Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Deregulation of CRAD-controlled cytoskeleton initiates mucinous colorectal cancer via β-catenin

Nature Cell Biology volume 20, pages 1303–1314 (2018)Cite this article

Subjects

Abstract

Epithelial integrity is maintained by the cytoskeleton and through cell adhesion. However, it is not yet known how a deregulated cytoskeleton is associated with cancer. We identified cancer-related regulator of actin dynamics (CRAD) as frequently mutated or transcriptionally downregulated in colorectal cancer. We found that CRAD stabilizes the cadherin–catenin–actin complex via capping protein inhibition. The loss of CRAD inhibits F-actin polymerization and subsequently disrupts the cadherin–catenin–actin complex, which leads to β-catenin release and Wnt signalling hyperactivation. In mice, CRAD knockout induces epithelial cell integrity loss and Wnt signalling activation, resulting in the development of intestinal mucinous adenoma. With APC mutation, CRAD knockout initiates and accelerates mucinous and invasive adenoma development in the colorectum. These results define CRAD as a tumour suppressor, the inactivation of which deregulates the cytoskeleton and hyperactivates Wnt signalling thus initiating mucinous colorectal cancer. Our study reveals the unexpected roles of an actin cytoskeletal regulator in maintaining epithelial cell integrity and suppressing tumorigenesis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: CRAD inactivation in CRC.
Fig. 2: Positive regulation of the actin polymerization by CRAD-inhibited CPs.
Fig. 3: Loss of CRAD-activated Wnt signalling through disruption of the CCA complex.
Fig. 4: Inhibition of CRC cell proliferation by CRAD.
Fig. 5: Intestinal adenoma development induced by CRAD KO.
Fig. 6: Accelerated intestinal tumorigenesis as a result of CRAD heterogeneous KO.
Fig. 7: Mucinous intestinal tumorigenesis by CRAD KO.
Fig. 8: Increased mucin deposition by CRAD KO.

Similar content being viewed by others

Data availability

Microarray data that support the findings of this study have been deposited in GEO (accession code GDS2947). Mass spectrometry data used in this study is available from Supplementary Table 2. The CRAD expression data in CRC cells were derived from the cBioportal using the TCGA Research Network (http://cancergenome.nih.gov/) and Genetech datesets. The dataset derived from this resource that supports the findings of this study is available in Oncomine (https://www.oncomine.org/resource). CRAD expression data were also derived from cBioportal (http://www.cbioportal.org/) and COSMIC database (https://cancer.sanger.ac.uk/cosmic). Source data for Figs. 1–8 and Supplementary Figs. 1–8 have been provided as Supplementary Table 4. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

References

  1. Mege, R. M., Gavard, J. & Lambert, M. Regulation of cell-cell junctions by the cytoskeleton. Curr. Opin. Cell Biol. 18, 541–548 (2006).

    CAS  PubMed  Google Scholar 

  2. Gumbiner, B. M. & Kim, N. G. The Hippo-YAP signaling pathway and contact inhibition of growth. J. Cell Sci. 127, 709–717 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Oda, H. & Takeichi, M. Evolution: structural and functional diversity of cadherin at the adherens junction. J. Cell Biol. 193, 1137–1146 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Maiden, S. L. & Hardin, J. The secret life of alpha-catenin: moonlighting in morphogenesis. J. Cell Biol. 195, 543–552 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Bianchini, J. M. et al. Reevaluating αE-catenin monomer and homodimer functions by characterizing E-cadherin/αE-catenin chimeras. J. Cell Biol. 210, 1065–1074 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Edwards, M. et al. Capping protein regulators fine-tune actin assembly dynamics. Nat. Rev. Mol. Cell Biol. 15, 677–689 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Bear, J. E. et al. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109, 509–521 (2002).

    CAS  PubMed  Google Scholar 

  8. Xue, B. & Robinson, R. C. Guardians of the actin monomer. Eur. J. Cell Biol. 92, 316–332 (2013).

    CAS  PubMed  Google Scholar 

  9. Schafer, D. A., Jennings, P. B. & Cooper, J. A. Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides. J. Cell Biol. 135, 169–179 (1996).

