Cholesterol and steroid synthesis pathways may be involved in the inhibition of osteosarcoma cell viability by calcium-sensing receptor antagonism

Introduction

Osteosarcoma, the most common primary malignant bone tumor, affects primarily children and adolescents. It is highly invasive and prone to distant metastasis, making it clinically challenging (Mirabello, Troisi & Savage, 2009; Ying et al., 2023). Currently, the standard treatment for osteosarcoma involves surgical resection in combination with neoadjuvant and adjuvant chemotherapy. While this multimodal approach has significantly improved patient survival over the past few decades, recurrence and metastasis remain major obstacles, and the five-year survival rate remains low (Marina et al., 2004; Mehrvar et al., 2023). Therefore, the identification of new therapeutic targets and effective drug regimens is essential for improving patient outcomes.

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor widely expressed in various tissues that is primarily responsible for regulating calcium ion (Ca²⁺) homeostasis (Alqudah et al., 2022; Brown & MacLeod, 2001). CaSR plays a crucial role in the development and progression of multiple malignant tumors, with its activation or inhibition influencing tumor cell proliferation, apoptosis, invasion, and metastasis (Chakravarti et al., 2009; Wolffs et al., 2025). In bone-related diseases, the role of CaSR is particularly significant. CaSR is aberrantly expressed in osteosarcoma cells and may regulate tumor growth and invasiveness through intracellular calcium signaling pathways (Tuffour et al., 2021). However, its precise role in the progression of osteosarcoma and its potential as a therapeutic target are still poorly understood.

Recent studies indicate that calcium signaling, particularly via the CaSR, plays an important role in lipid metabolism. CaSR activation regulates intracellular calcium levels through pathways such as Gq/11 and Gi/o, thereby affecting lipid synthesis and inducing the expression of lipogenic genes in adipocytes and hepatocytes (He et al., 2011b). Calcium availability also influences cholesterol biosynthesis by modulating SREBP transcription factors and key enzymes such as β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase (Wang et al., 2017). Disruption of calcium homeostasis may aberrantly activate SREBPs and enhance cholesterol synthesis, whereas calcium-dependent kinases like AMPK can suppress this process (Jeon, 2016). These insights suggest a potential interplay between CaSR-mediated calcium signaling and lipid–cholesterol metabolic regulation. Although direct evidence remains limited, this relationship implies that CaSR may modulate related metabolic pathways and therefore merits further investigation.

Previous studies have shown that CaSR expression is upregulated in osteosarcoma cells, suggesting its involvement in tumor proliferation and invasiveness (Zhao et al., 2020). Although these studies established a connection between CaSR activity and osteosarcoma cell behavior, the functional consequences of CaSR modulation and the underlying molecular mechanisms remain poorly understood. CaSR antagonists, or negative allosteric modulators of the calcium-sensing receptor, have demonstrated therapeutic potential in several diseases by interfering with calcium-dependent signaling pathways (Kim & Wysolmerski, 2016; Yang et al., 2023). Investigating their transcriptomic effects in osteosarcoma may therefore provide new insights into CaSR-mediated regulatory networks and enhance our understanding of the molecular mechanisms driving tumor progression.

This study was designed as an exploratory investigation to evaluate the effects of CaSR antagonism on human osteosarcoma cells and to uncover associated molecular mechanisms using transcriptomic analysis. Rather than assessing therapeutic selectivity or safety, our aim was to gain mechanistic insights into CaSR-regulated pathways and to identify potential targets for future therapeutic development in osteosarcoma.

