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. 2014 Mar 15;74(6):1682-93.
doi: 10.1158/0008-5472.CAN-13-1729. Epub 2014 Jan 21.

FoxO transcription factors promote AKT Ser473 phosphorylation and renal tumor growth in response to pharmacologic inhibition of the PI3K-AKT pathway

Aifu Lin  1 , Hai-Long Piao, Li Zhuang, Dos D Sarbassov, Li Ma, Boyi Gan
Affiliations

FoxO transcription factors promote AKT Ser473 phosphorylation and renal tumor growth in response to pharmacologic inhibition of the PI3K-AKT pathway

Aifu Lin et al. Cancer Res. .

Abstract

The PI3K-AKT pathway is hyperactivated in many human cancers, and several drugs to inhibit this pathway, including the PI3K/mTOR dual inhibitor NVP-BEZ235, are currently being tested in various preclinical and clinical trials. It has been shown that pharmacologic inhibition of the PI3K-AKT pathway results in feedback activation of other oncogenic signaling pathways, which likely will limit the clinical utilization of these inhibitors in cancer treatment. However, the underlying mechanisms of such feedback regulation remain incompletely understood. The PI3K-AKT pathway is a validated therapeutic target in renal cell carcinoma (RCC). Here, we show that FoxO transcription factors serve to promote AKT phosphorylation at Ser473 in response to NVP-BEZ235 treatment in renal cancer cells. Inactivation of FoxO attenuated NVP-BEZ235-induced AKT Ser473 phosphorylation and rendered renal cancer cells more susceptible to NVP-BEZ235-mediated cell growth suppression in vitro and tumor shrinkage in vivo. Mechanistically, we showed that FoxOs upregulated the expression of Rictor, an essential component of MTOR complex 2, in response to NVP-BEZ235 treatment and revealed that Rictor is a key downstream target of FoxOs in NVP-BEZ235-mediated feedback regulation. Finally, we show that FoxOs similarly modulate the feedback response on AKT Ser473 phosphorylation and renal tumor growth by other phosphoinositide 3-kinase (PI3K) or AKT inhibitor treatment. Together, our study reveals a novel mechanism of PI3K-AKT inhibition-mediated feedback regulation and may identify FoxO as a novel biomarker to stratify patients with RCC for PI3K or AKT inhibitor treatment, or a novel therapeutic target to synergize with PI3K-AKT inhibition in RCC treatment.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: The authors disclose no potential conflicts of interest.

