Calcineurin Homologous Protein 2 Acts as a Conditional Modulator of Migration and Proliferation in Human Bone Sarcoma Cells

TL;DR

This study shows that CHP2 helps bone sarcoma cells adapt to stress by supporting NHE1 activity, which promotes their migration and growth.

Contribution

CHP2 is identified as a conditional modulator of NHE1-dependent migration and proliferation in bone sarcoma cells under metabolic stress.

Findings

Serum deprivation caused sarcoma cells to temporarily reduce then recover migration and proliferation.

NHE1 inhibition reduced migration and slightly reduced proliferation in sarcoma cells.

CHP2 silencing under stress conditions significantly reduced NHE1 activity and cell migration/proliferation.

Abstract

This study investigates how human bone sarcoma cells respond to metabolic stress induced by serum deprivation. In two sarcoma cell lines, migration and proliferation were initially reduced but later recovered, whereas non-malignant osteoblastic cells showed sustained inhibition. Sarcoma cells primarily expressed the Na+/H+ exchanger NHE1, and inhibition of NHE1 decreased migration and moderately reduced proliferation. Although serum deprivation did not change CHP2 protein levels, silencing CHP2 reduced NHE1 activity under stress conditions and significantly decreased both migration and proliferation. Together, these findings indicate that bone sarcoma cells can adapt to serum deprivation through an NHE1-dependent mechanism and that CHP2 contributes to this response under metabolic stress.

Background/Objectives: Sarcomas are malignant bone tumors for which current therapeutic approaches provide limited improvement in patient survival. Cancer-associated extracellular acidosis, driven in part by Na+/H+ exchanger 1 (NHE1) activity, promotes malignant cell migration and proliferation while exerting inhibitory effects on normal bone-forming cells. Calcineurin Homologous Protein 2 (CHP2) is a binding partner and functional regulator of NHE1 that is preferentially expressed in cancer cells; however, its role in bone sarcoma biology remains undefined. Methods: In this study, we examined how serum deprivation, used as a model of metabolic stress, affects migration and proliferation in human bone sarcoma cell lines (143B and SW1353) and investigated the contribution of NHE1 and CHP2 to these behaviors. Results: Serum deprivation induced a time-dependent adaptive response in sarcoma cells, characterized by transient suppression followed by recovery of migratory and proliferative capacity, whereas both migration and proliferation were consistently inhibited in non-malignant osteoblastic (hFOB) cells. Both sarcoma cell lines predominantly expressed NHE1, and pharmacological inhibition of NHE1 activity with zoniporide significantly reduced migration and moderately reduced proliferation. Serum deprivation did not significantly alter CHP1 or CHP2 protein expression; however, silencing of CHP2 reduced NHE1 activity under serum-deprived conditions and significantly decreased migration and proliferation in both sarcoma cell lines. Conclusions: Together, these findings demonstrate that NHE1 activity is a critical determinant of migratory capacity in bone sarcoma cells and that CHP2 supports malignant migration and proliferation. Under stress conditions, CHP2 action is associated with NHE1 activity, supporting CHP2 as a conditional modulator of malignant behavior in human bone sarcoma cells.

Figures

Effect of serum deprivation on NHE1 activity following CHP2 silencing in 143B and SW1353 cells. (A) NHE activity in 143B cells was measured after 72 h of serum deprivation, with or without CHP2 silencing by shRNA. (B) NHE activity in SW1353 cells was measured after 72 h of serum deprivation, with or without CHP2 silencing by shRNA. Bars and error bars represent the mean ± standard error. The number of independent experiments performed under identical conditions is shown in parentheses. * p < 0.05 and p < 0.01 indicate statistically significant differences relative to serum control. A representative immunoblot showing CHP2 and actin expression under shCHP2 and empty vector conditions is shown in Figure 10.

Effect of CHP2 Silencing on Cell Migration in 143B and SW1353 Cells. (A) Representative images illustrating migratory inhibition in 143B cells following CHP2 silencing (shCHP2). (B) Quantification of migratory inhibition in 143B cells following CHP2 silencing. The area of the specified field occupied by cells is expressed as a percentage relative to control cells. Bars and error bars represent the mean ± standard error. A representative immunoblot showing CHP2 and actin expression under shCHP2 and empty vector conditions is shown. (C) Representative images illustrating migratory inhibition in SW1353 cells following CHP2 silencing (shCHP2). (D) Quantification of migratory inhibition in SW1353 cells following CHP2 silencing. A representative immunoblot showing CHP2 and actin expression under shCHP2 and empty vector conditions is shown. The number of independent experiments performed under identical conditions is shown in parentheses. p < 0.01 indicates a statistically significant difference from control by two-tailed t-test. The original immunoblots figures can be found in Supplementary File S1.

Full text

1. Introduction

Primary bone tumors are neoplasms that originate in the bone, with osteosarcoma and chondrosarcoma being among the more common cancers of the malignant tumor types [1]. Osteosarcoma primarily affects children and adolescents, and is the sixth most common cancer in young adults, with a survival rate of 65% [1]. Despite aggressive chemotherapy and radiotherapy, osteosarcoma frequently metastasizes to the lungs, and relapse remains common [1]. Chondrosarcoma, the third most common form of primary bone cancer, typically presents in adults and arises from malignantly transformed chondrocytes that overproduce cartilage matrix [1]. Chondrosarcomas are characteristically resistant to chemotherapy and radiation [1]. As a result, the current standard of care for primary bone cancers has not significantly improved patient survival over the past three decades [1].