    CAS  PubMed  Google Scholar 

  10. Taoka, M. et al. V-1, a protein expressed transiently during murine cerebellar development, regulates actin polymerization via interaction with capping protein. J. Biol. Chem. 278, 5864–5870 (2003).

    CAS  PubMed  Google Scholar 

  11. George, J. et al. Comprehensive genomic profiles of small cell lung cancer. Nature 524, 47–53 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Ambros, V. R., Chen, L. B. & Buchanan, J. M. Surface ruffles as markers for studies of cell transformation by Rous sarcoma virus. Proc. Natl Acad. Sci. USA 72, 3144–3148 (1975).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. de Curtis, I. & Meldolesi, J. Cell surface dynamics - how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton. J. Cell Sci. 125, 4435–4444 (2012).

    PubMed  Google Scholar 

  14. Gauthier, N. C., Masters, T. A. & Sheetz, M. P. Mechanical feedback between membrane tension and dynamics. Trends Cell Biol. 22, 527–535 (2012).

    CAS  PubMed  Google Scholar 

  15. Pollard, T. D. & Cooper, J. A. Actin, a central player in cell shape and movement. Science 326, 1208–1212 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Bryce, N. S. et al. Cortactin promotes cell motility by enhancing lamellipodial persistence. Curr. Biol. 15, 1276–1285 (2005).

    CAS  PubMed  Google Scholar 

  17. Montross, W. T., Ji, H. & McCrea, P. D. A beta-catenin/engrailed chimera selectively suppresses Wnt signaling. J. Cell Sci. 113, 1759–1770 (2000).

    CAS  PubMed  Google Scholar 

  18. Kwiatkowski, A. V. et al. In vitro and in vivo reconstitution of the cadherin–catenin–actin complex from Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 107, 14591–14596 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Maiden, S. L. et al. Specific conserved C-terminal amino acids of Caenorhabditis elegans HMP-1/α-catenin modulate F-actin binding independently of vinculin. J. Biol. Chem. 288, 5694–5706 (2013).

    CAS  PubMed  Google Scholar 

  20. Hong, S., Troyanovsky, R. B. & Troyanovsky, S. M. Binding to F-actin guides cadherin cluster assembly, stability, and movement. J. Cell Biol. 201, 131–143 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Braga, V. M. & Yap, A. S. The challenges of abundance: epithelial junctions and small GTPase signalling. Curr. Opin. Cell Biol. 17, 466–474 (2005).

    CAS  PubMed  Google Scholar 

  22. Tanaka, Y. et al. Frequent β-catenin mutation and cytoplasmic/nuclear accumulation in pancreatic solid-pseudopapillary neoplasm. Cancer Res. 61, 8401–8404 (2001).

    CAS  PubMed  Google Scholar 

  23. Peterson, L. W. & Artis, D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat. Rev. Immunol. 14, 141–153 (2014).

    CAS  PubMed  Google Scholar 

  24. van Es, J. H. et al. Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nat. Cell Biol. 7, 381–386 (2005).

    PubMed  Google Scholar 

  25. Moser, A. R., Pitot, H. C. & Dove, W. F. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247, 322–324 (1990).

    CAS  PubMed  Google Scholar 

  26. Bara, J., Forgue-Lafitte, M. E., Maurin, N., Flejou, J. F. & Zimber, A. Abnormal expression of gastric mucin in human and rat aberrant crypt foci during colon carcinogenesis. Tumour Biol. 24, 109–115 (2003).

    CAS  PubMed  Google Scholar 

  27. Sachs, N. & Clevers, H. Organoid cultures for the analysis of cancer phenotypes. Curr. Opin. Genet. Dev. 24, 68–73 (2014).

    CAS  PubMed  Google Scholar 

  28. Podolsky, D. K., Fournier, D. A. & Lynch, K. E. Human colonic goblet cells. Demonstration of distinct subpopulations defined by mucin-specific monoclonal antibodies. J. Clin. Invest. 77, 1263–1271 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Yamachika, T. et al. Establishment and characterization of a human colonic mucinous carcinoma cell line with predominant goblet-cell differentiation from liver metastasis. Pathol. Int. 55, 550–557 (2005).

    CAS  PubMed  Google Scholar 

  30. van Es, J. H. et al. Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435, 959–963 (2005).

