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Secreted IGFBP5 mediates mTORC1-dependent feedback inhibition of IGF-1 signalling

Nature Cell Biology volume 18, pages 319–327 (2016)Cite this article

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Abstract

The PI(3)K–Akt–mTORC1 pathway is a highly dynamic network that is balanced and stabilized by a number of feedback inhibition loops1,2. Specifically, activation of mTORC1 has been shown to lead to the inhibition of its upstream growth factor signalling. Activation of the growth factor receptors is triggered by the binding of their cognate ligands in the extracellular space. However, whether secreted proteins contribute to the mTORC1-dependent feedback loops remains unclear. We found that cells with hyperactive mTORC1 secrete a protein that potently inhibits the function of IGF-1. Using a large-scale, unbiased quantitative proteomic platform, we comprehensively characterized the rapamycin-sensitive secretome in TSC2−/− mouse embryonic fibroblasts, and identified IGFBP5 as a secreted, mTORC1 downstream effector protein. IGFBP5 is a direct transcriptional target of HIF1, which itself is a known mTORC1 target3. IGFBP5 is a potent inhibitor of both the signalling and functional outputs of IGF-1. Once secreted, IGFBP5 cooperates with intracellular branches of the feedback mechanisms to block the activation of IGF-1 signalling. Finally, IGFBP5 is a potential tumour suppressor, and the proliferation of IGFBP5-mutated cancer cells is selectively blocked by IGF-1R inhibitors.

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Figure 1: Cells with hyperactivated mTORC1 secrete a protein factor(s) that blocks IGF-1 signalling.
Figure 2: Expression of IGFBP5 is transcriptionally regulated by HIF1α.
Figure 3: IGFBP5 is a potent inhibitor of IGF-1 signalling.
Figure 4: IGFBP5 is a major mediator of mTORC1-dependent feedback inhibition of IGF-1 signalling.
Figure 5: mTORC1 orchestrates feedback inhibition of IGF-1 signalling by promoting HIF1α-dependent expression of IGFBP5.

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Acknowledgements

We thank J. Goldstein, J. Blenis, M. White, M. Cobb and B. Tu for critically reviewing the manuscript. We thank J. Minna and D. Kwiatkowski for providing critical reagents, and N. Williams, H. Wang, H. Chen and L. Wu for input and technical guidance. This work was supported in part by grants from the UT Southwestern Endowed Scholar Program (to Y.Y. and R.K.B.), the Cancer Prevention and Research Institute of Texas (CPRIT R1103 to Y.Y. and CPRIT RP130513 to R.K.B.), the Welch Foundation (I-1800 to Y.Y. and I-1568 to R.K.B.), National Institutes of Health (NIH; GM114160 to Y.Y.), American Cancer Society (Research Scholar Grant, RSG-15-062-01-TBE, and Institutional Research Grant, IRG-02-196-07, to Y.Y.), and a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund (to R.K.B.). Y.Y. is a Virginia Murchison Linthicum Scholar in Medical Research and a CPRIT Scholar in Cancer Research. R.K.B. is the Michael L. Rosenberg Scholar in Medical Research.

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  1. Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235, USA

    Ming Ding, Richard K. Bruick & Yonghao Yu

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Contributions

Y.Y. conceived the project. M.D., R.K.B. and Y.Y. designed the experiments. M.D. and Y.Y. performed the experiments. R.K.B. provided input on HIF1-related experiments. Y.Y. wrote the manuscript with input from all co-authors.

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Correspondence to Yonghao Yu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1

(A) LDH activity in the whole cell lysates and conditioned media from TSC2−/− MEFs was quantified using a coupled enzymatic colorimetric assay. LDH is a cytosolic enzyme that is found in nearly all living cells. (B) Chronic rapamcyin treatment (20 nM) of TSC2−/− MEFs led to Akt activation. For site-specific phosphorylation, pAkt(S473), pS6K(T389) and pS6(S235/236) levels were analyzed. (C) Heat-inactivated CM from TSC2−/− MEFs lost its ability to inhibit IGF-1 signalling. CM was collected from TSC2−/− MEFs and was heated at 95 °C for 5 min. CM was then mixed with IGF-1 (40 ng ml−1) and added to wt MEFs, the WCL of which were analyzed. For site-specific phosphorylation, pIGF-1R(Y1135/1136) and pAkt(S473) levels were analyzed. (D) Proteins identified from the two SILAC experiments. (E) Identification of FGF21 as a downstream target of mTORC1 in the extracellular space. Extracted ion chromatogram is shown for the corresponding light (rapamycin-treated, blue) and heavy (control, yellow) ions of a peptide (ALKPGVIQILGVK) from FGF21. This peptide showed a dramatic decrease after rapamycin treatment. (F) Identification of an IGFBP5 peptide (HMEASLQEFK). The MS2 spectrum from which the peptide was identified (matched b- and y- ions are highlighted in blue and red, respectively) is shown. (G) Re-introducing TSC2 into TSC2−/− MEFs led to dramatic downregulation of IGFBP5 in CM. For site-specific phosphorylation, pAkt(S473) levels were analyzed. (H) Rapamycin treatment also downregulated the expression of IGFBP5 in RT-4 cells. RT-4 cells were starved for 24 h, during which cells were treated with 20 nM rapamycin. For site-specific phosphorylation, pS6K(T389) and pS6(S235/236) levels were analyzed. (I) Insulin stimulation of MCF7 cells resulted in the accumulation of IGFBP5 in CM, which was blocked by concurrent rapamycin treatment. Cells were starved for 12 h, and were then treated with insulin (100 nM) for 12 h, in the absence or presence of rapamycin (20 nM). For site-specific phosphorylation, pAkt(S473) and pS6(S235/236) levels were analyzed.