Materials and Methods

Results

CaSR antagonism regulates the expression of cholesterol synthesis and steroid synthesis pathway genes in Saos-2 cells

To investigate the effect of a CaSR antagonist on human osteosarcoma cells, Saos-2 cells were treated with five µM NPS-2143 and transcriptome sequencing was performed. We identified 927 differentially expressed genes between the NPS-2143–treated group and the control group, of which 378 genes showed increased expression and 549 genes showed decreased expression (Fig. 2A). KEGG enrichment analysis of the differentially expressed genes showed that the steroid biosynthesis pathway (hsa00100, P-adjust < 0.0001, Fig. 2C) had the highest degree of enrichment, the enriched genes including LIPA (lipase A, lysosomal acid type, LIPA), LBR (lamin B receptor, LBR), HSD17B7 (hydroxysteroid 17-beta dehydrogenase 7, HSD17B7), LSS (lanosterol synthase, LSS), EBP (EBP cholestenol delta-isomerase, EBP), DHCR24 (24-dehydrocholesterol reductase, DHCR24), CYP51A1 (cytochrome P450 family 51 subfamily A member 1, CYP51A1), FDFT1 (farnesyl-diphosphate farnesyltransferase 1, FDFT1), SQLE (squalene epoxidase, SQLE), and MSMO1 (methylsterol monooxygenase 1, MSMO1). Reactome enrichment analysis of the differentially expressed genes showed that the cholesterol biosynthesis pathway (R-HSA-191273, P-adjust < 0.0001, Fig. 2E) had the highest degree of enrichment, the enriched genes including FDPS (farnesyl diphosphate synthase, FDPS), LBR, SREBF1 (sterol regulatory element binding transcription factor 1, SREBF1), HSD17B7, MSMO1, SQLE, MVD (mevalonate diphosphate decarboxylase, MVD), LSS, EBP, DHCR24, CYP51A1, SREBF2(sterol regulatory element binding transcription factor 2, SREBF2), FDFT1, HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase, HMGCR), HMGCS1 (3-hydroxy-3-methylglutaryl-CoA synthase 1, HMGCS1), MVK (mevalonate kinase, MVK), IDI1 (isopentenyl-diphosphate delta isomerase 1, IDI1), and ACAT2 (acetyl-CoA acetyltransferase 2, ACAT2).

CaSR antagonism promotes expression of genes for cholesterol synthesis and steroid synthesis pathways in human osteosarcoma cells

Seven genes shared by cholesterol synthesis and steroid synthesis pathways were selected for validation by qRT-PCR. Compared with the control group, the NPS-2143-treated MG-63 cell group had increased mRNA levels for CYP51A1 (P = 0.021), DHCR24 (P = 0.017), LSS (P = 0.005), MSMO1 (P = 0.028), and SQLE (P = 0.014) (Fig. 4A). The NPS-2143-treated Saos-2 cell group had increased mRNA levels for CYP51A1 (P = 0.022), DHCR24 (P = 0.029), LSS (P = 0.043), MSMO1 (P = 0.045), and FDFT1 (P = 0.002) (Fig. 4B) compared with the control group, which was consistent with the sequencing results.

Discussion

CaSR is a G-protein-coupled receptor that is distributed in a wide variety of tissues and is involved in the regulation of Ca²⁺ homeostasis. In recent years, accumulating evidence has shown that CaSR plays an important role in the occurrence and development of various malignant tumors, and that its activation or inhibition may affect the proliferation, apoptosis, invasion, and metastasis of tumor cells (Chakravarti et al., 2009; Ying et al., 2023). In some malignant cancers, such as breast, prostate, and parathyroid cancers, abnormal expression of CaSR is associated with highly aggressive and metastatic tumor cells (Chakravarti et al., 2009; Ying et al., 2023). CaSR is therefore a potential anti-tumor target, and CaSR antagonists, as negative regulators, show promise in regulating tumor cell proliferation and invasion.

