Role of IRE1-XBP1 arm in transendothelial migration of TNBC brain metastatic cells
Munir, Maliha Nuzhat
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Introduction: Breast cancer represents the second most frequent 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. However, the mechanisms that facilitate brain metastasis are not fully understood. TNBC cells have high levels of endoplasmic reticulum (ER) stress and show activation of the IRE1 pathway of the Unfolded Protein Response (UPR). Our lab previously demonstrated that MDA-231/BR cells, the brain metastatic subline of MDA-MB-231, have high endogenous levels of IRE1 activity suggesting that UPR pathway supports cancer cell survival under ER stress. My research investigates the involvement of the UPR IRE1 arm in cell survival and transendothelial migration of MDA-231/BR. Methods: Characterization of MDA-231/BR cells under different ER stress inducers and the IRE1α RNAse inhibitor MKC8866 was performed by Western blot experiments and viability assays. shXBP1 clones were established and assessed for successful downregulation of sXBP1 by Western blot detection. Cell migration was assessed by real time cell analysis and transendothelial Boyden-chamber migration assay. Cytokines secreted from MDA-231/BR under induced ER stress and MKC8866 were determined. Brain endothelial cells were subjected to MDA-231/BR conditioned media to determine changes in cell viability and differences in gene expression of tight junctional proteinsby qPCR. Preliminary in vivo analysis was performed to assess the effect of XBP1 silencing on MDA-231/BR brain metastasis. Results: MDA-231/BR cells showed constitutive activation of the IRE1 RNase arm which wasfurther induced by serum starvation and thapsigargin. The IRE1 RNase inhibitor MKC8866 completely blocked basal and induced IRE1 RNAse activity. In contrast to serum starvation, Tg treatment activated the pro-apoptotic PERK pathway which was confirmed by reduced cell viability and increased caspase 3/7 activity. MDA-231/BR cell migration studies under treatment with MKC8866 have shown a reduction in cell migration through endothelial layers. Knockdown of XBP1 however, did not consistently reduce endothelila transmigration of MDA-231/BR cells. ER stress-induced- MDA-231/BR cells show increased secretion of pro-metastatic cytokines IL-6, VEGF-A and thrombospondin-1. Brain endothelial cells exposed to MDA-231/BR conditioned media showed increased expression of tight junctional protein occludin under IRE1 RNase inhibition. In vivo data showed no significant difference in survival of mice in control versus doxycycline treated group. Conclusion: This study showed that the study of ER stress is dependent on different types of stressors used. Induction of ER stress leads to secretion of pro-inflammatory cytokines which can indirectly affect the endothelial cells to become more permeable and allow migration of breast cancer cells.