Research Grants Awarded
The 3D telomeric signature(s) of DCIS
Detection, Diagnosis and Prognosis
Background: The three-dimensional (3D) organization of the normal interphase nucleus is crucial to genome stability. Alterations in the nuclear structure, particularly with regard to the territories that telomeres inhabit, are causal to chromosomal rearrangements and are found in tumor cells. While telomeres of normal nuclei do not overlap, telomeres of tumor cells form aggregates and fusions. The exact definition of the transition from a normal mammary epithelial cell to ductal carcinoma in situ (DCIS) and thence to invasive ductal carcinoma (IDC) is the focus of this proposal. Preliminary 3D data suggest the presence of telomeric aggregates in breast cancer, with an increase in number and intensity in IDC compared with DCIS. Telomeric signatures may also differ in hereditary (BRCA1 and BRCA2 mutation carriers) compared with non-hereditary breast cancer. The deregulated expression of the c-Myc oncoprotein induces the nuclear remodeling of telomeres and chromosomes with subsequent breakage-bridge fusion cycles and chromosomal rearrangements. c-myc is frequently amplified and/or overexpressed in BRCA1-related breast cancer. Therefore, in hereditary breast cancers, we will specifically examine c-Myc-dependent changes in 3D telomeric signatures. Methods: We will examine 150 samples: 50 normal epithelial mammary samples form healthy individuals, 50 samples from non-hereditary breast cancers that contain normal, DCIS and IDC in one tissue section, and 25 samples each of BRCA1 and BRCA2 carriers with breast cancer that contain normal and tumor tissue in one section (DCIS is usually not seen, but will be included wherever possible). Tissue sections will be analyzed by quanitative 3D telomere FISH, imaged in 3D at a resolution of 107 nm in the x,y and 200 nm in the z direction. Images will be 3D reconstructed and quantitated using TeloView, a software we have specifically developed for this purpose. Statistical comparisons will be performed using ANOVA and other tests where appropriate. Hypotheses 1. DCIS has a specific 3D signature that distinguishes it from normal cells and from tumor cells. 2. There are transition stages from normal to DCIS and from DCIS to IDC that we can determine using 3D imaging and the 3D order of telomeres in the nuclei of respective tissues. 3. c-Myc deregulation plays an important role in the remodeling of telomeres, particularly in hereditary breast cancer. 4. The measurements and quantitation of the 3D telomeric organization can provide insights into the mechanisms by which non-invasive cancers transition to invasive and possibly metastatic states. Aims 1. Investigate, using 3D imaging, the 3D nuclear organization of telomeres in 100 age- and grade-matched tissue samples that contain normal, DCIS and invasive tumor cells. Normal mammary epithelial tissue from healthy individuals will be included. 2. Quantitate, using a novel 3D software (TeloView), the 3D organization of telomeres. 3. Using the 3D organization of telomeres, we will define 3D telomere-based criteria to measure the transition between normal and DCIS, and between DCIS and IDC. 4. Determine the role c-Myc plays in telomeric aggregate formation, especially in hereditary breast cancer. Significance: Mechanistically, the understanding of 3D telomeric changes will be crucial for the prevention of malignant transformation. From a preventive perspective, the definition of such nuclear alterations could become an essential tool in the detection of the phenotype of non-invasive breast tumor cells that are likely to progress to invasive cancer. Finally, therapy could be based on treatments that stabilize telomeres and their nuclear positions.
Breast cancer can be sub-divided into different stages, and each of these stages has been characterized by pathologists, clinicians and researchers. What is lacking, however, is reliable information about the transitional changes that occur when cells move from one state to the other, that is when breast cancer cells become aggressive and result in live threatening disease. Once such transition markers, or signatures, have been identified, patients can be followed more closely, disease progression can be monitored more efficiently, treatments can be decided in a patient-specific manner, and the overall outcome will be manageable in a very precise manner. The focus of this study is to determine, by high resolution imaging methods, the characteristics of breast cancer cells that develop into highly aggressive tumor cells which in turn can invade tissues and settle at distinct sites (i.e. form metastases) and have the potential to ultimately kill the patient. The imaging methods we use involve high resolution three-dimensional imaging of patient samples. Our preliminary data suggest that this method is sensitive enough to distinguish between normal and tumor cells and to visualize differences between invasive and non-invasive breast cancer. Using tissue collections and tissue banks, we have identified key samples for our study. Using tissue sections prepared from the above samples, we will visualize nuclei of each sample. Nuclei are the control centers of the cell; like the engine of a car, they contain all components that allow the car to drive – or that permit the cell to function properly. Once a nucleus exhibits a disorganized composition of its functional components, the cell will not behave as normal cells do. Such cells may turn into tumor cells. We focus on one component of the nucleus; this component is called a telomere. Telomeres are the ends of chromosomes, and chromosomes are the carriers of our genetic information. Telomeres protect the chromosomes from degradation. Similar to a cap, they protect them from fusions with other chromosomes. We have shown that tumor cell nuclei show a disorganized organization of telomeres, and we will use this finding to fine-map the changes that occur when breast cancer cells become invasive. We will conclude this study of breast cancer by identifying specific three-dimensional telomeric signatures that define stages and transitions between stages, which could suggest specific outcomes and possible treatments.