Abstract
Breast ductal carcinoma in situ (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.
Highlights
Ductal carcinoma in situ (DCIS) of the breast results from the proliferation and accumulation of atypical epithelial cells that remain restricted to the duct and is a non-obligate precursor to invasive breast cancer
After 12 days in 3D reconstituted basement membrane (rBM) overlay culture, the MCF10A cells form a uniform population of acinar structures as previously described [15,32] whereas the three DCIS models form larger and less uniform structures (Fig. 1A)
Comprehensive gene expression profiling has been used extensively for molecular classification of invasive breast tumors, but relatively fewer studies have been performed in DCIS [20,21,22,23,24,25,26,27,28,40,41]
Summary
Ductal carcinoma in situ (DCIS) of the breast results from the proliferation and accumulation of atypical epithelial cells that remain restricted to the duct and is a non-obligate precursor to invasive breast cancer. All women diagnosed with DCIS undergo at least surgical resection, and many of them are subjected to adjuvant radiation and anti-estrogen therapy Following this aggressive treatment, 5– 30% of these DCIS cases will locally recur, with half of these recurrences being to invasive ductal carcinoma (IDC) [1]. In addition to the problem of identifying additional therapeutic targets in these recurrent cases of DCIS, there is the issue of over-treatment in the majority of DCIS cases that would remain indolent in the absence of surgery and radiation [5,6]. Another challenge that DCIS presents is the heterogeneity of the lesions [7,8]. In addition preventive studies could be performed in women at lower risk because their DCIS is likely to remain indolent [11]
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