Urologic Oncology: Seminars and Original Investigations
Original articleDifferential mTOR pathway profiles in bladder cancer cell line subtypes to predict sensitivity to mTOR inhibition
Introduction
Bladder cancer is the fifth most common malignancy in the United States, with over 70,000 estimated new cases annually [1]. An accumulating number of genomic studies have identified distinct subtypes of bladder cancer stratified based on molecular and histological features [2], [3], [4], [5]. The general consensus characterizes bladder cancer into 2 unique subtypes, basal/squamous-like and luminal/papillary-like, but with some groups having identified up to 5 subtype assignments [6]. This stratification has guided subsequent investigations demonstrating differential responses of molecular subtypes to multimodal therapies with mechanistic target of rapamycin (mTOR), EGFR, VEGFR, FGFR3, and HER2/ERBB2 representing targets of particular interest [3], [7], [8]. Specifically, activating alterations of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway is frequently observed in invasive bladder cancer but targeted therapy with mTOR inhibition has had limited success [9], [10], [11]
Our group previously identified mTOR complex 2 (mTORC2) as a major regulator of bladder cancer invasion [12]. mTORC2 is distinct from the second mTOR-containing complex, mTOR complex 1 (mTORC1), by kinase activity, activation of downstream targets, and subunit composition (mTORC1 contains mTOR, Raptor, mLST8, PRAS40 and Deptor, whereas mTORC2 contains mTOR, Rictor, mSIN1, mLST8, Protor, and Deptor) [13]. mTORC1 positively regulates protein synthesis to regulate cell growth through activation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), p70 ribosomal S6 kinase 1 (S6K1), and ribosomal protein S6 [14]. By contrast, phosphorylation of Ser-473 in Akt, a target of mTORC2 activity, is generally understood to regulate various biological processes such as metabolism, cell survival, and cytoskeletal organization.
Presently, we used bladder cancer cell line models previously characterized into different molecular subtypes to determine if subtype stratification and expression of mTOR signaling components can be used as a predictor of sensitivity to mTORC1 and 2 inhibition using next generation mTOR inhibitors, Torin-2 and KU-0063794 [15]. Prior studies evaluating the efficacies of mTOR inhibitors have not considered possible differences in mTOR signaling activity among cell line models. We show that subsets of cell lines representing luminal (RT4 and RT112), basal (SCaBER and 5637), and a third subtype designated “non-type” (T24 and J82), bladder cancers exhibit differential expression levels and/or phosphorylation of Akt isoforms, 4E-BP1, mTOR, and Rictor. Upon treatment with Torin-2 or KU-0063794, cells with low to undetectable levels of Akt Ser-473 or Thr-308 phosphorylation were least sensitive to cytotoxicity induced by mTOR inhibition. Immunoblot analysis of the effects of mTOR inhibition on mTOR signaling targets revealed dose and time-dependent inhibition of Akt, S6, and 4E-BP1. However, ribosomal protein S6 phosphorylation of both serine residue series, Ser-235/236 and Ser-240/244, was inhibited to a greater extent in basal and luminal cell subtypes. These findings suggest that differential gene expression and protein activity associated with mTOR signaling influences bladder cancer cell line sensitivity to mTOR inhibition. Urothelial carcinomas with basal or nontype molecular features may be best suited for targeted mTOR therapies, either alone or in combination with other agents.
Section snippets
Cell culture and reagents
RT4, SCaBER, J82, and T24 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA). RT112 and 5637 cells were a kind gift from Dr P. Szlosarek (Queen Mary University of London, London, England). Cells were grown in RPMI-1640 (Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Gibco) and were maintained at 37°C in a humidified atmosphere containing 5% CO2. The dual mTORC1/2 inhibitors, Torin-2 (cat. no. 4248) and KU-0063794 (cat.
mTOR-associated signaling components are differentially expressed among bladder cancer cell line subtypes
Genomic alterations to the PI3K/Akt/mTOR pathway frequently occur in bladder cancer and we have previously shown that increased mTORC2 activity is a critical driver of bladder cancer cell invasion [3], [9], [10], [11], [12]. Several studies have identified distinct molecular subtypes of bladder cancer including basal-like and luminal-like subtypes, and these subtypes have been shown to respond differentially to chemotherapy [2], [3], [4], [5], [6], [7], [8]. A similar approach using human
Discussion
The recent enhanced understanding of bladder cancer biology has been fueled by numerous genomic studies characterizing this common disease [2], [3], [4], [5]. Further, the identification of different molecular subtypes with differential sensitivities to chemotherapeutic agents has significant implications to the clinical treatment and management of bladder cancer [7], [8]. Using molecular subtype classification in bladder cancer cell line models, we identified that basal, luminal and nontype
Acknowledgments
This work was supported by a grant from the Urology Care Foundation Research Scholars Program to A.M.H.
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