The regulation of pH is crucial in maintaining normal cellular and tissue homeostasis [2]. Under physiological conditions, extracellular pH is higher than intracellular pH, and bone resorption and formation remain in equilibrium at this pH range [3]. In cancer, however, this balance becomes disrupted. Malignant cells commonly develop a reversed pH gradient, in which the extracellular microenvironment becomes more acidic while intracellular pH remains relatively alkaline [4]. Extracellular acidification is a hallmark of malignancy and has been shown to promote the proliferation, survival, invasion, and migration of tumor cells [4]. In bone, these changes are particularly consequential: acidic extracellular pH stimulates osteoclast activity and increases bone resorption [5], while simultaneously inhibiting osteoblast-mediated mineralization [3]. In chondrocytes, extracellular acidosis increases cell volume and disrupts growth regulation [6]. Together, these events weaken bone integrity and may contribute to the pathological features observed in sarcoma patients.

A major driver of this altered pH landscape is the Sodium–Proton Exchanger-1 (NHE1), a ubiquitously expressed transporter that exchanges extracellular Na^+^ for intracellular H^+^ [7,8]. NHE1 plays a central role in intracellular pH homeostasis by extruding protons generated through high metabolic activity, thereby maintaining the relatively alkaline intracellular pH required for cell survival and rapid proliferation [7,9]. In cancer cells, including those of primary bone tumors, overactivity of NHE1 increases proton efflux, generating extracellular acidosis and supporting cellular processes such as adhesion, proliferation, migration, and matrix degradation [7]. For bone biology specifically, alkaline phosphatase activity required for mineralization functions optimally near pH 7.4 [3]. When extracellular pH decreases, alkaline phosphatase activity declines, and bone mineralization becomes impaired, a feature frequently observed in osteosarcoma [7].

Recent work in other tumor systems has shown that NHE1 does not function in isolation but operates within a larger pH-regulatory network that supports cancer cell metabolism [10,11]. By preventing intracellular acidification, NHE1 enables glycolytic tumor cells to maintain high metabolic flux and tolerate the substantial proton burden associated with lactate production [12,13]. This process also contributes to acidification of the extracellular space, creating an environment that enhances invasion and promotes tumor progression [11,14]. Although these metabolic and pH-regulating mechanisms have been documented in several cancers, they remain largely unexplored in primary bone tumors.

NHE1 activity is tightly regulated by interacting proteins, among which the calcineurin homologous protein (CHP) family plays a key role. CHP are calcium-binding regulatory cofactors, with CHP1 and CHP2 being the primary isoforms involved in NHE1 stabilization, trafficking, and activation [15]. CHP1 is an essential cofactor for NHE1 function, as a depletion of CHP1 results in a decreased expression level of NHE1 through posttranslational processes [16]. Previous studies have shown CHP1 to be the predominant isoform in normal cells, where CHP2 is highly expressed in tumor cells and present as a protective factor against serum deprivation-induced cell death [15]. CHP2 protein is usually absent in normal tissues, except in some, such as the intestine, but is commonly found in cancer cells. This suggests CHP2 may contribute to the transformation of normal cells into malignant ones [17,18]. High levels of CHP2 mRNA are also found in malignantly transformed cells, suggesting that microenvironments that resemble cancer cells may induce a higher expression of CHP2 [17].

The role that CHP2 plays in cancer cells and how CHP2 might contribute to NHE1 overactivation remains unclear; however, previous studies have shown that CHP1 and CHP2 share the same binding site on NHE1 [17]. CHP2 has a higher affinity for calcium compared to CHP1, and its expression is inducible in serum-deprived cells [19]. In microenvironments that mimic cancer cells, serum deprivation is shown to result in higher activity with the CHP2/NHE1 complex compared to that of CHP1/NHE1, resulting in extracellular acidosis [17]. In addition, in cancerous cells, there seems to be a serum-independent constitutive activation of NHE1 through CHP2 binding [17].

Changes in pH dynamics have not been studied in primary bone cancer cells, and the potential of NHE1 as a molecular target for primary bone cancer treatment has not been determined. Our hypothesis is that cell migratory and proliferation abilities seen in primary bone cancer cells are due to NHE1 overactivity, and the inhibition of NHE1 activity will allow cancer cells to return to normal physiological activity and prevent migration. We determined that NHE1 is the predominant isoform of NHE present in the primary bone cancer cell lines understudy. Incubation with zoniporide for 24 h, a potent and specific NHE1 inhibitor, effectively inhibited the migration of both cancer cell lines. Additionally, silencing of CHP2 resulted in a significant decrease in both migration and proliferation of the cancer cell lines understudy. Thus, NHE1 inhibition through CHP2-silencing may serve as a potential pharmacological strategy for the treatment of primary bone sarcoma by reducing the high migration and proliferation rates characteristic of aggressive sarcoma cells.

2. Materials and Methods

2.1. Cell Culture

Osteoblast hFOB (hFOB 1.19) cells (ATCC, Manassas, VA, USA) were cultured in a 1:1 mixture of Ham’s F12 Medium and Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 0.3 mg/mL G418 (Gibco, Waltham, MA, USA) and 10% fetal bovine serum (R&D Systems, Minneapolis, MN, USA). Undifferentiated hFOB cells were maintained at 34 °C in a humidified 95% air/5% CO_2_ environment and sub-cultured weekly by trypsinization with 0.1% trypsin/0.5 mM EGTA in PBS. Differentiation of hFOB cells was induced by incubation of cells at 37 °C for 96 h [20]. The osteosarcoma 143B cells (ATCC, Manassas, VA, USA) and the grade II chondrosarcoma SW1353 cells (ATCC, Manassas, VA, USA) were cultured in DMEM supplemented with 10% Fetal Bovine Serum, 50 IU/mL penicillin, and 50 μg/mL streptomycin (Gibco, Waltham, MA, USA). 143B and SW1353 cells were incubated at 37 °C in a humidified 95% air/5% CO_2_ environment and sub-cultured weekly by trypsinization with 0.1% trypsin/0.5 mM EGTA in PBS (Gibco, Waltham, MA, USA). Passages from 3 to 20 were used for hFOB cells, passages from 7 to 30 were used for 143B cells, and passages from 8 to 30 were used for SW1353 cells. In experiments assessing the effects of serum deprivation on cell migration and proliferation, the control condition corresponds to cells maintained in standard serum-containing medium (non–serum-deprived control).

