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The Effect of Stroma on the Tumorigenic Potential of Pre-malignant Mammary Epithelia
Background: There is increasing evidence that the stroma can modulate the tumorigenic potential of malignant mammary epithelia. However, the specific effects of stroma on pre-malignant mammary epithelia have not been investigated. Fifty percent of human breast carcinomas express elevated levels of normal Ha-Ras protein, and expression of oncogenic Ras is one of the components required for transformation of human mammary epithelial cells (HMEC). We will utilize HMEC expressing oncogenic Ha-Ras (HMEC-Ras) as a model of pre-malignant epithelia to study the effect of stroma on tumor progression. Objective/Hypothesis: We will compare the effects of human mammary fibroblasts (HMF), which are the major cellular component of stroma, on the tumorigenicity of pre-malignant HMEC-Ras. Normal HMF (derived from women undergoing reduction mammoplasty), high-risk HMF (derived from women harboring BRCA1/2 mutations), and cancer-associated HMF (derived from women with breast cancer) will be utilized. We hypothesize that high-risk and carcinoma-associated HMF promote the tumorigenicity of HMEC-Ras. Specific Aims: 1) To determine the effect of normal, high-risk, and carcinoma-associated HMF on cellular events associated with tumor progression including epithelial-to-mesenchymal transition (EMT), proliferation, and apoptosis of HMEC-Ras in vitro. 2) To determine the effect of normal, high-risk, and carcinoma-associated HMF on the tumorigenicity of HMEC-Ras in vivo. 3) To determine the mechanism by which fibroblasts promote the tumorigenicity of pre-malignant mammary epithelia. Study Design: 1) The effect of HMF on the behavior of HMEC-Ras in vitro will be assessed utilizing novel 2D and 3D matrigel co-culture systems. 2) HMEC-Ras will be injected either alone or in combination with HMF directly into surgically exposed mammary fat pads of nude mice and tumorigenicity will be assessed by monitoring tumor incidence, latency, volume, and weight. 3) Microarray analysis of normal vs high-risk and cancer-associated HMF will be conducted to identify similarities in gene expression between high-risk and cancer-associated HMF so as to identify key signaling pathways implicated in HMF-mediated mammary tumor progression. Potential Outcomes and Benefits: A better understanding of epithelial-stromal interactions in mammary tumor progression could lead to the development of new biomarkers for early detection as well as new therapeutic strategies for intervention prior to progression to malignancy.
Although the majority of breast cancers arise within the cells that make up the breast ducts (epithelial cells), there is increasing evidence that the stroma, the cells and proteins surrounding the breast ducts, can influence the progression of breast cancer. Specifically how the human breast stroma modulates the onset of breast cancer or its progression from a pre-malignant (non-cancerous) to a malignant (cancerous) state is unknown. One of the proteins often involved in the conversion of human mammary epithelial cells (HMEC) from a normal to a malignant state is Ha-Ras. This protein is elevated in over fifty percent of breast cancers. We will utilize HMEC expressing Ha-Ras (HMEC-Ras) to examine how the stroma can cause these cells to progress from a pre-malignant to a malignant state. We will compare the effects of human mammary fibroblasts (HMF), which are the major cellular component of stroma, on the behavior of HMEC-Ras both in tissue culture dishes and in the mammary glands of mice. Normal HMF (derived from women undergoing reduction mammoplasty), high-risk HMF (derived from women harboring BRCA1 or BRCA2 mutations), and cancer-associated HMF (derived from women with breast cancer) will be utilized for these studies. We predict that high-risk and cancer-associated HMF will promote the progression of HMEC-Ras to malignancy. We will test this prediction by 1) examining the effect of HMF on the ability of HMEC-Ras to change their shape, grow, multiply, and survive when both cell types are combined in tissue culture dishes; 2) examining the effect of HMF on the ability of HMEC-Ras to form tumors when injected into the mammary glands of mice. We will then determine how HMF modulate HMEC-Ras by looking for similarities in the gene expression profiles of high-risk and cancer-associated HMF to identify key proteins implicated in mediating mammary tumor progression by HMF. One of the most important aspects of these studies is that they will be conducted with pre-malignant cells, that is, cells that are not yet cancerous. A better understanding of how the stroma can cause these cells to become cancerous could lead to the development of new biomarkers for early detection as well as new therapeutic strategies for intervention prior to the development of cancer, both of which are critical for breast cancer prevention.