PI Billy W. Day, PhD and Jean J. Latimer, PhD

Title “Quantitative proteomics of nuclear matrix proteins in novel human ductal carcinoma in situ model systems”
Summary Background: Ductal carcinoma in situ (DCIS) is the earliest identifiable breast cancer lesion. Because DCIS is a pre-invasive malignancy, a better understanding of if and how it may progress to invasive disease will allow determination of which patients to treat aggressively and avoid unnecessary aggressive procedures. Once determined, these differentially expressed proteins may be used as biomarkers or therapeutic targets, as well as help determine the paths by which normal cells progress to DCIS plus provide a better understanding of breast carcinogenesis and ways to prevent it. Tumor grade, size and presence of necrosis are currently used to make clinical decisions regarding DCIS.

These have not proven to be good predictors, however, as low-grade DCIS often progresses to invasive disease. Proteins that hold promise for a better understanding of DCIS are those in the nuclear matrix (NMPs). The nucleus is a cellular landmark in the pathology of cancer. NMPs in part help to determine the nuclear shape and processes, have been identified as informative markers of disease states in a variety of cancers, and their detection in the serum and urine supports investigation of them in DCIS. NMPs have been investigated in invasive breast cancer, and several have been identified as unique to malignant specimens. Investigation of the NMPs in DCIS is therefore warranted. Prof. Latimer has over the past decade derived several unprecedented DCIS- and breast reduction mammoplasty-derived cell lines, generated without the use of transforming agents from clinically well-defined patients. In combination with the Day lab’s modern proteomics technologies, we will thoroughly investigate DCIS. The DCIS samples, both invasive and non-invasive, gave rise to cell lines from tumors and nontumor adjacent tissue, and Prof. Latimer also has cell lines derived from contralateral non-diseased breast samples. All of the lines are characterized by karyotype, array-based comparative genomic hybridization, growth rates, breast epithelial markers, and functional DNA repair capacity.

Hypothesis: Detectable differences exist between invasive and noninvasive DCIS at the protein level, particularly in the nucleus, and these differences are distinct from those demonstrated at the nucleic acid level.

Specific Aims: In this 2-year study, we will characterize these lines by examining their differential protein expression. In Aim 1, we will grow cultures of the various cells in quantities necessary for replete proteomics analyses, isolate NMPs from each, then employ two of our several proteomic techniques, namely difference two dimensional gel electrophoresis (DIGE) and isobaric tags for relative and absolute protein quantitation (iTRAQ), each followed by mass spectrometric protein identification. These two methods offer orthogonal means of quantitation to help verify results. In Aim 2, the results will be further evaluated with Western blotting and RNAi.

Study Design: By comparing the DCIS sample with the pathologically normal adjacent tissue, in addition to the contralateral normal breast sample, we will be able to determine markers specifically expressed by DCIS and investigate their role in the disease, including invasiveness.

The mammoplasty cultures will be used as truly normal comparative controls.

Impact: If we understand the changes in the cells that give rise to DCIS, we may be able to identify the origins of breast cancer and develop preventative agents

Source DOD Synergy Award (only 10 were given) $800,000

Term 2-year study