The urokinase receptor (u-PAR) contributes to colon cancer invasion and metastasis partly by accelerating proteolysis, u-PAR transcription is >10 fold higher in invasive colon cancer and we are interested in identifying molecules upstream of transcription that regulate its expression. Since the protein tyrosine kinase Src activity is elevated > 8 fold in metastatic colon cancer and because transfection of Src into colonic epithelial cells renders them invasive, we hypothesize (Specific Aim # 1), that u-PAR expression is regulated by this protein tyrosine kinase. This will be tested by determining the effect of (a) a Src inhibitor (PP2) (b) a constitutively active or (c) dominant negative Src on u-PAR expression in cultured or genetically-induced (mucin 2 -/-) colon cancer and correlating u-PAR levels with Src activity in cultured and resected colon cancers. Our preliminary studies implicate a footprinted region (-148/-124) bound with Sp1/Sp3 that regulates constitutive and Src-inducible u-PAR expression. To determine the mechanism by which Src regulates u-PAR expression via this Sp1/Sp3 -bound region (Specific Aim # 2), we will determine if Src (a) increases Sp1 expression (b) alters Sp1 phosphorylation to increase its DNA binding (c) increases histone acetylation at this footprinted region thereby promoting chromatin relaxation and Sp1/Sp3 binding or (d) increases the trans-acting activity of Sp1/Sp3. Our preliminary data indicate that Src regulates u-PAR expression in part through the Sp1/Sp3-bound -148/-124 region.
In Specific Aim # 3, we will exploit this information in translational studies by determining the ability of a bisanthracycline WP631 (which blocks Sp1/Sp3 binding to the -148/-124 region) alone, or combined with a Src inhibitor (PP2), to suppress u-PAR expression and colon cancer invasiveness in vitro. Studies on u-PAR expression, to date, have employed in vitro techniques which provide no information on promoter requirements for tissue-specific u-PAR expression and ignore the role of chromatin in regulating this gene.
In Specific Aim # 4, transgenic mice harboring a LacZ reporter regulated by 5' deleted u-PAR promoter fragments will be employed to determine (a) the minimal promoter sequence and (b) the role of the -148/-124 region required for u-PAR expression in the placenta and genetically-induced colon cancer (tissues characterized by their high u-PAR expression). Additionally, the sensitivity of transgene expression to Src inhibition will be determined.
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