Receptor for Activated C Kinase 1 promotes cervical cancer lymph node metastasis via glycolysis-dependent AKT/mTOR signaling pathway

Abstract

Background Cervical cancer (CC), an aggressive squamous cell carcinoma, is characterized by early lymph node metastasis and extremely poor prognosis. We previously showed aberrant glycolysis in patients with CC. The upregulation of Receptor for Activated C Kinase 1 (RACK1) is associated with CC lymph node metastasis. However, its role in mediating aerobic glycolysis in CC lymph node metastasis remains unclear. Methods 104 cervical cancer and 31 cervicitis tissue were enrolled. RACK1, insulin-like growth factor 1 receptor (IGF1R), POU class 2 homeobox 2 (POU2F2) and Hexokinase 2 (HK2) expression in cervical cancer patients were detected by Immunohistochemistry analysis. 1H-NMR experiment was used for metabolomics of cell supernatant after RACK1 knockdown. Tube formation assay, Transwell assay, determination of glucose consumption and lactate production assay were performed to detected MS751 and SiHa cells lymphangiogenesis, migration, invasion, glucose consumption and lactate production. Chromatin immunoprecipitation-PCR assay and Dual-Luciferase Reporter Assay was performed to detect the binding among POU2F2 and RACK1. Co-immunoprecipitation and Immunofluorescence staining were performed to detect the binding among RACK1 and GF1R. Xenograft footpad lymph node metastasis was performed using nude mice. Xenograft tumor and metastatic lymph node tissues of mice were experienced immunohistochemistry and hematoxylin–eosin staining. Results Here, 1H-NMR analysis revealed significantly correlated RACK1 expression with glycolysis/gluconeogenesis pathway. Additionally, RACK1 knockdown inhibited aerobic glycolysis and lymphangiogenesis in vitro and suppressed CC lymph node metastasis in vivo. Furthermore, AKT/mTOR signaling was identified as a critical RACK1-regulated pathway that led to increased lymphangiogenesis in CC. Co-immunoprecipitation, immunofluorescence, and Western blot showed that RACK1 activated AKT/mTOR signaling by interacting with IGF1R. POU2F2 binds to RACK1 promoter and regulates RACK1 transcription, thus functionally contributing to glycolysis and lymphangiogenesis in CC. Importantly, administration of 2-deoxy-D-glucose, which attenuates glycolysis, inhibited RACK1-induced lymphangiogenesis in CC. The positive correlations between RACK1, IGF1R, POU2F2, and HK2 were further confirmed in CC tissues. Conclusion RACK1 plays an important role in CC tumor lymph node metastasis by regulating IGF1R/AKT/mTOR signaling mediated glycolysis pathways. Targeting the POU2F2/ RACK1/IGF1R/AKT/mTOR signaling pathway may provide a novel strategy for CC treatment.