2.2. Measurement of Na+/H+ Exchange Activity

Prior to the measurement of NHE activity, 1.3 × 10^6^ cells were seeded on glass coverslips placed in a 35 mm culture dish and allowed to reach confluency. NHE activity was measured by spectrofluorometry using an intracellular pH (pHi) sensitive dye, 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF-AM) (Invitrogen, Carlsbad, CA, USA), as previously described [21]. Cells grown on glass coverslips were incubated with 10 µM of BCECF-AM in sodium-containing buffer (115 mM NaCl, 5 mM KCl, 1.1 mM CaCl_2_, 1.54 mM MgCl_2_, 30 mM HEPES, pH 7.4) for 20 min at room temperature. Cells were excited at 490 and 440 nm, and emitted light was measured at 520 nm using a computer-controlled spectrofluorometer (PTI QuantaMaster 40, Horiba Scientific, Piscataway, NJ, USA). The ratio of fluorescence was used to measure pHi. Na^+^/H^+^ exchange activity was estimated as the initial rate of Na^+^-dependent pHi recovery after an acid load imposed by the H^+^/K^+^ ionophore (Nigericin, Tocris Bioscience, Minneapolis, MN, USA) in sodium-free solution (20 µg/mL Nigericin, 115 mM Choline-Cl, 5 mM KCl, 1.1 mM CaCl_2_, 1.54 mM MgCl_2_, 30 mM HEPES, pH 7.4) in the absence of CO_2_/HCO_3_^−^.

2.3. Western Blot

Protein expression levels of CHP1 and CHP2 were determined by Western blot. To extract proteins, hFOB, 143B, and SW1353 cells were washed in ice-cold PBS and gently scraped into membrane buffer (containing in mM: 150 NaCl, 50 Tris-HCl, pH 7.4, 5 EDTA, supplemented with protease inhibitors). Cells were then homogenized by sonication and centrifuged at 12,000× g for 30 min at 4 °C. Resultant supernatants, which contain total membrane protein fractions, were collected, and protein content was quantified by the Bradford method. Equal amounts of protein were loaded into each well on 15% SDS-PAGE gel. After transferring proteins onto PVDF membranes, they were blotted with CHP1 goat 1:10,000 (Santa Cruz Biotechnology, Dallas, TX, USA) or CHP2 rabbit 1:5000 (Sigma, St. Louis, MO, USA) polyclonal antibodies. Membranes were rinsed and incubated with appropriate HRP-conjugated secondary antibodies 1:250 (Jackson ImmunoResearch Inc., West Grove, PA, USA), then visualized with Pierce^TM^ ECL Western blotting Substrate (Thermo Fischer Scientific, Waltham, MA, USA). Signal intensity was measured using ImageJ (version 1.52r) [22].

2.4. Silencing of CHP2

Short hairpin RNA constructs targeting human CHP2 (shCHP2) were obtained by cloning the two target sequences corresponding to human CHP2 (395–417 and 526–548 bp) separately into the MSCV-LTRmiR30-PIG vector (Open Biosystems, Huntsville, AL, USA) according to the manufacturer’s instructions. Both constructs showed comparable knockdown efficiency, and the 526–548 bp shCHP2 construct was used for most experiments. For proliferation studies, EGFP was substituted with a 6-His tag in the shCHP2-containing plasmid. All plasmids were sequenced, verified, and transfected into 143B and SW1353 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

2.5. Wound-Healing Assay for Cell Migration

2.5.1. Live Cell Imaging

Cells were seeded on 35 mm cell culture dishes at 50% confluence. Treatments were given to cells 24 h after seeding. A “wound” was introduced by scratching the dishes with a 200 μL plastic pipette tip, and cells were imaged at five different time points: 0, 3, 6, 9, 12, and 24 h after the initial “wound” introduction. The bright-field images of live cells were collected with Nikon Eclipse TE300 (Nikon Corp., Tokyo, Japan) equipped with the Nikon 5X NA0.75 and using pco.edge 4.2 camera (PCO AG, Kelheim, Germany) controlled by μManager software (version 1.4.22) [23].

2.5.2. Fixed Cell Imaging

Cells were grown on 9 mm glass coverslips (VWR International, Radnor, PA, USA), and all cell lines were grown until 80% confluence prior to treatment [24]. Treatments were given to cells 24 h before the introduction of the “wound”, which was made by scratching the coverslips with a 200 μL plastic pipette tip [24]. Cells were fixed with 4% paraformaldehyde at 0 h and 9 h following introduction of the “wound” for 143B cells, and cells were fixed at 0 h and 24 h following introduction of the “wound” for SW1353 and hFOB 1.19 cells [24]. Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining (Southern Biotech, Birmingham, AL, USA), and actin was visualized by Alexa Fluor^TM^ 546 phalloidin (Thermo Fischer Scientific, Waltham, MA, USA). Images were collected via Nikon Eclipse TE300 (Nikon Corp., Tokyo, Japan) with the Nikon 20X NA0.75 objective controlled by μManager software (version 1.4.22) [23]. DAPI and Alexa Fluor^TM^ 546 Phalloidin were consequently excited via 360/40 and 555/28 bandpass filters, respectively. The emission light was collected via 450/30 or 600/40 bandpass emission filters using pco.edge 4.2 camera (PCO AG, Kelheim, Germany).

