Introduction
Osteosarcoma is the most common primary malignant bone tumor, predominantly affecting children and adolescents, characterized by its highly aggressive nature and a propensity for early metastasis, particularly to the lungs (1). Despite advancements in chemotherapy regimens, the prognosis for patients, especially those with metastatic disease or a poor response to chemotherapy, remains dismal. Previous studies have elucidated the role of genetic factors in modulating these clinical outcomes (2,3). Among these, mutations in the tumor suppressor gene TP53 and polymorphisms in the glutathione S-transferase P1 (GSTP1) gene have been implicated.
p53, known as the ‘guardian of the genome,’ is a tumor suppressor protein that plays a crucial role in preventing cancer formation. Under normal conditions, p53 regulates the cell cycle and promotes apoptosis or cell repair in response to DNA damage (4,5). Mutations in the TP53 gene, which encodes the p53 protein, are among the most common mutations found in human cancers. These mutations often result in a loss of normal p53 function and can confer oncogenic properties to the protein, contributing to tumor progression and resistance to chemotherapy (6,7). The missense hot-spot mutation R175H was specifically selected for detailed analysis in the present study as it is a canonical ‘hot-spot’ allele with well-documented gain-of-function effects that promote chemoresistance in sarcoma models, and since institutional molecular prescreening performed between 2017 and 2022 identified R175H in 22% of sequenced osteosarcoma samples, the highest single-allele frequency observed locally (7), making it highly relevant to the patient population of the present study (8–11).
GSTP1 is part of the GST family, which plays a significant role in detoxification by conjugating glutathione to a wide range of substrates, including chemotherapy agents. This process can lead to the inactivation of drugs intended to kill cancer cells, contributing to chemotherapy resistance (12). High levels of GSTP1 expression have been associated with poor responses to chemotherapy. Furthermore, GSTP1 expression is often upregulated in response to oxidative stress and chemotherapy, which can further enhance resistance to treatment (13,14). Whereas earlier pharmacogenetic studies centred on the germline Ile105Val (rs1695) polymorphism, protein-level assessment, such as that undertaken in the present study, captures genotype-independent mechanisms, including somatic copy-number gain, promoter methylation, nuclear factor erythroid 2-related factor 2 (NRF2)-driven transcription and post-translational stabilisation (15,16). This integrative read-out may better reflect the functional detoxification capacity of the tumor.
Despite extensive investigation into the roles of mutant p53 or GSTP1 expression in osteosarcoma, the findings have been inconsistent and have often failed to account for the complex interplay between a specific TP53 hot-spot and GSTP1 expression (17,18). The present study, therefore, investigates whether the R175H-mutant p53 and upregulation of GSTP1 are jointly associated with histological chemoresponse and metastatic behaviour in osteosarcoma within a uniformly treated cohort.
Discussion
The present study findings reveal a significant and robust correlation between GSTP1 expression and mutant p53 expression in chemotherapy response. The tumor suppressor protein p53, encoded by the TP53 gene, plays a crucial role in regulating cellular responses to stress, particularly DNA damage. p53 is often referred to as the ‘guardian of the genome’ due to its role in preventing genomic instability by inducing cell cycle arrest, DNA repair, apoptosis or senescence in response to cellular stressors (24–26). The correlation between p53 status (wild-type vs. mutant-type) and response to chemotherapy in cancer treatment is complex and multifaceted; its status in cancer cells significantly affects the effectiveness of chemotherapy and radiation therapy, two main pillars of cancer treatment (27,28).
The present study cohort consisted predominantly of male adolescent cohort patients (mean age, 18 years), with tumors chiefly in the distal femur. High amputation rates (63.9%) reflected site-specific surgical constraints, yet the histopathological markers, mutant p53 and GSTP1, proved far more informative for treatment response than for metastatic behaviour. Both proteins were upregulated in every case with a poor chemotherapy response (IRS≥6), showing strong, significant associations with chemoresistance. This dichotomy underscores that chemo-protective mechanisms (e.g., defective apoptosis via mutant p53 or drug detoxification via GSTP1) operate independently of the biological pathways driving metastatic spread, highlighting the value of these markers for predicting chemoresponsiveness across clinical subgroups while signaling the need for additional molecular profiling to clarify determinants of metastasis in osteosarcoma.
The observation that mutant p53 and GSTP1 expression specifically predicts chemoresponse but not metastasis is consistent with the distinct prognostic roles of other established histopathological parameters. Beyond the Huvos score, which remains the standard measure of tumor necrosis, parameters such as post-treatment mitotic index and microscopic vascular invasion (MVI) are also prognostically significant. For example, a high mitotic index (≥10 mitoses/10 high-power field) in the resection specimen is an independent predictor of poor survival, reflecting a failure to achieve cell cycle arrest post-treatment (29). This aligns with the present molecular findings, as both indicate a chemoresistant phenotype. The prognostic utility of mitotic rate appears specific to post-treatment evaluation, as its value in pre-treatment biopsies has been shown to be limited (30).
