Exosome-derived ANXA9 functions as an oncogene in breast cancer

Breast cancer (BCA) is one of the most prevalent cancers among women. Emerging evidence has revealed that Annexin A-9 (ANXA9) plays a crucial function in the development of some cancers. Notably, ANXA9 has been reported to be a new prognostic biomarker for gastric and colorectal cancers. However, its expression and biological function in BCA have not yet been investigated. Using online bioinformatics tools such as TIMER, GEPIA, HPA, and UALCAN, we predicted ANXA9 expression and its correlation with the clinicopathological characteristics of BCA patients. RT-qPCR and western blot were utilized to measure ANXA9 mRNA and ANXA9 protein expression in BCA patient tissues and cells. BCA-derived exosomes were identified by transmission electron microscopy. Functional assays were employed to evaluate the biological role of ANXA9 in BCA cell proliferation, migration, invasion, and apoptosis. A tumor xenograft in vivo model was utilized to assess the role of ANXA9 in tumor growth in mice. Bioinformatics and functional screening analysis revealed that ANXA9 was highly expressed in BCA patient tissues, with median ANXA9 expression 1.5- to 2-fold higher than in normal tissues (p < 0.05). RT-qPCR confirmed that ANXA9 expression in BCA tissues was around 1.5-fold higher than the adjacent normal tissues (p < 0.001). ANXA9 expression in different subtypes of BCA also showed a difference, and ANXA9 was found to be mostly significantly upregulated in luminal BCA relative to normal tissues or other histological subtypes (p < 0.001). Moreover, ANXA9 expression was elevated in different races, ages, clinical stages, node metastasis status, and menopause status groups relative to the normal group (p < 0.001). Furthermore, ANXA9 was found to be secreted by BCA tissue-derived exosomes and its expression was upregulated 1- to 7-fold in BCA cells treated with exosomes (p < 0.001), while its expression in MCF10A cells was not significantly altered by treatment with exosomes (p > 0.05). ANXA9 silencing induced a significant decrease of around 30% in the colony number of BCA cells (p < 0.01). The number of migrated and invaded BCA cells also decreased by around 65 and 68%, respectively, after silencing ANXA9 (p < 0.01). Tumor size was significantly reduced (nearly half) in the LV-sh-ANXA9 group relative to the LV-NC group in the xenograft model (p < 0.01), suggesting that ANXA9 silencing repressed tumor progression in BCA progression in vitro and in vivo. In conclusion, exosome-derived ANXA9 functions as an oncogene that facilitates the proliferation, migration, and invasiveness of BCA cells and enhances tumor growth in BCA development, which may provide a new prognostic and therapeutic biomarker for BCA patients.