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Open Access Highly Accessed Research article

Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response

Anika Nagelkerke12*, Johan Bussink1, Hilda Mujcic34, Bradly G Wouters34, Steffi Lehmann5, Fred CGJ Sweep2 and Paul N Span12

Author Affiliations

1 Department of Radiation Oncology, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands

2 Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands

3 Ontario Cancer Institute, Campbell Family Research Institute, University Health Network, Departments of Radiation Oncology and Medical Biophysics, University of Toronto, 610 University Ave., Toronto, ON M5G 2M9, Canada

4 Maastricht Radiation Oncology (MaastRo) Lab, GROW-School for Oncology and Developmental Biology, University of Maastricht, Universiteitssingel 50/23, 6229 ER Maastricht, The Netherlands

5 Department of Cell Biology, NCMLS, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands

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Breast Cancer Research 2013, 15:R2  doi:10.1186/bcr3373

Published: 7 January 2013

Abstract

Introduction

The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined.

Methods

A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion.

Results

Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids.

Conclusions

Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.