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TSC-insensitive Rheb mutations induce oncogenic transformation through a combination of constitutively active mTORC1 signalling and proteome remodelling

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Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1 that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, tuberous sclerosis complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving an increased rate of anaerobic glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through differential interactions with 5′ AMP-activated protein kinase (AMPK) which modulate its activity. Our findings suggest that unique, personalized, combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.

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Data availability

All RAW MS-based proteomics data have been deposited to ProteomeXchange via the PRIDE partner repository with the identifier PXD017006 and can be accessed at https://www.ebi.ac.uk/pride/archive/login.

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Acknowledgements

We would like to thank Professor Andrew Tee (Cardiff University, Cardiff, UK) and Dr. Brendan Manning (Harvard Medical School, Boston, MA, USA) for their valuable suggestions in regard to the GAP assay.

Funding

This work was initiated with funding from the UK Biotechnology and Biological Sciences Research Council (to CGP) and then supported by SAHMRI. SDP acknowledges support by a Scholarship from the Australian Government Research Training Program (RTP). JX acknowledges a SAHMRI early/mid-career seed funding grant. LAS is supported by a Principal Cancer Research Fellowship awarded by Cancer Council’s Beat Cancer project on behalf of its donors, the state Government through the Department of Health, and the Australian Government through the Medical Research Future Fund.

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Author notes

  1. Jianling Xie and Stuart P. De Poi contributed equally to this work.

Authors and Affiliations

  1. Lifelong Health, South Australian Health and Medical Research Institute, Adelaide, SA, 5001, Australia

    Jianling Xie, Stuart P. De Poi, Wenru Pan & Christopher G. Proud

  2. School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK

    Jianling Xie & Christopher G. Proud

  3. Department of Molecular and Biomedical Sciences, University of Adelaide, Adelaide, SA, 5005, Australia

    Stuart P. De Poi & Christopher G. Proud

  4. Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, 2006, Australia

    Sean J. Humphrey

  5. Lysosomal Health in Ageing, Lifelong Health, South Australian Health and Medical Research Institute, Adelaide, SA, 5001, Australia

    Leanne K. Hein & Timothy J. Sargeant

  6. Institute for Photonics and Advanced Sensing, School of Biological Sciences, University of Adelaide, Adelaide, SA, 5005, Australia

    John B. Bruning

  7. Adelaide Medical School, University of Adelaide, Adelaide, SA, 5005, Australia

    Wenru Pan

  8. Flinders Health and Medical Research Institute, Flinders University, Bedford Park, SA, 5042, Australia

    Luke A. Selth

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  1. Jianling Xie
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All animal work was conducted in accordance with the National Health and Medical Research Council’s Care and use of Animals for Scientific Purposes guidelines and with approval (No. SAM339) from the South Australian Health and Medical Research Institute’s animal ethics committee.

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18_2021_3825_MOESM1_ESM.tif

Supplementary file1 (TIF 1892 KB) Supplementary Figure S1 Activation by Rheb mutants of signalling downstream of mTOR is blocked by AZD8055 but not AZD6244. Related to Fig. 1. (a) Immunoblot analysis of HEK293 cells transfected with the indicated Rheb mutants. Cells were treated with 1 µM AZD8055 1 h prior to harvest. (b) Immunoblot of lysates from HEK293 cells that had been transfected with a pcDNA3.1 empty vector (EV) or vector encoding wild-type Rheb or the indicated mutant. After 24 h, cells were treated with either DMSO or 5 µM AZD6244 for 2 h. All figures are representative images of three independent replicate experiments. (c) Immunoblot of lysates from HEK293 cells that had been transfected with a pcDNA3.1 empty vector or vector encoding wild-type Rheb or the indicated mutant. After 24 h, the medium was replaced with fresh medium either with or without FBS. After 16 h, cells were lysed. Immunoblots were probed with antibodies for the indicated proteins or phosphorylation sites. (d) Quantification of Supplementary Fig. S1c. P-ERK and total ERK were normalised to β-actin and the ratio of P-ERK:ERK graphed). Data are means ± S.D.; n = 3. (e) Immunoblots of lysates from HEK293 cells previously transfected with either the indicated Rheb mutant and a pcDNA3.1 empty vector or the indicated Rheb mutant and vectors encoding FLAG-TSC1 and FLAG-TSC2. After 24 h, the medium was replaced with fresh DMEM without FBS for 16 h followed by D-PBS for 1 h. (f) Quantification of Supplementary Fig. S1e. P-S6K1 Thr389 (top) and P-4EBP1 Ser65 (bottom) normalized to Tubulin loading control for means ± S.D.; n = 3. Data were analysed using Student’s t-test where *: 0.01 ≤ P < 0.05; **: 0.001 ≤ P < 0.01; ***: P < 0.001.

18_2021_3825_MOESM2_ESM.tif

Supplementary file2 (TIF 787 KB) Supplementary Figure S2: Rheb Mutants co-immunoprecipitate with TSC1/2 and mTORC1. Related to Fig. 2. (a) HEK293 cells were transfected with indicated vectors for FLAG-Rheb for 48 h, for both (a) and (b) Rheb and associated proteins were pulled down from cell lysates and both immunoprecipitates and input lysates were subjected to immunoblotting analysis for the indicated proteins.

