Among the 64 subjects with metastatic lesions, 47 had bone-involvement metastasis and 17 did not have bone metastasis. The demographic and clinical characteristics of the study participants based on bone involvement and bone only metastasis are outlined in Table 2.

Among subjects with bone-involvement metastasis, the most common locations were the vertebrae (70.2%), costae and/or sternum (57.4%), and pelvis (27.7%). Most of the subjects with bone metastases were in T4 and N3 stages, with 50.0% having 2–5 metastatic sites. Osteoblastic-type lesions were the most common, accounting for 71.4% of cases, as shown in Fig. 1.

DEG analysis was performed by comparing gene expression levels in the bone-involvement metastasis group with the non-metastasis group (Table A2). A comparison of the expression levels of the top 40 genes that differed between the two groups can be seen in the volcano plot in Fig. 2a. Based on the DEG analysis, the CXCL8 gene was downregulated and the CXCR4 gene was upregulated, as detailed in Table 3.

DEG analysis was conducted by comparing subjects with only bone metastasis to those without metastasis (Table A3). The volcano plot in Fig. 2b illustrates the comparison of the expression levels of the top 40 genes that differed between the two groups. The CXCR4 gene was found to be upregulated in subjects with bone metastasis only, as shown in Table 4.

In this study, most subjects were male (75.8%), with an average age of 48 years at diagnosis. This finding is consistent with previous studies in Indonesia that have reported that nasopharyngeal carcinoma cases occur predominantly in males.^[16] Adham et al.^[17] reported that men were 2.4 times more likely to be diagnosed with nasopharyngeal cancer than women. A significant portion of the subjects in this study (73.4%) had bone metastasis, followed by lung and liver metastasis. This aligns with previous reports suggesting that bone is the most frequently involved organ in nasopharyngeal cancer.^[18,19] Primary bone metastasis is the most common finding in patients who present with metastasis at the time of diagnosis, accounting for 64%–67% cases.^[20] Most subjects with bone metastasis were male, with metastases primarily located in the vertebrae (70.2%), costae and sternum (57.4%), and pelvis (27.7%). These findings correspond with a previous report stating that the thoracic and lumbar vertebrae, sternum, costae, ilium, and femur are the most common metastatic sites.^[20] This is associated with a greater presence of red bone marrow and more vascularization at those sites, which facilitate the homing of cancer cells.^[21] The majority of primary bone metastasis lesions in this study were identified at 2–5 sites (50.0%). This aligns with previous studies showing that primary metastasis in nasopharyngeal cancer typically involves only one organ but with multiple lesions.^[22]

The most frequent type of bone metastasis lesion in this study was osteoblastic (71.4%). The radiological findings of this study, however, slightly differed from those of previous studies. In a study by Sham et al.^[23] radiological images of bone metastasis in nasopharyngeal cancer showed lytic lesions (66%), mixed lesions (12.8%), and sclerotic lesions (21.2%). These differences likely stem from variations in criteria, as Sham et al.^[23] defined bone metastasis as a radiological abnormality accompanied by pain resulting from the metastasis.

Analysis of DEGs found that CXCL8 expression levels decreased, while CXCR4 expression levels significantly increased in the group with bone metastasis involvement. When comparing between the bone-only metastasis group and the non-metastasis group, only CXCR4 showed a significant difference gene expression.

The CXCR4 gene is involved in adhesion, invasion, and the migration of cancer cells to bone^[11], as seen in breast, nasopharyngeal, kidney, lung, prostate, pancreatic, and ovarian cancers and melanoma. Higher levels of CXCR4 expression in NPC linked to tumor growth, angiogenesis, metastasis, and treatment resistance. Additionally, increased CXCR4 expression levels are linked to epithelial-mesenchymal transition (EMT) in various types of cancer. In ovarian cancer, the upregulation of CXCR4 is accompanied by increased expression levels of vimentin and snail, along with decreased expression levels of E-cadherin. EMT initiates metastasis, enabling cancer cells to migrate to other organs through the systemic circulation.^[24,25]

The binding of CXCR4 to its ligand, CXCL12, triggers a conformational change in the receptor molecule. This binding activates G proteins, leading to the conversion of GDP to GTP. The GTP molecule then binds to the α and βγ subunits, causing them to dissociate. The βγ subunit activates two signaling pathways: phospholipase C-β (PLC-β) and PI3K pathways. Meanwhile, the α subunit affects gene transcription and expression through the PI3K-AKT-NF-κB, MEK1/2, and ERK1/2 pathways.^[25] As a result, various cellular changes occur, including an increase in intracellular calcium levels, gene transcription, chemotaxis, cell survival, and cell proliferation.^[25]

CXCR4 expression has been linked to bone metastasis, especially in breast cancer.^[26-29] The role of CXCR4 in bone metastasis involves effects on the processes of cancer cell adhesion, invasion, and migration to the bone. Increased expression levels of CXCR4 are linked to the development of bone metastases in breast cancer.^[11] The findings of this study also confirmed the role of CXCR4 in bone metastasis in nasopharyngeal cancer.

CXCL8 is typically challenging to detect under physiological conditions.^[30] This chemokine plays a significant multifunctional role in modulating tumor proliferation, invasion, and migration through autocrine or paracrine signaling. The binding of CXCL8 to CXCR1/2 activates intracellular signaling pathways that regulate angiogenesis and cell proliferation, migration, survival, invasion, and motility.^[31] Increased expression levels of CXCL8 have been reported in nasopharyngeal cancer tissue.^[32]

CXCL8 has been shown to have different effects during the process of bone metastasis. Iguchi et al.^[33] reported that in an in vivo study involving lung cancer cells, CXCL8 has an inhibitory effect on the occurrence of bone metastasis. This effect arises from its inhibition of osteoclast resorption, which is a necessary initial step for bone metastasis to occur. Bone resorption allows cancer cells to penetrate the mineralized matrix within bone tissue. Other studies of breast cancer have shown that increased levels of IL-8 are associated with osteolytic bone metastasis due to heightened osteoclast formation and the stimulation of RANKL production through its binding to CXCR1.^[34] A study of the role of IL-8 in multiple myeloma reported that increased CXCL8 levels are linked to cell proliferation, osteoclast formation, and anti-apoptotic effects.^[35-37] In this study, a decrease in CXCL8 expression levels was observed in the group with bone-involvement metastasis. IL-8 promotes the maturation and differentiation of osteoclasts, thus enhancing bone resorption processes. The reduced expression levels of CXCL8 shifts the balance of bone remodeling toward a dominance of osteoblastic activity.^[34,38]

This study is the first to simultaneously evaluate the role of the expression levels of multiple genes by measuring mRNA levels in patients with nasopharyngeal cancer, specifically focusing on bone metastases. The study’s limitations included a retrospective design, small subgroup subjects, and a focus solely on the primary tumor by examining cancer cell gene expression levels without assessing the host microenvironment in metastases.