EXCEED THE SPACE PROVIDED. We have demonstrated a reciprocal function for downregulation of AKT activation and inhibition of cell survival signaling by Smad 3. Moreover, we have shown that autocrine Smad 3 carries out an apoptotic function, in part, by repressing the oncogenic survival protein, survivin. Autocrine Smad 3 function appears to involve attenuation of cell survival signaling. An important objective of the project is to determine the function of the Smad3/AKT node in response to cellular stresses such as growth factor and nutrient deprivation as well as hypoxia. As malignant progression associated with loss of TGFp signaling occurs, the ability to respond to environmental stress is lot. In contrast, we have found that GFDS enhances the expression Smad 3 which transcriptionally represses survivin expression and at the same time reduces AKT activation as well as downstream activation of NFicB, another well known survival signaling mediator in cells with functional TGFp signaling. Thus, this control node appears to be an important means by which cells communicate and react to the environment. As such, it would appear to be a ripe target for disruption during malignant progression. To learn more about how this stress response node controls death and survival we propose to utilize knockdown models described above that are specific for Smad2 and SmadS in Specific Aim 1 to further test the hypothesis that Smad 3, but not Smad 2 is responsible for this mechanism of control by determining the roles that preformed cytoplasmic complexes containing SmadS bound to transcription factor complexes responsible for control of inhibition the cell cycle by TGFp play in carrying out the function of the Smad3/AKT node activation. Finally, in Specific Aim 3 we will determine the relationship between the Smad3/AKT Specific Aim II will, therefore, also test the hypothesis that AKT promotes survivin expression through the phosphorylation of kBa and thus loss of Smad 3 not only released survivin from transcriptional repression, but also increases its transcription through the restoration of AKT control node and malignant progression in vivo using orthotopic implantation and analysis of resulting changes in metastasis of colon cancer cells in athymic mice.
Aim 3 will utilize SmadS and Smad2 knockdown cells to determine whether the SmadS/AKT node is linked to invasion and/or metastasis using orthotopic implantation models of colon cancer.
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