Conversely, and in contrast to the present p53 and GSTP1 results, MVI is a powerful predictor of metastatic progression; its presence is strongly associated with higher rates of metastasis, local recurrence and mortality. This risk is markedly amplified in patients who are also poor chemo-responders, whose 5-year survival has been reported to be as low as 24% (31). This comparison reinforces the conclusion that the molecular mechanisms of chemoresistance, indicated in the present cohort by mutant p53 and GSTP1 expression, appear to be distinct from the biological pathways, such as MVI, that primarily govern metastatic dissemination.
This lack of association contrasts sharply with a recent study that estimated that the association of GSTP1 upregulation and mutant TP53 led to to a roughly two-fold increase in metastatic risk; therefore, the null finding of the present study most plausibly reflects the limited cohort size and relatively short follow-up, which together reduce power to capture late-emerging metastases (32). Future multicentre studies with larger samples and extended surveillance will be required to resolve this discrepancy.
Mutations in the TP53 gene often result in the loss of wild-type p53 function, which disrupts the ability of a cell to undergo apoptosis in response to DNA damage induced by chemotherapy (33). This loss of function can occur through various mechanisms, including disruptions in DNA binding, which prevents p53 from activating its target genes involved in cell cycle arrest and apoptosis (34,35). Mutant p53 can modulate the tumor microenvironment to create conditions that support tumor survival and growth, as well as resistance to chemotherapy (36). This includes promoting angiogenesis, immune evasion and the secretion of factors, such as IL-6, IL-8, HGF or IGF-1, that support tumor cell survival (37,38).
p53 mutations have been linked to increased expression of multidrug resistance proteins, such as P-glycoprotein, which can actively efflux chemotherapy agents from cancer cells, reducing their effectiveness (39). Additionally, p53 mutations can modulate the expression of genes involved in DNA repair, allowing cancer cells to survive and proliferate despite the genotoxic effects of chemotherapy (25).
Mutant p53 proteins often acquire gain-of-function (GOF) properties that contribute to tumor progression and chemotherapy resistance. These GOF properties include enhanced drug efflux, metabolism, promotion of survival, inhibition of apoptosis, increased DNA repair, suppression of autophagy, enhanced microenvironmental resistance and induction of a stem-like phenotype. For example, specific p53 mutations, such as R175H and R273H, have been shown to increase resistance to chemotherapeutic agents, such as cisplatin and etoposide, by disrupting the normal p53-mediated apoptosis pathway (40,41).
These mutations can also affect the expression of various genes involved in metabolism and drug effects, further contributing to resistance. Specifically, in osteosarcoma, the R270C p53 mutant does not suppress the transcriptional activation function of wild-type p53, leading to resistance to doxorubicin, a common chemotherapy agent (42). Additionally, the expression of mutant p53 is associated with increased expression of ONZIN (Plac8), which contributes to osteosarcoma metastasis through the CXCL5-MAPK signaling pathway (43).
GSTP1 is a critical enzyme involved in the detoxification of xenobiotics and carcinogens by catalyzing the conjugation of glutathione to reactive intermediates. This enzyme plays a crucial role in cellular defense mechanisms against toxic and carcinogenic compounds. However, its expression in cancer cells has been associated with resistance to chemotherapy, affecting the efficacy of treatment various types of malignancies, including breast, ovarian, lung, colorectal, prostate, gastric and hepatic cancers, as well as leukemias, where elevated GSTP1 levels have been consistently linked to reduced sensitivity to commonly used chemotherapeutic agents (44,45).
GSTP1 is a phase II metabolic enzyme that catalyzes the conjugation of reduced glutathione to various substrates, including chemotherapy agents, leading to their detoxification and excretion. Upregulation of GSTP1 in osteosarcoma cells has been associated with an increased capacity to deactivate chemotherapy drugs, thereby reducing their cytotoxicity and effectiveness (46). A study indicated that osteosarcoma cells with increased GSTP1 expression exhibit resistance to various chemotherapeutic drugs, including doxorubicin, cisplatin and methotrexate, which are commonly used in the treatment of osteosarcoma (47).
The GSTP1 mechanism contributes to chemotherapy drug resistance by its ability to conjugate glutathione to chemotherapy drugs, thereby neutralizing their cytotoxic effects. This conjugation increases the solubility of the drugs, leading to their rapid elimination from cells and reducing the effects of their cytotoxic chemotherapy. For example, GSTP1 has been shown to detoxify platinum-based compounds, such as cisplatin and oxaliplatin, which are commonly used in the treatment of various types of cancer (13,48). Additionally, GSTP1 can modulate signaling pathways associated with cell survival and apoptosis. For instance, GSTP1 has been shown to inhibit the pathway of Jun N-terminal kinase, a protein involved in critical mediation of apoptosis or pro-apoptotic signaling, thereby promoting the survival of cancer cells from chemotherapy (49).
By contrast, pharmacogenetic investigations have centred on the germline Ile105Val (rs1695) polymorphism of GSTP1, yet its association with chemoresponse and survival has been inconsistent (50). Measuring GSTP1 at the protein level provides a more integrative read-out, as expression reflects not only such coding variants but also somatic copy-number changes, promoter methylation, NRF2-driven transcription and post-translational stabilisation during oxidative stress; this broader regulatory capture may explain why expression status, but not Ile105Val genotype alone, was robustly associated with chemoresistance.
