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A pan-cancer analysis reveals nonstop extension mutations causing SMAD4 tumour suppressor degradation

Nature Cell Biology volume 22, pages 999–1010 (2020)Cite this article

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Abstract

Nonstop or stop-loss mutations convert a stop into a sense codon, resulting in translation into the 3′ untranslated region as a nonstop extension mutation to the next in-frame stop codon or as a readthrough mutation into the poly-A tail. Nonstop mutations have been characterized in hereditary diseases, but not in cancer genetics. In a pan-cancer analysis, we curated and analysed 3,412 nonstop mutations from 62 tumour entities, generating a comprehensive database at http://NonStopDB.dkfz.de. Six different nonstop extension mutations affected the tumour suppressor SMAD4, extending its carboxy terminus by 40 amino acids. These caused rapid degradation of the SMAD4 mutants via the ubiquitin–proteasome system. A hydrophobic degron signal sequence of ten amino acids within the carboxy-terminal extension was required to induce complete loss of the SMAD4 protein. Thus, we discovered that nonstop mutations can be functionally important in cancer and characterize their loss-of-function impact on the tumour suppressor SMAD4.

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Fig. 1: Nonstop mutations in cancer.
Fig. 2: Nonstop extension mutations abolish SMAD4 protein, but not mRNA expression in pancreatic and colon cancer cells.
Fig. 3: Nonstop mutations inhibit SMAD4 function in vitro and in vivo, requiring a peptide bond between SMAD4 and extension.
Fig. 4: Suppression by the SMAD4 extension is transferable, mediated by a degron of ten amino acids and proteasome dependent.
Fig. 5: SMAD4 protein but not mRNA expression is lost in endogenous SMAD4 nonstop mutant HEK293T and PANC-1 cell lines.
Fig. 6: Endogenous SMAD4 nonstop extension mutation compromises TGF-β/SMAD4-dependent transcriptional regulation.
Fig. 7: SMAD4 nonstop extension mutants retain multimerization properties and are degraded by the ubiquitin–proteasome pathway.

Data availability

The datasets used for the analysis of nonstop mutations in this study were obtained from the publicly available COSMIC database (https://www.sanger.ac.uk/cosmic). A searchable resource containing details of the nonstop mutations in different cancer entities is provided in the NonStopDB database accessible at http://NonStopDB.dkfz.de. All other data generated or analysed as part of this study are included within this published article and its supplementary files. Source data are provided with this paper.

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Acknowledgements

This research was funded by the German Research Foundation (DFG Di 1421/9-1) and the foundation for the project was laid by a DKFZ NCT 3.0 Integrative Project in Cancer Research (NCT3.0_2015.54 DysregPT). We thank the COSMIC database team for providing and maintaining this large and important resource. The authors are grateful to P. Frey, V. Penucci, G. Turchiano, T. Cathomen (Freiburg) and G. Stoecklin (Mannheim) for support with setting up the project and helpful discussions, and to A. Hecht (Freiburg) for providing cell lines. We thank T. Tuschl (New York), Addgene (Boston), M.B. Menon (New Delhi), R. Hedge (Cambridge) and B. Korn (Heidelberg) for providing plasmids. We also thank the Lighthouse Core Facility at the Center for Translational Cell Research (ZTZ) in Freiburg for support with the flow cytometry experiments, and the Freiburg Galaxy team with B. Grüning and R. Backofen for providing this resource. We are grateful to H. Binder and D. Hauschke (Freiburg) for expert statistical advice.