    PubMed  Google Scholar 

  31. Hugen, N., Brown, G., Glynne-Jones, R., de Wilt, J. H. & Nagtegaal, I. D. Advances in the care of patients with mucinous colorectal cancer. Nat. Rev. Clin. Oncol. 13, 361–369 (2016).

    CAS  PubMed  Google Scholar 

  32. Brabletz, T. et al. Nuclear overexpression of the oncoprotein beta-catenin in colorectal cancer is localized predominantly at the invasion front. Pathol. Res. Pract. 194, 701–704 (1998).

    CAS  PubMed  Google Scholar 

  33. Vermeulen, L. et al. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat. Cell Biol. 12, 468–476 (2010).

    CAS  PubMed  Google Scholar 

  34. Goentoro, L. & Kirschner, M. W. Evidence that fold-change, and not absolute level, of beta-catenin dictates Wnt signaling. Mol. Cell 36, 872–884 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Voloshanenko, O. et al. Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells. Nat. Commun. 4, 2610 (2013).

    PubMed  Google Scholar 

  36. Hulsken, J., Birchmeier, W. & Behrens, J. E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton. J. Cell Biol. 127, 2061–2069 (1994).

    CAS  PubMed  Google Scholar 

  37. Fagotto, F., Funayama, N., Gluck, U. & Gumbiner, B. M. Binding to cadherins antagonizes the signaling activity of beta-catenin during axis formation in Xenopus. J. Cell Biol. 132, 1105–1114 (1996).

    CAS  PubMed  Google Scholar 

  38. Brannon, A. R. et al. Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions. Genome Biol. 15, 454 (2014).

    PubMed  PubMed Central  Google Scholar 

  39. Okada, K. et al. Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1. J. Cell Biol. 189, 1087–1096 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Kufe, D. W. Mucins in cancer: function, prognosis and therapy. Nat. Rev. Cancer 9, 874–885 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Edgar, R., Domrachev, M. & Lash, A. E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Forbes, S. A. et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 39, D945–D950 (2011).

    CAS  PubMed  Google Scholar 

  43. Gao, J. et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, pl1 (2013).

    PubMed  PubMed Central  Google Scholar 

  44. Rhodes, D. R. et al. ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia 6, 1–6 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Cerami, E. et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 (2012).

    PubMed  Google Scholar 

  46. Jung, H. Y. et al. PAF and EZH2 induce Wnt/β-catenin signaling hyperactivation. Mol. Cell 52, 193–205 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Jun, S. et al. PAF-mediated MAPK signaling hyperactivation via LAMTOR3 induces pancreatic tumorigenesis. Cell Rep. 5, 314–322 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    CAS  PubMed  Google Scholar 

  49. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    CAS  PubMed  Google Scholar 

  50. Symons, M. H. & Mitchison, T. J. Control of actin polymerization in live and permeabilized fibroblasts. J. Cell Biol. 114, 503–513 (1991).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank J.A. Cooper for his insightful comments and recommendations regarding the experiment and result analysis. We thank S.-H. Lee, H.N. Suh, S.H. Lee and K.-S. Park for helpful comments on the manuscript. This work was supported by the Cancer Prevention Research Institute of Texas (grant no. RP140563 to J-.I.P.), the National Institutes of Health (grant nos R01 CA193297-01 (to J-.I.P.), 5R01 GM107079 (to P.D.M.) and R01 GM126048 (to W.W.)), the Department of Defense Peer Reviewed Cancer Research Program (grant no. CA140572 to J-.I.P.), a Duncan Family Institute for Cancer Prevention and Risk Assessment Grant (IRG-08-061-01; to J-.I.P.), a Center for Stem Cell and Developmental Biology Transformative Grant (MD Anderson Cancer Center to J-.I.P.), an Institutional Research Grant (MD Anderson Cancer Center; to J-.I.P.), a New Faculty Award (MD Anderson Cancer Center Support Grant; to J-.I.P.), a Metastasis Research Center Grant (MD Anderson; to J-.I.P.) and a Uterine SPORE Career Enhancement Program (MD Anderson; to J-.I.P.). The core facility (DNA sequencing and Genetically Engineered Mouse Facility) was supported by the MD Anderson Cancer Center Support Grant (CA016672).

