Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Mar;11(3):282-93.
doi: 10.1158/1541-7786.MCR-12-0558. Epub 2013 Jan 14.

Molecular dissection of AKT activation in lung cancer cell lines

Yanan Guo  1 , Jinyan Du, David J Kwiatkowski
Affiliations

Molecular dissection of AKT activation in lung cancer cell lines

Yanan Guo et al. Mol Cancer Res. 2013 Mar.

Abstract

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 in HCC2429 was PI3K- and mTOR complex 2 (mTORC2)-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were 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.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest. None.

Figures

Figure 1
Figure 1. Lung cancer cell lines with high pAKT(S473) under serum starvation conditions
A–D. Immunoblots are shown for 19 lung cancer cell lines after 24 h of serum starvation (−) or 30 min after serum addback after serum starvation (+), for multiple components of the AKT-mTOR signaling pathway. Thirteen cell lines show constitutive, high levels of pAKT(S473) during serum starvation. Six can be seen to be null (H1650, H1781, H157, HCC2935 and HCC461) or markedly reduced (H2444) in PTEN expression. Four cell lines are controls and show normal regulation of pAKT(S473) levels.
Figure 2
Figure 2. Both AKT1 and AKT2are activated in HCC2429 cells, effects of PI3K inhibitors
A, B. HCC2429 cells were serum starved for 24 h or grown in regular conditions followed by immunoprecipitation with either AKT1 (A) or AKT2 (B) antibodies. Both whole cell lysates (WCL) and protein immunoprecipitates (IP) were analyzed by immunoblotting. Note that pAKT(S473) is present in IPs done for either AKT1 or AKT2. C, D. HCC2429 cells were serum starved for 24 h or grown in regular conditions in the presence of PI3K inhibitors LY294002 (LY) or Wortmannin (Wort) for indicated time and doses. Immunoblots are shown. Note that LY294002 treatment for 24 hours caused significant cell death of serum-starved HCC2429 cells, leading to reduced protein loading in that lane (C, lane 6).
Figure 3
Figure 3. AKT activation in HCC2429 is mTORC2 dependent
A. HCC2429 cells were treated with mTOR inhibitor PP242 (2.5uM) or Torin1 (250nM) for 24 h in the presence or absence of serum, and analyzed by immunoblotting. B. HCC2429 cells were treated with rapamycin for either 1 h or 24 h with the indicated doses in the presence or absence of serum, and analyzed by immunoblotting. C. HCC2429 cells were treated with rapamycin for the indicated times and doses in the presence or absence of serum for 24 h followed by immunoprecipitation (IP) with mTOR antibody, and analysis by immunoblotting.
Figure 4
Figure 4. Tyrosine kinase phosphorylation profiling of HCC2429 cells shows active SRC and Notch pathways
A heat map is shown for cell lysates analyzed by Luminex immunosandwich assays for phosphorylation of tyrosine kinases. Normalized results are shown with the color scale indicating relative increase or decrease in phospho-protein levels relative to controls, and expressed as the multiple of the standard deviation of the row. HCC2429, H3255, HCC15 and HCC1833 cell lines were serum starved for 24 h or maintained in regular growth media. Some HCC2429 cell preparations were also treated with rapamycin (10nM) for 24 h, as indicated.
Figure 5
Figure 5. AKT activation in HCC2429 is SRC- but not Notch-dependent
A. HCC2429 cells were treated with Rapamycin (20nM) alone or together with Notch inhibitor DAPT (15uM) for 24 h in the presence or absence of serum, and cell lysates were analyzed by immunoblotting. Note that HES1 is not seen in cells treated with DAPT. pAKT(S473) shows no major change in response to DAPT. B. HCC2429 cells were treated with Notch inhibitor Compound E at the indicated doses for 24 h in the presence or absence of serum. Note that HES1 levels are absent in cells treated with Compound E but there is no effect on pAKT(S473) levels. C. Cells were treated with Notch inhibitor DAPT or Compound E for 48 h at the indicated doses. Cell numbers were determined by the MTT assay and normalized to untreated cells. D–E. HCC2429 cells were treated with SRC inhibitor PP2 or Dasatinib at the indicated doses for 24 h in the absence (D) or presence (E) of serum. F. Cells were treated with SRC inhibitor PP2 or Dasatinib for 48 h at the indicated doses. Cell numbers were determined by the MTT assay and normalized to untreated cells.
Figure 6
Figure 6. SRC inhibitors and Torin1 synergistically inactivate AKT and reduce cell survival
A. HCC2429 cells were treated with SRC inhibitors PP2 (10uM), Dasatinib (1uM), and mTOR inhibitor Torin1 (25nM), or combinations of these drugs for 24 h in the absence of serum, and analyzed by immunoblotting. B–C. HCC2429, HCC15, and H3255 cells were treated with SRC inhibitor PP2 (B) or Dasatinib (C) together with mTOR inhibitor Torin1 for 48 h at the indicated doses. Cell numbers were determined by the MTT assay and normalized to untreated cells.
Figure 7
Figure 7. Synergistic effects of Dasatinib and mTOR inhibitors on HCC2429 xenografts
HCC2429 cells were injected into both flanks of Scid (C.B-17) mice to generate tumors. When tumors were palpable, mice were treated with Placebo, Dasatinib (5mg/kg), Torin2 (10mg/kg) or Dasatinib(5mg/kg) + Torin2 (10mg/kg) by oral gavage 5 days a week. N = 3 – 5 mice per treatment group. Mice were sacrificed 12, 14, 17, and 17 days after initiation of treatment for vehicle, Dasatanib, Torin2, and combined treatment mice, respectively. A. Representative HCC2429 xenograft tumors. B. Relative tumor volume during treatment. C. Tumor weight at the end of treatment. D. Tumor volume at the end of treatment. E. IHC staining for pSRC(Y416), pAKT(S473), and pS6(S240/244)after 3 days of treatment, in a separate set of Scid mice.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, 2008. CA Cancer J Clin. 2008;58(2):71–96. - PubMed
    1. Sos ML, Michel K, Zander T, Weiss J, Frommolt P, Peifer M, et al. Predicting drug susceptibility of non-small cell lung cancers based on genetic lesions. J Clin Invest. 2009;119(6):1727–40. - PMC - PubMed
    1. Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature. 2008;455(7216):1069–75. - PMC - PubMed
    1. Thomas RK, Baker AC, Debiasi RM, Winckler W, Laframboise T, Lin WM, et al. High-throughput oncogene mutation profiling in human cancer. Nat Genet. 2007;39(3):347–51. - PubMed
    1. Weir BA, Woo MS, Getz G, Perner S, Ding L, Beroukhim R, et al. Characterizing the cancer genome in lung adenocarcinoma. Nature. 2007;450(7171):893–8. - PMC - PubMed

Publication types

MeSH terms