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Breast Cancer Invasion via Cell Growth and Survival Signaling Pathways
The signaling pathways that control breast cancer invasion and metastasis are not well understood. Interestingly, the gene expression profiles that define metastatic breast cancer are also found in a subset of noninvasive DCIS (Ductal Carcinoma in situ), suggesting that the mutational events that confer uncontrolled growth and survival may also predispose cells to invasion. Indeed, two of the critical signaling pathways that regulate cell proliferation and survival also regulate cell motility: the Ras/MAPK (mitogen-activated protein kinase) and PI(3)K (phosphatidyl-inositol 3 kinase)/Akt pathways. Objective: The objective of this proposal is to elucidate the specific subset of proteins in the PI(3)K/Akt and/or Ras/MAPK pathways that mediate breast cancer invasion. Specific Aims: 1) Generate an in vitro breast cancer model system with defined mutations that recapitulate those that occur in human breast cancer. 2) Identify and mechanistically characterize proteins that interact with the PI(3)K and/or MAPK pathways to induce invasion by performing a siRNA screen and biochemical analyses. Study Design: A breast cancer model system will be generated by introducing defined genetic elements into explanted human mammary epithelial cells. The modified cell lines will differ only in expression of a single breast cancer gene, and will be phenotypically and biochemically compared for invasion through extracellular matrix and activation of the PI(3)K and Ras pathways. In collaboration with the ICCB-Longwood Screening Facility, invasive mammary epithelial cells will be transfected with a siRNA library targeting human signaling proteins and screened for loss of invasive function. The relationships between the candidates and components of the PI(3)K and MAPK pathways will be examined using genetic epistasis analysis. We will also evaluate the candidates for their effect on anchorage independent growth in soft agar to determine if they function in cell growth and survival, or are specific to cell invasion. Key novel regulators will be biochemically analyzed to determine their mechanism of action. Outcomes and Benefits of the Research: Together, these experiments will uncover signaling pathways that cooperate with universal proliferation and survival proteins to induce breast cancer invasion. These novel regulators may be practical therapeutic targets and knowledge of their activities during invasion may improve assessment of the risk of DCIS progression to invasive cancer.
The spread of breast cancer to distant sites is the leading cause of breast cancer illness and death, but its molecular details are poorly understood. When the cells lining the mammary duct amass mutations that cause them to grow and survive unchecked, they fill the lumen of the duct, causing DCIS (Ductal Carcinoma in situ). Breast cancer spread begins when DCIS cells invade out of the duct, into the surrounding tissue. Interestingly, the gene expression signatures that define metastatic breast cancer are also found in noninvasive DCIS. Thus, the genetic background and mutations that confer uncontrolled growth and survival in DCIS may be one and the same as those that induce invasion. Some established growth/survival regulators are commonly mutated in breast cancer and have recently been found to also regulate cell motility. The objective of this proposal is to identify and characterize the proteins that cause these canonical growth/survival proteins to induce breast cancer invasion. As protein expression, interaction, and involvement in tumorigenesis varies among species and cancers, we will generate a human mammary cell culture model with defined mutations that recapitulate those that occur in human breast cancer. We will apply a technology that reduces the expression of individual genes in the model cells to assess the role of known growth/survival regulators in invasion. This technology will then be employed in a high throughput manner to thousands of genes to impartially identify the specific activators, effectors, and cooperating proteins that specify invasion. The role of the identified genes in invasion will be independently verified. The role of these genes in cell growth and survival will also be assayed to determine if they specifically regulate invasion, or are also involved in other tumor processes. Lastly, key novel proteins will be studied in detail to determine their mechanism of action. Therapeutic targeting of canonical growth/survival proteins often causes unwanted side effects due to their critical roles in many tissues. These experiments will uncover genes that cooperate with growth/survival pathways to induce breast cancer invasion, which may be more practical therapeutic targets. This work may also lead to more accurate assessment of the risk of DCIS progression to invasive cancer and thus, better patient management.