Y-Box Binding Protein-1 Induces the Expression of CD44 and CD49f Leading to Enhanced Self-Renewal, Mammosphere Growth, and Drug Resistance

Experimental Medicine Program, University of British Columbia, Canada.
Cancer Research (Impact Factor: 9.33). 03/2010; 70(7):2840-51. DOI: 10.1158/0008-5472.CAN-09-3155
Source: PubMed


Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor expressed in >40% of breast cancers, where it is associated with poor prognosis, disease recurrence, and drug resistance. We questioned whether this may be linked to the ability of YB-1 to induce the expression of genes linked to cancer stem cells such as CD44 and CD49f. Herein, we report that YB-1 binds the CD44 and CD49f promoters to transcriptionally upregulate their expressions. The introduction of wild-type (WT) YB-1 or activated P-YB-1(S102) stimulated the production of CD44 and CD49f in MDA-MB-231 and SUM 149 breast cancer cell lines. YB-1-transfected cells also bound to the CD44 ligand hyaluronan more than the control cells. Similarly, YB-1 was induced in immortalized breast epithelial cells and upregulated CD44. Conversely, silencing YB-1 decreased CD44 expression as well as reporter activity in SUM 149 cells. In mice, expression of YB-1 in the mammary gland induces CD44 and CD49f with associated hyperplasia. Further, activated mutant YB-1(S102D) enhances self-renewal, primary and secondary mammosphere growth, and soft-agar colony growth, which were reversible via loss of CD44 or CD49f. We next addressed the consequence of this system on therapeutic responsiveness. Here, we show that paclitaxel induces P-YB-1(S102) expression, nuclear localization of activated YB-1, and CD44 expression. The overexpression of WT YB-1 promotes mammosphere growth in the presence of paclitaxel. Importantly, targeting YB-1 sensitized the CD44(High)/CD24(Low) cells to paclitaxel. In conclusion, YB-1 promotes cancer cell growth and drug resistance through its induction of CD44 and CD49f.