18_2021_3825_MOESM3_ESM.tif

Supplementary file3 (TIF 696 KB) Supplementary Figure S3: Rheb mutants do not drive faster proliferation in fully supplemented media. Related to Fig. 3. (a) MTT assay data for HEK293 cells transiently expressing the indicated Rheb mutants in fully supplemented medium. Data are means ± S.D.; n = 3. (b) BrdU incorporation data for HEK293 cells transiently expressing the indicated Rheb mutants in fully supplemented medium. (c) Table summarizing the phenotypes of various Rheb mutants as determined in Figs. 1d, e, 2c, d, 3a and b.

18_2021_3825_MOESM4_ESM.tif

Supplementary file4 (TIF 1142 KB) Supplementary Figure S4: Characterisation of NIH3T3 cells stably over-expressing Rheb mutants. Related to Figs. 3 and 4. (a) Sanger sequencing results of monoclonal cell lines selected for use in the in vivo study. Cells were grown for 8 weeks before DNA was extracted and sequenced using primers specific to the inserted plasmid. (b) Survival curve showing the number of days post-injection until tumour volume reached 60 mm3. Statistics were calculated using the Mantel-Cox logrank test where @: P < 0.001. (c) Quantification of DAB stain in Fig. 4d for P-rpS6:rpS6 ratio. (d) As in (c) for P-ERK1/2:ERK1/2 ratio. Error bars indicate SD for three independent experiments. For panels c and d, data are means ± S.D.; n = 3. Statistical significance was determined by Student’s t-test where *: 0.01 ≤ P < 0.05; **: 0.001 ≤ P < 0.01.

18_2021_3825_MOESM5_ESM.tif

Supplementary file5 (TIF 2923 KB) Supplementary Figure S5: MS analysis of proteome changes between fully supplemented and serum starved NIH3T3 cells. Related to Fig. 5. NIH3T3 cells stably over-expressing Rheb-WT, T23M or E40K were either cultured in full growth medium (with 10% FBS) or starved of serum for 72 h before being subjected to MS analysis. (a) Heat-map illustrates six proteins whose abundances differ among the cell groups cultured in fully supplemented growth medium. (b) Cluster analysis of proteins that are either upregulated when cultured in full growth medium or when serum starved. (c) Heat-map illustrates four different protein clusters among the treatment groups.

18_2021_3825_MOESM6_ESM.tif

Supplementary file6 (TIF 852 KB) Supplementary Figure S6: Validation of MS data. Related to Fig. 5 a) RT-qPCR analysis of cDNA generated from whole cell RNA extracts from NIH3T3 cells stably expressing the indicated Rheb vector grown in fully supplemented media (+FBS) or starved of serum for 72 h (-FBS) using primers specific for PKM2. b) As in a) with primers for EEF2. c) Immunoblot analysis of NIH3T3 cells stably expressing the indicated Rheb mutant. Cells were grown in fully supplemented media or starved of serum for 72 h, as indicated. d) Quantification of c) normalized to β-actin. e) Proteins from tumour tissue (from Fig. 4) lysates were separated on SDS-PAGE gels, indicated proteins were immunoblotted with their corresponding antibodies. f) The indicated protein ratios in e) were quantified. g) Quantification of Fig. 6a for ratio of P-eEF2:eEF2. For panels XXX, data are means ± S.D.; n = 3. Statistical significance was determined by 2-way ANOVA with multiple comparisons to WT + FBS where *: 0.01 ≤ P < 0.05; **: 0.001 ≤ P < 0.01; ***: P < 0.001.

18_2021_3825_MOESM7_ESM.tif

Supplementary file7 (TIF 1161 KB) Supplementary Figure S7: CA-Rheb mutants drive an increase in migration and invasion in trans-well assays. Related to Fig. 6. a) Transwell migration and invasion assays of 1.5 × 104 HEK293 cells transfected with vectors encoding the indicated Rheb mutant. For invasion assays, transwells were pre-coated with Matrigel. Cells were treated with 0.5 µg/µl for the duration of the experiment. Cells were allowed to migrate or invade for 72 h. b) Quantification of a) for migration. c) Quantification of a) for invasion. d) Immunoblot analysis of NIH3T3 cells. Cells were either grown in fully supplemented media or serum starved for 2h prior to harvest as indicated. As a positive control for AMPK activation, one well had media preplaced for media lacking glucose for 1 h prior to the addition of 2-deoxyglucose (2-DG) for 2 h. e) NIH3T3 cells stably expressing the indicated Rheb mutants were treated with AZD8055 or DMSO for 1h as indicated. f) NIH3T3 cells stably expressing the indicated Rheb mutants were treated with either 1 µM AZD8055 or DMSO, as indicated, for 1 h. Lysates were collected, and western blot analysis performed with the indicated antibodies. For panels b and c, data are means ± S.D.; n = 3. Statistical significance was determined by Student’s t-test where *: 0.01 ≤ P < 0.05; **: 0.001 ≤ P < 0.01; ***: P < 0.001.

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Xie, J., De Poi, S.P., Humphrey, S.J. et al. TSC-insensitive Rheb mutations induce oncogenic transformation through a combination of constitutively active mTORC1 signalling and proteome remodelling. Cell. Mol. Life Sci. 78, 4035–4052 (2021). https://doi.org/10.1007/s00018-021-03825-7

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