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Research Grants Awarded
Molecular and functional characterization of breast cancer heterogeneity
The biology of breast cancer remains poorly understood especially at late stages and new and improved molecular based therapeutic strategies are desperately needed. Recent studies have demonstrated the existence of cells with stem cell like properties in breast carcinomas that were able to regenerate a phenotypically heterogeneous tumor in xenograft assays. We determined the gene expression and genetic profiles of distinct cell populations purified from breast carcinomas and normal breast tissue using cell surface markers CD24 and CD44. A gene signature specific for CD44+ cells was enriched for known stem cell markers, and for CD24+ with known differentiation markers. The CD24+ or CD44+ cell populations from the same tumor were clonally related but not always identical, and epigenetically distinct. Surprisingly the number of CD24+ cells was dramatically increased in distant metastases compared to matched primary invasive tumors. Systemic network analyses determined that the TGFBeta pathway is specifically active in CD44+ cells and its inhibition induced a more epithelial phenotype in cells cultured in vitro. We hypothesize that breast cancer CD24+ and CD44+ cells are clonally related, but not always identical, and both cell types may contribute to metastatic progression. Furthermore, the two cell types have different requirement for growth factors and therapeutic sensitivities. We will pursue following specific aims: to determine the clonal relationship between CD44+ and CD24+ cancer cells during breast tumor progression and to determine if CD24+ and CD44+ breast cancer cells have distinct therapeutic sensitivity. We will investigate if CD24+ and CD44+ tumor cells are genetically identical and/or clonally related within primary breast tumors and between primary tumors and distant metastases. We will purify these cells from distant metastases and analyze them using SNP arrays to identify areas of copy number gains and losses. Subsequently we will perform immuno-FISH analyses of distant metastases and matched primary invasive tumors using BACs corresponding to areas of copy number changes, CD44 and CD24 antibodies to determine which clone and cell type is responsible for metastatic progression. Similar immuno-FISH analyses will be performed in a larger set of primary invasive tumors to determine how consistently the CD24+ and CD44+ cell populations are different genetically. We will also perform differentiation assays in vitro to determine if CD44+ cells could be converted into CD24+ cells and if differentiation ability is correlated with clonal mono or heterogeneity. To determine the clinical relevance of the distinct cellular phenotypes of CD24+ and CD44+ breast cancer cells, we will analyze their cellular response in vitro to current breast cancer therapeutic agents. The completion of this project will help us to understand mechanisms of tumor progression and metastases and to potentially improve therapeutic regiments.
Due to the inevitable fatality of advanced breast cancer, understanding of the biology of tumor progression to metastases is crucial for the development of targeted and more effective therapies. Emerging evidence suggests that breast cancers are composed of a mix of distinct cell populations, each with differing abilities to grow and spread, and with varying sensitivities to anti-cancer drugs. It was proposed some time ago that a subpopulation of tumor cells exists that has stem cell like properties. These putative breast cancer stem cells should be able to form tumors, replicate themselves, lack some features of mature cells, and have the ability to regenerate all the cell populations of the original tumor. This theory was strengthened by recent data describing a method to enrich for these cells. It is also proposed that the recurrence, distant spread, and therapeutic resistance of primary breast tumors may potentially be due to the existence and characteristics of these cancer stem cells. Our preliminary data support this hypothesis, but also indicate another possible ways of tumor progression, where two or more populations of tumor cells diverge from each other and undergo parallel progression to metastases. The goals of this project are to test the cancer stem cell and parallel progression models by analyzing human breast cancer samples and to determine clinical relevance of different tumor cell populations. In the first aim we will purify more differentiated and putative stem cells from sets of matched distant metastases and identify areas of genetic differences. We then will perform analysis of chromosomal alteration of distant metastases and matched primary invasive tumors using DNA probes corresponding to areas of these genetic changes, specific antibody will be used to differentiate more differentiated and putative stem cells. Similar analysis will be performed in a larger set of primary invasive tumors to determine how consistently the more differentiated and putative stem cells populations are different genetically. We will also perform assays to determine if putative stem cells could be converted into more differentiated cells in vitro and if differentiation ability is correlated with clonal mono or heterogeneity. To determine the clinical relevance of the distinct phenotypes of more differentiated and putative stem cells, we will analyze their cellular response in vitro to current breast cancer therapeutic agents. Role of putative breast cancer stem cells in breast cancer formation, metastatic spreading, and the development of therapeutic resistance is virtually unknown. Furthermore, the breast cancer stem cell hypothesis has not been evaluated in terms of how it fits together with the known genetic heterogeneity of breast tumors. Thus the completion of this project will improve our understanding of the mechanisms of breast tumor progression and metastasis and potentially identify more effective cancer therapeutic approaches.