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. 2017 Nov 30;7(1):16684.
doi: 10.1038/s41598-017-16687-6.

Profiling of the Transcriptional Response to All-Trans Retinoic Acid in Breast Cancer Cells Reveals RARE-independent Mechanisms of Gene Expression

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Free PMC article

Profiling of the Transcriptional Response to All-Trans Retinoic Acid in Breast Cancer Cells Reveals RARE-independent Mechanisms of Gene Expression

Krysta Mila Coyle et al. Sci Rep. .
Free PMC article

Abstract

Retinoids, derivatives of vitamin A, are key physiological molecules with regulatory effects on cell differentiation, proliferation and apoptosis. As a result, they are of interest for cancer therapy. Specifically, models of breast cancer have varied responses to manipulations of retinoid signaling. This study characterizes the transcriptional response of MDA-MB-231 and MDA-MB-468 breast cancer cells to retinaldehyde dehydrogenase 1A3 (ALDH1A3) and all-trans retinoic acid (atRA). We demonstrate limited overlap between ALDH1A3-induced gene expression and atRA-induced gene expression in both cell lines, suggesting that the function of ALDH1A3 in breast cancer progression extends beyond its role as a retinaldehyde dehydrogenase. Our data reveals divergent transcriptional responses to atRA, which are largely independent of genomic retinoic acid response elements (RAREs) and consistent with the opposing responses of MDA-MB-231 and MDA-MB-468 to in vivo atRA treatment. We identify transcription factors associated with each gene set. Manipulation of the IRF1 transcription factor demonstrates that it is the level of atRA-inducible and epigenetically regulated transcription factors that determine expression of target genes (e.g. CTSS, cathepsin S). This study provides a paradigm for complex responses of breast cancer models to atRA treatment, and illustrates the need to characterize RARE-independent responses to atRA in a variety of models.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Microarray analysis identifies disparate transcriptional responses to ALDH1A3 manipulation and atRA treatment in MDA-MB-231 and MDA-MB-468 cells. (a) Overlap of ALDH1A3-upregulated genes in MDA-MB-231 (ALDH1A3 cDNA/scramble vector) compared to MDA-MB-468 (scramble vector/ALDH1A3 shRNA knockdown). (b) Overlap of genes upregulated by 100 nM atRA in MDA-MB-231 and MDA-MB-468. Comparison of ALDH1A3-upregulated and atRA-upregulated genes in (c) MDA-MB-231 and (d) MDA-MB-468). (e) Sequence logos generated from all RAREs identified in associated gene lists.
Figure 2
Figure 2
DAC treatment does not align atRA-induced transcriptional profiles. Hierarchical clustering (heatmap.2, gplots) of microarray expression values from MDA-MB-231 and MDA-MB-468 cells treated with atRA, DAC, or both demonstrate that the use of DAC did not align the RA-inducible transcriptional profiles in these cell lines. Genes which were commonly upregulated in both cell lines are indicated by * on the right-hand side, while limited clusters of genes which displayed DAC-permissive atRA inducibility are indicated by lowercase Roman numerals. These genes are described in more detail in Supplementary Table S3.
Figure 3
Figure 3
Decitabine does not restore atRA inducibility of specific genes between cell lines. (a) MDA-MB-231 and MDA-MB-468 cells were treated with atRA, DAC, and/or TSA and relative expression of GDF15, CDH5, and SCEL were determined by qPCR. A two-way analysis of variance was used to compare the effect of atRA treatment to the effects of DAC and/or TSA treatment (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001). (b) β-values representing the relative methylation (Illumina HM450 arrays) of distinct CpG sites in MDA-MB-231 and MDA-MB-468 cells treated with DAC are compared within 1500 bp of the transcription start site (TSS) (n = 3, GSE103425).
Figure 4
Figure 4
IRF1 is an atRA-inducible transcription factor. (a) PASTAA analysis of transcription factor affinities identified disparate transcription factors associated with atRA-inducible genes in MDA-MB-231 as compared to MDA-MB-468. (b) qPCR was used to detect expression of IRF1 in MDA-MB-231 and MDA-MB-468 cells. (c) Relative expression of IRF1 following atRA and DAC treatment in MDA-MB-231 and MDA-MB-468 cells was determined by qPCR. A two-way analysis of variance was used to compare the effect of atRA treatment to the effect of DAC treatment (*p < 0.05, **p < −0.01). (d) The expression of STAT1 was measured in MDA-MB-231 following atRA treatment and compared using a paired student’s t-test.
Figure 5
Figure 5
IRF1 expression is required for CTSS expression in MDA-MB-231 cells. (a) shRNA knockdowns of IRF1 were generated in MDA-MB-231 and MDA-MB-468. Values were compared using a one-way analysis of variance with repeated measures (n = 4). (b) CTSS expression was measured by qPCR and compared between MDA-MB-231 and MDA-MB-468 cells using a paired student’s t-test (n = 4). (c) CTSS expression was measured in shRNA knockdowns following treatment with atRA and/or DAC. A two-way analysis of variance was used to determine the effect of IRF1 knockdown compared to atRA/DAC treatment (n = 4). (d) MDA-MB-231 and MDA-MB-468 cells were treated with DAC and/or TSA and CTSS expression was measured by qPCR. Values were compared with a two-way analysis of variance (n = 4). (e) β-values representing the relative methylation (Illumina HM450 arrays) of distinct CpG sites in MDA-MB-231 and MDA-MB-468 cells treated with DAC are compared within 1500 bp of the transcription start site (TSS) (n = 3, GSE103425). For all statistical comparisons, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6
IRF1 expression strongly correlates with CTSS and GBP4 expression in breast cancer patient tumors. The expression of IRF1 target genes CTSS, GBP4, TNFSF10, and RARRES3 in 421 breast cancer patient tumors are plotted against IRF1 expression. Correlation and significance are indicated for each plot.

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