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    Awarded Grants
    Protein Signatures of Microinvasive (ER-) and Adjacent Noninvasive (ER+) Cell Clusters During the Progression of In Situ Lesions to Invasive Carcinomas

    Scientific Abstract:
    Protein Signatures of Microinvasive (ER-) and Adjacent Noninvasive (ER+) Cell Clusters During the Progression of in situ Lesions to Invasive Carcinomas Disruption of myoepithelial cells (ME) and the basement membrane is a prerequisite for human ductal carcinoma in situ (DCIS) invasion into the stroma, and this process is first characterized by microinvasive cell clusters overlying focal disruptions in these layers. We have previously shown that 86.4% of these cell clusters are negative for estrogen receptor (ER) expression, while adjacent cells bound by an intact ME layer express high levels of ER. Additional differences may furnish important clues as to the biological mechanisms governing the initial stages of tumor invasion. Our aims are to show that (1) microinvasive and noninvasive cell clusters possess distinct protein expression profiles, not confined to the ER, but (2) including tumor and metastasis suppressors, and (3) that cleavage of oligosaccharide chains will increase the yield of identifiable proteins. We will generate surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) proteomics spectra on 50 pairs of paraffin-embedded microdissected ER+ and ER- tissues recovered from the same duct in 50 separate patients, then purify and identify any differentially expressed proteins using powerful, multidimensional separation techniques with a special emphasis on the identification of tumor suppressors, metastasis suppressors, proteases, growth factors and their receptors. Incubation of tissue lysates with endoglycosidases to cleave oligosaccharide chains that limit the efficiency of protein identification using Expasy tools that search the Swiss-Prot database is anticipated to greatly improve the yield of identifiable proteins, currently limited to 25%. Additional validation of all findings will be accomplished with freshly frozen microdissected tissues from 10 patients. The determination of protein differences between these noninvasive and microinvasive cell clusters, and the protein identities, could provide major benefits for all patients diagnosed with DCIS, as there is no way to differentiate between dangerous cancers and less threatening lesions. If the protein markers for the initiation of invasion could be identified, lesions that are likely to develop into invasive carcinomas could be detected and treated early, as swift diagnosis and management is the hallmark of effective therapy, and patients with in situ lesions could avoid the agonizing decisions often associated with radical therapeutic options.

    Lay Abstract:
    Protein Signatures of Microinvasive (ER-) and Adjacent Noninvasive (ER+) Cell Clusters During the Progression of in situ Lesions to Invasive Carcinomas Human ductal carcinoma in situ (DCIS) is a form of breast cancer in which the breast duct remains intact, so tumor cells are trapped inside the duct, unable to escape and spread into the surrounding tissue. DCIS may eventually progress to an invasive cancer, but the majority will remain non-threatening lesions, which complicates the determination of an appropriate treatment regimen for breast cancer patients. We will study cells during the earliest stages of invasion, called microinvasion, when the first few cells have broken through the biological barriers that separate them from the surrounding tissues, thereby beginning the process of metastasis. At the same time, we will study neighboring cells from the same duct that have failed to disrupt these barriers. We will use a process called microdissection, in which small clusters of cells are removed from the duct, and we hope to determine what property of the invasive cells gives them the ability to charge through the biological roadblocks before them. We suspect the differences between these cell clusters will provide important clues as to how and why some cells invade while others do not. We will obtain 50 pairs of microdissected cell clusters from 50 separate patients, with each sample pair composed of invasive and noninvasive cells from the same duct of the same patient. We will analyze these samples using an emerging technology known as SELDI-TOF-MS, then utilize powerful purification techniques to help determine the identity of these proteins. We will also attempt to improve upon this existing technology by using a new technique to remove sugar molecules from the proteins. These improvements to protein analysis methods may benefit all cancer researchers. During the final phase of this project, we will verify and enhance our findings by a repeat analysis using 10 pairs of freshly frozen tissue samples. These findings could provide major benefits for all patients diagnosed with DCIS, as there is currently no way to differentiate between dangerous tumors and less threatening lesions. If the protein signatures of early invasion could be identified, lesions that are likely to develop into invasive carcinomas could then be detected and treated early, as swift diagnosis and management is the hallmark of effective therapy, and patients with non-threatening lesions could avoid the agonizing decisions often associated with radical therapeutic options.