Author information

Authors and Affiliations

  1. Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Youn-Sang Jung, Sohee Jun, Jie Zhang, Mrinal Srivastava, Moon Jong Kim, Esther M. Lien, Joan Shang, Junjie Chen & Jae-Il Park

  2. Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA

    Wenqi Wang

  3. Program in Genetics and Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Junjie Chen, Pierre D. McCrea & Jae-Il Park

  4. Graduate School of Biomedical Sciences at Houston, The University of Texas Health Science Center and MD Anderson Cancer Center, Houston, TX, USA

    Junjie Chen, Pierre D. McCrea & Jae-Il Park

  5. Department of Molecular Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Pierre D. McCrea

  6. Department of Pathology, The University of Texas McGovern Medical School, Houston, TX, USA

    Songlin Zhang

Authors
  1. Youn-Sang Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wenqi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sohee Jun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mrinal Srivastava
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Moon Jong Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Esther M. Lien
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Joan Shang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Junjie Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Pierre D. McCrea
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Songlin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jae-Il Park
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.-S.J. and J.-I.P. conceived the experiments. Y.-S.J., W.W., S.J., J.Z., M.S., M.J.K., E.M.L., J.S. and J.-I.P. performed the experiments. Y.-S.J., J.C., P.D.M., S.Z. and J.-I.P. analysed the data. Y.-S.J. and J.-I.P. wrote the manuscript.

Corresponding author

Correspondence to Jae-Il Park.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Genetic inactivation of CRAD in cancer.

(a), Genetic inactivation of CRAD in CRC. cBioportal database analysis of CRAD in CRC. CRAD shows 10.18 % incidence rate of truncated mutants (Nonsense + Frameshift) in CRC. APC, DCC, CTNNB1, and AXIN2 were analyzed as the positive control. HPRP1 served as negative control. Total 502 CRC cases were analyzed. (b), Genetic alteration of CRAD in human cancer. cBioportal analysis. (c), Potent epigenetic suppression of CRAD promoter. Upregulated CRAD expression by the inhibition of methylation. IECs and CRC cells were treated with 5-Azacytidine (5-AZA; 20 μM) for 24 hr. n = 3 independent experiments; Error bars: average +/− S.D.; NS: not significant (P > 0.05); Two-sided unpaired t-test. (d), COSMIC analysis of CRAD mutations in CRC cell lines.

Supplementary Figure 2 Positive regulation of the actin polymerization by CRAD-induced capping protein inhibition.

(a and b), Morphological comparison of IECs and CRC cells. IF staining (a). Quantification of the cell area of IECs and CRC cells by AxioVision software (b; n = 5 independent experiments). (c and d), Depletion of endogenous CRAD by shRNAs. FHC cells were transfected with shRNAs (c). shCRAD#1 stably expressing IECs were analyzed for IB (d). IB was performed once. (e-g), Cell shrinkage by CRAD depletion. 48 hr after transfection, cells were analyzed for IF staining (e) and cell area quantification (f; n = 5 (independent experiments). Cells were infected with lenti-shCRAD for 48 hr. After infection, cells were selected by puromycin (2 μg/ml) treatment for 72 hr. The cell morphology was analyzed by IF staining for Phalloidin (g). (h-k), Extended cell morphology by CRAD in CRC cells. 48 h after transfection (Vec [empty vector] or FLAG-CRAD), cells were analysed for IF staining (actin, h; Phalloidin, k) and cell area quantification (i; n = 5 independent experiments). The cell morphology was monitored using blight field microscope (j). (l), Physical interaction of CRAD with CPs. Purified FLAG-Actin, FLAG-CPs, and HA-CRAD proteins were used for co-IP. These results recapitulate in vitro interaction between CRAD and CPs performed using GST recombinant proteins. Experiment was performed three times with similar results. (m-o), Decreased F-actin formation by CRAD depletion. Quantification of the F-actin in the CRAD depleted (m), or the CRAD ectopic expressed condition (n and o). The level of F-actin and G-actin was examined by immunoblots (n) and quantified by the subsequent ImageJ analysis (m and o; n = 3 independent experiments). After measurement, F-actin was normalized by G-actin. (p), Coomassie Brilliant Blue (CBB) staining of the purified recombinant proteins. Each recombinant protein was expressed in E. coli and purified, followed by GST cleavage. Experiment was performed once. (q), Inhibition of interaction between CAPZs and actin by CRAD. Co-IP assay using purified recombinant proteins (see Fig. 2m) was quantified by ImageJ. n = 3 independent experiments). (r and s). No effect of A511V-CRAD on Wnt/β-catenin signaling. AXIN2 qRT-PCR (r; n = independent experiments) and IB (s) using WT-CRAD or A511V-CRAD transfected CRC cells. IB was performed three times with similar results. Representative images are shown; Scale bars indicate 20 μm; Error bars: mean +/− S.D.; NS: not significant (P > 0.05); Two-sided unpaired t-test.