Supplementary Figure 2 Gene ontology analysis of the rapamycin-sensitive proteins (abundances decreased by at least 32-fold after rapamycin treatment).

(A) Cellular compartment analysis. (B) Molecular function analysis. For clear presentation, only results from SILAC experiment #1 were considered.

Supplementary Figure 3 A schematic indicating the possible model of HIF1-regulated expression of IGFBP5.

The design of various primers (P1-P4) capturing potential HIF1α binding sites on IGFBP5 is shown.

Supplementary Figure 4

(A) Proteins secreted from TSC2−/− MEFs inhibits the growth of MCF7 cells in a co-culture system. MCF7 cells were labeled with red florescent protein (DsRed), and were grown with GFP-labeled either TSC2+/+ or TSC2−/− MEFs. Cells were grown in DMEM supplemented with IGF-1. Scale bars, 40 μm. IGF-1 protects cells from staurosporine, (B), or etoposide (C)-induced apoptosis. This effect was abolished when IGFBP5 was co-added to the culture media. The asterisk indicates cleaved PARP1. For site-specific phosphorylation, pIGF-1R(Y1135/1136) and pAkt(S473) levels were analyzed. (D) IGFBP5 accounts for a major fraction of the IGF-1-inhibitory activity in CM from TSC2−/− MEFs. CM from TSC2+/+, shGFP TSC2−/− or shIGFBP5 TSC2−/− cells were mixed with IGF-1, and were added to recipient cells (wild type MEFs). WCL were analyzed by immunoblotting experiments using the indicated antibodies. The results were quantified using ImageJ. For site-specific phosphorylation, pIGF-1R(Y1135/1136) and pAkt(S473) levels were analyzed. *P < 0.05 (two-tailed Student t-test), NS = Not significant, n = 3 independent biological replicate experiments. Error bars represent s.d. (E) IGFBP5 mediates the mTORC1-dependent IGF-1-inhibitory activity in TSC2−/− MEFs. RNAi was used to knock down IGFBP5 in TSC2−/− MEFs. Where indicated, control knock down cells (shGFP TSC2−/− MEFs) were treated with rapamycin (20 nM for 24 h). The results were quantified using ImageJ. For site-specific phosphorylation, pIGF-1R(Y1135/1136), pAkt(S473) and pS6K(T389) levels were analyzed. pIGF-1R levels were normalized using total IGF-1R levels (note the rapamycin treatment increases total IGF1-R levels). **P < 0.01 (two-tailed Student t-test), NS = Not significant. n = 4 independent biological replicate experiments. Error bars represent s.d.

Supplementary Figure 5

(A) CM from NCI-H1435 cells does not contain detectable IGFBP5 signals. CM from HEK293T cells that ectopically express IGFBP5 is used as the control. (B) Molt-4 cells (an acute lymphoblastic leukemia cell line that harbors a K135fs*13 mutation of the IGFBP5 gene) is more sensitive to IGF-1R inhibitors (BMS-536924 and BMS-754807), compared to Gefitinib and Sunitinib (48 h treatment). P < 0.001 (two-way ANOVA test). n = 9 independent biological replicate experiments. Error bars represent s.d. (C) The expression of wt-IGFBP5 in Molt-4 cells (to a level similar to that in IGFBP5-wt cells, e.g. T47D and RT-4) leads to reduced proliferation of these cells (grown in DMEM supplemented with 10% FBS). BMS-536924 treatment (500 nM) was used as the control. (D) The proliferation of IGFBP5-mutated (NCI-H1435), but not IGFBP5-wt (A549, NCI-H1693, HCC4017 and HCC15) NSCLC cells is sensitive to IGF-1R inhibitors (BMS-536924 and BMS-754807). None of these cells were sensitive to Sunitinib, a multi-RTK inhibitor, which, however, does not block IGF-1R.

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Ding, M., Bruick, R. & Yu, Y. Secreted IGFBP5 mediates mTORC1-dependent feedback inhibition of IGF-1 signalling. Nat Cell Biol 18, 319–327 (2016). https://doi.org/10.1038/ncb3311

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