S6K1 and 4E-BP1 are independent regulated and control cellular growth in bladder cancer.

Roman Nawroth,Arndt Hartmann,Wolfgang A Schulz,Bernd J Krause,Robert Stoehr,Juergen E Gschwend,Margitta Retz,Florian Stellwagen

doi:10.1371/journal.pone.0027509

Abstract

Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and the mitogen activated protein kinase (MAPK) signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC). However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK.

Highlights

Bladder cancer is the fifth most common cancer world-wide with an estimated 357.000 new cases and 145.000 deaths in 2010 [1]
Urothelial carcinoma (UC) representing 90% of all bladder tumors are a heterogenous entity comprised of papillary tumors and solid invasive carcinomas which require radical treatment once they have progressed into the muscular layer of the bladder
Constructs, transfection and infection siRNA oligonucleotides against 4E-BP1, substrates of mTORC1 are the kinases p70S6K1 (S6K1) and control were designed by and purchased from Qiagen GmbH and Akt 1,22,23 stealth siRNA oligonucleotides from Invitrogen

Summary

Introduction

Bladder cancer is the fifth most common cancer world-wide with an estimated 357.000 new cases and 145.000 deaths in 2010 [1]. Radical cystectomy with pelvic lymph node dissection is the standard of care for muscle-invasive bladder cancer. With this approach 5-year progression-free survival probabilities of 65–68% can be achieved across all tumor stages [3,4]. Better knowledge about aberrant activation of cell signaling pathways that are involved in tumorigenesis of the bladder might provide suitable molecular targets for novel therapies [6,7,8]. One such pathway is the PI3K/Akt/mTOR signaling pathway that has been linked to tumorigenesis in many tissues [9,10]

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Results

Conclusion