Brain Metastasizing Breast Cancer Cell Secretome is Modulated by Endoplasmic Reticulum Stress

Abstract

Introduction Breast cancer is the second leading cause of brain metastasis. 30% of the patients suffering from Triple Negative Breast Cancer (TNBC) develop brain metastasis which confers poor prognosis to breast cancer patients. The mechanisms that facilitate brain metastasis are not fully understood. TNBC cells have high levels of endoplasmic reticulum stress (ER stress) and show activation of the IRE1 pathway of the Unfolded Protein Response (UPR). We showed that MDA-231/BR cells, the brain metastatic subline of MDA-MB-231, have high endogenous levels of IRE1 activity suggesting this UPR pathway to support survival under ER stress. Our research investigates the role of the UPR IRE1 arm in formation of TNBC brain metastasis. Here, we present the effect of ER stress induction and IRE1 inhibition on PERK activation and cytokine secretion in MDA-231/BR. Methods We used the brain metastatic triple-negative cell model MDA-231/BR to study the influence of IRE1 inhibition on other UPR pathways. ER stress was induced by serum-starvation or pharmacologically with Thapsigargin (Tg). MKC8866, a highly selective non-competitive inhibitor of IRE1 RNAse and splicing activity, was used at 10 μM. Successful inhibition IRE1 RNAse activity was assessed by Western blot detection of the spliced XBP1 protein (sXBP1). Modulation of the PERK activity upon ER stress induction and MKC8866 inhibition was determined by Western blot and with caspase-3/7 apoptosis assays. Cell viability and cell migration was assessed by real time cell analysis. Cytokines secreted form MDA-231/BR were determined. Brain endothelial cells were subjected to MDA-231/BR conditioned media and cell viability assays. Gene expression changes of tight junctional proteins claudin-3, claudin-5 and occludin were quantified by qPCR. Results MDA-231/BR cells show constitutive activation of the IRE1 RNAse arm which can be further induced upon serum starvation as well as low concentrations of Thapsigargin (Tg). The IRE1 RNAse inhibitor MKC8866 (10 μM) completely represses sXBP1 under basal and induced ER stress. However, the stressors show different downstream effects: unlike serum starvation, only Tg treatment activates the pro-apoptotic PERK pathway. This is confirmed by reduced cell viability and increased apoptosis upon Tg treatment. Cytokine analysis from culture supernatants of ER stress-induced MDA-231/BR cells showed increased secretion of pro-metastatic cytokines, some were independent of IRE1 activity. Brain endothelial cells exposed to MDA-231/BR conditioned media did not show altered cell viability nor changes in tight-junctional protein expression suggesting direct cell contacts to influence endothelial phenotypes. Conclusions This study showed that moderate ER stress induction by serum starvation, in contrast to pharmacological ER stress induction, does not induce pro-apoptotic UPR activity in MDA-231/BR cells. Increased MDA-231/BR secreted cytokines are not affecting brain endothelial cell viability, proliferation or expression of tight junction proteins.