Author information

Author notes

  1. Jochen Maurer

    Present address: Department of Gynecology, RWTH Aachen University Hospital, Aachen, Germany

  2. These authors contributed equally: Sonam Dhamija, Chul Min Yang.

Authors and Affiliations

  1. Division of Cancer Research, Department of Thoracic Surgery, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, German Cancer Consortium (DKTK) Partner Site Freiburg, Freiburg, Germany

    Sonam Dhamija, Chul Min Yang, Ksenia Myacheva, Angela Wieland, Mahmoud Abdelkarim, Yogita Sharma, Marisa Riester & Sven Diederichs

  2. Division of RNA Biology and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Sonam Dhamija, Jeanette Seiler, Ksenia Myacheva, Maiwen Caudron-Herger, Matthias Groß & Sven Diederichs

  3. Department of Visceral Surgery, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, German Cancer Consortium (DKTK) Partner Site Freiburg, Freiburg, Germany

    Jochen Maurer

  4. National Center for Tumor Diseases (NCT), Heidelberg, Germany

    Sven Diederichs

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Contributions

S. Diederichs conceived of and designed the study. S. Dhamija, C.M.Y., M.C.-H., J.M. and S. Diederichs designed the experiments and analysed the data. S. Dhamija, C.M.Y., J.S., K.M., A.W., M.A., M.R., M.G. and J.M. performed the experiments. M.C.-H. and Y.S. generated the NonStopDB database. S. Dhamija, C.M.Y. and S. Diederichs prepared the figures and wrote the manuscript.

Corresponding author

Correspondence to Sven Diederichs.

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S.Diederichs is a co-owner of siTOOLs Biotech, Munich, which is not related to this work. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Nonstop extension mutations abolish SMAD4 protein, but not mRNA expression in pancreatic and colon cancer cells.

a, b, SMAD4 protein expression (bottom: western blot, top: intensities normalized to β-actin and WT) after transfection of empty vector (EV), SMAD4 wildtype (WT) or mutant expression vectors into pancreatic cancer cells PANC-1 (a) and colorectal cancer cells HCT116 (b). The nonsense SMAD4 C1333T mutant was used for comparison. c, d, Exogenous SMAD4 mRNA expression (RT-qPCR, exogenous transcripts (EF1a)) and total SMAD4 mRNA after transfection of EV, WT and mutants into PANC-1 (c) and HCT116 (d) cells normalized to the housekeeping gene PPIA and represented relative to WT. e, f, SMAD4 protein expression (e: western blot, f: quantification) after transfection of EV, FLAG-HA tagged SMAD4 WT or mutants into BxPC3 cells. All panels: n=3 biologically independent experiments, mean ± SEM, ANOVA with ***p<0.001, **p<0.01, *p<0.05. Numerical source data and unprocessed blots are provided with the paper.

Source data

Extended Data Fig. 2 SMAD4 nonstop extension mutations suppress protein expression and function.

a-c, SMAD4 protein (a: western blot, b: quantification) and mRNA (c) expression after lentiviral transduction of EV, WT and mutants and selection of SW620 cells (n=3 biologically independent experiments). d-e, Expression of the SMAD4 targets PAI (d) and ADAM19 (e) quantified by RT-qPCR in transiently transfected BxPC3 cells treated with 10 ng/ml TGFβ (n=4 biologically independent experiments). f, SMAD4 transcript levels quantified by RT-qPCR (n=3 biologically independent experiments) for the T2A element fusion experiment presented in the main figure panels Fig. 3h–j. g, EYFP and Neomycin (Neo) mRNA levels were quantified by RT-qPCR after transfection of BxPC3 cells (n=3 biologically independent experiments). All panels: mean ± SEM, ANOVA with ***p<0.001, **p<0.01, *p<0.05. Numerical source data and unprocessed blots are provided with the paper.

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Extended Data Fig. 3 Identification of the minimal functional peptide sequence in the SMAD4 extension.

a, Schematic representation of WT and mutant SMAD4 with the terminal glycine residue (G592) indicated. b, SMAD4 protein expression determined by western blot after transfection of BxPC3 cells with WT SMAD4, T1657C mutant or T1657C with C-terminal Gly592Ala (G>A) / Gly592Val (G>V) mutations. c, Schematic representation of the SMAD4 extension amino acid sequence. The extension of 39 aa after the mutated stop codon was divided into four parts: A, B, C (10 aa each) & D (9 aa). These are indicated and this nomenclature is followed in the figure panels including main Fig. 4d. Further deletions of residues are indicated by subscripts showing the retained amino acid segments in the respective domains. d-i, SMAD4 protein expression determined by western blot (d,f,h) and quantified for replicates (e,g,i) in lysates from BxPC3 cells transfected with pEF-DEST51 empty vector (EV), WT SMAD4, SMAD4 with the full length extension (ABCD) or vectors expressing indicated truncations and deletions in the extension. All panels: n=3 biologically independent experiments, mean ± SEM, ANOVA with ***p<0.001, **p<0.01, *p<0.05. Numerical source data and unprocessed blots are provided with the paper.