Figures

Figure 1
Figure 1. NVP-BEZ235 treatment-induced AKT Ser473 phosphorylation is significantly compromised in FoxO deficient cells
(A and B) FoxO WT and KO MEFs were treated with 50 nM NVP-BEZ235 for different hours as indicated. Cell lysates were then analyzed by western blotting. (C) FoxO WT and KO MEFs were treated with NVP-BEZ235 for 24 hours with different concentrations as indicated. Cell lysates were then analyzed by western blotting. (D) UOK 101 cells infected with either control shRNA or FoxO1/FoxO3 shRNA were treated with 10 nM NVP-BEZ235 for different hours as indicated, and then subjected to western blotting analysis. (E) 786-O cells infected with either control shRNA or FoxO1/FoxO3 shRNA were treated with 50 nM NVP-BEZ235 for different hours as indicated, and then subjected to western blotting analysis.
Figure 2
Figure 2. FoxO deficiency promotes cell proliferation suppression and cell death induction under NVP-BEZ235 treatment
(A) FoxO WT and KO MEFs were treated with 0 (vehicle) or 50 nM NVP-BEZ235 for different days as indicated, and then subjected to cell proliferation analysis. (B) Bar graph showing the percentages of Annexin V staining of FoxO WT and KO MEFs which were treated with 0 or 50 nM NVP-BEZ235 for 24 hours. (C) UOK 101 cells infected with either control shRNA or FoxO3 shRNA were treated with 0 or 50 nM NVP-BEZ235 for different days as indicated, and then subjected to cell proliferation analysis. (D) Bar graph showing the percentages of Annexin V staining of UOK101 cells infected with either control shRNA or FoxO1/FoxO3 shRNA which were treated with 0 or 50 nM NVP-BEZ235 for 24 hours. (E) RCC4 cells infected with either control shRNA or FoxO3 shRNA were treated with 0 or 50 nM NVP-BEZ235 for different days as indicated, and then subjected to cell proliferation analysis. (F) 786-O cells infected with either control shRNA or FoxO1/FoxO3 shRNA were treated with 0 or 50 nM NVP-BEZ235 for different days as indicated, and then subjected to cell proliferation analysis.
Figure 3
Figure 3. FoxO deficiency potentiates NVP-BEZ235-induced renal tumor suppression in vivo
(A) The representative images of 786-O xenograft tumors infected with control shRNA or FoxO1/FoxO3 shRNA which were treated with vehicle or NVP-BEZ235 for 20 days. (B) Tumor volumes of different tumor and treatment groups at different days during treatment. (C) Tumor weights of different tumor and treatment groups at the end point (20 days after treatment). (D) Protein lysates obtained from different tumor and treatment groups at the end point were subjected to western blotting analysis as indicated. (E) Representative immunohistochemical images showing the staining of tumor samples obtained from different tumor and treatment groups at the end point. Scale bars: 50 um. (F) Bar graphs showing the relative mean intensities of immunohistochemical staining signals quantified by Image-pro plus 6.0 software (Media Cybernetics, Bethesda, MD).
Figure 4
Figure 4. FoxOs upregulate Rictor expression under NVP-BEZ235 treatment
(A and B) FoxO WT and KO MEFs were treated with 50nM NVP-BEZ235 for 24 hours, and then subjected to western blotting (A) or real-time PCR analysis (B). (C) 786-O cells infected with either control shRNA or FoxO1/FoxO3 shRNA were treated with 50 nM NVP-BEZ235 for 24 hours, and then subjected to western blotting analysis. (D) UOK 101 cells infected with either control shRNA or FoxO3 shRNA were treated with 50 nM NVP-BEZ235 for different hours as indicated, and then subjected to western blotting analysis. (E) Protein lysates obtained from different tumor and treatment groups at the end point were subjected to western blotting analysis as indicated. (F and G) FoxO WT and KO MEFs were treated with 0 or 50 nM NVP-BEZ235 for 24 hours, and then subjected to ChIP analysis to detect FoxO1/3 binding to Rictor promoter. Bar graph showing the relative enrichment determined by real-time PCR following ChIP analysis.
Figure 5
Figure 5. Rictor mediates FoxO regulation of AKT Ser473 phosphorylation, cell proliferation and survival in response to NVP-BEZ235 treatment
(A) FoxO WT and KO MEFs with stable expression of either empty vector (EV) or Rictor were treated with 0 or 50nM NVP-BEZ235 for 24 hours, cell lysates were then analyzed by western blotting. (B) FoxO WT and KO MEFs with stable expression of either empty vector (EV) or Rictor were treated with 0 or 50 nM NVP-BEZ235 for different days as indicated, and subjected to cell proliferation analysis. (C) FoxO WT and KO MEFs with stable expression of either empty vector (EV) or Rictor were treated with 0 or 50 nM NVP-BEZ235 for 24 hours. The apoptosis rates of the cells were then detected by Annexin V staining assay.
Figure 6
Figure 6. FoxO deficiency potentiates cell proliferation suppression and cell death induction in response to either BKM120 or MK-2206 treatment
(A and B) FoxO WT and KO MEFs were treated with vehicle, 50 nM BKM120 (A) or 50 nM MK-2206 (B) for 24 hours, and then subjected to western blotting analysis. (C and G) FoxO WT and KO MEFs were treated with 0 (vehicle), or 50 nM BKM120 (C), or 50 nM MK-2206 (G) for different days as indicated, and then subjected to cell proliferation analysis. (D and H) Bar graph showing the percentages of Annexin V staining of FoxO WT and KO MEFs which were treated with vehicle, or 50 nM BKM120 (D) or MK-2206 (H) for 24 hours. (E and I) UOK 101 cells infected with either control shRNA or FoxO3 shRNA were treated with vehicle, or 50 nM BKM120 (E), or MK-2206 (I) for different days as indicated, and then subjected to cell proliferation analysis. (F and J) 786-O cells infected with either control shRNA or FoxO3 shRNA were treated with vehicle or 50 nM BKM120 (F), or MK-2206 (J) for different days as indicated, and then subjected to cell proliferation analysis.
Figure 7
Figure 7. FoxO deficiency promotes BKM120 or MK-2206 treatment-induced renal tumor suppression in vivo
(A) The representative images of 786-O xenograft tumors infected with control shRNA or FoxO1/FoxO3 shRNA which were treated with vehicle, BKM120, or MK-2206 for 20 days. (B) Tumor weights of different tumor and treatment groups at the end point (20 days after treatment). (C and D) Tumor volumes of different tumor and treatment groups at different days during drug treatment. (E and F) Protein lysates obtained from different tumor and treatment groups at the end point were subjected to western blotting analysis as indicated.
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