The role of CaSR varies according to tumor type, cell microenvironment, and signaling pathway. CaSR can inhibit tumor growth by inhibiting cell proliferation and promoting apoptosis, but it can also promote cell proliferation, invasion and metastasis in some tumors (Brennan et al., 2013). In a variety of malignancies, abnormal activation of CaSR may promote tumor cell survival and invasion. CaSR antagonists can inhibit ERK1/2, PI3K/Akt, and Wnt/β-catenin signaling pathways, thereby reducing the proliferation of tumor cells and promoting apoptosis. Furthermore, during bone metastasis in breast cancer, CaSR promotes the adaptation of cancer cells to the bone microenvironment (Organista-Juarez et al., 2013), while CaSR antagonists may reduce the capacity of tumor cells to invade bone by reducing Ca²⁺ dependent signaling. Although CaSR antagonists exhibit antitumor activity in some tumors, in other tumor types CaSR may have an inhibitory effect and the use of CaSR antagonists may instead promote tumor growth (Saidak, Mentaverri & Brown, 2009). While CaSR exhibits divergent roles across cancer types, it has been shown to be upregulated in MG-63 osteosarcoma cells compared to normal osteoblasts in vitro, where it may modulate proliferation, apoptosis, and invasion via calcium signaling (Zhao et al., 2020). In this study, the MG-63 and Saos-2 cell lines were selected to validate the effects of CaSR antagonism on osteosarcoma cells. Our results confirm that CaSR antagonism inhibits the viability of osteosarcoma cells in vitro. Therefore, we suggest that CaSR antagonists have potential for use in the treatment of osteosarcoma.

We analyzed the transcriptomic effects of CaSR antagonism on osteosarcoma cells to identify pathways potentially involved in reduced cell viability. We found that a CaSR antagonist promoted the expression of genes associated with cholesterol and steroid synthesis in osteosarcoma cells. qRT-PCR analysis confirmed that the CaSR antagonist promoted the expression of the cholesterol and steroid synthesis-related genes, CYP51A1, DHCR24, LSS, and MSMO1 in MG-63 and Saos-2 cells. LSS (Lanosterol synthase) catalyzes one of the earliest steps in sterol biosynthesis by converting 2,3-oxidosqualene to lanosterol, a key intermediate in the production of cholesterol and other sterol compounds (Guo & Zhang, 2023). This process involves a series of complex chemical reactions, such as intramolecular nucleophilic addition, carbocation rearrangement, and proton transfer to form the tetracyclosterane structure and transforming the linear precursor into a steroid skeleton structure (Nes, 2011). The catalytic activity of LSS is essential in the homeostatic regulation of cholesterol and other sterols because lanosterene is the first tetra-closterane compound in the cholesterol synthesis pathway and is further converted into cholesterol and steroid hormones. This enzyme is mainly located in the endoplasmic reticulum, and its abundance and activity are regulated by feedback from the level of cholesterol to maintain the balance of intracellular sterol metabolism. CYP51A1 (Lanosterol 14α-demethylase) functions downstream of LSS in the early-to-mid stage of cholesterol biosynthesis. It is a cytochrome P450 enzyme that catalyzes the conversion of lanosterol to 4,4-dimethylcholesta-8(9),14,24-trien-3β-ol (Akhtar & Wright, 2015). Cytochrome P450 enzymes are a conserved protein family involved in the metabolism of organic compounds and the biosynthesis of steroids, lipids, and vitamins in eukaryotes. As part of this family, lanosterol 14α-demethylase plays a critical role in sterol biosynthesis by removing the C-14α-methyl group from lanosterol. This demethylation process is the initial control point for the biosynthesis of cholesterol and other sterols, and is critical for ensuring sterol hormone synthesis (Lepesheva & Waterman, 2007). MSMO1 (methylsterol monooxygenase 1) acts at the mid-stage of cholesterol biosynthesis and serves as a key enzyme in this pathway. It primarily catalyzes the three-step monooxygenation required for the demethylation of 4,4-dimethyl and 4alpha-methylsterols, which can be subsequently metabolized to cholesterol. This process is essential for subsequent cholesterol synthesis steps because the correct structure of cholesterol requires the removal of excess methyl groups (He et al., 2011a; Mazein et al., 2013). DHCR24 (δ24-cholesterol reductase, also known as 3β-hydroxysterol Δ24-reductase) is an enzyme that catalyzes the final step in the cholesterol biosynthesis pathway (mevalonate pathway), converting desmosterol to cholesterol (Bai et al., 2022; Fu & Wang, 2024; Zerenturk et al., 2013). Collectively, these four enzymes—LSS, CYP51A1, MSMO1, and DHCR24—cover sequential reactions from the early to the terminal stages of the cholesterol biosynthetic pathway, offering an integrated view of sterol metabolism and suggesting that CaSR signaling may intersect with multiple levels of cholesterol regulation in osteosarcoma cells. Cholesterol synthesis plays a key role in maintaining cell membrane stability, signal transduction, and cell metabolism. Accumulating evidence shows that disrupted cholesterol metabolism is closely related to the occurrence, development, and drug resistance of various malignant tumors. Tumor cells have a high demand for cholesterol to maintain the membrane lipid bilayer stability required for rapid proliferation and survival. At the same time, cholesterol is also a precursor of various steroid hormones and signaling molecules, which play an important role in cancer progression (Huang, Song & Xu, 2020). Inhibitors that target cholesterol synthesis, such as HMG-CoA reductase inhibitors (statins), have anti-tumor effects in multiple cancer types (Tutuska et al., 2020). While tumor cells can drive metabolically dependent proliferation by abnormally activating cholesterol synthesis pathways, our findings suggest that CaSR antagonism may modulate cholesterol biosynthesis-related pathways at the transcriptomic level.