2.6. Image Analysis

Live Cell Imaging: The area of the wound was calculated using the ImageJ software, where the “wound” was selected and measured [25]. The average area of the “wound” region was estimated by the collection of at least 7 fields of view. To calculate the % wound closure 24 h after the initial “wound” introduction, the following formula was used:

$$[eqn]$$

where “ $[eqn]$ ” represents the average area of the “wound” 24 h after the introduction of the “wound,” and “ $[eqn]$ ” represents the average area of the “wound” 0 h after the introduction of the “wound”.

Fixed Cell Imaging: Automatic detection of nuclei was performed using the QuPATH software (version 0.2.3) [26]. Cell detection parameters were optimized to maximize the number of nuclei in the image. The average number of cells in the “wound” region was estimated by the collection of at least 10 fields of view of the “wound” regions, while the average number of cells in undisturbed regions of the coverslip was assessed by the collection of at least six fields of view of non-“wounded” regions. To calculate the percentage of area occupied by nuclei, we used the formula:

$$[eqn]$$

where $[eqn]$ represents the average number of cells in the “wound” region, and $[eqn]$ represents the average number of cells in the “non-wounded” region.

2.7. Cell Proliferation Analysis

The 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay (Thermo Fischer Scientific, Waltham, MA, USA) was used to measure cell proliferation. Cells were labeled with 10 μM of EdU for 2 h [27] and then fixed and stained according to the manufacturer’s instructions. Click-iT^®^ reaction cocktail was prepared according to the manufacturer’s instructions. Nuclei were visualized by Hoechst 33342 staining and imaged via Nikon Eclipse TE300 (Nikon Corp., Tokyo, Japan) with the Nikon 20X NA0.75 objective as described previously. EdU positive nuclei were visualized by “click” reaction with Alexa Fluor488 azide (Thermo Fischer Scientific, Waltham, MA, USA).

For the proliferation study involving CHP2 silencing, the shCHP2 plasmid containing 6-His tag and pmCherry as an empty vector were used. Cells expressing shCHP2 were detected by immunostaining using the goat polyclonal 6-His antibody (Bethyl, Montgomery, TX, USA) and visualized using donkey-anti-goat DyLight^TM^ 649 (Jackson ImmunoResearch Inc, West Grove, PA, USA) as previously described [21]. Proliferation fraction was calculated using the formula:

$$[eqn]$$

where Proliferating Cells represent the number of EdU-positive cells (or EdU-positive cells that were successfully transfected), and Nuclei represents the number of cells (or number of transfected cells), yielding a corrected proliferation fraction.

2.8. Statistical Analysis

All data were analyzed using Microsoft Excel and GraphPad Prism (version 9.0.1, GraphPad Software, Boston, MA, USA). The experiments were repeated at least three times. Mean values were compared by two-tailed Student’s t-tests or one-way ANOVA followed by Tukey’s post hoc test. A statistical significance was defined as p < 0.05.

3. Results

3.1. Effect of Serum-Deprivation on Cell Migration of 143B and SW1353 Cells

The migration rate of 143B and SW1353 cells was determined in serum-deprived cells to study the cell characteristics in an environment that mimics the decrease in growth factors observed in cancer tissue [18]. Migration rate was measured by calculating % of wound closure after an initial wound introduction using live cell imaging. In the control condition (0 h serum-deprivation), the wound closed 24 h after wound introduction in both bone sarcoma cell lines. In 48 h serum-deprivation condition, 143B cells exhibited a decrease in migration by over 80% (Figure 1A,B); however, migration rate of 143B cells began to increase in 72 h of serum-deprivation when compared to 48 h serum-deprivation condition; 30% increase in migration rate was measured from 48 h to 72 h serum deprivation (Figure 1A,B). In SW1353 cells, 48 h of serum-deprivation also resulted in a decrease in migration rate, specifically by 40% (Figure 1D,E). However, the wound was completely closed in SW1353 cells serum-deprived for 72 h (Figure 1E).

To confirm the difference in migration rate measured at 0 h and 48 h of serum deprivation in both sarcoma cell lines, imaging of fixed cells was analyzed to assess wound closure by calculating % of area occupied by cells. The results obtained from fixed cells (Figure 1C,F) mirrored those obtained using live cells, where both sarcoma cell lines showed a decrease in migration after being serum-deprived for 48 h, with 143B being more sensitive to serum-deprivation (Figure 1C,F). Indeed, 24 h post-wound, 143B cells showed a 72% decrease in % of area occupied by cells in condition of 48 h serum-deprivation when compared with condition of serum-deprivation for 0 h (Figure 1C), and SW1353 cells showed 67% decrease in the % of area occupied by cells on serum-deprivation for 48 h when compared with cells on serum-deprivation for 0 h (Figure 1F).

3.2. Effect of Serum Deprivation on Cell Migration of hFOB Cells

Previous studies have shown that hFOB cells display distinct behaviors in differentiated and undifferentiated states, which can be induced by incubation at a permissive temperature [20]. Therefore, migration was assessed in both differentiated and undifferentiated hFOB cells using the same wound-healing assay. In differentiated hFOB cells, wounds did not close under any condition tested (0-, 48-, or 72 h serum deprivation; Figure 2A,B). Although differentiated cells in the 0 h condition showed faster early migration at 3 h post-wound, all conditions reached only ~30% wound closure by 24 h (Figure 2B). In contrast, undifferentiated hFOB cells demonstrated robust migration under control conditions, with complete wound closure by 24 h in the absence of serum deprivation (0 h; Figure 2D,E). Serum deprivation markedly reduced migration in these cells, with an ~80% decrease after 48 h and a further ~5% reduction after 72 h compared with 48 h (Figure 2E).

