Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive

Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive tumor growth in many cancer models although its role in urothelial carcinoma (UCC) has not been extensively explored. Reduced proliferation corresponded with reduced P-S6 levels by Western blot and effects were ablated by pretreatment of cells with mTOR-specific siRNA. No effects of rapamycin on apoptosis were recognized by TUNEL labeling or PARP cleavage. Administration of rapamycin to T24-xenografted mice resulted in a 55% reduction in tumor volume (= 0.03) and a 40% reduction in proliferation (< 0.01) compared with vehicle-injected mice. These findings show that mTOR pathway activation frequently occurs in UCC and that mTOR inhibition may be a potential means to reduce UCC growth. Bladder malignancy occurs in multiple forms the most common of which is usually urothelial carcinoma (UCC) which represents >90% of all bladder cancers.1 Approximately 30 to 50% of patients with invasive bladder malignancy into the muscular wall of the bladder will develop metastatic disease and die within 2 years of diagnosis.2 In addition virtually all patients diagnosed with distant UCC metastases will succumb to disease.3 Currently the standard treatment modality for muscle-invasive bladder malignancy is radical cystectomy; systemic chemotherapy is generally reserved for patients with metastatic disease although these treatment regimens provide only a limited long-term benefit with only rare reports of total remission.4 5 Arry-520 Arry-520 In light of these clinical outcomes identification of new therapeutic targets is needed to define potential additional treatment avenues for these patients. Activation of the mammalian target of rapamycin (mTOR) signaling pathway occurs in many cancers and has recently been shown to correlate with more aggressive disease behavior 6 7 8 9 although it has not been examined in great detail in UCC. Activation of mTOR occurs via a multistep process that includes upstream phosphoinositide-3 kinase (PI3K) and AKT activation leading to phosphorylation and inactivation of the tuberous sclerosis complex 1 and 2 (TSC1/TSC2) heterodimer.10 11 Inactivation of this heterodimer Arry-520 results in release of Rheb inhibition and subsequent mTOR activation by means of Rheb GTPase activity. Once activated mTOR can induce increased mRNA translation or regulate the actin cytoskeleton via differential association Rictor and Raptor proteins.10 11 Ultimately mTOR activity regulates the effects of a number of downstream molecules important in cellular growth including p70 S6 kinase-1 (S6K) and elongation-initiation factor 4E binding protein-1 (4E-BP1). Selective inhibition of the mTOR pathway can be achieved using rapamycin or rapamycin analogs temsirolimus (CCI-779 Wyeth Pharmaceuticals) and everolimus (RAD001 Novartis) which are currently in use in numerous clinical trials for solid tumors with encouraging results in patients with advanced renal cell carcinoma.12 13 To further investigate the potential role of mTOR signaling and inhibition in UCC of the bladder we used human malignancy specimens xenograft models and analysis to determine the effects of mTOR on cellular proliferation apoptosis tumor growth and clinical outcomes in this malignancy population. Materials and Methods Patient Specimens Permission for this study was obtained from The Cleveland Medical center Institutional Review Table. Specimens included archived paraffin blocks from patients who Arry-520 underwent radical cystectomy or cystoprostatectomy for muscle-invasive UCC (pathological stage pT2 or greater) between the years 1998 to 2007. All specimens were received in the surgical pathology suite on ice within 10 minutes postcystectomy and tissue was immediately placed into 10% buffered formalin for routine processing. Main bladder tumors that were either nonmetastatic (= 52) or metastatic to regional lymph nodes (= 69) Rps6kb1 were used for analysis. Paired lymph node metastases from your latter group were available in 59 cases for analysis. Patient demographics clinicopathologic features and outcomes are offered in Table 1. In addition noninvasive low- (= 20) and high-grade (= 20) papillary UCCs recognized on biopsy were used for comparison for P-mTOR and P-S6 expression. Table 1 Patient Demographics and Clinical Outcomes Immunohistochemistry Tissue microarrays were prepared from both nonmetastatic and.