Oncogenes and Tumor Suppressors Molecular Dissection of AKT Activation in Lung Cancer Cell Lines

AKT is a critical signaling node downstream of phosphoinositide 3-kinase (PI3K), which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non–small cell lung carcinoma cell lines under serum starvation conditions. We identified 13 lines, which showed persistent AKT activation in the absence of serum. In 12 of 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGF receptor (EGFR) or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT inHCC2429was PI3KandmTOR complex 2 (mTORC2)-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that bothNotch and SRCwere highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Furthermore, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Overexpression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features.Mol Cancer Res; 11(3); 282–93. 2012 AACR. Introduction Lung cancer is the leading cause of cancer-related deaths in both the United States and worldwide with a 5-year survival rate of only 15% (1). Lung cancer consists of several pathologic subtypes, including adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Those types not including small cell carcinoma are often grouped together as non–small cell lung carcinoma (NSCLC). Cancer genome sequencing efforts have identified many recurrent mutations that seem to contribute to human lung cancer development (2, 3). In adenocarcinoma, several proto-oncogenes, including KRAS and EGFR, are known to be mutated at significant frequency (3–5). Mutation of tumor suppressors including TP53, CDKN2A, PTEN, NF1, and LKB1 are also common in lung adenocarcinoma (3, 6). The PI3K/AKT/mTOR pathway, which plays multiple roles in cell growth, proliferation, and survival, is frequently deregulated in cancer. The most common mechanism of activation of this pathway in cancer is due to loss or reduced expression of PTEN in multiple cancer types (7, 8). However, several other activating mechanisms/mutations are known, including PIK3CA and AKT1 mutation, and activation of upstream tyrosine kinases including EGF receptor (EGFR; ref. 3). Phosphoinositide 3-kinase (PI3K) consists of a regulatory p85 subunit and a catalytic p110 subunit, which phosphorylates lipid inositides at the 30 position (9). PI3K activation results in the generation primarily of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 in turn recruits both AKT and PDK1 to the membrane, where PDK1 phosphorylates AKT at the T308 site. AKT is also phosphorylated at S473 by mTOR complex 2 (mTORC2) as a secondary and more strongly activating phosphorylation event (10). AKT is a serine/threonine kinase that is a central regulator of cell proliferation, survival, and metabolism by phosphorylating multiple protein substrates including TSC2, FOXO proteins, eNOS, BAD, and inhibitor of IkB kinase (IKK)-a (11, 12). Phosphorylation of TSC2 by AKT inactivates its Rheb GTPase activity, leading to activation of mTOR complex 1 (mTORC1; refs. 13, 14). mTORC1 phosphorylates a number of downstream proteins including the S6 kinases and eIF4E-binding proteins (4EBP; ref. 15). Together, these effects lead to ribosome and protein biogenesis, cell size increase, and preparation for cell division Authors' Affiliations: Division of Translational Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston; and The Broad Institute of Harvard University andMassachusetts Institute of Technology, Cambridge, Massachusetts Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: David J Kwiatkowski, Division of Translational Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115. Phone: 617-355-9005; Fax: 617-355-9016; E-mail: dk@rics.bwh. harvard.edu doi: 10.1158/1541-7786.MCR-12-0558 2012 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 11(3) March 2013 282 on June 19, 2017. © 2013 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from Published OnlineFirst January 14, 2013; DOI: 10.1158/1541-7786.MCR-12-0558