To confirm these findings, wound closure was evaluated in fixed cells at 0 h and 48 h serum deprivation. In differentiated hFOB cells, there was no significant difference in the percentage of area occupied between 0 h and 48 h (p > 0.05; Figure 2C). In undifferentiated hFOB cells, however, 48 h serum deprivation resulted in a significant decrease in the percentage of area occupied (−60%, p < 0.05) compared with 0 h (Figure 2F).

3.3. Effect of Serum-Deprivation on Cell Proliferation in 143B and SW1353 Cells

Proliferation of 143B and SW1353 cells was next assessed under serum deprivation. In both cell lines, 48 h serum deprivation reduced proliferation by approximately 20% in 143B cells (Figure 3A,B; p > 0.05) and 40% in SW1353 cells (Figure 3C,D; p < 0.05). In 143B cells, this reduction was transient; proliferation after 72 h serum deprivation returned to levels comparable to the 0 h condition (Figure 3B). In SW1353 cells, however, the decrease in proliferation persisted after 72 h serum deprivation (Figure 3D).

3.4. Effect of Serum-Deprivation on Cell Proliferation in hFOB Cells

Baseline proliferation differed between undifferentiated and differentiated hFOB cells under serum-replete conditions, with differentiated cells showing a lower proliferation fraction than undifferentiated cells (39% vs. 61% at 0 h serum deprivation, respectively). In differentiated hFOB cells, proliferation decreased by 77% after 48 h serum deprivation (p < 0.05; Figure 4A,B). Although a modest increase in proliferation was observed at 72 h compared with 48 h (p < 0.01; Figure 4B), proliferation remained lower than that of undifferentiated hFOB cells. In undifferentiated hFOB cells, proliferation decreased by 38% after 48 h serum deprivation (p < 0.01) and did not decrease further at 72 h (p < 0.05; Figure 4C,D). No significant difference in proliferation fraction was observed between 48 h and 72 h serum deprivation in undifferentiated hFOB cells.

3.5. Cell Migration of Zoniporide-Treated 143B, SW1353, and hFOB Cells

To assess the contribution of NHE activity to migration, NHE activity was measured following acute (15 min) treatment with zoniporide. In 143B cells, NHE activity was effectively inhibited at 10 nM zoniporide, with an IC_50_ of 0.537 nM (Figure 5A), consistent with reported inhibition of NHE1 [28]. Treatment with 10 nM zoniporide for 24 h resulted in a complete inhibition of NHE activity and reduced migration rate compared with vehicle-treated cells at 9 h post-wound (Figure 5B,C). The reduction in migration rate was measured as % of wound closure or % of area occupied by cells after an initial wound introduction (Figure 5B,C, respectively). By 24 h, migration rates were similar between zoniporide- and vehicle-treated 143B cells.

In SW1353 cells, NHE activity was effectively inhibited at 10 nM zoniporide, with an IC_50_ of 30 pM (Figure 5D), also within the reported range for NHE1 inhibition [28]. Treatment with 10 nM zoniporide for 24 h resulted in a complete inhibition of NHE activity and reduced migration compared with vehicle-treated cells at 24 h post-wound (Figure 5E,F; migration rate measured as in Figure 5B,C). The difference in migration was evident by 9 h and persisted through 24 h.

As for 143B and SW1353 cells, NHE activity was measured in hFOB cells after an acute, 15 min-long incubation with zoniporide. NHE activity was completely inhibited at 100 nM concentration of zoniporide, and the IC_50_ was 8.68 nM (Figure 6A), which is within the range of inhibition for NHE1 [28]. Treatment with zoniporide given at a concentration of 100 nM for 24 h resulted in a complete inhibition of NHE activity and in a reduction in migration rate of undifferentiated hFOB cells compared to vehicle-treated cells, 24 h post-wound (Figure 6B,C). Differentiated hFOB cells were not included in this set of experiments as the cells did not demonstrate a significant migration rate in the control condition.

3.6. Cell Proliferation in Zoniporide-Treated 143B, SW1353, and hFOB Cells

Zoniporide treatment (10 nM, 24 h) reduced proliferation in both 143B and SW1353 cells by approximately 15% (Figure 7A,B), reaching significance in SW1353 cells (p < 0.05) but not in 143B cells (p > 0.05). In undifferentiated hFOB cells, 100 nM zoniporide reduced proliferation by approximately 40% after 24 h (Figure 7C; p < 0.05). Proliferation was not assessed in differentiated hFOB cells due to their low baseline proliferative rate.

3.7. Protein Expression of CHP1 and CHP2 in 143B, SW1353, and hFOB Cells

In 143B and SW1353 cells, baseline CHP1 protein expression was detected and showed a change in SW1353 cells while remaining stable in 143 cells, following 72 h of serum deprivation compared with 0 h of serum deprivation (Figure 8A). CHP2 protein was also expressed in both 143B and SW1353 cells, with a similar trend in SW1353 cells, while remaining stable in 143 cells (Figure 8A). Importantly, none of these differences reached statistical significance in either sarcoma cell line after 72 h of serum deprivation (Figure 8B).

In hFOB cells, baseline CHP1 protein expression was detected at baseline and did not change significantly following 72 h of serum deprivation (Figure 8C,D). CHP2 protein was also expressed in hFOB cells and did not change significantly following 72 h of serum deprivation (Figure 8C,D).

3.8. Effect of CHP2 Silencing on NHE1 Activity Under Serum Deprivation Conditions

CHP2 is involved in NHE1 regulation, and its protein expression increases the cancerous characteristics observed in many cancer cell lines [19]. Therefore, we next examined whether CHP2 contributes to the regulation of NHE1 activity under serum deprivation in bone sarcoma cells. NHE1 activity was measured following 72 h of serum deprivation in bone sarcoma cell lines and non-malignant osteoblastic cells. In 143B cells, serum deprivation alone did not significantly alter NHE1 activity in control cells; however, silencing of CHP2 resulted in a significant reduction in NHE1 activity under serum-deprived conditions (Figure 9A). Notably, CHP2 silencing did not significantly affect NHE1 activity in serum-replete conditions, indicating that dependence on CHP2 emerges specifically under metabolic stress.