Source data

Extended Data Fig. 4 Translation stalling does not strongly contribute to the diminished expression of SMAD4 harboring nonstop extension mutations.

a, SMAD4 WT and nonstop extension mutations and a control missense mutant (G1082A) encoding vectors were subjected to coupled in vitro transcription/translation reactions (IVT). The IVT reactions were analyzed by western blotting and probed with anti-SMAD4 antibodies. b, Bands from (a) were quantified and presented as % of WT-SMAD4. c, SMAD4 transcripts from the IVT reactions were quantified by RT-qPCR, normalized to rabbit 18S rRNA and presented as relative expression normalized to WT. d, Schematic representation of the translation stalling in vivo reporters. e, Reporter plasmids containing no stalling sequence in the linker between GFP and mCherry (control), the positive control inducing stalling with 20 lysine codons ((AAA)20), a negative control stem loop or the SMAD4 extension were transfected into BxPC3 cells and analyzed by flow cytometry. Scatter plots show mCherry and GFP expression in transfected cells are shown. f, Median fluorescence intensities of mCherry and GFP in GFP-positive cells were determined and their means are depicted as relative mCherry / GFP ratios normalized to control. The gating strategy for flow cytometry is described in Supplementary Fig. 10. All panels: n=3 biologically independent experiments, mean ± SEM, ANOVA with ***p<0.001, **p<0.01, *p<0.05. Numerical source data and unprocessed blots are provided with the paper.

Source data

Extended Data Fig. 5 Solubility of SMAD4 nonstop extension mutants.

a-b, Stably transduced BxPC3 (a) and SW620 (b) cells were lysed in 1% NP-40 buffer. The insoluble pellet fraction was further solubilized in 2X SDS PAGE sample buffer (2% SDS). Equal volumes of NP-40-soluble and -insoluble fractions were separated and probed with SMAD4 antibodies, with β-actin as loading control (n=3 biologically independent experiments). c-d, Similar solubility analysis with the indicated EYFP fusion constructs transfected into BxPC3 (c) and SW620 (d) cells. All panels: n=3 biologically independent experiments. Unprocessed blots are provided with the paper.

Source data

Extended Data Fig. 6 Bortezomib- and mutation-mediated rescue of SMAD4 NSExt mutant expression.

a-b, SMAD4 band quantification of biological replicates for Bortezomib-mediated rescue in BxPC3 (main Fig. 4h, n=5 biologically independent experiments) and SW620 (main Fig. 4i, n=3 biologically independent experiments) cells normalized to α/β-Tubulin and relative to the DMSO-treated WT control. c-d, The lysines shown in Fig. 7f were mutated individually (K570R, K578R, K591R) or in combination (2K = K570R+K578R or 3K = K570R+K578R+K591R) and the protein levels were monitored in transiently transfected HEK293T cells. A representative result (c) with the immunoblot quantification data from three independent experiments is shown (d). All panels: mean ± SEM, ANOVA with ***p<0.001, **p<0.01, *p<0.05. Numerical source data and unprocessed blots are provided with the paper.

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Supplementary information

Supplementary Information

Supplementary Note 1 and Figs. 1–10.

Reporting Summary

Supplementary Tables

Supplementary Table 1 Oligonucleotides: a list of all of the oligonucleotide sequences used in this study (in the 5′–3′ direction). Supplementary Table 2 Antibodies: a list of all of the antibodies used in this study.

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Dhamija, S., Yang, C.M., Seiler, J. et al. A pan-cancer analysis reveals nonstop extension mutations causing SMAD4 tumour suppressor degradation. Nat Cell Biol 22, 999–1010 (2020). https://doi.org/10.1038/s41556-020-0551-7

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