Targeting NBL1/PI3K/AKT axis to enhance the chemosensitivity and radiosensitivity of esophageal squamous cell carcinoma

BACKGROUND: The development of radioresistance and chemoresistance remains a huge obstacle for the prognosis of esophageal squamous cell carcinoma (ESCC) patients. When investigating novel therapeutic targets involved in ESCC radioresistance and chemoresistance, we identified that ESCC patients expressing lower levels of neuroblastoma suppression of tumorigenicity 1 (NBL1) tended to have short overall survival. This research aimed to elucidate the functional roles of NBL1 in ESCC progression and further identify its downstream signaling pathway. METHODS: The expression level of NBL1 in ESCC tissues and non-tumor tissues was evaluated using enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC). The correlation between NBL1 expression and patient survival was analyzed using the Kaplan-Meier survival curve method. Knockdown and overexpression of NBL1 in ESCC cells were achieved via short hairpin RNA (shRNA) and lentiviral transduction, respectively. The survival capacity of NBL1-silenced cells under chemotherapy and radiotherapy was assessed through colony formation assay and Cell Counting Kit-8 (CCK-8) assay. Western blotting was employed to identify downstream signaling pathways of NBL1 involved in chemoresistance and radioresistance in ESCC, revealing that the PI3K-AKT (phosphoinositide 3-kinase-AKT signaling) pathway was activated in NBL1-silenced cells. Colony formation and CCK-8 assays were further applied to evaluate cell survival after combined treatment with chemotherapy and PI3K-AKT inhibitors (LY294002 or BYL719) or radiotherapy combined with LY294002/BYL719. Levels of (Bone Morphogenetic Protein) BMP2, 4, 6, and 7 were interfered with using siRNA in NBL1-knockdown cells, followed by qPCR validation and Western blot analysis to determine the expression of PI3K, AKT, and p-AKT proteins. A mouse xenograft model was established to investigate the antitumor efficacy of LY294002 and BYL719 in enhancing chemosensitivity and radiosensitivity in esophageal squamous cell carcinoma. RESULTS: Kaplan-Meier survival analysis revealed that ESCC patients with lower NBL1 expression had a shorter overall survival (P = 0.0141), suggesting NBL1 as a potential prognostic biomarker for ESCC. To investigate the functional role of NBL1 in ESCC progression, we performed knockdown of NBL1 expression in ESCC cells for functional assays. The results demonstrated that downregulation of NBL1 significantly promoted cell proliferation and migration, and enhanced resistance to multiple chemotherapeutic agents (chemoresistance) as well as radiotherapy (radioresistance). These phenotypes indicate that NBL1 may function as a tumor suppressor in ESCC. Mechanistically, both in vitro and in vivo studies confirmed that the PI3K-AKT signaling pathway was markedly activated in NBL1-knockdown ESCC cells. Further rescue experiments showed that treatment with PI3K-AKT pathway inhibitors (LY294002 or BYL719) significantly sensitized ESCC cells, particularly NBL1-silenced cells, to chemotherapy and radiotherapy. The qPCR results verified effective reduction in BMP2, 4, 6, and 7 levels following siRNA interference. Western blot analysis indicated that knockdown of BMP7 led to increased expression of PI3K, AKT, and p-AKT proteins. Taken together, these findings suggest that NBL1 may negatively regulate the PI3K-AKT signaling pathway through BMP2,4,7 thereby suppressing malignant progression and therapy resistance in ESCC. CONCLUSION: NBL1 functions as a tumor suppressor in ESCC. Its downregulation enhances radioresistance and chemoresistance in ESCC by activating the PI3K-AKT signaling pathway through BMP2,4,7. Inhibition of the PI3K-AKT pathway with LY294002 or BYL719 increases the cytotoxic effects of chemotherapy and radiation, offering a novel therapeutic strategy for ESCC patients, particularly those with low NBL1 expression.