Fig 2: BET inhibition and depletion repress the expression of RCAN1.4. a-b MDA-MB-231 cells and BT549 cells were treated with various concentrations of BRD4 inhibitor JQ1 for 24 h or 500 nM JQ1 for the indicated times. The RCAN1.4 mRNA levels were quantified using qRT–PCR(a). The cell lysates were prepared for immunoblots (b). c-d MDA-MB-231 and BT549 cells were transiently transfected with BRD4 siRNA for 48 h. The RCAN1.4 and BRD4 mRNA levels were quantified using qRT–PCR(c). The cell lysates were prepared for immunoblots (d). Blue star, RCAN1.1; Red closed circle, RCAN1.4. e BRD4 binding pattern in the promoter regions and in the SE regions of RCAN1.4. The cells were treated with DMSO or JQ1. The data were retrieved from Gene Expression Omnibus (GSE63581). f-g MDA-MB-231 and BT549 cells were treated with or without 200 nM JQ1 for 24 h. The cells were subjected to ChIP analysis using antibodies against BRD4. The association with the SE region (f) and promoter region (g) of RCAN1.4 was quantified by qPCR. An isotype-matched IgG was used as a negative control. h-i MDA-MB-231 and BT549 cells were treated with or without 200 nM JQ1 for 24 h. The cells were subjected to ChIP analysis using antibodies against H3K27ac. The association with the SE region (h) and promoter region (i) of RCAN1.4 was quantified by qPCR. j MDA-MB-231 and BT549 cells were treated with or without 200 nM JQ1 for 24 h. The cells were subjected to ChIP analysis using antibodies against H3K4me1. The association with the SE region of RCAN1.4 was quantified by qPCR. k-n Luciferase reporter assay of RCAN1.4 promote activity and E3 super-enhancer activity in MDA-MB-231 cells and BT549 cells treated with 200 nM JQ1 for 24 h (k, l), or treated with BRD4 siRNA for 48 h (m, n). Error bars represent mean ± SD, n = 3 biological independent samples. * P < 0.05, **P < 0.01. The P value in a, c, m, n was determined by one-way analysis ANOVA with Dunnett’s multiple comparisons test, the P value in f, g, h, i, j was determined by one-way ANOVA with Tukey’s multiple comparisons test, no adjustments were made for multiple comparisons. The P value in k, l was determined by a two-tailed unpaired Student’s t test. Data were representative of three independent experiments

Fig 3: Downregulation of RCAN1.4 and its clinical relevance in breast cancer. a The heatmap of 10 downregulated genes located on HSA21 in breast cancer. The FPKM values of 112 pairs of breast cancer invasive samples and adjacent normal samples were retrieved from TCGA database. b TCGA analysis showed the expression levels of RCAN1 transcript isoforms in breast cancer tissues and normal tissues using the ISOexpresso website (http://wiki.tgilab.org/ISOexpresso/). c Immunoblot results of RCAN1.4 and RCAN1.1 protein expression in 15 pairs of matched breast tissue. Blue star, RCAN1.1; Red closed circle, RCAN1.4. d-e Kaplan-Meier analyses of RFS based on RCAN1(215253_at) mRNA levels in all breast cancer patients (d) and Kaplan-Meier analyses of OS based on RCAN1(208370_at) mRNA levels in the breast cancer patients with lymph node positive subgroup (e) were performed by using the KM-plotter breast cancer database (http://kmplot.com/analysis). Auto select best cutoff was chosen to split RCAN1-high and low groups in the analysis. Univariate Cox proportional hazards regression was carried out to identify HR and 95% CI. f The representative images of strong RCAN1.4 staining in the matched adjacent normal cells (N) and weak staining in tumor cells (T) (Left). Quantitative IHC analysis of RCAN1.4 staining of primary breast tumours and adjacent normal breast tissues was shown (n = 99, right). The P value was determined by Wilcoxon matched-pairs signed rank test (two-sided). g The representative images for low and high expression of RCAN1.4 staining in 258 primary breast cancer tissues were shown (left). Kaplan-Meier plots of the overall survival of patients, stratified by protein expression of RCAN1.4 (right). h The expression of RCAN1.4 in breast cancer tissue without or with distant metastasis. The P value was determined by an unpaired non-parametric Mann-Whitney test (two-sided). Error bars in f, h represented lower hinge - 1.5 * IQR to upper hinge + 1.5 * IQR (where IQR is the inter-quartile range, or distance between the first and third quartiles)

