HMGB3 promotes brain metastasis in lung adenocarcinoma by recruiting SSBP1 for nuclear translocation to remodel mitochondrial metabolism

Background: Brain metastasis, a leading cause of death in patients with lung adenocarcinoma (LUAD), arises from tumor cells adapting to the brain's unique microenvironment of the brain through metabolic remodeling regulated by key oncogenes. Here, we aimed to determine the role of high mobility group protein B3 (HMGB3) in regulating tumor cell metabolism to promote the progression and brain metastasis in LUAD. Methods: In this study, a LUAD cell model predisposed to brain metastasis was developed in this study, and differentially expressed genes HMGB3 was identified. HMGB3 expression in LUAD was then validated through a series of in vitro and in vivo experiments, including western blotting, single-cell RNA sequencing (scRNA-seq), and immunohistochemistry (IHC). Tissue microarray (TMA) and LUAD brain metastasis clinical samples were used to explore the expression of HMGB3 and its clinical significance in LUAD. Additionally, we performed gene enrichment analysis on scRNA-seq and bulk RNA-seq, along with western blotting, to identify the pathways through which HMGB3 may cause brain metastasis. We then used coimmunoprecipitation and mass spectrometry to identify protein single-stranded DNA binding protein 1 (SSBP1), which interacts with HMGB3. Finally, gain and loss-of-function and rescue experiments were conducted to study SSBP1's roles in LUAD cells. Results: HMGB3 upregulation in LUAD primary tumors and brain metastases associated with poor prognosis independent of metastasis. HMGB3 enhanced the migration, invasion, and epithelial-mesenchymal transition (EMT) capabilities of LUAD cells in vitro and promotes the occurrence and development of brain metastasis in vivo. Mechanistically, HMGB3 recruited SSBP1, inducing its nuclear translocation and reprogramming mitochondrial metabolism, thereby increasing cytoplasmic reactive oxygen species (ROS) levels, which activated the phosphatidylinositol 3-kinase–protein kinase B (PI3K-Akt) pathway by downregulating phosphatase and tensin homolog (PTEN), enhancing tumor cell proliferation, migration, invasion, and EMT. Conclusions: This study demonstrated that HMGB3 functions as a driver gene in the brain metastasis of LUAD, promoting metabolic adaptation of tumor cells to the brain microenvironment through the regulation of mitochondrial metabolism, thereby offering potential therapeutic targets for LUAD brain metastases. Overall design: A xenograft model of brain metastasis was generated using 4-week-old male BALB/c nude mice. To address potential injection failures and allow humane euthanasia of severely ill mice, 10–12 mice were assigned to each group, ensuring that eight mice were used for the final analysis. Mice were anesthetized using 1% pentobarbital in phosphate-buffered saline (PBS), fixed supine, and disinfected. The left ventricle was located by gently pressing the chest (second to third intercostal spaces) and marked. A 1 mL syringe was prepared with a 200 µL air gap, filled with 200 µL of cell suspension containing cells (H2030) at a concentration of 5 × 10^4 cells/mL, and then air bubbles were removed. The syringe was inserted vertically 3–5 mm at the marked point; bright red blood flow confirmed left ventricle entry, and 100 µL of cell suspension was injected within 10 s. The needle was slowly withdrawn, and the bleeding was stopped by applying pressure. Mice were allowed to recover on a warming pad and housed in a specific pathogen free environment. Five weeks later, the mice were euthanized using CO2, then soaked in 75% ethanol, and dissected using sterile tools to extract the brain and isolate the tumor masses, which were then minced and enzymatically digested using collagenase I/II and trypsin. The digested tissue was neutralized with complete medium, centrifuged at 1,000 rpm for 5 min, then resuspended in complete medium, and plated in culture dishes. Cells are incubated at 37°C with 5% CO2 for 24–48 h, after which tumor cells with parental morphology were observed, medium was replaced, and cells are expanded. Once the cell number expanded to 106, the first-generation LUAD brain metastasis cells (Lbrm1) were subjected to a second round of intracardiac injection using the same method. After the next round of brain metastatic tumors formation, H2030-Lbrm2 cells were primarily cultured using the same method. Ultimately, we obtained H2030-Lbrm1, Lbrm2, Lbrm3, Lbrm4, Lbrm5, and Lbrm5, which were designated as H2030-Lbrm and used for in vitro experiments as they had highest brain metastasis properties. To downregulate the expression of HMGB3, HMGB3-targeting small interfering RNA (siRNA) siHMGB3: 5'-GCAGAUAAAGUGCGCUAUGAU-3' along with a negative control (siNC), were synthesized by Tsing Ke (Shanghai, China). The transfection experiments for siRNA were performed according to the manufacturer's guidelines for Lipo3000 as follows. Cells (H2030-Lbrm) were seeded in six-well plates at 70%–80% confluence. Transfection complexes including 250 µL Opti-MEM I, 7 µL lipofectamine 3000, and 15 pmol siRNA, incubated for 10–20 min, and added to each well. The medium was replaced after 6 h, and the cells were cultured for 48–72 h.