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. Plain Language Summary: Clear cell renal cell carcinoma (ccRCC) is the most aggressive and common type of kidney cancer. Despite improvements in surgery and existing drugs, many patients face high recurrence rates and limited treatment options. Through advanced data analysis and laboratory experiments, we identified a critical growth engine in kidney cancer cells called the ETS1/MYC axis. This pathway acts as a switch that drives rapid tumor cell multiplication. Our study found that patients with high levels of these factors typically have a poorer prognosis. To shut down this growth engine, we engineered a smart delivery system called ETS1@Lip-CAIX. This system consists of tiny lipid-based carriers (nanoparticles) loaded with a genetic drug (siRNA) designed to silence the ETS1 gene. To ensure the drug reaches its target, we attached a specialized GPS peptide (CaIX-P7) to the surface. This peptide specifically recognizes and binds to CA9, a protein that is overexpressed on the surface of kidney cancer cells but rarely found in healthy kidneys. We tested this nanomedicine using diverse models, including traditional cell cultures, patient-derived 3D organoids (mini-tumors that mimic real patient biology), and animal models. Results demonstrated that the targeted nanoparticles significantly inhibited tumor growth by disrupting the ETS1/MYC axis. Crucially, the treatment showed no toxicity to normal cells, indicating high safety and biocompatibility. This research identifies a novel therapeutic target and presents a precise, highly effective nanomedicine strategy. It provides a promising new avenue for personalized treatment of aggressive kidney cancer, potentially improving survival rates while minimizing side effects for patients. The illustration consists of three sections. The first section shows CA9 interacting with CalX-P7, leading to the formation of ETS1@Lip-CAIX using lipid precursors and aqueous peptides. The second section depicts the delivery of siRNA into a ccRCC cell, targeting ETS1, resulting in MYC downregulation. The third section illustrates the effect on patient-derived ccRCC organoids, showing cell death and an in vivo mouse model demonstrating tumor growth inhibition with ETS1@Lip-CAIX compared to a control. The diagram highlights the therapeutic approach targeting ccRCC using [email protected] @Lip-CAIX targets ccRCC: delivers siRNA, kills organoids, inhibits in mice, uses lipid precursors.