Louis, MO). and Mitogen-activated protein kinase (MAPK) pathways as well as IGF-I-induced Akt- and MAPK-dependent phosphorylation of paxillin, which relocated at dynamic focal adhesions and was necessary for promoting motility in bladder malignancy cells. Our results provide the first evidence for a role of the IGF-IR in motility and invasion of bladder malignancy cells and support the hypothesis that this IGF-IR may play a critical role in the establishment of the invasive phenotype in urothelial neoplasia. Thus, the IGF-IR may also serve as a novel biomarker for bladder malignancy. Bladder malignancy is usually a major epidemiological problem, whose incidence continues to rise each 12 months. The most recent cancer statistic1 has estimated 68,810 new cases with 14,100 estimated deaths in the United States. Bladder tumors show widely differing histopathological and clinical behavior,2 and this is usually a key problem in the management of bladder tumors. Isochlorogenic acid A The majority of bladder tumors (70%) are low-grade noninvasive papillary tumors that do not penetrate the epithelial basement membrane (Ta stage). The remainder comprises tumors that Isochlorogenic acid A have penetrated the basement membrane but not invaded the muscle mass layer of the bladder wall (T1 stage) and muscle-invasive tumors (T2, T3, and T4 stages).3 The insulin-like growth factor receptor I (IGF-IR) plays a critical role in cell growth both gene have severe growth retardation, being only 45% the size of wild-type littermates, and die shortly after birth primarily due to respiratory failure.6,7 These data suggest that the IGF-I/IGF-IR axis is critical for normal Mouse monoclonal to TNFRSF11B growth. The importance of the IGF-IR in transformation was initially suggested by experiments performed with cells derived from the experiments on tumor cell lines and epidemiological studies have confirmed that activation of the IGF-IR is usually involved in the development of many common neoplastic diseases, including carcinomas of lung, prostate, pancreas, liver, colon, and breast.8,10,11 The transforming capability of the IGF-IR most likely depends Isochlorogenic acid A on its ability to protect cancer cells from apoptosis.12,13,14,15 The IGF-IR has now become a very attractive target for cancer therapy, and in fact antibodies against the IGF-IR are currently in phase I clinical trials.16,17 Whether the IGF-IR contributes to the transforming phenotype of urothelial cells has not been clearly established, but recent data suggest that the IGF-IR is overexpressed in bladder malignancy.18 In this study, using 5637 and T24 urothelial carcinoma-derived cells, we established that activation of the IGF-IR plays a critical role in bladder malignancy by promoting migration, wound healing, and invasion of malignancy urothelial cells. We have also characterized the mechanism of action of the IGF-IR in malignancy urothelial cells and showed that IGF-IR-dependent cell motility and invasion required the activation of the Akt and MAPK pathway and Akt- and Extracellular-signal-related kinase 1 (ERK)-dependent activation of paxillin. Collectively, these results provide novel information toward a better understanding of the mechanisms that regulate tumor formation in bladder malignancy at the cellular and biochemical level and suggest that the IGF-IR may be critical for bladder malignancy. Materials and Methods Immunohistochemical Detection of the IGF-IR in Normal and Malignancy Bladder Tissue Specimens Immunohistochemical analysis of IGF-IR levels in bladder tissues was performed as previously explained.19 Formalin-fixed paraffin-embedded sections from Isochlorogenic acid A five invasive (T3/T4) urothelial cell carcinomas and adjacent normal tissues were obtained from the Pathology Tissue Bank of Thomas Jefferson University (Philadelphia, PA). Informed consent to use extra pathological specimens for research purposes was obtained from all five patients. Slides were incubated with 1:500 dilution of an anti-human IGF-IR polyclonal antibody (C-20; Santa Cruz Biotechnology, Santa Cruz, CA). The specificity of this antibody has been previously validated.18 Cells, Growth, and Migration Assays Invasive urothelial carcinoma-derived human 5637 and T24 cell lines were obtained from the American Type Culture Collection (Manassas, VA)..