破坏超级增强子驱动的肿瘤抑制基因 RCAN1.4 的表达可促进乳腺癌的恶性转化。

BACKGROUND: Super-enhancers (SEs) play a crucial role in cancer, which is often associate with activated oncogenes. However, little is known about how SEs facilitate tumour suppression. Individuals with Down syndrome exhibit a remarkably reduced incidence of breast cancer (BC), moving the search for tumor suppressor genes on human chromosome 21 (HSA21). In this study, we aim to identify and explore potential mechanisms by which SEs are established for tumor suppressor RCAN1.4 on HSA21 in BC. METHODS: In silico analysis and immunohistochemical staining were used to assess the expression and clinical relevance of RCAN1.4 and RUNX3 in BC. Function experiments were performed to evaluate the effects of RCAN1.4 on the malignancy of breast carcinoma in vitro and in vivo. ChIP-seq data analysis, ChIP-qPCR, double-CRISPR genome editing, and luciferase reporter assay were utilized to confirm RUNX3 was involved in regulating RCAN1.4-associated SE in BC. The clinical value of co-expression of RCAN1.4 and RUNX3 was evaluated in BC patients. RESULTS: Here, we characterized RCAN1.4 as a potential tumour suppressor in BC. RCAN1.4 loss promoted tumour metastasis to bone and brain, and its overexpression inhibited tumour growth by blocking the calcineurin-NFATc1 pathway. Unexpectedly, we found RCAN1.4 expression was driven by a ~ 23 kb-long SE. RCAN1.4-SE was sensitive to BRD4 inhibition, and its deletion decreased RCAN1.4 expression by over 90% and induced the malignant phenotype of BC cells. We also discovered that the binding sites in the SE region of RCAN1.4 were enriched for consensus sequences of transcription factor RUNX3. Knockdown of RUNX3 repressed the luciferase activity and also decreased H3K27ac enrichment binding at the SE region of RCAN1.4. Furthermore, abnormal SE-driven RCAN1.4 expression mediated by RUNX3 loss could be physiologically significant and clinically relevant in BC patients. Notably, we established a prognostic model based on RCAN1.4 and RUNX3 co-expression that effectively predicted the overall survival in BC patients. CONCLUSIONS: These findings reveal an important role of SEs in facilitating tumour suppression in BC. Considering that the combination of low RCAN1.4 and low RUNX3 expression has worse prognosis, RUNX3-RCAN1.4 axis maybe a novel prognostic biomarker and therapeutic target for BC patients.

背景：超级增强子（SEs）在癌症中起着至关重要的作用，通常与激活的癌基因有关。然而，对于 SE 如何促进肿瘤抑制知之甚少。唐氏综合征患者的乳腺癌（BC）发病率显著降低，促使人们在人类 21 号染色体（HSA21）上寻找肿瘤抑制基因。在这项研究中，我们旨在鉴定和探索 SE 在 BC 中建立 HSA21 上肿瘤抑制基因 RCAN1.4 的潜在机制。

方法：通过计算机分析和免疫组织化学染色评估 RCAN1.4 和 RUNX3 在 BC 中的表达和临床相关性。进行功能实验以评估 RCAN1.4 在体外和体内对乳腺癌恶性程度的影响。利用 ChIP-seq 数据分析、ChIP-qPCR、双 CRISPR 基因组编辑和荧光素酶报告基因检测实验证实 RUNX3 参与调节 BC 中 RCAN1.4 相关 SE。评估了 BC 患者中 RCAN1.4 和 RUNX3 共表达的临床价值。

结果：在此，我们将 RCAN1.4 鉴定为 BC 中的潜在肿瘤抑制因子。RCAN1.4 缺失促进肿瘤向骨骼和大脑转移，而其过表达通过阻断钙调神经磷酸酶-NFATc1 途径抑制肿瘤生长。出乎意料的是，我们发现 RCAN1.4 的表达受一个约 23kb 长的 SE 驱动。RCAN1.4-SE 对 BRD4 抑制敏感，其缺失使 RCAN1.4 表达降低 90%以上，并诱导 BC 细胞的恶性表型。我们还发现，RCAN1.4 SE 区域的结合位点富含转录因子 RUNX3 的保守序列。RUNX3 敲低抑制了荧光素酶活性，并降低了 SE 区域的 H3K27ac 富集结合。此外，BC 患者中由 RUNX3 缺失引起的异常 SE 驱动的 RCAN1.4 表达可能具有生理意义和临床相关性。值得注意的是，我们建立了一个基于 RCAN1.4 和 RUNX3 共表达的预后模型，该模型能够有效地预测 BC 患者的总生存率。

结论：这些发现揭示了 SE 在促进 BC 肿瘤抑制中的重要作用。鉴于低 RCAN1.4 和低 RUNX3 表达的组合具有更差的预后，RUNX3-RCAN1.4 轴可能成为 BC 患者的新型预后生物标志物和治疗靶标。