Effect of serum deprivation on NHE1 activity following CHP2 silencing in 143B and SW1353 cells. (A) NHE activity in 143B cells was measured after 72 h of serum deprivation, with or without CHP2 silencing by shRNA. (B) NHE activity in SW1353 cells was measured after 72 h of serum deprivation, with or without CHP2 silencing by shRNA. Bars and error bars represent the mean ± standard error. The number of independent experiments performed under identical conditions is shown in parentheses. * p < 0.05 and ** p < 0.01 indicate statistically significant differences relative to serum control. A representative immunoblot showing CHP2 and actin expression under shCHP2 and empty vector conditions is shown in Figure 10.

Effect of CHP2 Silencing on Cell Migration in 143B and SW1353 Cells. (A) Representative images illustrating migratory inhibition in 143B cells following CHP2 silencing (shCHP2). (B) Quantification of migratory inhibition in 143B cells following CHP2 silencing. The area of the specified field occupied by cells is expressed as a percentage relative to control cells. Bars and error bars represent the mean ± standard error. A representative immunoblot showing CHP2 and actin expression under shCHP2 and empty vector conditions is shown. (C) Representative images illustrating migratory inhibition in SW1353 cells following CHP2 silencing (shCHP2). (D) Quantification of migratory inhibition in SW1353 cells following CHP2 silencing. A representative immunoblot showing CHP2 and actin expression under shCHP2 and empty vector conditions is shown. The number of independent experiments performed under identical conditions is shown in parentheses. ** p < 0.01 indicates a statistically significant difference from control by two-tailed t-test. The original immunoblots figures can be found in Supplementary File S1.

In SW1353 cells, serum deprivation significantly increased NHE1 activity in control cells (Figure 9B). This serum deprivation–induced increase was abolished by CHP2 silencing, and NHE1 activity was significantly reduced in CHP2-silenced cells under serum-deprived conditions compared with control cells (Figure 9B). The effect of CHP2 silencing on NHE1 activity was measured in bone sarcoma cell lines to support subsequent experiments examining the role of CHP2 in migration and proliferation in these same cell lines.

In hFOB cells, serum deprivation did not significantly affect NHE1 activity in undifferentiated cells (108 ± 4% of serum control, p > 0.05, n = 5), whereas a significant increase in NHE1 activity was observed in differentiated hFOB cells (118 ± 6% of serum control, p < 0.05, n = 5). These results indicate that differentiation status influences the NHE1 response to serum deprivation in osteoblastic cells.

3.9. Cell Migration and Proliferation Mediated by CHP2 Silencing in 143B and SW1353 Cells

Silencing of CHP2 in both 143B and SW1353 cells significantly inhibited cell migration. The cell migration rate slowed down by 55% in 143B cells transfected with shCHP2 48 h post-transfection (p < 0.01) compared to cells transfected with an empty vector (Figure 10A,B). CHP2 protein expression was decreased in 143B cells by CHP2 silencing, as shown in Figure 9B. A similar effect was seen in SW1353 transfected with shCHP2, where cell migration was inhibited by 62% (p < 0.01) compared to those transfected with an empty vector (Figure 10C,D). CHP2 protein expression was decreased in SW1353 after transfection with the shCHP2 vector, as shown in 10D. Experiments were conducted in the presence of serum because serum deprivation significantly reduces cell migration, as shown in Figure 1.

Silencing of CHP2 significantly inhibited the proliferation of both 143B and SW1353 cells (Figure 11A,C). Cell proliferation decreased by 64% in 143B cells transfected with shCHP2 compared to cells transfected with empty vector (p < 0.01) (Figure 11B). Cell proliferation decreased by 37% in SW1353 cells transfected with shCHP2 compared to cells transfected with the empty vector (p < 0.001) (Figure 11D).

4. Discussion

In this study, we investigated how serum deprivation, used as a model of metabolic and growth factor stress encountered within the tumor microenvironment, affects migration and proliferation of the bone sarcoma cell lines, 143B and SW1353. We further examined the contribution of the NHE1 and its regulatory protein CHP2 to these cellular behaviors. Our findings demonstrate that bone sarcoma cells exhibit a time-dependent adaptive response to serum deprivation affecting both migration and proliferation, that NHE1 activity is a critical determinant of migratory capacity, and that CHP2 supports both migration and proliferation. These effects of CHP2 may be linked to NHE1 activity under stress conditions induced by serum deprivation. Together, these results identify CHP2 as a conditional regulator of malignant behavior in bone sarcoma cells.

4.1. Transient Migratory Responses of 143B and SW1353 Cells Following Serum Deprivation

Serum deprivation is commonly used to model metabolic and growth-factor stress in the tumor microenvironment and has been shown to alter malignant characteristics in a context- and time-dependent manner [29]. Due to their high metabolic demand, cancer cells frequently encounter nutrient-limited conditions, and sensitivity or resistance to such stress can influence tumor aggressiveness and prognosis [30,31]. In some cancer models, nutrient deprivation enhances therapeutic efficacy when combined with cytotoxic agents [32,33], whereas in others, prolonged deprivation promotes adaptive behaviors that support malignancy [29]. Consistent with this view, nutrient deprivation has been shown to modulate cancer cell behavior and enhance therapeutic responsiveness when combined with additional stressors, highlighting its utility as a model to study adaptive malignant responses rather than simple growth inhibition [32,33]. Thus, serum deprivation is used here to reveal stress-adaptive regulatory mechanisms rather than to model a uniform or static tumor microenvironment.

