Structural determinant of BST-2-mediated regulation of breast cancer cell motility: a role for cytoplasmic tail tyrosine residues.

Wasifa Naushad,Chioma M Okeoma,Wadie D Mahauad-Fernandez

doi:10.18632/oncotarget.22753

Wasifa Naushad, Chioma M Okeoma + Show 1 more

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https://doi.org/10.18632/oncotarget.22753

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Abstract

There is now irrefutable evidence that overexpression of the innate immunity protein―BST-2, in breast cancer cells is implicated in tumor growth and progression. The cellular mechanisms that control BST-2-mediated effect in tumor progression involve enhancement of cancer cell motility―migration/invasion. However, the distinct structural elements of BST-2 that mediate breast cancer cell motility remain unknown. Here, we used various motility assays and different variants of BST-2 to examine the cellular and structural mechanisms controlling BST-2-mediated cell motility. We show that BST-2 silencing in various cancer cell lines inhibits cell motility. Restoration of BST-2 expression using construct expressing wild type BST-2 rescues cell motility. Mutational analysis identifies the cytoplasmic tail of BST-2 as a novel regulator of cancer cell motility, because cell motility was significantly abrogated by substitution of the BST-2 cytoplasmic tail tyrosine residues to alanine residues. Furthermore, in a spheroid invasion model, BST-2-expressing tumor spheroids are highly invasive inside 3D Matrigel matrices. In this model, the spreading distance of BST-2-expressing spheroids was significantly higher than that of BST-2-suppressed spheroids. Collectively, our data reveal that i) BST-2-expressing breast cancer cells in spheroids are more motile than their BST-2-supressed counterparts; ii) BST-2 cytoplasmic tail regulates non-proteolytic (migration) and proteolytic (invasion) mechanisms of breast cancer cell motility; and iii) replacement of the tyrosine residues at positions 6 and 8 in the cytoplasmic tail of BST-2 with alanine residues inhibits cell motility.

Highlights

Breast cancer is responsible for ~450,000 deaths per year worldwide and over 40,000 in the US
Using three different readouts to compare the numbers of migrated shCTL versus shBST-2 cells, we show that silencing BST-2 expression (Supplementary Figure 1A–1C) in the aggressive 4T1 (Figure 1C, 1G, 1K, 1O) and 4T07 (Figure 1D, 1H, 1L, 1P) cells results in more significant decrease in cell migration compared to the less aggressive 168FARN cells (Figure 1E, 1I, 1M, 1Q)
Following silencing of BST2 expression with two different human BST-2-targeting shRNAs (Supplementary Figure 1D), we found that loss of BST-2 significantly reduces migration of MDA-MB-231 cells (Supplementary Figure 1)

Summary

Introduction

Breast cancer is responsible for ~450,000 deaths per year worldwide and over 40,000 in the US. Five distinct subtypes of breast cancer―including, Luminal A, Luminal B, HER2 enriched, basal, and claudin low subtypes are recognized to have clinical significance [1,2,3]. In all of these cancer subtypes, the level of the antiviral innate immunity factor BST-2 is elevated [4,5,6,7,8,9]. BST-2 is composed of an N-terminal cytoplasmic tail (CT), a transmembrane domain (TM), a coiled-coiled extracellular domain (ECD), and a C-terminal glycosylphosphatidylinositol (GPI)-anchor [12] in that order. BST-2 dimerization mediated by the ECD cysteine residues has been shown to enhance cell to cell and cell to ECM interaction, as well as in promoting cancer cell survival through the disruption of the anoikis machinery [9]

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