Supplementary Figure 3 Illustration of the working model of CRAD-induced F-actin polymerization.

CRAD inhibits CPs’ binding to the barbed (+) end, resulting in the increased actin polymerization.

Supplementary Figure 4 CRC cell growth inhibition by CRAD.

(a), The mutually exclusive expression between CRAD and AXIN2 in IECs and CRC. Oncomine analysis of CRAD and AXIN2 expression in CRC. (b), Inversed correlation between CRAD and β-catenin. IECs and CRC cells were fractionated into the nucleus and cytosol fractions using fractionation kit. WCL: whole cell lysates. (c and d), Decreased nuclear β-catenin by CRAD. IECs (c) and CRC cells (d) were transfected with shCtrl or shCRAD and Vec or CRAD, respectively. After 48 hr, cells were fractionated into the cytosolic and nucleus fractions, followed by IB. (e and f), Increased interaction between E-Cadherin and β-catenin by CRAD. Cell extracts from IEC (e) and CRC cell (f) were used for IP analysis. Binding between E-Cadherin and β-catenin was quantified by ImageJ. n = 3. (g), Increased interaction between E-cadherin and catenins by CRAD. HCT116 cells were transiently transfected with FLAG-CRAD plasmid and analyzed for IF staining using Super Resolution microscope (see Fig. 3p). E-cadherin intensity was quantified by ZEN software (Zeiss). n = 10. (h and i), Blockade of the actin cytoskeleton increases Wnt/β-catenin reporter activity. CCD-841CoN cells were transfected with β-catenin reporter plasmids (pMEGA-TOP/FOP-FLASH). 24 hr after, cells were treated with Cytochalasin D (Cyto D; 2 μM; barbed [+] end inhibition)51, Latrunculin B (Lat B; 1 μM; monomeric G-actin inhibition)52, and Jasplakinolide (2 μM; a stabilizer of actin cytoskeleton) for 72 hr. β-catenin transcription activity was measured by TOP/FOP luciferase activity (h). CCD-841CoN cells were treated with indicated reagents as same concentration of (h). 72 h after treatment, cells were collected for qRT-PCR (i). n = 3 independent experiments. j, Illustration of the working model: the molecular mechanism of CRAD loss-induced hyperactivation of Wnt/β-catenin signaling. In normal epithelial cells, CRAD inhibits CPs, which results in actin polymerization. Subsequently, increased F-actin stabilizes complex formation composed of E-cadherin-catenins-F-actin. In the absence of CRAD, F-actin is disrupted by CPs, which leads to destabilization of E-cadherin-catenin complex. Disturbance of E-cadherin-catenin complex releases β-catenin into the cytosols and the nucleus, which transactivates Wnt/β-catenin target genes. Error bars: average +/− S.D.; Two-sided unpaired t-test; Centre: Average.

Supplementary Figure 5 CRC cell growth inhibition by CRAD.

