Abstract 4405: Annexin A1 regulates lenvatinib resistance through mediating STAT3/S100A6 signaling cascade and M2 macrophage polarization in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most commonly diagnosed cancer and third leading cause of cancer-related deaths worldwide. Lenvatinib has been approved by the FDA for front-line treatment of HCC patients at advanced stages. However, the survival benefit of lenvatinib is modest due to the development of acquired drug resistance. To understand its underlying mechanism, we have established lenvatinib resistant (LenR) HCC patient derived xenograft model (PDTX) via administration of four rounds of lenvatinib and the corresponding tumors from LenR and mock counterparts were analyzed by single cell RNA-sequencing (scRNA-seq). SCENIC analysis revealed that Annexin A1 (ANXA1) is enriched in LenR tumor. This finding was similar in patients with LenR HCC when compared to the sensitive group. An in vitro hepatocyte differentiation model showed that the expression of ANXA1 peaked during the intermediate stages of liver development but was absent in mature hepatocytes but upregulated in HCC tumor tissues, indicating its role in the regulation of cancer stemness in HCC. Further functional analysis showed that ANXA1 drives cancer stem cell-driven lenvatinib resistance, as evidenced by the in vitro and in vivo limiting dilution and apoptosis assays. Through in silico analysis, SOX2 was predicted to be an upstream regulator of ANXA1, which was further validated using chromatin immunoprecipitation-qPCR and luciferase promoter assays in LenR and SOX2-silenced cells. To investigate potential downstream targets of ANXA1, differential gene expression (DEG) analysis was conducted on the ANXA1High and ANXA1Low groups within the LenR tumors. The results revealed S100A6 was the most significantly upregulated gene in the ANXA1High subpopulation, suggesting a possible regulatory relationship between ANXA1 and S100A6 expression. Further GESA analysis of DEGs of the ANXA1High to ANXA1Low group further showed that ANXA1 transcriptionally regulates S100A6 mediated through STAT3 activation. Consistently, a positive correlation between STAT3 and ANXA1/S100A6 levels was observed in the TCGA-LIHC cohort. Interestingly, ANXA1 secreted from HCC cells also drives cancer stemness and drug resistance in an autocrine manner. Analysis of the same set of scRNA-seq data revealed that tumor microenvironment (TME) in LenR tumors was altered by increasing the M2/M1 ratio of tumor-associated macrophages (TAM). Likewise, enhanced infiltration of M2 macrophages was observed in LenR PDTX and mouse HCC tumors. Using an in vitro assay with conditioned media, LenR-HCC-derived ANXA1 induced macrophage polarization towards the M2 phenotype. In conclusion, ANXA1 may regulate lenvatinib resistance intrinsically via the promotion of cancer stemness and extrinsically via reshaping immunosuppressive TME by inducing M2 macrophage polarization. Citation Format: Carmen Oi Leung, Catherine Yu Gu, Terence Kin Lee. Annexin A1 regulates lenvatinib resistance through mediating STAT3/S100A6 signaling cascade and M2 macrophage polarization in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4405.