Fig 4: The tumor suppressive effects of RCAN1.4 in breast cancer cells by inhibition of CaN-NFATC1 signaling. a, b Immunoblot in MDA-MB-231 and BT549 cells expressing sgRNAs targeting human RCAN1.4 (a), and in HCC1806 cells stably overexpressing RCAN1.4 (b). Blue star, RCAN1.1; Red closed circle, RCAN1.4. c The transwell cell migration and invasion assay of the indicated MDA-MB-231, BT549 and HCC1806 cells was shown. The migration or invasion of control cells was set as 100%. d 2 × 105 luciferase-tagged WT or RCAN1.4-knockout MDA-MB-231 cells were injected intracardially. BLI images showed representative mice in each group (upper). Normalized metastasis BLI signals from mice (n = 8, bottom). e Kaplan-Meier survival curve of mice were calculated (n = 8). f-g Tumour growth of the indicated BT549 cells in BALB/c nude mice (n = 7 mice per group). Tumor volumes (f) and tumour weights upon autopsy on day 21(g) were calculated. h The indicated MDA-MB-231, BT549 and HCC1806 cells were stained with fluorescent antibodies against NFATc1 (green) or with DAPI (blue). Statistics of the percentage of NFATc1 in the nucleus were determined in the indicated samples (n = 30 cells per group). i-j qRT-PCR monitoring target gene expression in RCAN1.4-knockout MDA-MB-231 and BT549 cells or RCAN1.4-overexpression HCC1806 cells (n = 3 biological independent samples). Expression levels were normalized for GAPDH. Average KO/WT ratios of genes expression in MDA-MB-231and BT549 cells (i), and average OV/vector ratios of genes expression in HCC1806 cells (j) were quantified. k The indicated MDA-MB-231 and BT549 cells were treated with 5 μM CsA for 24 h and the nuclear levels of NFATC1 were detected by Immunoblot. l-n MDA-MB-231-RCAN1.4 KO cells or BT549-RCAN1.4 KO cells were transfected with CaN siRNAs and NFATC1 siRNAs for 48 h. qRT-PCR was used to monitor IGF1 expression (l, n = 3 biological independent samples). Quantification of migratory and invasive cells of images per group was shown (m-n, n = 3 biological independent samples). Error bars represent mean ± SD. **P < 0.01. The P value in c was determined by one-way ANOVA with Dunnett’s multiple comparisons test, the P value in l, m, n was determined by one-way ANOVA with Tukey’s multiple comparisons test, no adjustments were made for multiple comparisons. The P value in d, f, g, h was determined by a two-tailed unpaired Student’s t test. The P value in e was assessed using the log-rank test. All data are representative of three independent experiments

Fig 5: Correlations among RCAN1.4 and RUNX3 expression in breast cancer tissues. a,b Three pairs of breast cancer tissues and matched normal breast tissues were subjected to ChIP analysis using antibody against H3K27ac. The association with the supper-enhancer of RCAN1.4 (a) and promoter region of RCAN1.4 (b) were quantified by qPCR. c The mRNA expression of RCAN1.4 (left) and RUNX3 (right) in three pairs of breast cancer tissues and matched normal tissues. d Kaplan-Meier analyses of OS, RFS and DMFS based on RUNX3 (204197_at) mRNA levels using the KM-plotter breast cancer database (http://kmplot.com/analysis). Auto select best cutoff was chosen in the analysis. e Kaplan-Meier plots of the overall survival of patients, stratified by both mRNA expression of RUNX3 and RCAN1.4. The data were retrieved from breast cancer patients of TCGA database. f The representative images of strong RUNX3 staining in the matched adjacent normal cells (N) and weak staining in tumor cells (T) (left). Quantitative IHC analysis of RUNX3 staining in primary breast tumors and adjacent normal breast tissues was shown (n = 99, right). g The representative images for low and high expression of RUNX3 staining in 258 primary breast cancer tissues were shown (left). Kaplan-Meier plots of the overall survival of patients, stratified by protein expression of RUNX3(right). h The Spearman correlation of RUNX3 with RCAN1.4 expression in breast cancer patient tumors. i Kaplan-Meier plots of the overall survival of patients, stratified by protein expression of RUNX3 and RCAN1.4. j Proposed model of RUNX3 deficiency-mediated disruption of SE-driven RCAN1.4 expression to promote the malignancy of breast carcinoma. During the mammary epithelial cell differentiation, the tumor suppressor gene RCAN1.4 expression is driven by a ~ 23 kb-long super-enhancer region, which is located ~ 266 kb away with the epigenetic signature of active enhancers H3K4me1 and H3K27ac. This SE is occupied by the key transcription factor RUNX3 and physically interacts with the RCAN1.4 locus via DNA looping. In breast cancer cells, the loss of RUNX3 results in disruption of super-enhancer-driven RCAN1.4 expression, which promotes the malignancy of breast carcinoma. The error bars represent mean ± SD, n = 3 biological independent samples. **P < 0.01. The P value in a, b was determined by one-way ANOVA with Tukey’s multiple comparisons test, no adjustments were made for multiple comparisons. The P value in c was determined by a two-tailed unpaired Student’s t test. The P value in f was determined by Wilcoxon matched-pairs signed rank test (two-sided), and the error bars represented lower hinge - 1.5 * IQR to upper hinge + 1.5 * IQR (where IQR is the inter-quartile range, or distance between the first and third quartiles). Data in a, b, c were representative of three independent experiments