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***test. significantly, positive correlations between the expressions of MUC1, EGFR, and IL-6 were found in 20 cervical cancer patients after chemotherapy. Mining TCGA data sets also uncovered the expressions of MUC1-EGFR-IL-6 Zoledronic Acid correlates with poor disease-free survival in chemo-treated cervical cancer patients. Collectively, our work has demonstrated that the MUC1-EGFR-CREB/GR axis stimulates IL-6 expression to induce CSCs enrichment and importantly, this effect can be abrogated by erlotinib, uncovering a novel strategy to treat paclitaxel-resistant cervical cancer. test. ***test. **gene in HeLa229/TR (Supplementary Fig. S3A) and SiHa/TR (Supplementary Fig. S3F) cells through CRISPR/Cas9. Remarkably, MUC1 deficiency resulted in a substantial reduction in not only mRNA expression of IL-6 (Supplementary Fig. S3B left) and production of IL-6 (Supplementary Fig. S3B Zoledronic Acid right) but also spheres number (Supplementary Fig. S3C) and colonies number (Supplementary Fig. S3D) in HeLa229/TR cells. These data suggest that paclitaxel-induced CSCs was mediated by MUC1. We next examined the effect of knockout on activation of EGFR and found that the pEGFR was significantly decreased in MUC1-deficient HeLa229/TR cells (Supplementary Fig. S3A). In accordance, the levels of pEGFR, IL-6, the number of spheres, and the number of colonies were significantly reduced upon treatment of erlotinib Zoledronic Acid in HeLa229 TR/CTL cells, but not in HeLa229 TR/CRISPR cells (Supplementary Fig. S3ACD). Moreover, IL-6-neutralizing Zoledronic Acid antibody effectively abrogated the paclitaxel-induced sphere formation in HeLa229 TR/CTL cells, but not in HeLa229 TR/CRISPR cells (Supplementary Fig. S3E). A similar experimental strategy was employed with SiHa/TR cells, which showed an analogous association among MUC1 expression, EGFR activation, IL-6 expression, and CSCs enrichment (Supplementary Fig. S3FCI). To further verify the role of the MUC1-EGFR-IL-6 axis in paclitaxel-resistance, we knocked down in HeLa229 parental cells and found that chemotherapy-induced pEGFR expression was abolished (Supplementary Fig. S4A). In addition, paclitaxel failed to stimulate IL-6 expression in erlotinib-treated cells (Supplementary Fig. S4B). Furthermore, we applied the conditional medium from HeLa229/shCTL or HeLa229/shMUC1-B cells with or without paclitaxel treatment to the cultures of HeLa229/shCTL and HeLa229/shMUC1-B cells (Supplementary Fig. S4C upper). Zoledronic Acid Paclitaxel-treated HeLa229/shCTL conditional medium significantly expanded the CD133+ cells population in HeLa229/shCTL cells, but not in HeLa229/shMUC1-B cells (Supplementary Fig. S4C lower). These data suggested that MUC1 promotes CSCs enrichment through stimulating IL-6-mediated autocrine effect. Accordingly, paclitaxel failed to induce spheres and colonies formation in the presence of erlotinib or IL-6 neutralizing antibody in HeLa229/shCTL cells (Supplementary Fig. S4DCE). Altogether, these results demonstrate that MUC1 activates EGFR to promote IL-6 expression and CSCs enrichment. EGFR induces IL-6 transcription through CREB and GR binding sites To determine how MUC1-EGFR is involved in IL-6 regulation, we conducted immunofluorescence staining in HeLa229P and HeLa229/TR cells that were treated with or without erlotinib (Supplementary Fig. S5A), as well as HeLa229/shMUC1-B cells that re-expressed MUC1 and were treated with or without paclitaxel respectively (Supplementary Fig. S5B). Consistent with our previous report, we found that paclitaxel treatment increased both MUC1 and EGFR in the nucleus, and this effect was blocked by treatment with erlotinib. In light of many studies showing that MUC1 and EGFR act as transcription coactivators, we investigated whether MUC1 and EGFR in the nucleus might Rabbit Polyclonal to BRS3 participate in the transcriptional regulation of IL-6. Chromatin immunoprecipitation (ChIP) showed that paclitaxel induced the binding of EGFR to IL-6 promoter around the region of +386 to +504 (Fig. ?(Fig.3a).3a). This effect of paclitaxel was completely abolished by EGFR inhibitor or MUC1 depletion (Fig. ?(Fig.3b3b upper). Interestingly, we found that MUC1 bound to the ABCB1 promoter, as expected, but not IL-6 promoter (Fig. ?(Fig.3b3b middle). The H3K27Ac acts as a transcriptional activation control (Fig. ?(Fig.3b3b lower). The results suggested that the effect of MUC1 on IL-6 was mediated by EGFR, which directly bound to IL-6 promoter. We carried out luciferase assay.