De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer

Sylvain Lefort,Gian Luca Negri,Davide Pellacani,Susanna Tan,Amal El-Naggar,Martin Hirst,Connie J Eaves,Shane Colborne,Gregg B Morin,Poul H Sorensen,Barry Gusterson

doi:10.1038/s41418-021-00836-6

Abstract

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRASG12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.

Highlights

Mammalian Y-box binding protein-1 (YB-1) is a member of the family of DNA/RNA binding proteins with a conserved cold-shock domain (CSD)
To investigate the possibility that these effects might be operative during MDA-MB-231-induced tumour formation in vivo, we performed RNAseq and proteomic analyses of cells isolated from the small tumours derived from MDA-MB-231 cells transduced with shYB-1, and compared the results to data obtained from simultaneously harvested control tumours (Appendix Fig. S4A–D)
Here we show that increased expression of YB-1 characteristic of poor prognosis breast cancers is associated with elevated KRAS or AKT activity and an activated stress response programme

Summary

INTRODUCTION

Mammalian Y-box binding protein-1 (YB-1) is a member of the family of DNA/RNA binding proteins with a conserved cold-shock domain (CSD). If and how YB-1 might play a role in the initial stages of malignant transformation of human cells has remained unknown, both because of the lack of patients’ samples of viable tissue at this stage and of in vivo models of de novo cancer development from cells isolated directly from normal human tissue To overcome this constraint, we have taken advantage of a system we have described recently that allows invasive ductal carcinomas (IDCs) to be reproducibly and rapidly obtained from freshly isolated normal human mammary cells transduced with a lentiviral vector encoding a KRASG12D cDNA [10, 11]. Human cells were sorted after staining with anti-human specific antibodies directed against EpCAM and CD298 (Biolegend) with simultaneous depletion of mouse cells stained with anti-mouse-specific

MATERIALS AND METHODS

RESULTS

DISCUSSION