Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Present address: CRUK Beatson Institute, Bearsden, Glasgow, UK.
BMC Cancer. 2021 Dec 4;21(1):1296. doi: 10.1186/s12885-021-08955-6.
Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Treatment options for TNBC patients are limited and further insights into disease aetiology are needed to develop better therapeutic approaches. microRNAs' ability to regulate multiple targets could hold a promising discovery approach to pathways relevant for TNBC aggressiveness. Thus, we address the role of miRNAs in controlling three signalling pathways relevant to the biology of TNBC, and their downstream phenotypes.
To identify miRNAs regulating WNT/β-catenin, c-Met, and integrin signalling pathways, we performed a high-throughput targeted proteomic approach, investigating the effect of 800 miRNAs on the expression of 62 proteins in the MDA-MB-231 TNBC cell line. We then developed a novel network analysis, Pathway Coregulatory (PC) score, to detect miRNAs regulating these three pathways. Using in vitro assays for cell growth, migration, apoptosis, and stem-cell content, we validated the function of candidate miRNAs. Bioinformatic analyses using BC patients' datasets were employed to assess expression of miRNAs as well as their pathological relevance in TNBC patients.
We identified six candidate miRNAs coordinately regulating the three signalling pathways. Quantifying cell growth of three TNBC cell lines upon miRNA gain-of-function experiments, we characterised miR-193b as a strong and consistent repressor of proliferation. Importantly, the effects of miR-193b were stronger than chemical inhibition of the individual pathways. We further demonstrated that miR-193b induced apoptosis, repressed migration, and regulated stem-cell markers in MDA-MB-231 cells. Furthermore, miR-193b expression was the lowest in patients classified as TNBC or Basal compared to other subtypes. Gene Set Enrichment Analysis showed that miR-193b expression was significantly associated with reduced activity of WNT/β-catenin and c-Met signalling pathways in TNBC patients.
Integrating miRNA-mediated effects and protein functions on networks, we show that miRNAs predominantly act in a coordinated fashion to activate or repress connected signalling pathways responsible for metastatic traits in TNBC. We further demonstrate that our top candidate, miR-193b, regulates these phenotypes to an extent stronger than individual pathway inhibition, thus emphasizing that its effect on TNBC aggressiveness is mediated by the coordinated repression of these functionally interconnected pathways.
三阴性乳腺癌（TNBC）是乳腺癌（BC）中最具侵袭性的亚型。TNBC 患者的治疗选择有限，需要进一步深入了解疾病的发病机制，以开发更好的治疗方法。microRNA 调节多个靶标的能力为探索与 TNBC 侵袭性相关的途径提供了一种有前途的发现方法。因此，我们研究了 microRNA 在调控与 TNBC 生物学相关的三个信号通路及其下游表型中的作用。
为了鉴定调控 WNT/β-catenin、c-Met 和整合素信号通路的 microRNA，我们采用高通量靶向蛋白质组学方法，研究了 800 个 microRNA 对 MDA-MB-231 TNBC 细胞系中 62 种蛋白质表达的影响。然后，我们开发了一种新的网络分析方法——通路核心调控（PC）评分，以检测调控这三个通路的 microRNA。我们使用体外细胞生长、迁移、凋亡和干细胞含量测定实验，验证候选 microRNA 的功能。我们还使用 BC 患者数据集进行生物信息学分析，评估 microRNA 的表达及其在 TNBC 患者中的病理相关性。
我们鉴定出六个协同调控这三个信号通路的候选 microRNA。通过 miRNA 功能获得实验定量测定三种 TNBC 细胞系的细胞生长情况，我们将 miR-193b 鉴定为增殖的强而一致的抑制剂。重要的是，miR-193b 的作用强于单个通路的化学抑制作用。我们进一步证明，miR-193b 诱导 MDA-MB-231 细胞凋亡、抑制迁移并调节干细胞标志物。此外，miR-193b 在被归类为 TNBC 或基底型的患者中的表达最低，与其他亚型相比。基因集富集分析表明，miR-193b 的表达与 TNBC 患者中 WNT/β-catenin 和 c-Met 信号通路活性的降低显著相关。
通过整合 miRNA 介导的作用和网络上的蛋白质功能，我们表明 microRNA 主要以协调的方式激活或抑制负责 TNBC 转移特征的连接信号通路。我们进一步证明，我们的首选候选 microRNA，miR-193b，在某种程度上比单个通路抑制更能调节这些表型，从而强调其对 TNBC 侵袭性的影响是通过对这些功能上相互关联的通路的协调抑制来介导的。
