A10 knockdown inhibited AKT and mTOR phosphorylation in OSa cells but had almost no effects on total AKT and mTOR levels, while such effects were abated by an AKT agonist, SC-79, indicating that S100A10 activated the AKT/mTOR signaling in OSa cells. In addition, SC-79 also reversed the impact of S100A10 inhibition on glycolysis, cell proliferation, migration, invasion and apoptosis in OSa cells. Therefore, it was further demonstrated that the AKT/mTOR signaling contributed to S100A10-mediated glycolysis and malignant features in OSa cells.

Conclusion

In summary, our findings provide evidence that S100A10-induced acceleration of glycolysis and activation of the AKT/mTOR signaling may contribute to OSa carcinogenesis in vitro. Therefore, S100A10 may be a promising therapeutic target for OSa treatment.

Click here for additional data file.