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

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.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.Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.Breast cancer mortality is caused foremost by the spread of cancer cells within the host in a process called metastasis [1]. Before tumor cells can metastasize, the tumor will need to invade, seek access to the lymphatic or vascular system and colonize the metastatic site [2,3]. Insights in this process will aid in the prevention of cancer metastasis and help improve prognosis.An important characteristic of most solid tumors is the presence of hypoxic regions [4-6]. Absent or inadequate vasculature within the tumor causes disruption of the supply of blood and consequentially an impaired delivery of oxygen and nutrients and an impaired removal of carbon dioxide and waste products. Several studies found low oxygen tension in tumors to be an adverse prognostic marker in different tumor types [7-10]. In addition, endogenous hypoxia-related markers, such as carbonic anhydrase-IX