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

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.

References

  1. 1.

    Frischmeyer, P. A. et al. An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science 295, 2258–2261 (2002).

    CAS  PubMed  Google Scholar 

  2. 2.

    Klauer, A. A. & van Hoof, A. Degradation of mRNAs that lack a stop codon: a decade of nonstop progress. Wiley Interdiscip. Rev. RNA 3, 649–660 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Arribere, J. A. et al. Translation readthrough mitigation. Nature 534, 719–723 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Vidal, R. et al. A stop-codon mutation in the BRI gene associated with familial British dementia. Nature 399, 776–781 (1999).

    CAS  PubMed  Google Scholar 

  5. 5.

    Doucette, L. et al. A novel, non-stop mutation in FOXE3 causes an autosomal dominant form of variable anterior segment dysgenesis including Peters anomaly. Eur. J. Hum. Genet. 19, 293–299 (2011).

    PubMed  Google Scholar 

  6. 6.

    Hollingsworth, T. J. & Gross, A. K. The severe autosomal dominant retinitis pigmentosa rhodopsin mutant Ter349Glu mislocalizes and induces rapid rod cell death. J. Biol. Chem. 288, 29047–29055 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Shibata, N. et al. Degradation of stop codon read-through mutant proteins via the ubiquitin–proteasome system causes hereditary disorders. J. Biol. Chem. 290, 28428–28437 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Sun, J. et al. Functional analysis of a nonstop mutation in MITF gene identified in a patient with Waardenburg syndrome type 2. J. Hum. Genet. 62, 703–709 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Bock, A. S. et al. A nonstop variant in REEP1 causes peripheral neuropathy by unmasking a 3′UTR-encoded, aggregation-inducing motif. Hum. Mutat. 39, 193–196 (2018).

    CAS  PubMed  Google Scholar 

  10. 10.

    Pang, S. et al. A novel nonstop mutation in the stop codon and a novel missense mutation in the type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing, respectively, nonclassic and classic 3beta-HSD deficiency congenital adrenal hyperplasia. J. Clin. Endocrinol. Metab. 87, 2556–2563 (2002).

    CAS  PubMed  Google Scholar 

  11. 11.

    Zhang, G. et al. Genetic spectrum of dyschromatosis symmetrica hereditaria in Chinese patients including a novel nonstop mutation in ADAR1 gene. BMC Med. Genet. 17, 14 (2016).

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    McInerney-Leo, A. M. et al. Mutations in LTBP3 cause acromicric dysplasia and geleophysic dysplasia. J. Med. Genet. 53, 457–464 (2016).

    PubMed  Google Scholar 

  13. 13.

    Duis, J. et al. KIF5A mutations cause an infantile onset phenotype including severe myoclonus with evidence of mitochondrial dysfunction. Ann. Neurol. 80, 633–637 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Banfai, Z. et al. Novel phenotypic variant in the MYH7 spectrum due to a stop-loss mutation in the C-terminal region: a case report. BMC Med. Genet. 18, 105 (2017).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    El-Agnaf, O. M. et al. Effect of the disulfide bridge and the C-terminal extension on the oligomerization of the amyloid peptide ABri implicated in familial British dementia. Biochemistry 40, 3449–3457 (2001).

    CAS  PubMed  Google Scholar 

  16. 16.

    Ghiso, J. et al. A newly formed amyloidogenic fragment due to a stop codon mutation causes familial British dementia. Ann. NY Acad. Sci. 903, 129–137 (2000).

    CAS  PubMed  Google Scholar 

  17. 17.

    Bremond-Gignac, D. et al. Identification of dominant FOXE3 and PAX6 mutations in patients with congenital cataract and aniridia. Mol. Vis. 16, 1705–1711 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Iseri, S. U. et al. Seeing clearly: the dominant and recessive nature of FOXE3 in eye developmental anomalies. Hum. Mutat. 30, 1378–1386 (2009).

    CAS  PubMed  Google Scholar 

  19. 19.

    Moalla, M. et al. Nonstop mutation in the Kisspeptin 1 receptor (KISS1R) gene causes normosmic congenital hypogonadotropic hypogonadism. J. Assist Reprod. Genet. 36, 1273–1280 (2019).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Ameri, A. et al. A nonstop mutation in the factor (F)X gene of a severely haemorrhagic patient with complete absence of coagulation FX. Thromb. Haemost. 98, 1165–1169 (2007).

    CAS  PubMed  Google Scholar 

  21. 21.

    Inoue, K. et al. Translation of SOX10 3′ untranslated region causes a complex severe neurocristopathy by generation of a deleterious functional domain. Hum. Mol. Genet. 16, 3037–3046 (2007).

