The theme of this project has been the role of TGF-beta in the regulation of the biological behavior of colon cancer cells. The basic hypothesis of the last cycle of the project was that loss of autocrine negative TGF-beta was a significant determinant of malignant progression. During the last cycle of the project this hypothesis was confirmed by showing (1) repression of autocrine TGF-beta rather than loss of response to exogenous TGF-beta leads to malignant progression in vitro and in vivo, (2) an important mechanism of loss of autocrine TGF-beta was determined to be loss of the type II TGF-beta receptor (designated RII) by mutation in cell lines and primary tissues and (3) reconstitution of autocrine TGF-beta activity by RII expression reverses malignancy. Mutational inactivation of RII was found in colon cancer patients and cell lines with DNA repair defects leading to microsatellite instability (designated RER+). It is important to determine whether TGF-beta receptor expression is also defective in non-RER disease. Preliminary evidence has identified a subset of non-RER cell lines and tumors which shows a lack of RII expression while a second set shows reduced expression of RI expression which results in the elimination of detectable autocrine TGF-beta activity. The hypothesis that epigenetic mechanisms are responsible for these subsets will be tested in this cycle of the project. Other preliminary evidence indicates that loss of autocrine TGF- beta leads to the acquisition of growth factor independence for DNA synthesis. Growth factor independence is mediated in part by increased autocrine positive TGF-alpha activity. Exogenous TGF-beta and mitogen treatment have been shown to modulate cell cycle elements controlling cyclin dependent kinase activation in opposite directions. Therefore, loss of autocrine TGF-beta along with the associated changes in autocrine TGF-alpha activity may lead to impaired ability of the cell to modulate its cell cycle control elements. The hypothesis that loss of autocrine TGF-beta leads to an imbalance favoring activated cyclin dependent kinases over their inhibitors will be tested in this cycle of the project.
Specific Aims for addressing the hypothesis referred to above are: 1. Determination of the autocrine mechanisms of TGF- beta in maintaining control of cell cycle transit; 2. Characterization of control of expression of RI and RII; and 3. Characterization of epigenetic disruption of RI and RII expression.
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