Abstract 2279: Akt inhibitor SC-66 leads to inhibition of mTOR pathway in cervical cancer cell line C33A.
Authors: Rashmi Ramachandran , DeSelm Carl , Schwarz Julie K. 
Published: August 2012

Abstract
Background We observed previously that overexpression of activated Akt is associated with poor prognosis in cervical cancer patients treated with concurrent cisplatin and pelvic irradiation (communicated). Several inhibitors of the PI3K/Akt/mTOR pathway have been developed, and many are being tested in clinical trials. The purpose of this study was to test the allosteric Akt inhibitor, SC-66 in cervical cancer cell lines and to determine drug sensitivity and inhibition of downstream pathway components. Experimental Design Eight cervical cancer cell lines (CaSki, C33A, C41, HeLa, HT-3, ME-180, SiHa and SW756) were assessed for activation status of Akt (Ser473, Thr308 and Thr450), downstream targets of Akt (FOXO-1, GSK3-α, PRAS40) and mTOR (mTOR, ribosomal S6 kinases (p70S6K), (eukaryotic initiation factor 4E)-binding protein, 4E-BP1 and S6) pathway by western blot. Among these cell lines tested, one with high Akt (C33A) was chosen for further experiments. Cell lines were treated with the allosteric Akt inhibitor SC-66, which interferes with PH domain binding to PIP3 leading to Akt ubiquitination. The dose and time course analysis were done by determining the cell viability using Alamar Blue and EC50 using PRIZM software. Akt and mTOR pathway inhibition were assessed by analyzing phosphorylation of various molecules of the pathway with or without SC-66 (6-10µg/ml) by western blot. Results Expression of activated Akt (S473, T450 and T308) was prominent in C33A. Since C33A expressed high pAkt levels we checked for alterations in the upstream regulators PIK3CA and PTEN (Phosphatase and tensin homolog). C33A harboured G263A (R88Q) alteration in exon1 of PIK3CA and PTEN protein expression was undetected. C33A was found to be the most sensitive cell line towards SC-66 treatment, with an EC50 value of 0.486µg/ml followed by ME-180 (0.82µg/ml), SW756 (1µg/ml) and SiHa (2.5µg/ml). To test the effects of SC-66 on the Akt/mTOR pathway, C33A cells were treated with doses from 6-10µg/ml of SC-66 for 3h and all the mTOR pathway targets were inhibited effectively at 7µg/ml, and Akt substrates between 7-8µg/ml. Akt activates mTOR which in turn phosphorylates p70S6K and 4E-BP1 ultimately resulting in cell growth and proliferation. SC-66 is effective in blocking essentially all these steps of signaling in C33A, a cell line with high activated Akt. Conclusions SC-66 potently inhibits proliferation of cervical cancer cell lines. C33A, a cervical cancer cell line with high levels of pAkt, is highly sensitive to SC-66, resulted in inhibition of phosphorylation of mTOR, 4EBP1 and p70S6K. Since high levels of pAkt are associated with poor outcome after concurrent chemo-radiation in cervical cancer, SC-66 could prove to be an effective therapeutic adjunct in these patients.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2279. doi:1538-7445.AM2012-2279