    CAS  PubMed  Google Scholar 

  22. 22.

    Rebelo, A. P. et al. Cryptic amyloidogenic elements in the 3′ UTRs of neurofilament genes trigger axonal neuropathy. Am. J. Hum. Genet. 98, 597–614 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Diederichs, S. et al. The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding RNA and synonymous mutations. EMBO Mol. Med. 8, 442–457 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Crona, J. et al. Somatic mutations and genetic heterogeneity at the CDKN1B locus in small intestinal neuroendocrine tumors. Ann. Surg. Oncol. 22, S1428–S1435 (2015).

    PubMed  Google Scholar 

  25. 25.

    Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502, 333–339 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Zirn, B., Wittmann, S. & Gessler, M. Novel familial WT1 read-through mutation associated with Wilms tumor and slow progressive nephropathy. Am. J. Kidney Dis. 45, 1100–1104 (2005).

    CAS  PubMed  Google Scholar 

  27. 27.

    Bailey, M. H. et al. Comprehensive characterization of cancer driver genes and mutations. Cell 173, 371–385.e18 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Hata, A., Lo, R. S., Wotton, D., Lagna, G. & Massague, J. Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4. Nature 388, 82–87 (1997).

    CAS  PubMed  Google Scholar 

  29. 29.

    Bardeesy, N. et al. Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer. Genes Dev. 20, 3130–3146 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Forbes, S. A. et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 45, D777–D783 (2017).

    CAS  PubMed  Google Scholar 

  31. 31.

    Biankin, A. V. et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 491, 399–405 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Sharma, Y. et al. A pan-cancer analysis of synonymous mutations. Nat. Commun. 10, 2569 (2019).

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Shihab, H. A. et al. Predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden Markov models. Hum. Mutat. 34, 57–65 (2013).

    CAS  PubMed  Google Scholar 

  34. 34.

    Caudron-Herger, M. & Diederichs, S. Mitochondrial mutations in human cancer: curation of translation. RNA Biol. 15, 62–69 (2018).

    PubMed  Google Scholar 

  35. 35.

    Futreal, P. A. et al. A census of human cancer genes. Nat. Rev. Cancer 4, 177–183 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Ong, C. K. et al. Exome sequencing of liver fluke-associated cholangiocarcinoma. Nat. Genet. 44, 690–693 (2012).

    CAS  PubMed  Google Scholar 

  37. 37.

    Fleming, N. I. et al. SMAD2, SMAD3 and SMAD4 mutations in colorectal cancer. Cancer Res 73, 725–735 (2013).

    CAS  PubMed  Google Scholar 

  38. 38.

    Giannakis, M. et al. RNF43 is frequently mutated in colorectal and endometrial cancers. Nat. Genet. 46, 1264–1266 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Zehir, A. et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat. Med. 23, 703–713 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Giannakis, M. et al. Genomic correlates of immune-cell infiltrates in colorectal carcinoma. Cell Rep. 15, 857–865 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Fullerton, P. T. Jr., Creighton, C. J. & Matzuk, M. M. Insights into SMAD4 loss in pancreatic cancer from inducible restoration of TGF-β signaling. Mol. Endocrinol. 29, 1440–1453 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Watanabe, Y. et al. TMEPAI, a transmembrane TGF-β-inducible protein, sequesters Smad proteins from active participation in TGF-β signaling. Mol. Cell 37, 123–134 (2010).

    CAS  PubMed  Google Scholar 

  43. 43.

    Parada, C., Li, J., Iwata, J., Suzuki, A. & Chai, Y. CTGF mediates Smad-dependent transforming growth factor β signaling to regulate mesenchymal cell proliferation during palate development. Mol. Cell. Biol. 33, 3482–3493 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Koinuma, D. et al. Promoter-wide analysis of Smad4 binding sites in human epithelial cells. Cancer Sci. 100, 2133–2142 (2009).

    CAS  PubMed  Google Scholar 

  45. 45.

    Doronina, V. A. et al. Site-specific release of nascent chains from ribosomes at a sense codon. Mol. Cell. Biol. 28, 4227–4239 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Koren, I. et al. The eukaryotic proteome is shaped by E3 ubiquitin ligases targeting C-terminal degrons. Cell 173, 1622–1635.e14 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Lin, H. C. et al. C-terminal end-directed protein elimination by CRL2 ubiquitin ligases. Mol. Cell 70, 602–613.e3 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Dupont, S. et al. FAM/USP9x, a deubiquitinating enzyme essential for TGFβ signaling, controls Smad4 monoubiquitination. Cell 136, 123–135 (2009).

