Department of Medical Oncology, Longyan First Affiliated Hospital of Fujian Medical University, Longyan, Fujian, PR China.
J Pathol Clin Res. 2023 Sep;9(5):378-390. doi: 10.1002/cjp2.334. Epub 2023 Jun 9.
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.
乳腺癌（BCA）是女性中最常见的癌症之一。新出现的证据表明，膜联蛋白 A-9（ANXA9）在某些癌症的发展中起着关键作用。值得注意的是，已经报道 ANXA9 是胃癌和结直肠癌的新预后生物标志物。然而，其在 BCA 中的表达和生物学功能尚未得到研究。我们使用在线生物信息学工具，如 TIMER、GEPIA、HPA 和 UALCAN，预测了 ANXA9 在 BCA 患者组织中的表达及其与临床病理特征的相关性。使用 RT-qPCR 和 Western blot 测量了 BCA 患者组织和细胞中 ANXA9 mRNA 和 ANXA9 蛋白的表达。通过透射电子显微镜鉴定了源自 BCA 的外泌体。使用功能测定评估了 ANXA9 在 BCA 细胞增殖、迁移、侵袭和凋亡中的生物学作用。体内肿瘤异种移植模型用于评估 ANXA9 在小鼠肿瘤生长中的作用。生物信息学和功能筛选分析表明，ANXA9 在 BCA 患者组织中高表达，中位 ANXA9 表达水平比正常组织高 1.5-2 倍（p<0.05）。RT-qPCR 证实，与相邻正常组织相比，BCA 组织中 ANXA9 的表达高 1.5 倍以上（p<0.001）。不同亚型的 BCA 中 ANXA9 的表达也存在差异，与正常组织或其他组织学亚型相比，腔 BCA 中 ANXA9 的表达显著上调（p<0.001）。此外，与正常组相比，不同种族、年龄、临床分期、淋巴结转移状态和绝经状态组中 ANXA9 的表达升高（p<0.001）。此外，发现源自 BCA 组织的外泌体分泌 ANXA9，并且用外泌体处理的 BCA 细胞中 ANXA9 的表达上调 1-7 倍（p<0.001），而用外泌体处理的 MCF10A 细胞中 ANXA9 的表达没有明显改变（p>0.05）。沉默 ANXA9 导致 BCA 细胞集落数量减少约 30%（p<0.01）。沉默 ANXA9 后，迁移和侵袭的 BCA 细胞数量分别减少约 65%和 68%（p<0.01）。在异种移植模型中，与 LV-NC 组相比，LV-sh-ANXA9 组的肿瘤大小显著减小（近一半）（p<0.01），这表明 ANXA9 沉默抑制了 BCA 进展中体外和体内的肿瘤进展。总之，外泌体衍生的 ANXA9 作为一种癌基因发挥作用，促进了 BCA 细胞的增殖、迁移和侵袭，并增强了 BCA 发展中的肿瘤生长，这可能为 BCA 患者提供了新的预后和治疗生物标志物。
