Oncogenomics and Epigenetics Unit, IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.
Department of Molecular Biology, International Institute of Molecular and Cell Biology in Warsaw, Księcia Trojdena 4, 02-109, Warsaw, Poland.
Breast Cancer Res. 2018 Jun 19;20(1):59. doi: 10.1186/s13058-018-0990-2.
As crucial regulators of the immune response against pathogens, macrophages have been extensively shown also to be important players in several diseases, including cancer. Specifically, breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (inhibitors of differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like breast cancer. Moreover, ID4 favours angiogenesis by enhancing the expression of pro-angiogenic cytokines interleukin-8, CXCL1 and vascular endothelial growth factor. In the present study, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer.
We performed IHC analysis of ID4 protein and macrophage marker CD68 in a triple-negative breast cancer series. Next, we used cell migration assays to evaluate the effect of ID4 expression modulation in breast cancer cells on the motility of co-cultured macrophages. The analysis of breast cancer gene expression data repositories allowed us to evaluate the ability of ID4 to predict survival in subsets of tumours showing high or low macrophage infiltration. By culturing macrophages in conditioned media obtained from breast cancer cells in which ID4 expression was modulated by overexpression or depletion, we identified changes in the expression of ID4-dependent angiogenesis-related transcripts and microRNAs (miRNAs, miRs) in macrophages by RT-qPCR.
We determined that ID4 and macrophage marker CD68 protein expression were significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 messenger RNA (mRNA) levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signalling, the activation of an angiogenic programme in macrophages. This programme includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine granulin, whose enhanced expression in macrophages confers increased angiogenic potential.
These results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer.
作为对抗病原体免疫反应的关键调节者，巨噬细胞也被广泛证明是多种疾病（包括癌症）的重要参与者。具体来说，乳腺癌巨噬细胞严格控制着血管生成开关和恶性转化的进展。ID4 是 ID（分化抑制剂）家族蛋白的成员之一，与基底样乳腺癌中的干细胞样表型和预后不良相关。此外，ID4 通过增强促血管生成细胞因子白细胞介素 8、CXCL1 和血管内皮生长因子的表达来促进血管生成。在本研究中，我们研究了 ID4 蛋白是否在调节乳腺癌中肿瘤相关巨噬细胞的活性的同时发挥其促血管生成功能。
我们对三阴性乳腺癌系列中 ID4 蛋白和巨噬细胞标志物 CD68 的免疫组化分析。接下来，我们使用细胞迁移实验来评估在乳腺癌细胞中调节 ID4 表达对共培养巨噬细胞迁移能力的影响。对乳腺癌基因表达数据存储库的分析使我们能够评估 ID4 预测具有高或低巨噬细胞浸润的肿瘤亚组的生存能力的能力。通过在乳腺癌细胞中培养条件培养基，这些细胞中 ID4 的表达通过过表达或耗尽来调节，我们通过 RT-qPCR 鉴定了 ID4 依赖性血管生成相关转录物和 microRNAs（miRNAs，miRs）在巨噬细胞中的表达变化。
我们确定 ID4 和巨噬细胞标志物 CD68 蛋白表达在一系列三阴性乳腺癌中显著相关。有趣的是，ID4 信使 RNA（mRNA）水平在具有高巨噬细胞浸润的肿瘤亚组中能够强有力地预测生存。体外和体内迁移实验表明，乳腺癌细胞中 ID4 的表达刺激巨噬细胞的迁移。在分子水平上，乳腺癌细胞中 ID4 蛋白的表达通过旁分泌信号控制巨噬细胞中血管生成程序的激活。该程序包括增加与血管生成相关的 mRNAs 和减少抗血管生成 miR-15b/107 组的成员。有趣的是，这些 miRNA 控制细胞因子 granulin 的表达，其在巨噬细胞中的表达增强赋予了更强的血管生成潜力。
这些结果揭示了 ID4 在决定乳腺癌中肿瘤相关巨噬细胞行为方面的关键作用。
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