Abstract
IntroductionThe 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.MethodsA 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.ResultsUsing 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.ConclusionsThus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.
Highlights
The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis
Breast cancer cell migration in transwell assays To study the role of the unfolded protein response (UPR) in the process of breast cancer cell migration, a number of models for in vitro metastasis were used
We provide evidence that apart from the established involvement of the HIF-pathway in the induction of cancer cell spread, the UPR is a second manner in which hypoxia is implicated in breast cancer cell migration
Summary
The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is 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. 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. The common denominator of most, if not all, of these factors is that they are either directly or indirectly influenced by the action of the family of master transcription regulators during hypoxic conditions: the hypoxia-inducible factor (HIF)-family [18]
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