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    • "This finding was confirmed by the clearance of Notch1 NICD immunostaining from the nuclei of T315-treated MDA-MB-231 cells (Figure 2B), as well as the dose-dependent suppressive effect of T315 on Notch reporter activity (Figure 2C) and the expression of various Notch downstream targets (Figure 2D), including HES1 [24],c-Myc[25],Nestin[26],and ABCG2 [27], in both MDA-MB-231 and SUM-159 cells. It is noteworthy that T315-mediated down-regulation of these Notch targets was accompanied by parallel decreases in the expression of CSCassociated epithelial-mesenchymal transition regulators, including zinc finger E-box binding homeobox 1 (ZEB1) [28], Twist [29],a n dY box-binding protein 1 [30], which were also reported to be downstream effectors of ILK [13] [31] (Figure 2D). Moreover, T315 was equipotent in downregulating other CSC-associated markers, including the Wnt signaling effector β-catenin and the hedgehog signaling effector Bmi1 (Figure 2D). "
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    ABSTRACT: Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6-driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6-driven Notch1 activation by ILK in IL-6-producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7(IL-6) cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase-mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6-induced breast CSCs. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Neoplasia (New York, N.Y.) 06/2015; 105(6). DOI:10.1016/j.neo.2015.06.001 · 4.25 Impact Factor
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    • "Similar to Sox2, Y-box binding protein-1 (YB-1) is a transcription factor that has been found in embryonic stem cells, mammary progenitor cells and BC cells [21-23]. Found in 40% of BC tumors [24], YB-1 is believed to promote the tumorigenesis of BC, since it has been shown to enhance mammosphere formation in vitro, and transcriptionally up-regulate the expressions of a large cassette of stem cell-associated proteins including CD44, CD49f (α6 integrin), c-Met, EGFR, Her-2, Cyclin D1, MDR-1, and p110α [22,25-28]. ln other cell types, it has been shown that YB-1 can also suppress gene expression, such as those encoding Fas and granulocyte macrophage colony-stimulating factor [29,30]. "
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    ABSTRACT: Background Sox2, a transcription factor and an embryonic stem cell marker, has been implicated in the pathogenesis of breast cancer (BC). YB-1 is another transcription factor that has been shown to promote stemness in BC cells. Methods Western blotting, quantitative PCR, and siRNAs were used to query the regulatory relationships between YB-1, Sox2, and their downstream targets. Chromatin immunoprecipitation was used to detect YB-1 interactions at the Sox2 promoter. Mammosphere and soft agar assays were used to assess the phenotypic consequences of YB-1 knockdown. Results Here, we report that YB-1 regulates Sox2. YB-1 was found to bind to the SOX2 promoter and down-regulate its expression in MCF7 and ZR751. The regulatory interaction between YB-1 and Sox2 was drastically different between the two phenotypically distinct cell subsets, purified based on their differential response to a Sox2 reporter. They are referred to as the reporter unresponsive (RU) cells and the reporter responsive (RR) cells. Upon siRNA knockdown of YB-1, RU cells showed an increase in Sox2 expression but no change in Sox2 reporter activity; in contrast, RR cells exhibited increased expression and reporter activity of Sox2. Correlating with these findings, YB-1 knockdown induced a differential response in the expression of genes known to be regulated by both Sox2 and YB-1 (e.g. CCND1 and ITGA6). For instance, in response to YB-1 knockdown, CCND1 and ITGA6 expression were decreased or unchanged in RU cells but paradoxically increased in RR cells. Compared to RU cells, RR cells were significantly more resistant to the suppression of mammosphere formation due to YB-1 knockdown. Importantly, mammospheres derived from parental MCF7 cells treated with YB-1 siRNA knockdown exhibited higher expression levels of SOX2 and its downstream targets. Conclusions To conclude, in a subset of BC cells, namely RR cells, YB-1 regulates Sox2 to coordinately maintain stemness and tumorigenic properties.
    BMC Cancer 05/2014; 14(1):328. DOI:10.1186/1471-2407-14-328 · 3.36 Impact Factor
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    • "YB-1 is known to increase the resistance of cancer cells to a number of chemotherapeutic drugs (reviewed in [1]) and can promote tumour cell invasiveness and EMT (reviewed in [2]). YB-1 is also a driver of the stem-cell-like phenotype [48]. Therefore it appears that YB-1 and these miRNAs are opposing in their actions. "
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    ABSTRACT: The nucleic acid-binding protein YB-1, a member of the cold-shock domain protein family, has been implicated in the progression of breast cancer and is associated with poor patient survival. YB-1 has sequence similarity to LIN28, another cold-shock protein family member, which has a role in the regulation of small noncoding RNAs (sncRNAs) including microRNAs (miRNAs). Therefore, to investigate whether there is an association between YB-1 and sncRNAs in breast cancer, we investigated whether sncRNAs were bound by YB-1 in two breast cancer cell lines (luminal A-like and basal cell-like), and whether the abundance of sncRNAs and mRNAs changed in response to experimental reduction of YB-1 expression. RNA-immunoprecipitation with an anti-YB-1 antibody showed that several sncRNAs are bound by YB-1. Some of these were bound by YB-1 in both breast cancer cell lines; others were cell-line specific. The small RNAs bound by YB-1 were derived from various sncRNA families including miRNAs such as let-7 and miR-320, transfer RNAs, ribosomal RNAs and small nucleolar RNAs (snoRNA). Reducing YB-1 expression altered the abundance of a number of transcripts encoding miRNA biogenesis and processing proteins but did not alter the abundance of mature or precursor miRNAs. YB-1 binds to specific miRNAs, snoRNAs and tRNA-derived fragments and appears to regulate the expression of miRNA biogenesis and processing machinery. We propose that some of the oncogenic effects of YB-1 in breast cancer may be mediated through its interactions with sncRNAs.
    PLoS ONE 11/2013; 8(11):e80171. DOI:10.1371/journal.pone.0080171 · 3.23 Impact Factor
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