Consistent with these observations, our results revealed a biphasic migratory response to serum deprivation in both 143B and SW1353 cells. Migration was initially suppressed following 48 h of serum deprivation but significantly increased after 72 h, indicating a transition from serum-dependent to serum-independent migratory behavior. Similar time-dependent adaptations have been reported in breast cancer models, where prolonged serum deprivation promotes recovery and enhancement of migratory capacity [29]. Importantly, resistance to nutrient deprivation has been associated with more aggressive tumor phenotypes and poorer clinical outcomes, as demonstrated in colorectal cancer cells [30]. Consistent with this, metabolic adaptation to nutritional stress has been directly linked to increased tumor aggressiveness and poorer prognosis, supporting the interpretation that recovery of migratory capacity following prolonged serum deprivation reflects an adaptive malignant phenotype [30,34].

These findings suggest that prolonged serum deprivation selects for or induces adaptive mechanisms that allow bone sarcoma cells to regain and enhance migratory capacity under metabolically stressful conditions.

4.2. Differential Effects of Serum Deprivation on Migration in Non-Malignant hFOB Cells

In contrast to cancer cells, serum deprivation typically suppresses migration and induces cell cycle arrest or apoptosis in non-malignant cells [35,36,37]. Although fewer studies have examined serum deprivation in hFOB cells, previous work has established distinct behavioral differences between differentiated and undifferentiated hFOB cells [38]. Undifferentiated hFOB cells exhibit greater migratory capacity than differentiated cells, consistent with their progenitor-like phenotype [38]. In our study, serum deprivation significantly inhibited migration in both undifferentiated and differentiated hFOB cells, with a more pronounced effect in undifferentiated cells. Differentiated hFOB cells exhibited minimal migratory capacity even under prolonged observation. These results underscore a fundamental distinction between malignant and non-malignant cells: whereas cancer cells can adapt to prolonged serum deprivation and regain migratory behavior, non-cancerous osteoblastic cells remain sensitive to nutrient stress and do not acquire serum-independent migration.

4.3. Transient Proliferative Responses of Bone Sarcoma Cells to Serum Deprivation

Serum deprivation can also differentially affect proliferative signaling pathways in cancer cells. Previous studies have demonstrated that nutrient deprivation may activate compensatory pathways, such as MEK/ERK signaling or metabolic reprogramming, to sustain proliferation under stress [39,40]. In hepatoma and pancreatic cancer models, prolonged deprivation promotes metabolic adaptations that support continued cell growth [40]. In agreement with these reports, both 143B and SW1353 cells displayed an initial reduction in proliferation after 48 h of serum deprivation, followed by recovery or enhancement after 72 h. In 143B cells, proliferation after 72 h returned to levels comparable to serum-replete conditions. This temporal pattern closely mirrored the migratory response, indicating coordinated adaptation of proliferative and motility programs during prolonged stress.

The longer-term proliferative responses of 143B and SW1353 cells showed some differences, which likely reflect intrinsic biological heterogeneity between these two tumor cell models. Osteosarcoma and chondrosarcoma cells differ in differentiation state, metabolic regulation, and signaling network organization, and therefore may not be expected to respond identically to metabolic stress. Importantly, migration and proliferation represent related but distinct cellular programs. Wound healing assays reflect not only proliferative contributions but also cytoskeletal remodeling, adhesion dynamics, and metabolic adaptation. Accordingly, a sustained reduction in proliferation in SW1353 cells does not necessarily conflict with their migratory behavior but may instead indicate partial uncoupling of motility and cell cycle progression under stress. Similar context-dependent divergence between migration and proliferation has been reported in cancer models exposed to metabolic or growth-factor limitation [41,42,43,44,45].

By contrast, serum deprivation decreased proliferation in both undifferentiated and differentiated hFOB cells, consistent with the well-established sensitivity of non-malignant cells to nutrient stress [39]. Differentiated hFOB cells also exhibited lower baseline proliferation compared to undifferentiated cells, in agreement with previous studies [38]. While our proliferation assays primarily assessed newly synthesized DNA, future studies will be required to examine cell cycle arrest and DNA damage responses in greater detail.

4.4. Inhibition of NHE1 Reduces Migration and, to a Lesser Extent, Proliferation in Bone Sarcoma Cells

Extracellular acidification driven by NHE1 activity is widely recognized as a facilitator of cancer cell migration and invasion [7]. We identified NHE1 as the predominant Na^+^/H^+^ exchanger isoform expressed in 143B and SW1353 cells and demonstrated that pharmacological inhibition of NHE1 with zoniporide significantly reduced migration in both cell lines. The IC_50_ values for zoniporide were within the reported range for selective NHE1 inhibition, supporting the conclusion that NHE1 mediates this effect [28]. Our findings are consistent with prior studies showing that NHE1 inhibition reduces migration and invasiveness in 143B cells and other cancer models [7]. In this study, cariporide inhibited NHE1 and subsequently decreased the elevated intracellular pH seen in 143B cells, which was further associated with an inhibition of reactive oxygen species-induced migration [7]. Importantly, inhibition of NHE1 also reduced proliferation in both sarcoma cell lines, although this effect was less pronounced than the effect on migration. Similar dissociations between migration and proliferation have been reported in other cancer systems, suggesting that NHE1 plays a dominant role in regulating motility, while proliferative control involves additional mechanisms [18,46]. This distinction is consistent with broader mechanistic analyses identifying NHE1 as a key regulator of cytoskeletal dynamics and localized pH signaling during tumor cell migration [47,48].

