RNA-Seq of Human Breast Ductal Carcinoma <em>In Situ</em> Models Reveals Aldehyde Dehydrogenase Isoform 5A1 as a Novel Potential Target

<div><p>Breast ductal carcinoma <em>in situ</em> (DCIS) is being found in great numbers of women due to the widespread use of mammography. To increase knowledge of DCIS, we determined the expression changes that are common among three DCIS models (MCF10.DCIS, SUM102 and SUM225) compared to the MCF10A model of non-tumorigenic mammary epithelial cells in three dimensional (3D) overlay culture with reconstituted basement membrane (rBM). Extracted mRNA was subjected to 76 cycles of deep sequencing (RNA-Seq) using Illumina Genome Analyzer GAIIx. Analysis of RNA-Seq results showed 295 consistently differentially expressed transcripts in the DCIS models. These differentially expressed genes encode proteins that are associated with a number of signaling pathways such as integrin, fibroblast growth factor and TGFβ signaling, show association with cell-cell signaling, cell-cell adhesion and cell proliferation, and have a notable bias toward localization in the extracellular and plasma membrane compartments. RNA-Seq data was validated by quantitative real-time PCR of selected differentially expressed genes. Aldehyde dehydrogenase 5A1 (ALDH5A1) which is an enzyme that is involved in mitochondrial glutamate metabolism, was over-expressed in all three DCIS models at both the mRNA and protein levels. Disulfiram and valproic acid are known to inhibit ALDH5A1 and are safe for chronic use in humans for other disorders. Both of these drugs significantly inhibited net proliferation of the DCIS 3D rBM overlay models, but had minimal effect on MCF10A 3D rBM overlay models. These results suggest that ALDH5A1 may play an important role in DCIS and potentially serve as a novel molecular therapeutic target.</p> </div

ALDH5A1 as a target in DCIS models.

<p>(<i>A, B</i>) Effect of DSF and VPA on proliferation of MCF10A (<i>open circles</i>), MCF10.DCIS (<i>closed circles</i>), SUM102 (triangles) and SUM225 (<i>squares</i>) 3D rBM cultures. Cells were incubated for 72 hours with the indicated concentrations of drugs or with DMSO as a vehicle control and subjected to an MTT assay. Sigmoidal dose-response curves were plotted using nonlinear regression analysis. Data represent the mean ± SEM of three independent experiments (each performed in triplicate). (<i>C</i>) Effect of DSF and VPA on 3D rBM cultures of SUM102-mRFP cells. Cells were incubated for 8 days with the indicated concentrations of drugs or with DMSO as a vehicle control and DIC images are shown. Size bar, 90 µm.</p

Significant canonical pathways associated with differentially expressed genes.

<p>The list of genes identified as consistently differentially expressed in all models of DCIS were uploaded into Ingenuity Pathway Analysis software. A Fisher’s exact test was used to test the statistical significance with a significance level of 0.05.</p

ALDH5A1 is over-expressed in DCIS models.

<p>Whole cell lysates from 3D rBM overlay cultures of MCF10A, MCF10.DCIS, SUM102 and SUM225 were probed for ALDH5A1 (upper panel) and for GAPDH (lower panel) as a loading control. Densitometry indicated that ALDH5A1 levels in the lysates of MCF10.DCIS, SUM102, and SUM225 were 2.7, 3.1, and 7.3-fold over that in MCF10A in this representative experiment. In three independent analyses of MCF10.DCIS 3D rBM cultures, the mean increase in ALDH5A1 over control was 3.8-fold.</p

Comparison of the changes in expression of selected genes detected by RNA-Seq and qRT-PCR.

<p>The values represent log₂fold change in MCF10.DCIS, SUM102 and SUM225 compared to expression in MCF10A. Positive values indicate up-regulation (highlighted in green) and negative values indicate down-regulation (highlighted in red).</p

Significant biological processes associated with the differentially expressed genes as identified by Genomatix.

<p>The differentially expressed genes in all models of DCIS in comparison to MCF10A were analyzed by WebGestalt2 tool. A Fisher’s exact test was used to determine statistical significance with a significance level of 0.05. The <i>p</i>-values and the number of genes associated with a particular function or process are indicated in bold in the respective boxes.</p

The molecular and cellular functions and diseases and disorders associated with the consistently differentially expressed genes derived from RNA-Seq data analysis.

<p>The values in the second column represent the statistical significance score (<i>p </i>value). The number of differentially expressed genes in the dataset are indicated in the third column.</p

Genes with the largest fold change in expression identified in the list of those differentially expressed.

<p>The values in the Table refer to log₂ based fold changes. For any differentially expressed gene, if its log₂fold change value is infinity (or minus infinity) in any model, it was assigned a value of 10 (or −10). The significantly differentially over-expressed in DCIS in comparison to non-tumorigenic MCF10A include anterior gradient homolog 2 <i>(AGR2</i>), grainyhead like (Drosophila) <i>(GRHL2)</i>, apolipoprotein D (<i>APOD</i>), chemokine (C-C motif) ligand 20 (<i>CCL20</i>), cation transport regulator homolog 1 (E. coli) (<i>CHAC1</i>), claudin 4 (<i>CLDN4</i>), lipocalin 2 (<i>LCN2</i>), lipase member H (<i>LIPH</i>), fucosyltransferase 3 (<i>FUT3</i>), and cAMP responsive element binding protein 5 (<i>CREB5</i>). The genes which were found be significantly down-regulated include alcohol dehydrogenase 1B (class I), (<i>ADH1B</i>), RNA, Ro-associated Y5 (<i>RNY5</i>), transforming growth factor, beta-induced (<i>TGFBI</i>), podoplanin (<i>PDPN</i>), tolloid-like 1 (<i>TLL1</i>), dermatopontin (<i>DPT</i>), cell adhesion molecule 3 (<i>CADM3</i>), aspartoacylase (<i>ASPA</i>), Duffy blood group, chemokine receptor (<i>DARC</i>) and nidogen 1 (<i>NID1</i>).</p

RNA-Seq analysis of MCF10A and DCIS models (<i>A</i>) Differential interference contrast (DIC) images of 12-day 3D rBM overlay cultures of MCF10A, MCF10.DCIS, SUM102, and SUM225 cells.

<p>Scale bar, 200 µm. (<i>B</i>) Unsupervised hierarchical clustering of the transcript profile from the eight RNA-Seq samples. (<i>C</i>) Cluster dendrogram of RNA-Seq samples based on differentially expressed genes. (<i>D</i>) Venn diagram of differential expression results (with a p-value <0.001 and cut-off fold change of 4) showing the overlap between the genes expressed by different models of DCIS in comparison to MCF10A. There are a total of 295 genes that are consistently differentially expressed between the MCF10A and the three DCIS models: MCF10.DCIS, SUM102 and SUM225. The expansion shows that 156 transcripts were decreased, 63 transcripts were increased, and 76 transcripts were differentially expressed in all three models but the direction of change was variable.</p

Significant consistently differentially expressed genes involved in various types of cancers.

<p>The number of focus genes and the p-values are indicated in the table.</p