三阴性乳腺癌（TNBC）是一个重大的临床挑战。化疗仍然是大部分TNBC患者的主要治疗方法，但药物耐药和肿瘤复发频繁发生。迫切需要确定TNBC的新型分子靶点，并开发对这种侵袭性疾病有效的治疗方法。我们进行了免疫组化实验，检查了TNBC样本中HER3的表达。利用蛋白质印迹实验评估了蛋白质的表达和激活情况。利用细胞生长（MTS）测定法确定细胞增殖和存活情况。分析TCGA数据库，将HER3 mRNA的表达与TNBC患者的临床结果相关联。使用特异性shRNA来抑制HER3的表达。利用IncuCyte系统监测细胞生长和迁移。使用LIVE/DEAD细胞成像检测活细胞和死细胞。我们用ELISA、流式细胞术和共免疫沉淀实验验证了我们的4A7抗-HER3单克隆抗体对HER3的识别能力。在裸鼠中建立了原位移植瘤模型，以确定TNBC细胞形成肿瘤的能力，并测试我们的4A7抗体是否能增强紫杉醇的抗肿瘤活性。 在测试的TNBC样本和细胞系中观察到HER3的过度表达。TCGA数据库的分析发现，在肿瘤中HER3 mRNA表达高的TNBC患者的总生存期（OS）和无复发生存期（RFS）明显较低。特异性抑制HER3显著抑制了TNBC细胞的增殖和乳腺球体形成，以及体内肿瘤的生长。我们的4A7抗体消除了HER3配体HER3配体HER-3蛋白mAb 4A7分别通过ELISA、流式细胞术和共免疫沉淀实验验证。在TNBC细胞中，4A7与EGFR酪氨酸激酶抑制剂（TKI）吉非替尼的联合应用显著降低了磷酸化HER3、EGFR、Akt和ERK1/2的水平，并具有明显的生长抑制和细胞死亡作用。此外，4A7与紫杉醇的联合应用在体外和体内对TNBC具有显著的抗肿瘤活性。 我们的数据表明，增加的HER3是TNBC的有效治疗靶点，我们的抗-HER3 mAb（4A7）可以增强吉非替尼或紫杉醇在TNBC中的疗效。© 2023 BioMed Central Ltd., Springer Nature的一部分。

Triple negative breast cancer (TNBC) represents a significant clinical challenge. Chemotherapy remains the mainstay for a large part of TNBC patients, whereas drug resistance and tumor recurrence frequently occur. It is in urgent need to identify novel molecular targets for TNBC and develop effective therapy against the aggressive disease.Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. IncuCyte system was utilized to monitor cell growth and migration. LIVE/DEAD Cell Imaging was to detect live and dead cells. HER3 recognition by our anti-HER3 monoclonal antibody (mAb) 4A7 was verified by ELISA, flow cytometry, and co-immunoprecipitation assays. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our mAb 4A7 could potentiate the antitumor activity of paclitaxel in vivo.Elevated expression of HER3 was observed in approximately half of the TNBC specimens and cell lines tested. Analyses of TCGA databases found that the TNBC patients with high HER3 mRNA expression in the tumors showed significantly worse overall survival (OS) and relapse-free survival (RFS) than those with low HER3 expression. Specific knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Our mAb 4A7 abrogated heregulin (a ligand for HER3), but not SDF-1 (a ligand for CXCR4)-induced enhancement of TNBC cell migration. Combinations of 4A7 and the EGFR-tyrosine kinase inhibitor (TKI) gefitinib dramatically decreased the levels of phosphorylated HER3, EGFR, Akt, and ERK1/2 in TNBC cells and potently induced growth inhibition and cell death. Moreover, 4A7 in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo.Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of gefitinib or paclitaxel in TNBC.© 2023. BioMed Central Ltd., part of Springer Nature.