MIS is a member of the TGFbeta family. MIS receptor expression inversely correlates with growth during various stages of mammary gland morphogenesis and MIS inhibits breast cancer cell growth both in vitro and in vivo. MIS strongly induces the expression of IRF-1, a tumor suppressor, lost in 41% of high nuclear grade breast tumors. MIS by virtue of inducing IRF-1 intersects with signaling by interferon-gamma (IFN-gamma), a cytokine with anti-tumor activity, and enhances IFN-GAMMA-induced gene expression and apoptosis in breast cancer cells suggesting a functional interaction between these two signaling molecules. Another gene identified as a MIS-inducible gene was BTG2, a growth inhibitory gene that maps to the locus 1q32. In the rat mammary gland, BTG2 declines precipitously during pregnancy and BTG2 protein was absent in approximately 61% of human breast tumors compared to matched normal glands suggesting that BTG2 might represent one of the tumor suppressors of the breast residing within 1q23-32, a region that presents LOH in breast cancer. Thus we propose to test the following hypotheses: (1) MIS may regulate normal mammary epithelial cell morphogenesis and suppress the tumorigenicity of breast cancer cells. (2) MIS may enhance the differentiation and anti-tumor effects of IFN-gamma in the breast. (3) IRF-1 and BTG2 may be regulators of differentiation in mammary epithelial cells and effective suppressors of tumorigenic characteristics in breast cancer cells. These hypotheses will be tested in the following aims:
Aim 1 : Test the effect of MIS +/- IFNgamma on mammary epithelial acinar morphogenesis and tumor phenotypes of breast cancer cells. The human mammary epithelial cell line, MCF10A, when cultured on extracellular matrix, forms acini recapitulating several aspects of glandular architecture in vivo, while breast cancer cells form nonpolar, undifferentiated colonies. We will determine (1) whether MIS (MIS+/-IFNgamma), will disrupt the morphogenesis of MCF10A, (2) revert breast cancer cells from forming undifferentiated nonpolar colonies when plated on Matrigel to normal differentiated acini, and (3) reverse the tumorigenicity of breast cancer cells. We will utilize knockdown of IRF-1 & BTG2 to correlate the biologic effects with the molecular effects to establish the mechanistic link.
Aim 2 : Test the anti-tumor effect of MIS(+/-IFN-gamma) on human breast cancer cell xenografts grown in immune suppressed mice. We will test the hypothesis that MIS enhances the anti-tumor activity of IFN-gamma against human breast tumor xenografts grown in mice.
Aim 3 : Test the anti-tumor effect of MIS+/-IFN-gamma in a transgenic spontaneous mammary carcinoma mouse model. Spontaneous mammary tumor models allow the study of stage-specific responses to anti-tumor agents, further defining appropriate timing for intervention with these compounds. We will test the hypotheses that MIS (+/-IFN-gamma) treatment will suppress tumor initiation/progression, and cause the regression of established spontaneously arising mammary tumors in mice.
Aim 4 : Characterize the role of BTG2, IRF-1 and CEACAM1 in breast cancer. To test the hypothesis that aberrant regulation of IRF-1, CEACAM1 and BTG2 enables some breast tumors to overcome growth control, and to validate that these genes are indeed important targets in human breast cancer, we will determine if their expression is deregulated in breast tumors and correlate expression with clinical characteristics.
Aim 5 : Characterize the tumor suppressive effects of IRF-1 and BTG2 in breast cancer cells. We will test the hypothesis that ectopic expression of IRF-1 and BTG2 in breast cancer cells will restore a normal differentiated morphogenetic phenotype in carcinoma cells and revert its tumorigenic characteristics.
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