BACKGROUND Breast cancer metastasis is driven by a profound remodeling of the cytoskeleton that enables efficient cell migration and invasion. Anillin is a unique scaffolding protein regulating major cytoskeletal structures, such as actin filaments, microtubules, and septin polymers. It is markedly overexpressed in breast cancer, and high anillin expression is associated with poor prognosis. The aim of this study was to investigate the role of anillin in breast cancer cell migration, growth, and metastasis. METHODS CRISPR/Cas9 technology was used to deplete anillin in highly metastatic MDA-MB-231 and BT549 cells and to overexpress it in poorly invasive MCF10AneoT cells. The effects of anillin depletion and overexpression on breast cancer cell motility in vitro were examined by wound healing and Matrigel invasion assays. Assembly of the actin cytoskeleton and matrix adhesion were evaluated by immunofluorescence labeling and confocal microscopy. In vitro tumor development was monitored by soft agar growth assays, whereas cancer stem cells were examined using a mammosphere formation assay and flow cytometry. The effects of anillin knockout on tumor growth and metastasis in vivo were determined by injecting control and anillin-depleted breast cancer cells into NSG mice. RESULTS Loss-of-function and gain-of-function studies demonstrated that anillin is necessary and sufficient to accelerate migration, invasion, and anchorage-independent growth of breast cancer cells in vitro. Furthermore, loss of anillin markedly attenuated primary tumor growth and metastasis of breast cancer in vivo. In breast cancer cells, anillin was localized in the nucleus; however, knockout of this protein affected the cytoplasmic/cortical events, e.g., the organization of actin cytoskeleton and cell-matrix adhesions. Furthermore, we observed a global transcriptional reprogramming of anillin-depleted breast cancer cells that resulted in suppression of their stemness and induction of the mesenchymal to epithelial trans-differentiation. Such trans-differentiation was manifested by the upregulation of basal keratins along with the increased expression of E-cadherin and P-cadherin. Knockdown of E-cadherin restored the impaired migration and invasion of anillin-deficient breast cancer cells. CONCLUSION Our study demonstrates that anillin plays essential roles in promoting breast cancer growth and metastatic dissemination in vitro and in vivo and unravels novel functions of anillin in regulating breast cancer stemness and differentiation.

中文翻译：

---

Anillin通过涉及细胞干性和分化控制的非经典机制来调节乳腺癌细胞的迁移，生长和转移。

背景技术乳腺癌的转移是由细胞骨架的深刻重塑驱动的，其使得有效的细胞迁移和侵袭成为可能。Anillin是一种独特的支架蛋白，可调节主要的细胞骨架结构，如肌动蛋白丝，微管和septin聚合物。它在乳腺癌中明显过表达，而高的Anillin表达与不良预后相关。这项研究的目的是调查Anillin在乳腺癌细胞迁移，生长和转移中的作用。方法CRISPR / Cas9技术被用于在高度转移的MDA-MB-231和BT549细胞中消耗Anillin，并在侵袭性较弱的MCF10AneoT细胞中过表达。通过伤口愈合和基质胶侵袭试验检测了Anillin耗竭和过表达对体外乳腺癌细胞运动的影响。通过免疫荧光标记和共聚焦显微镜评估肌动蛋白细胞骨架的组装和基质粘附。通过软琼脂生长测定法监测体外肿瘤的发展，而使用乳球形成测定法和流式细胞术检查癌症干细胞。通过向NSG小鼠中注射对照和贫去Anillin的乳腺癌细胞来确定Anillin敲除对体内肿瘤生长和转移的影响。结果功能丧失和功能获得的研究表明，阿尼林在体外加速乳腺癌细胞的迁移，侵袭和锚定非依赖性生长是必要和充分的。此外，茴香醚的丧失显着减弱了体内乳腺癌的原发性肿瘤生长和转移。在乳腺癌细胞中，阿尼林位于细胞核内。然而，该蛋白的敲除影响了细胞质/皮层事件，例如肌动蛋白细胞骨架的组织和细胞基质的粘附。此外，我们观察到了贫乏Anillin的乳腺癌细胞的全球转录重编程，导致其干细胞抑制和间充质向上皮转分化的诱导。这种转分化通过基底角蛋白的上调以及E-钙黏着蛋白和P-钙黏着蛋白的表达增加来体现。降低E-钙粘着蛋白恢复了受损的Anillin缺乏乳腺癌细胞的迁移和侵袭。