CA9-Targeted Liposomal Delivery of siETS1 Inhibits Clear Cell Renal Cell Carcinoma Progression by Disrupting the ETS1/MYC Regulatory Axis.

Background: Clear cell renal cell carcinoma (ccRCC) is a highly aggressive malignancy with a high rate of recurrence and limited therapeutic options. Carbonic anhydrase IX (CA9) is characteristically overexpressed on the surface of ccRCC cells, making it a promising target for site-specific drug delivery. However, identifying the key molecular drivers of ccRCC progression and developing efficient, targeted nanomedicines remain critical challenges in current research. Methods: Bioinformatics analysis of TCGA and single-cell RNA sequencing data was used to elucidate the ETS1/MYC axis. Direct transcriptional regulation of MYC by ETS1 was experimentally validated by chromatin immunoprecipitation-quantitative PCR (ChIP-PCR) and dual-luciferase reporter assays. An optimized CA9-targeting peptide, CaIX-P7, was designed via computational modeling and mutation screening, with affinity validated by surface plasmon resonance (SPR). siETS1-loaded liposomal nanoparticles (LNPs) were prepared using microfluidics and surface-functionalized with CaIX-P7 (ETS1@Lip-CAIX). The nanoparticles were characterized for size, zeta potential, and encapsulation efficiency. Therapeutic efficacy was evaluated in ccRCC cell lines (786-O, A-498), patient-derived organoids (PDO), and nude mouse xenograft models. Results: Single-cell analysis identified ETS1 and MYC as synergistically activated transcription factors within tumor epithelial cells. Mechanistically, ChIP-PCR and dual-luciferase assays demonstrated that ETS1 promotes MYC transcription through this specific binding site, establishing ETS1 as a direct transcriptional activator of MYC. The optimized peptide CaIX-P7 demonstrated superior binding affinity to CA9 (Kd=52.96 nM) compared to its precursor. The engineered ETS1@Lip-CAIX nanoparticles exhibited a stable size of 154.8 nm and high siRNA encapsulation efficiency (89.1%). Systematic evaluation revealed that ETS1@Lip-CAIX effectively silenced the ETS1/MYC axis, leading to significant growth inhibition across all models, including patient-derived 3D organoids and in vivo xenografts, showed no discernible morphological alterations or pathological damage in major organs. Conclusion: This study identifies the ETS1/MYC axis as a novel therapeutic target in ccRCC. We further successfully developed a CA9-targeted nanoplatform, ETS1@Lip-CAIX, which exhibits robust anti-tumor efficacy by disrupting this newly discovered regulatory hub. These findings provide a foundation for future translational studies of ccRCC.