Abstract

BackgroundSorting Nexin 27 (SNX27) belongs to a family of sortin nexins and possesses a unique binding domain at the C-terminus which mediates protein-protein interaction in intracellular trafficking, membrane remodeling, organelle motility, and tight junctions. However, its role in cancer development, especially in vivo, remains largely unknown.MethodsWe have generated a stable SNX27 knockdown clone in a highly aggressive breast cancer cell line MDA-MB-231 using an inducible lentiviral shRNA system. Cell migration and proliferation of SNX27 knockdown (KD) cells were compared with wild-type (WT) cells by MTT and wound healing assay, respectively. The differences in colony formation between SNX27-KD and WT cells were detected by soft agar culture and matrigel 3D culture. Furthermore, tumor growth was examined in a xenograft nude mouse model using SNX27-KD and WT MDA-MB-231 cells. The critical EMT (epithelial-mesenchymal transition) regulators were examined in vitro and in vivo.ResultsThe wound healing assay showed that SNX27 knockdown significantly decreased cell motility and proliferation. Colony formation in soft agar showed that the SNX27 knockdown cells formed significantly fewer and smaller colonies than the parental MDA-MB-231 cells. Western blots and immunostaining showed that knockdown of SNX27 led to increased expression of E-cadherin and β-catenin proteins, which facilitate adhesion formation and reverse EMT. EMT is a cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal cells, promoting carcinoma invasion. The expression levels of Vimentin, the transcription factor of EMT, and tight junction protein Claudin-5, were significantly diminished in the SNX27 knockdown cells. The expression of PCNA, the cell proliferation marker, was increased in SNX27-KD cells transfected with E-cadherin siRNA. In a xenograft nude mouse model, we found that knockdown of SNX27 significantly inhibited tumor growth. The tumors from mice with SNX27-KD cells showed less proliferation compared to tumors from mice injected with wildtype cells. The increase in E-cadherin and β-catenin and decrease in Vimentin and Claudin-5 were observed in tumors of mice injected with SNX27-KD cells.ConclusionsOur data have demonstrated that SNX27 plays a crucial role in tumor growth in vitro and in vivo.

Highlights

  • Sorting Nexin 27 (SNX27) belongs to a family of sortin nexins and possesses a unique binding domain at the C-terminus which mediates protein-protein interaction in intracellular trafficking, membrane remodeling, organelle motility, and tight junctions

  • A whole set of the Epithelial-mesenchymal transition (EMT) Antibody Sampler Kit was purchased from Cell Signalling (Danvers, MA), including monoclonal antibodies to human β-catenin, E-cadherin, N-Cadherin, Vimentin and TCF-β/ZEB1 raised in rabbit

  • The expressional level of SNX27 in different breast cancer cell lines In order to compare the difference in expression of SNX27 in different malignant breast cancer cell lines, we collected 7 breast cancer cell lines with different levels of aggression

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Summary

Introduction

Sorting Nexin 27 (SNX27) belongs to a family of sortin nexins and possesses a unique binding domain at the C-terminus which mediates protein-protein interaction in intracellular trafficking, membrane remodeling, organelle motility, and tight junctions. SNXs rescue transmembrane proteins from the lysosomal degradative pathway and facilitate their recycling to other cellular compartments as well as play roles in membrane trafficking, cell signaling, membrane remodeling, organelle motility, ion channel regulation and receptor recycling [1, 3]. The sorting nexin 27 (SNX27), which contains a PSD95, Dlg, ZO-1 (PDZ)-binding motif, promotes recycling of internalized transmembrane proteins from endosomes to the plasma membrane by linking PDZ-dependent cargo recognition to retromer-mediated transport or regulate of endosome-to-plasma membrane recycling of transmembrane [4, 5]. Cell-cell adhesion is mediated by a variety of membrane proteins, such as classical E or N-cadherins, claudins, and βcatenin [13,14,15]. Whether SNX27 affects breast cancer has not been explored

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