    CAS  PubMed  Google Scholar 

  49. 49.

    Klampfl, T. et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N. Engl. J. Med. 369, 2379–2390 (2013).

    CAS  PubMed  Google Scholar 

  50. 50.

    Falini, B. et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N. Engl. J. Med. 352, 254–266 (2005).

    CAS  PubMed  Google Scholar 

  51. 51.

    Mair, B. et al. Gain- and loss-of-function mutations in the breast cancer gene GATA3 result in differential drug sensitivity. PLoS Genet. 12, e1006279 (2016).

    PubMed  PubMed Central  Google Scholar 

  52. 52.

    Doma, M. K. & Parker, R. RNA quality control in eukaryotes. Cell 131, 660–668 (2007).

    CAS  PubMed  Google Scholar 

  53. 53.

    Liang, M. et al. Ubiquitination and proteolysis of cancer-derived Smad4 mutants by SCFSkp2. Mol. Cell. Biol. 24, 7524–7537 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Knudson, A. G. Jr Mutation and cancer: statistical study of retinoblastoma. Proc. Natl Acad. Sci. USA 68, 820–823 (1971).

    PubMed  Google Scholar 

  55. 55.

    Berger, A. H., Knudson, A. G. & Pandolfi, P. P. A continuum model for tumour suppression. Nature 476, 163–169 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Inoue, K. & Fry, E. A. Haploinsufficient tumor suppressor genes. Adv. Med. Biol. 118, 83–122 (2017).

    PubMed  PubMed Central  Google Scholar 

  57. 57.

    Izeradjene, K. et al. Kras G12D and Smad4/Dpc4 haploinsufficiency cooperate to induce mucinous cystic neoplasms and invasive adenocarcinoma of the pancreas. Cancer Cell 11, 229–243 (2007).

    CAS  PubMed  Google Scholar 

  58. 58.

    Xu, X. et al. Haploid loss of the tumor suppressor Smad4/Dpc4 initiates gastric polyposis and cancer in mice. Oncogene 19, 1868–1874 (2000).

    CAS  PubMed  Google Scholar 

  59. 59.

    Alberici, P. et al. Smad4 haploinsufficiency in mouse models for intestinal cancer. Oncogene 25, 1841–1851 (2006).

    CAS  PubMed  Google Scholar 

  60. 60.

    Ozawa, H. et al. SMAD4 loss is associated with cetuximab resistance and induction of MAPK/JNK activation in head and neck cancer cells. Clin. Cancer Res. 23, 5162–5175 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Cui, Y. et al. Genetically defined subsets of human pancreatic cancer show unique in vitro chemosensitivity. Clin. Cancer Res. 18, 6519–6530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Afgan, E. et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 46, W537–W544 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Polycarpou-Schwarz, M. et al. The cancer-associated microprotein CASIMO1 controls cell proliferation and interacts with squalene epoxidase modulating lipid droplet formation. Oncogene 37, 4750–4768 (2018).

    CAS  PubMed  Google Scholar 

  64. 64.

    Langlais, C. et al. A systematic approach for testing expression of human full-length proteins in cell-free expression systems. BMC Biotechnol. 7, 64 (2007).

    PubMed  PubMed Central  Google Scholar 

  65. 65.

    Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004).

    CAS  PubMed  Google Scholar 

  66. 66.

    Ulrich, A., Andersen, K. R. & Schwartz, T. U. Exponential megapriming PCR (EMP) cloning—seamless DNA insertion into any target plasmid without sequence constraints. PLoS ONE 7, e53360 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Juszkiewicz, S. & Hegde, R. S. Initiation of quality control during poly(A) translation requires site-specific ribosome ubiquitination. Mol. Cell 65, 743–750.e4 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Richardson, C. D. et al. CRISPR–Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat. Genet. 50, 1132–1139 (2018).

    CAS  PubMed  Google Scholar 

  69. 69.

    Zawel, L. et al. Human Smad3 and Smad4 are sequence-specific transcription activators. Mol. Cell 1, 611–617 (1998).

    CAS  PubMed  Google Scholar 

  70. 70.

    Menon, M. B. et al. Endoplasmic reticulum-associated ubiquitin-conjugating enzyme Ube2j1 is a novel substrate of MK2 (MAPKAP kinase-2) involved in MK2-mediated TNFα production. Biochem. J. 456, 163–172 (2013).

    CAS  PubMed  Google Scholar 

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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.

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Authors

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. Source data

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. Source data

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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