WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells.

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G(1) arrest. The G(1) arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G(1) arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and beta-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G(1) arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer.

Expression of Wnt pathway components frizzled and disheveled in colon cancer arising in patients with inflammatory bowel disease.

Mutations in apc which lead to activation of the Wnt signaling pathway are a hallmark of sporadic colon cancers but occur infrequently in colon cancers arising in patients with inflammatory bowel disease (IBD). There is evidence, however, that other components of the Wnt pathway may be altered in IBD-related colon cancer. In this study, we examined the expression the Wnt pathway components frizzled (Fz), the cell surface receptor, and disheveled (DVL), a family of cytoplasmic signal transduction molecules, in IBD and IBD-related colon cancer. Paraffin sections of normal and malignant colon tissues were obtained from patients with a history of ulcerative colitis and from controls with sporadic colon cancer. Tissue sections were stained with antibodies directed against Fz1/2 receptors and DVL1, DVL2 and DVL3 and antigen expression visualized by immunohistochemistry. Fz1/2 receptors were minimally expressed in normal IBD mucosa, were not expressed in IBD colon cancer, but exhibited strong expression in dysplastic tissues adjacent to the cancers. DVL1 was not expressed in IBD normal mucosa or normal mucosa from non-IBD patients, but was expressed in all cancers. DVL2 and DVL3 were expressed in all normal mucosa samples tested, and in sporadic colon cancer, but were not expressed in colon cancers arising in IBD patients. The characteristics of Fz and DVL expression in IBD tissues reported herein provides evidence of the importance of Wnt signaling in IBD and IBD-related colon cancer and, specifically, the significance of non-APC components of this pathway. Fz may serve as a marker for dyspasia in IBD patients and DVL1 is a potential therapeutic target for IBD-related colon cancer.

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1) in human melanoma and identification of the therapeutic potential of resveratrol as an APE/Ref-1 inhibitor.

Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1) is a multifunctional protein involved in DNA base excision repair and redox regulation of many transcription factors. In different melanoma cell lines, we found that both nucleus and cytoplasm exhibited higher levels of Ref-1 compared with normal melanocytes. Similar increases of Ref-1 expression, detected by immunohistofluorescence, were also evident in nevi and malignant melanoma biopsies compared with normal skin, which were predominantly localized in the nucleus. Using recombinant adenovirus Adref-1, encoding full-length Ref-1, we transiently overexpressed APE/Ref-1 in human melanocytes, which protected these cells from UVB-induced apoptosis and increased foci formation in culture. Ref-1 overexpression also protected melanoma cells from cisplatin- or H2O2-induced apoptosis, whereas increased apoptosis was observed with Ref-1 antisense construct infection. These observations suggested that intracellular Ref-1 levels played an important role in sensitization of melanoma cells to apoptosis. Electrophoretic mobility shift assay results showed that in both cultured primary and metastatic melanomas DNA-binding activities of activator protein-1 and nuclear factor-kappaB were significantly diminished or shifted when anti-APE/Ref-1 antibody was added to deplete APE/Ref-1 from the binding complexes. Induced nuclear factor-kappaB transcriptional activities were also evident after Ref-1 overexpression. Furthermore, using three-dimensional molecular structure modeling and virtual screening, we found that resveratrol, a natural compound found in fruits and vegetables, docks into a druggable pocket of Ref-1 protein. In vitro studies revealed that resveratrol inhibited, in a dose-dependent manner, Ref-1-activated activator protein-1 DNA-binding activities as well as Ref-1 endonuclease activities and rendered melanoma cells more sensitive to dacarbazine treatment.

EF-Tu binding peptides identified, dissected, and affinity optimized by phage display.

The highly abundant GTP binding protein elongation factor Tu (EF-Tu) fulfills multiple roles in bacterial protein biosynthesis. Phage-displayed peptides with high affinity for EF-Tu were selected from a library of approximately 4.7 x 10(11) different peptides. The lack of sequence homology among the identified EF-Tu ligands demonstrates promiscuous peptide binding by EF-Tu. Homolog shotgun scanning of an EF-Tu ligand was used to dissect peptide molecular recognition by EF-Tu. All homolog shotgun scanning selectants bound to EF-Tu with higher affinity than the starting ligand. Thus, homolog shotgun scanning can simultaneously optimize binding affinity and rapidly provide detailed structure activity relationships for multiple side chains of a polypeptide ligand. The reported peptide ligands do not compete for binding to EF-Tu with various antibiotic EF-Tu inhibitors, and could identify an EF-Tu peptide binding site distinct from the antibiotic inhibitory sites.