Notably, the sensitivity of undifferentiated hFOB cells to NHE1 inhibition, in contrast to differentiated osteoblastic cells, indicates that NHE1 function is particularly important in progenitor-like cellular states characterized by high migratory and metabolic plasticity. This stage-dependent requirement for NHE1 is consistent with its established role in regulating cytoskeletal dynamics, intracellular pH homeostasis, and undifferentiated, highly motile cellular phenotypes [49,50,51,52,53].

4.5. CHP2 Protein Levels Are Not Strongly Regulated by Serum Deprivation

CHP2 is preferentially expressed in cancer cells and exhibits a higher binding affinity for NHE1 than CHP1 [18,19]. To determine whether serum deprivation regulates CHP expression, protein levels of CHP1 and CHP2 were assessed following prolonged (72 h) deprivation. While CHP2 has been primarily studied in cancer cells, its expression in bone-derived cells remains poorly defined. Here, we show that CHP2 is detectable in immortalized hFOB osteoblasts, extending its expression beyond malignant contexts.

Although serum deprivation did not result in statistically significant changes in CHP1 or CHP2 protein abundance, consistent trends toward altered expression of both proteins were observed across the different cell lines under serum-deprived conditions compared with serum-containing controls. While these differences did not reach statistical significance, they suggest that serum deprivation may influence CHP expression in a cell-type–dependent manner. These findings indicate that serum deprivation does not strongly regulate CHP2 at the level of protein abundance, but may differentially modulate CHP expression depending on cellular context, consistent with prior studies showing that although nutrient stress alters CHP2 function, its protein expression increases modestly over prolonged serum deprivation in non–small cell lung cancer cells [18]. In the same study, CHP1 protein expression was also unaffected by serum deprivation [18]. Together, these observations suggest that the effects of CHP on NHE1 activity and downstream cellular behaviors are mediated primarily through functional mechanisms, such as modulation of NHE1 activity, stability, or stress responsiveness, rather than through transcriptional or translational upregulation.

4.6. Silencing of CHP2 Impairs Migration and Proliferation in Bone Sarcoma Cells

CHP2 has previously been implicated in promoting malignant characteristics across multiple cancer types [18,19,54,55,56]. In our study, silencing of CHP2 significantly reduced migration in both 143B and SW1353 cells, phenocopying the effects of NHE1 inhibition. This supports the existence of a functional CHP2–NHE1 axis that is required for efficient cancer cell migration.

In addition to its effects on migration, CHP2 silencing significantly inhibited proliferation in both sarcoma cell lines. This effect was more pronounced than that observed with NHE1 inhibition alone, suggesting that CHP2 may influence proliferation through both NHE1-dependent pH regulation and additional stress-adaptive pathways. Although the phenotypic overlap between NHE1 inhibition and CHP2 silencing strongly supports a functional CHP2–NHE1 axis, the more pronounced effect of CHP2 silencing on proliferation suggests that CHP2 may additionally influence NHE1-independent stress-adaptive pathways. Previous studies demonstrating enhanced proliferation upon CHP2 overexpression and reduced proliferation following CHP2 knockdown further support this interpretation [17,18,54,56,57]. Together, these results identify CHP2 as a key regulator of both migratory and proliferative capacity in bone sarcoma cells, with these effects particularly linked to NHE1 function under conditions of serum deprivation–induced stress.

4.7. Integrated Model and Implications

Direct measurements of NHE1 activity support this model. Serum deprivation did not uniformly increase NHE1 activity across cell types: NHE1 activity was increased in SW1353 cells and differentiated hFOB cells but remained unchanged in 143B cells and undifferentiated hFOB cells. Importantly, silencing of CHP2 reduced NHE1 activity only under serum-deprived conditions, indicating that serum deprivation imposes a CHP2-dependent regulatory state on NHE1 function. Notably, the absence of a measurable increase in total NHE1 activity does not preclude a requirement for NHE1 in migration, as localized or stress-maintained NHE1 function may be sufficient to support leading-edge pH regulation [49,50,58]. These findings demonstrate that although inducible increases in NHE1 activity are cell-state dependent, maintenance of NHE1 activity under stress requires CHP2 and is sufficient to support migratory behavior.

Collectively, our data support a model in which NHE1 activity is a dominant determinant of migratory capacity in bone sarcoma cells, while CHP2 functions as a stress-dependent regulator that may enable NHE1-dependent behavior and supports proliferative fitness. Although serum deprivation does not uniformly increase NHE1 activity, both 143B and SW1353 cells require NHE1 activity to migrate efficiently, and both depend on CHP2 to maintain this function under stress. These findings suggest that targeting CHP2 may offer a strategy to disrupt malignant migration and proliferation while avoiding the broader systemic effects associated with direct NHE1 inhibition. Given the limited efficacy and significant toxicity of current osteosarcoma treatments, further investigation of the CHP2–NHE1 regulatory axis may provide new opportunities for therapeutic intervention.

5. Conclusions

This study demonstrates that bone sarcoma cells exhibit a time-dependent adaptive response to serum deprivation that supports recovery of migratory and proliferative capacity under metabolic stress. Our findings identify NHE1 as a key regulator of migration and establish CHP2 as a stress-dependent modulator of NHE1 function. Although serum deprivation does not uniformly increase NHE1 activity, maintenance of NHE1 function under stress requires CHP2, and CHP2 expression is necessary to sustain malignant cell migration and proliferation. Together, these results define a functional CHP2–NHE1 regulatory axis that contributes to stress-adaptive behavior in bone sarcoma cells. Importantly, these conclusions are based on studies in the 143B and SW1353 cell lines, and the distinct responses observed between these models indicate that regulation of migration and proliferation under serum deprivation is cell-type dependent. Future work will be needed to determine the relevance of these findings across additional sarcoma subtypes and to define how the CHP2-NHE1 complex interacts with key signaling pathways regulating migration and proliferation.