In addition, in the present study, contrary results were found between GSTP1 expression and mutant p53 expression regarding metastasis events in patients with osteosarcoma at Cipto Mangunkusumo General Hospital, showing no significant associations. Several studies have explored the genetic and molecular underpinnings of GSTP1 expression in osteosarcoma, particularly its potential role in metastasis. A comprehensive study conducted by Li et al (50) investigated the role of genes involved in metabolic and transport pathways, including GSTP1, in osteosarcoma survival after chemotherapy. The findings revealed no significant association between GSTP1 expression and metastatic disease in patients with osteosarcoma. Similarly, a pharmacogenetic analysis of primary and metastatic osteosarcoma failed to identify GSTP1 among the genes whose expression is associated with metastasis (51).
The mechanistic role of GSTP1 in cancer metastasis remains unclear. Although its detoxification function could theoretically influence metastatic potential by altering the tumor microenvironment or modulating responses to chemotherapy, current studies have not established a direct mechanistic link between GSTP1 expression and metastatic behavior in osteosarcoma cells. Some investigations have examined GSTP1 in relation to chemoresistance and apoptosis, but they have not directly addressed its impact on metastasis. Although GSTP1 participates in detoxification and has been implicated in chemotherapy resistance, its direct contribution to osteosarcoma metastasis remains uncertain as available evidence has not demonstrated a clear association between GSTP1 expression and metastatic progression. The complexity of metastasis, which involves multiple genetic and environmental factors, likely diminishes the influence that any single gene, including GSTP1, may exert on metastatic outcomes (46,52).
The complexity of cancer metastasis, which involves many genetic and environmental factors, likely diminishes the potential impact of a single gene, such as GSTP1, on metastasis outcomes. Other studies suggest various mechanisms and pathways that could be more directly involved in the osteosarcoma metastasis process. For example, research has highlighted the importance of signaling pathways such as PI3K/AKT/mTOR and their roles in tumor growth and metastasis in osteosarcoma. These pathways may offer a more direct link to metastatic behavior than GSTP1 (53,54).
Several studies have investigated the correlation between p53 mutations and metastasis in osteosarcoma, with varying conclusions. For example, a study by Wunder et al (55) found no evidence that p53 mutations predict the development of metastases in patients with high-grade osteosarcoma. The p53 mutation status did not differentiate between patients who presented with localized or metastatic disease. Similarly, research by Overholtzer et al (56) reported that the p53 mutation status was concordant between primary tumors and matched metastases in patients with osteosarcoma, suggesting that p53 mutations are early events in tumorigenesis rather than drivers of metastasis.
Mutant p53 plays a critical role in osteosarcoma metastasis. The R270C mutant p53, equivalent to human R273C, has been shown to bind to chromatin near the transcription start sites of various genes, altering their expression. This mutant form exhibits a different binding profile from wild-type p53 and does not suppress the transcriptional activation function of the wild-type protein in osteosarcoma cells. Although the deletion of this mutant p53 reduces tumor growth, it does not affect invasion or prevent metastasis in vivo. This suggests that while mutant p53 contributes to tumor growth, it may not directly affect the metastatic ability of osteosarcoma cells (42).
The clinical implication based on the results of the present study is that the expression of mutant p53 and GSTP1 can be used as for diagnostic purposes in patients with osteosarcoma, thereby serving as a predictor of chemotherapy response, prognosis and decision-making in the management of affected patients.
The present study focused on a specific population of patients with osteosarcoma, providing valuable insights within a defined geographic and demographic context, thus yielding relevant data for that population. Additionally, this study was more feasible to implement due to its cross-sectional design, which eliminates the need for long-term follow-up. Conducted at a single point in time, this study offers a faster and more cost-effective approach compared with longitudinal studies.
A key limitation of this study is its cross-sectional design, which involves a single data collection point, thereby providing only a snapshot of the population at a particular moment. This limitation is particularly pertinent in research involving dynamic biological processes, such as chemotherapy response or metastasis development, where longitudinal changes and trends may be crucial to understanding the underlying mechanisms. Moreover, the relatively small, single-centre cohort and the retrospective design may reduce statistical power, introduce selection bias and limit the generalisability of the study findings.
In conclusion, the present study found a significant association between mutant p53 expression and GSTP1 expression and poor chemotherapy response in patients with osteosarcoma. Meanwhile, the association between mutant p53 expression and GSTP1 expression and the occurrence of metastasis was not significant in patients with osteosarcoma. Further research should focus on longitudinal studies that track the expression levels of GSTP1 and mutant p53 from initial diagnosis to the development of metastasis and the treatments provided. Further research requires comprehensive genomic profiling studies to explore the interactions of GSTP1 and mutant P53 with other genes, as well as the signaling pathways involved in metastasis and chemotherapy response in osteosarcoma, to provide a more comprehensive understanding of the biology of this disease.