Basal-like triple-negative breast cancers (TNBCs) display poor prognosis, have a high risk of tumor recurrence, and exhibit high resistance to drug treatments. The TNBC aggressive features are largely due to the high proportion of cancer stem cells present within these tumors. In this study, we investigated the interplay and networking pathways occurring between TGFβ family ligands in regulating stemness in TNBCs. We found that TGFβ stimulation of TNBCs resulted in enhanced tumorsphere formation efficiency and an increased proportion of the highly tumorigenic CD44^high/CD24^low cancer stem cell population. Analysis of the TGFβ transcriptome in TNBC cells revealed bone morphogenetic protein4 (BMP4) as a main TGFβ-repressed target in these tumor cells. We further found that BMP4 opposed TGFβ effects on stemness and potently decreased cancer stem cell numbers, thereby acting as a differentiation factor in TNBC. At the molecular level, we found that TGFβ inhibition of BMP4 gene expression is mediated through the Smad pathway and cyclin D1. In addition, we also found BMP4 to act as a pro-differentiation factor in normal mammary epithelial cells and promote mammary acinar formation in 3D cell culture assays. Finally, and consistent with our in vitro results, in silico patient data analysis defined BMP4 as a potential valuable prognosis marker for TNBC patients.

基底样三阴性乳腺癌（TNBC）预后差，肿瘤复发风险高，并且对药物治疗的抵抗力高。TNBC的侵袭性特征很大程度上归因于这些肿瘤中存在的癌症干细胞比例很高。在这项研究中，我们调查了TGFβ家族配体之间在调节TNBCs干中的相互作用和网络通路。我们发现TNGFs的TGFβ刺激导致增强的肿瘤球形成效率和高致癌性CD44^高/ CD24^低的比例增加癌症干细胞群体。对TNBC细胞中TGFβ转录组的分析显示，骨形态发生蛋白4（BMP4）是这些肿瘤细胞中主要的TGFβ抑制靶标。我们进一步发现BMP4对抗TGFβ对干细胞的作用，并有效降低了癌症干细胞的数量，从而在TNBC中起分化因子的作用。在分子水平上，我们发现TGFβ对BMP4基因表达的抑制作用是通过Smad途径和细胞周期蛋白D1介导的。此外，我们还发现BMP4在正常的乳腺上皮细胞中起促分化因子的作用，并在3D细胞培养试验中促进乳腺腺泡的形成。最后，与我们的体外研究结果一致，计算机病历患者数据分析将BMP4定义为TNBC患者潜在的有价值的预后标志物。