Abstract: Background Aberrant expression of XIST, a long noncoding RNA initiating X chromosome inactivation (XCI) during early embryogenesis, is a common feature of breast cancer (BC). However, the roles of post-XCI XIST in breast carcinogenesis remain elusive. Methods In this study, we examined the expression of XIST in human BC cell (BCC) lines across the spectrum of BC subtypes. We then investigated the effect of knockdown (KD) of aberrantly expressed XIST in luminal and triple-negative (TN) BCCs on tumor growth, cancer stem cell (CSC) activities, and global gene expression. We identified the most significantly altered genes and pathways in ALDH bulk tumor cells and ALDH+ CSCs upon XIST KD and validated the roles of these genes in regulating ALDH+ epithelial (E) versus CD24−/loCD44+/hi mesenchymal (M) CSCs. Lastly, we conducted miRNA array and luciferase reporter assays to define the molecular mechanisms of XIST in CSC regulation. Results Doxycycline (DOX) induced XIST KD markedly inhibits spheroid/colony forming capacity, tumor growth and tumor-initiating potential. This phenotype is attributed to impaired E-CSC in luminal and E- and M-CSC activities in TN BCCs. Gene expression profiling demonstrates that XIST KD most significantly affects cytokine-cytokine receptor interactions, resulting in markedly suppressed expression of proinflammatory cytokines IL-6 and IL-8 in the bulk of tumor cells. Exogenous IL-6, but not IL-8, rescues the reduced sphere-forming capacity and proportion of ALDH+ CSCs in luminal and TN BCCs following XIST KD. This suggests a mechanism whereby XIST regulates IL-6 production by bulk tumor cells, which then acts in a paracrine manner on ALDH+ CSCs that display elevated IL-6 receptor (IL6R) expression. XIST functions as a molecular sponge for MicroRNA let-7a-2-3p to derepress IL-6 expression, which in turn promotes self-renewal of ALDH+ CSCs by inducing STAT3 activation and expression of key CSC factors including c-MYC, KLF4 and SOX9. Conclusions This study supports a novel role of XIST by derepressing let-7 controlled paracrine IL-6 proinflammatory signaling to promote CSC self-renewal.

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