Annilin regulates breast cancer cell migration, growth and metastasis by non‐canonical mechanisms involving control of cell stemness and differentiation.

Abstract

Breast cancer metastasis is driven by profound remodeling of the intracellular 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 metastasis. 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 a combination of wound healing and Matrigel invasion assays. Assembly of the actin cytoskeleton and matrix adhesions was evaluated by immunofluorescence labeling and confocal microscopy. In vitro tumor development was monitored by soft agar grow, whereas cancer stem cells were examined using a mammoshpere formation assay and flow cytometry. The effects of anillin knockout on tumor growth and metastasis in vivo was determined by injecting control and anillin‐depleted breast cancer cells into NGS mice. The 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, such as 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 upregulation of basal keratins along with increased expression of E‐cadherin and P‐cadherin. Knockdown of E‐cadherin reversed attenuated migration and invasion of anillin‐deficient breast cancer cells. 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.