FABP4 Regulates Cell Proliferation, Stemness, Apoptosis, and Glycolysis in Colorectal Cancer via Modulating ROS/ERK/mTOR Pathway
Ying-Chao Gao, Yuanyuan Wang, Xin Wang, Jianwei Ma, Wei Ming, Na Li, Zengren Zhao
Abstract
Background: Colorectal cancer is a common digestive tract malignancy. This study aimed to expound the functional role of fatty-acid-binding protein 4 (FABP4) and the potential underlying mechanisms in the development of colorectal cancer. Methods: Several techniques were utilized to investigate the role of FABP4 in colorectal cancer. FABP4 mRNA expression was quantified using Real time-quantitative PCR (RT-qPCR). Cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), sphere formation assays and flow cytometry evaluated cell growth, stemness, and apoptosis in SW480 and HT29 cells. Glycolysis was assessed via extracellular acidification rate (ECAR) , lactate production, glucose uptake, adenosine triphosphate (ATP)/adenosine 5'-diphosphate (ADP) ratio, and Glut1 and Elevated lactate dehydrogenase A (LDHA) protein expression. Reactive oxygen species (ROS) levels were analyzed by flow cytometry. Western blot measured the protein expression of FABP4, Proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, Glut1, LDHA, stemness makers (Sox2, Oct4, and ALDHA1), and extracellular regulated protein kinase (ERK)/mammalian target of rapamycin (mTOR) pathway proteins. <i>In vivo</i> experiments, BALB/c nude mice (n = 12) were inoculated with 200 μL HT29 cells (5 × 10<sup>6</sup> cells) transfected with sh-FABP4 or short hairpin (sh)-negative control (NC), forming two groups with 6 mice each. The <i>in vivo</i> mice tumor model allowed for evaluating FABP4's impact on tumor growth. Results: FABP4 was significantly upregulated in colorectal cancer tissues and cells (<i>p</i> < 0.05). FABP4 knockdown markedly inhibited cell proliferation, stemness, and glycolysis, while promoting apoptosis in these cells (<i>p</i> < 0.05). Additionally, FABP4 depletion led to a significant increase in ROS level (<i>p</i> < 0.05). However, N-acetyl-L-cysteine (NAC) (<i>p</i> < 0.05), a ROS scavenger, mitigates these effects. Furthermore, the effects of FABP4 depletion on cell growth, stemness, glycolysis, and apoptosis in colorectal cancer cells were also retarded by NAC (<i>p</i> < 0.05). Notably, FABP4 knockdown also suppressed the ERK/mTOR pathway, suggesting its regulation via ROS (<i>p</i> < 0.05). <i>In vivo</i> study results showed, FABP4 depletion significantly curbed tumor growth in colorectal cancer (<i>p</i> < 0.05). Conclusions: These results suggest that FABP4 depletion inhibits colorectal cancer progression by modulating cell growth, stemness, glycolysis and apoptosis. This regulation occurs through the ROS/ERK/mTOR pathway.