Open Access Dissertation
Date of Award
Raymond R. Mattingly
Mammary ductal carcinoma in situ (DCIS) is being found in great numbers of women due to the widespread use of mammography. The molecular and genetic changes underlying the progression from normal breast tissue to DCIS are not clearly understood. The goal of the present study was to determine gene expression changes in different DCIS models (MCF10.DCIS, SUM102 and SUM225) in comparison to normal breast epithelial cells (MCF10A) that may enable us to identify novel markers of disease progression and potential therapeutic targets. We cultured the cells in three dimensional reconstituted basement membrane (3D rBM) for RNA extraction and used the purified RNA for whole genome expression analysis by Affymetrix GeneChipÂ® U133A 2.0 Arrays. We found 157 genes that were consistently differentially expressed between MCF10A and different DCIS models. Pathway analysis showed that a subset of differentially expressed genes in DCIS is strongly linked to glutamate metabolism and others involved in dysregulation of various pathways such as IGF-1 signaling, integrin signaling and fatty acid biosynthesis. To further enrich the expression data and identify low abundance transcripts we employed next generation sequencing (RNA-Seq) using Illumina Genome Analyzer GAIIx. Analysis of the sequencing data showed 295 consistently differentially expressed transcripts in the DCIS models. We found that these differentially expressed genes encode proteins that are associated with a number of signaling pathways such as integrin, fibroblast growth factor and transforming growth factor beta 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. We further mined our sequencing data to explore common frameworks in the promoter regions of differentially expressed genes. We found significant enrichment of several common frameworks present in promoters of genes like ELF3, CCL20, NFATC4, RAP1GAP, SPRY4 and PDGFB. We also validated the expression data from microarray and RNA Seq with quantitative real-time PCR (qRT-PCR) of selected differentially expressed genes. The qRT-PCR results showed better concordance with sequencing than with microarray results.
We further characterized ALDH5A1, which encodes the enzyme aldehyde dehydrogenase 5A1 that is involved in mitochondrial glutamate metabolism. We found that ALDH5A1 protein is over-expressed in all three DCIS models. Further, two different drugs, disulfiram and valproic acid, that target ALDH5A1 significantly inhibited growth and proliferation in the DCIS models, but had minimal effect on MCF10A. Our results suggest that ALDH5A1 may play an important role in DCIS and additional studies are warranted to evaluate the potential repurposing of disulfiram and valproic acid to treat DCIS.
Kaur, Hitchintan, "Analysis of gene expression alterations in premalignant progression from normal mammary epithelium to ductal carcinoma in situ by multiple orthogonal tools" (2012). Wayne State University Dissertations. 569.