Although cholesterol is essential for maintaining cell membrane integrity, signal transduction, and cell metabolism, its excessive production can disrupt cellular homeostasis. Cholesterol biosynthesis involves multiple redox processes, and excessive cholesterol synthesis leads to the accumulation of reactive oxygen species, which in turn triggers oxidative stress (Guo et al., 2024). Excessive cholesterol synthesis can also lead to mitochondrial dysfunction, DNA damage, protein oxidation, and lipid peroxidation, which ultimately affect cell proliferation and promote apoptosis (Liu, 2024; Xiao et al., 2023). In addition, excess intracellular cholesterol can alter cell membrane composition, increase membrane rigidity and destroy lipid rafts, inhibit key pathways such as PI3K/Akt/mTOR and MAPK/ERK, reduce cell reactivity to growth signals, and limit proliferation (Xiao et al., 2023). Excess cholesterol can be oxidized to form oxysterols, which can activate a variety of signaling pathways leading to the production of pro-inflammatory cytokines. Cholesterol overload also reduces autophagy activity, leading to cell dysfunction. Autophagy is a cellular process in which damaged organelles and proteins are degraded and recycled to maintain cell homeostasis. Accumulation of too much cholesterol impairs autophagy, leading to a build-up of cell debris and organelle dysfunction. This damage can reduce cell proliferation and trigger cell death pathways (Guo et al., 2024; Xiao et al., 2023). These results suggest that excessive cholesterol synthesis may contribute to the activation of cell death pathways and the suppression of cell viability. This finding provides preliminary evidence that CaSR antagonists could exert anti-tumor effects by modulating cholesterol biosynthesis, offering a potential mechanistic link warranting further investigation. This study has several limitations. All experiments were conducted in vitro using osteosarcoma cell lines, which may not fully reflect the complexity of tumor behavior in vivo. Additionally, the absence of in vivo or clinical validation restricts the translational relevance of our findings. Further studies involving animal models and patient-derived samples are warranted to verify the mechanistic insights and assess the therapeutic potential of CaSR antagonists in osteosarcoma. Another limitation is the lack of non-malignant control cells, which precludes evaluation of the compound’s selectivity for tumor versus normal cells. Future work will incorporate normal osteoblasts or mesenchymal stem cells to examine the effects of NPS-2143 on non-tumor cell viability.

Conclusions

This study demonstrates that CaSR antagonism reduces the viability of human osteosarcoma cells in vitro. Transcriptomic analysis revealed upregulation of key genes involved in cholesterol and steroid biosynthesis following treatment with NPS-2143. These findings suggest that targeting CaSR may disrupt lipid metabolism and represent a potential therapeutic strategy for osteosarcoma. Further in vivo studies are needed to validate these results and explore their translational relevance.

Supplemental Information