Effects Of Endoplasmic Reticulum Protein Homeostasis On The Regulation Of Stem Cells From Different Origins And Breast Cancer

Objective To explore the role and underlying mechanisms of UPR and ERAD in the tumorigenesis of multiple tumors and breast cancer stem cell functions.Methods Western blot and immunohistochemistry were used to examine the expression level and activation of UPR related molecules and p97 in different tumor cells and tissues under normal and pathological stress conditions; MTS cell proliferation assay and flow cytometry were used to evaluate tumor cell proliferation and apoptosis exposed to inhibitors targeting ER stress signaling pathways and p97 under normal and stress conditions; Adenoviral infection, RNA interference (RNAi). flow cytometry, mammosphere culture and Transwell invasion assay were used to investigate the role of ER stress inhibitors, p97 inhibitors and p97 expression level in the breast cancer stem cells in vitro; Subcutaneous xenograft tumor model, immunohistochemistry and flow cytometry were used to examine the influence of UPR and p97 inhibitors on the function of breast cancer stem cell in vivo; Microarray. real-time qPCR. western blot and RNAi were used to elucidate the mechanism of p97 inhibitors on the function of breast cancer stem cell.Results Significant difference of ER stress related molecules expression and activation level was observed among different tumor cells and tissues under normal and stress conditions. UPR and p97 inhibitors decreased tumor cell proliferation with different efficacy, however, only p97 inhibitors induced obvious cell apoptosis under normal condition.ER stress inhibitors caused significant apoptosis under stress conditions.P97 expression level was positively correlated with that of SOX2 and CD44-/CD24-population in breast cancer cells and tissues. Alteration of p97 expression level in breast cancer cells resulted in corresponding changes in breast cancer stem cell functions.CD44-/CD24" percentage, mammosphere formation and in vitro invasion ability were inhibited when breast cancer cells were exposed to p97 inhibitors. P97 inhibitors could significantly reduce the MDA-MB-231 formed xenografts growth, breast cancer sternness and recurrence. The cell proliferation, apoptosis and breast cancer stemness were affected obviously after treated with p97 inhibitor, Eer I,plus Doxorubicin or Bortezomib. Mechanically, p97 increased SKP2 and Sox2 expression level via downregulating C/EBP-8 and upregulating p53 expression levels, then resulted in maintenance of breast cancer stem cell functions.Conclusion Three UPR branches signaling pathway and ERAD affected the adherent breast cancer cells and CSCs to different extent, and p97 represents a prime target in the UPR crucial for both cancer growth and the CSC population maintenance.