E3 ubiquitin ligase FBXL8 promotes malignant progression of intrahepatic cholangiocarcinoma by ubiquitination-mediated degradation of p53

Intrahepatic cholangiocarcinoma (ICC) is characterized by high invasiveness and extremely poor prognosis, and till now, its effective molecular targets for targeted therapy remain scarce. Dysregulation of ubiquitination modification is a crucial mechanism underlying tumorigenesis and progression. E3 ubiquitin ligase FBXL8 exerts tissue-specific functions in tumors, yet its expression profile, clinical value and molecular mechanisms in ICC remain unclear. This study aims to systematically investigate the expression characteristics of FBXL8 in ICC, analyze its correlation with clinicopathological parameters, clarify its regulatory effects on the biological behaviors of ICC cells and elucidate its potential molecular mechanisms, thereby providing novel scientific evidence and potential targets for prognostic evaluation and molecular targeted therapy of ICC.

Bioinformatic analysis was performed to examine the differential expression of FBXL8 in ICC tissues and adjacent normal tissues based on the TCGA and GEO databases. Tissue microarrays containing cancerous tissues and paired adjacent normal tissues from 79 ICC patients were collected, and immunohistochemical staining was applied to detect the protein expression of FBXL8. According to the results, the patients were divided into a high-expression group (n=44) and a low-expression group (n=35). Pearson χ² test was used to analyze the correlation between FBXL8 expression and clinicopathological features, and Kaplan-Meier test and Log-rank test were employed for survival analysis. In human ICC cell lines RBE and SNU-1079, 2 Fbxl8-targeted siRNAs (siFbxl8#1 and siFbxl8#2) were transfected to downregulate FBXL8 expression. CCK-8 assay was conducted to measure cell proliferative activity, and Transwell assay was carried out to evaluate cell migratory ability. The UbiBrowser database was used to predict potential substrate proteins of FBXL8. Co-immunoprecipitation (Co-IP) was conducted to verify the interaction between FBXL8 and candidate substrates. Western blotting was used to detect the changes in substrate protein expression after intervention with the proteasome inhibitor MG132, and in vivo ubiquitination assay was performed to confirm the regulatory effect of FBXL8 on the ubiquitination modification of substrate proteins.

Bioinformatic analysis showed that the expression level of FBXL8 was significantly higher in ICC tissues (n=35) than in adjacent normal tissues (n=9) in the TCGA database (t=13.57, P<0.001). Two datasets from the GEO database, GSE26566 (ICC: n=104, adjacent: n=59) and GSE107943 (ICC: n=30, adjacent: n=27), further validated the above results (t=3.47, P<0.001; t=8.74, P<0.001). Immunohistochemical staining revealed that both the positive expression rate and staining intensity of FBXL8 protein were markedly elevated in ICC tissues compared with adjacent normal tissues. Clinicopathological analysis indicated that high FBXL8 expression was significantly correlated with tumor diameter ≥3 cm (χ²=16.528, P<0.001), lymph node metastasis (χ²=4.090, P=0.043), TNM stage Ⅲ-Ⅳ (χ²=11.446, P<0.001) and poor tumor differentiation (χ²=26.404, P<0.001), whereas no significant correlation was observed with gender, age, or vascular and perineural invasion. Survival analysis demonstrated that the patients with high FBXL8 expression had a significantly shorter overall survival than those with low expression (χ²=19.47, P<0.001). Cell functional experiments showed that effective silencing of FBXL8 expression in RBE cells by siRNA significantly reduced the [OD (450)] value, and the absorbance value was decreased from 1.15±0.05 in the control group to 0.73±0.04 in the siFbxl8#1 group (q=19.42, P<0.001) and 0.70±0.04 in the siFbxl8#2 group (q=23.69, P<0.001). The number of migrated RBE cells was also remarkably reduced, decreasing from 899.67±85.13 in the control group to 344.00±67.02 in the siFbxl8#1 group (q=7.46, P<0.05) and 355.00±51.00 in the siFbxl8#2 group (q=27.49, P<0.01). Consistent results were observed after FBXL8 silencing in SNU-1079 cells. Mechanistically, p53 was identified as the top-ranked potential substrate of FBXL8. Co-IP assay confirmed the endogenous interaction between FBXL8 and p53. FBXL8 overexpression reduced p53 protein level, while such degradation was blocked after the addition of MG132, with p53 protein levels recovering. In vivo ubiquitination assay further verified that FBXL8 overexpression enhanced the ubiquitination level of p53.

FBXL8 is highly expressed in ICC tissues, and its high expression is closely associated with adverse clinicopathological characteristics of ICC patients, including increased tumor volume, lymph node metastasis, advanced TNM stage and poor differentiation, serving as a potential biomarker for poor prognosis in the patients. FBXL8 promotes the proliferation and migration of ICC cells and drives the malignant progression by ubiquitinating and degrading p53 protein through the ubiquitin-proteasome pathway. Therefore, FBXL8 may be regarded as a potential target for molecular targeted therapy of ICC, providing a new direction for precision treatment of the disease.