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  • br A br SKBrM br Sham

    2020-08-28


    A
    SKBrM3
    Sham BCF
    RNAseq
    Common:
    B
    ShamBCF ShamBCF HMGA2 survival 100
    HERPUD1
    MBOAT2
    RELN -free 60
    GBP1
    GCNT1 Relapse
    HSPH1
    PIM1
    SKBrM3
    HMGA2Low
    HMGA2High
    C
    Sham BCF Sham BCF
    HMGA2
    -tubulin
    RNA
    D ShamBCF E 5 *
    StainingIntensity 4
    Sham
    H
    I
    F
    *** * Sham BCF Sham BCF
    HMGA2expression (log
    8 met met HMGA2
    HMGA2mRNA 2
    -tubulin
    BCF
    No-metBrain- Lung-
    6 BCF
    CSC populaiton **
    SKBrM3 MCF7-
    shXIST
    SKBrM3
    Sham
    Sham BCF 
    BCF
    BrM
    SKB 2
    L Sham BCF Sham BCF
    HMGA2
    -tubulin
    shScr shCav3.2
    J
    Sham K
    BCF
    ns
    SKBrM3
    Sham
    BCF 
    M Sham
    BCF BCF
    % CSCpopulaiton 6
    % CSCpopulaiton 4
    Control HMGA2  ns
    Vehicle Ethosuximide 
    cofactor known to enhance tumourigenesis and metastasis of multiple cancer types by reprograming stem TAK 242 [32,33]. Therefore, we exam-ined if BCF also affects the stem cell population. BCF treated cells indeed significantly reduced stem cell population and sphere forming ability of brain-tropic cancer cells without affecting stemness of neuron cells (Fig. 4H-I, Supplementary 3D). In addition, silencing HMGA2 in brain-tropic cells diminished stem cell population to an extent similar to BCF 
    treatment (Supplementary Fig. 3E-F), while ectopic expression of HMGA2 in the parental cells, SKBr3 and MDA-MB-231, significantly in-creased the CSCs population (Supplementary Figs. 3G-H). Ectopic ex-pression of HMGA2 also abrogated the suppressive effect on CSCs by BCF (Fig. 4J), suggesting that this effect on CSCs is mediated through downregulation of HMGA2. Additionally, the suppressive effect on HMGA2 and CSC was abrogated by ethosuximide treatment or Cav3.2 r> knockdown (Fig. 4K-L and Supplementary Fig. 3I\\K). Previous reports suggest that calcium-influx activates CAMKII to phosphorylate and de-grade β–catenin, which is a known upstream regulator of HMGA2 [34,35]. Therefore, we examined the possibility that calcium influx by BCF controls HMGA2 via β-catenin. Indeed, BCF strongly upregulated phosphorylated β-catenin level while decreasing the total β-catenin in CAMKII dependent manner (Fig. 4M). These results suggest that BCF suppress CSC population by decreasing HMGA2 expression through CAMKII-mediated β-catenin degradation.
    3.4. BCF suppress exosomal miR-1246 expression
    As shown in Fig. 2, the tumour suppressive effect of BCF is remark-ably more prominent in vivo compared to its effect in cell based study in vitro. Therefore, we hypothesised that, in addition to its direct effect on tumour cells, BCF modulates cell-cell communication between tu-mour cells and stroma in the tumour microenvironment to amplify the suppressive effect in vivo. Exosomal vesicles or exosomes are known to be shed by tumour cells and carry components for cell-cell communication to promote tumour progression [36]. To examine the effect of BCF on exosomal RNA expression, we first isolated exosomes from SKBrM3 conditioned medium (CM) and confirmed the particle size by electron microscopy (Supplementary Fig. 4A). We then per-formed expression profile analysis of exosomal microRNAs from Sham- or BCF-treated SKBrM3 cells (Fig. 5A). We found that miR-1246 showed the highest extent of downregulation upon BCF treatment. The suppressive effect of BCF on miR-1246 was verified in both 231-BrM and SKBrM3 cells (Fig. 5B). In addition, the exosomes isolated from the blood of mice that were treated with BCF showed significantly diminished levels of miR-1246 (Fig. 5C). Consistent with these results, higher miR1246 expression was correlated with worse survival when TCGA dataset was examined (Fig. 5D). We also found that miR1246 ex-pression is significantly higher in brain metastatic lesions compared to other metastatic sites (Fig. 5E). Similarly, serum level of miR-1246 was also elevated in patients with brain metastasis (Fig. 5F). In addition, the expression of both intracellular and exosomal miR-1246 was higher in brain tropic cells in vitro (Fig. 5G). Previous reports indicate that miR-1246 promotes tumour angiogenesis through intracellular and extracel-lular mechanisms [37,38]. Therefore, it is plausible that BCF suppresses angiogenesis in brain microenvironment by decreasing exosomal miR-1246 levels. To test this possibility, we treated human brain microvascu-lar endothelial cells (HBMEC) with conditioned medium (CM) or exosomes prepared from BCF-treated brain metastatic cells. BCF treated CM or exosomes significantly decreased miR-1246 expression and tube-forming ability of HBMEC (Fig. 5H and supplementary 4B-C). To further verify the role of exosomal miR-1246 in angiogenesis, we prepared exosomes from MDA-MB-231 cells overexpressing miR-1246. Indeed, treatment with exosomes from 231-miR1246 cells increased the tube formation ability of HBMEC (Fig. 5I). Additionally, we ectopically expressed miR-1246 directly in HBMEC. These cells acquired higher tube forming ability concomitant with decreased expression of TSP2, which is a known target of miR-1246 (Supplementary Fig. 4D-E). We then performed immunohistochemical analysis for the brain tumours that were treated with BCF using mouse-specific anti-CD31 antibody