Purpose: Besides its therapeutic effects, chemotherapeutic agents also enhance the malignancy of treated cancers in clinical situations. Recently, epithelial-mesenchymal transition (EMT) has attracted attention in studies of tumor progression. We aimed to test whether transient Adriamycin treatment induces EMTand apoptosis simultaneously in cancer cells, clarify why the same type of cells responds differentially (i.e., apoptosis, EMT) to Adriamycin treatment, and elucidate the role of Twist1, the master regulator of EMT, in this process. Experimental Design: In unsynchronized MCF7 cells or cells synchronized at different phases, apoptosis, EMT, and concurrent events [multidrug resistance (MDR) and tumor invasion] after Adriamycin or/and Twist1 small interfering RNA treatment were examined in vitro and in vivo. The Adriamycin-induced Twist1 expression and the interaction of Twist1 with p53-Mdm2 were examined by immunoblotting and immunoprecipitation, respectively. Results: We showed in vitro that Adriamycin induced EMTand apoptosis simultaneously in a cell cycle^dependent manner. Only the cells undergoing EMT displayed enhanced invasion and MDR. Twist1 depletion completely blocked the mesenchymal transformation, partially reversed MDR, and greatly abolished invasion induced by Adriamycin. Also, we confirmed in vivo that Twist1 RNA interference improved the efficacy of Adriamycin for breast cancers. Further, Twist1 reduction in Adriamycin-treated cells promoted p53-dependent p21induction and disrupted the association of p53 with Mdm2. Conclusions: Our studies show the diverse responses to Adriamycin treatment in cells at different phases, suggest an unrecognized role of EMT in regulating MDR and invasion, and show the efficacy of Twist1RNA interference in Adriamycin-based chemotherapies for breast cancer.

The epithelial-mesenchymal transition (EMT) induced by chemotherapeutic agents promotes malignant tumor progression; however, the mechanism underlying the drug-induced EMT remains unclear. In this study, we reported that miR-448 is the most downregulated microRNA following chemotherapy. Suppression of miR-448 correlated with EMT induction in breast cancer in vitro and in vivo. With the use of chromatin immunoprecipitation-seq analysis, we demonstrated that miR-448 suppression induces EMT by directly targeting special AT-rich sequence-binding protein-1 (SATB1) mRNA, leading to elevated levels of amphiregulin and thereby, increasing epidermal growth factor receptor (EGFR)-mediated Twist1 expression, as well as nuclear factor jB (NF-jB) activation. On the other hand, we also found that the adriamycin-activated NF-jB directly binds the promoter of miR-448 suppressing its transcription, suggesting a positive feedback loop between NF-jB and miR-448. Furthermore, all patients who received cyclophosphamide (CP), epirubicin plus taxotere/CP, epirubicin plus 5-fluorouracil chemotherapy showed miR-448 suppression, an increased SATB1, Twist1 expression and acquisition of mesenchymal phenotypes. These findings reveal an underlying regulatory pathway, in which the autoregulation between NF-jB and miR-448 is important for restrain miR-448 suppression upon chemotherapy and may have a role in the regulation of chemotherapy-induced EMT. Disruption of the NF-jB-miR-448 feedback loop during clinical treatment may improve the chemotherapy response of human breast cancers in which EMT is a critical component. Chemotherapy is a systemic treatment that destroys reproducing cells, but it cannot differentiate between normal and cancerous cells. Side effects occur when normal cells become damaged. Apart from the side effects of cancer chemotherapy, unexpected 'opposite effects' of chemotherapy, which enhance the critical steps in malignancy rather than inhibiting them, 1-3 have attracted progressively more attention, suggesting that novel strategies should be developed to reverse these opposite effects and render chemotherapy more effective.Tumor cells progress from non-invasive to malignant phenotypes via a series of critical steps that involve morphological changes referred to as the epithelial-mesenchymal transition (EMT). 4 EMT is a process originally observed during the embryonic development, in which cells lose epithelial characteristics. [5][6][7][8][9][10] With respect to the chemotherapy, recent studies have demonstrated a close link between EMT and insensitivity to chemotherapeutic agents. Hiscox et al. 11 showed increased b-catenin expression and elevated levels of transcription of b-catenin target genes known to be involved in tumor progression and EMT in the tamoxifen (TAM)-resistant MCF7 cells. Kajiyama et al. 12 identified an association between chronic paclitaxel resistance and induction of EMT in epithelial ovarian carcinomas. In addition, we previously showed that transient adriamycin treatmentinduced EMT and apoptosi...

Cushing’s disease results from corticotroph adenomas of the pituitary that hypersecrete adrenocorticotropin (ACTH), leading to excess glucocorticoid and hypercortisolism. Mutations of the deubiquitinase gene USP8 occur in 35–62% of corticotroph adenomas. However, the major driver mutations in USP8 wild-type tumors remain elusive. Here, we report recurrent mutations in the deubiquitinase gene USP48 (predominantly encoding p.M415I or p.M415V; 21/91 subjects) and BRAF (encoding p.V600E; 15/91 subjects) in corticotroph adenomas with wild-type USP8. Similar to USP8 mutants, both USP48 and BRAF mutants enhance the promoter activity and transcription of the gene encoding proopiomelanocortin (POMC), which is the precursor of ACTH, providing a potential mechanism for ACTH overproduction in corticotroph adenomas. Moreover, primary corticotroph tumor cells harboring BRAF V600E are sensitive to the BRAF inhibitor vemurafenib. Our study thus contributes to the understanding of the molecular mechanism of the pathogenesis of corticotroph adenoma and informs therapeutic targets for this disease.