(a and b), Suppression of shCRAD-induced cell hyperproliferation by β-catenin inhibition in IECs. FHC (shCtrl and shCRAD) cells were treated with iCRT14 for 14 days and stained with crystal violet (a). Cell proliferation was quantified by measurement of absorbance at 590 nm (b; n = 3 independent experiments). (c-e), Engrailed-LEF1 inhibits CRAD depletion-induced IEC hyperproliferation. 1,000 cells of CCD-841CoN were seeded onto tissue culture plate. Cells were transfected with indicated plasmids. Eng-LEF1 was used as a dominant-negative mutant blocker of β-catenin-mediated gene transactivation53,54. 48 h after transfection, cells were incubated in 37 °C tissue culture CO2 chamber for 14 days. Colony forming was visualized by crystal violet staining (c) and quantified (d and e; n = 3 independent experiments). (f-h), CRC cell growth inhibition by CRAD expression. CRC cells (Vec [control] and CRAD expressing) were analyzed for cell counting (f; n = 3 independent experiments), crystal violet staining (g), and quantification (h). (i-o), β-catenin rescues CRAD-induced CRC cell growth inhibition. CRC cells were transfected with CRAD or-catenin plasmids and analyzed for crystal violet staining (i,k,m), quantification (j, l, n), and cell counting (o). (p and q), CRC cell growth inhibition by CPI motif-containing CRAD mutants. CRAD (full length, ΔCPI, and M1-M4)-transfected CRC cells were analysed for quantification of cell proliferation. HCT116 (p); SW620 cells (q). Representative images are shown; Error bars: mean +/− S.D.; NS: not significant (P > 0.05); Two-sided unpaired t-test.

Supplementary Figure 6 Intestinal adenoma development by CRAD KO.

(a), CRISPR/Cas9-mediated targeting of CRAD alleles. Exon2 of CRAD was targeted using gRNAs. gRNA and Cas9 mRNA were injected into the pronuclei of C57BL/B6 mouse embryos. (b), CRAD expression in the small intestine. IHC of mouse intestine. (c), Validation of CRAD KO. The cell lysates were extracted from the small intestine of six independent CRAD KO mice for IB. (d), Sequencing analysis of potential off-target genes of CRAD gRNAs. No mutations in 14 off-target genes were detected. (e), Increased Wnt signaling target genes in CRAD KO mouse. CRAD KO-induced tumors were analyzed for qRT-PCR of Hippo, Notch, Shh, BMP, and Wnt signaling pathway target genes. n = 3 independent mice. f,g, Tumor development in the lung and pancreas of CRAD KO mice. Development of the early lesion of SCLC. The multiple sites displaying the early lesion of SCLC (black arrows; f) and the pancreatic tumors (g; i and ii) were observed. Scale bars indicate 100 μm (f) or 20 μm (g). (h), Disruption of epithelial cell integrity. Cytokeratin 19 (CK19). Arrows: Villi not expressing CK19. (i and j), Analysis of inflammation in CRAD KO mouse. Small intestine samples of WT and CRAD KO mouse were stained with H&E (i; n = 8 mice). Intestinal inflammation (foci size > 200 μm) was assessed by counting (j). Scale bars indicate 200 μm. (k-n), Cell hyperproliferation in CRAD KO small intestine. Phospho-histone H3 (k) and Ki67 (n) staining and quantification (l and m; n = 10 crypts). Scale bars indicate 20 μm. o, Analysis of apoptosis in CRAD KO-induced tumor lesion. WT and CRAD KO mice were analyzed for cleaved Caspase-3 (Casp3). Scale bars indicate 20 μm. (p), Abnormal differentiation of IECs by CRAD KO. WT and CRAD KO small intestine were immunostained with ChgA (arrows). Scale bars indicate 20 μm. (q), Disorganized cell adhesion in CRAD KO mice. Cells were stained with E-cadherin. Scale bars indicate 20 μm. (r), Increased β-catenin target genes in the intestinal adenoma of CRAD KO mice. IHC for CD44. Scale bars indicate 20 μm. Representative images of three independent experiments; Error bars: mean +/− S.D.; NS: not significant (P > 0.05); Two-sided unpaired t-test.

Supplementary Figure 7 Accelerated intestinal tumorigenesis by CRAD heterogeneous KO.

(a), Heterozygous mutation of CRAD gene in CRC. Zygosity analysis of CRAD mutation in CRC patient samples using COSMIC and cBioportal databases. Of note, the frequency of heterozygous mutation is higher than that of homozygous mutation. (b-e), IHC of the non-tumor region of the colorectum of APCMIN and APCMIN:CRAD+/− mice (4mo of age). Cyclin D1 (b); Ki67 (d). Quantification (c; n = 9 crypts, (e); n = 10 crypts). (f), Micro-invasion by CRAD KO. Micro-invasion was observed in the tumor region of APCMIN:CRAD+/− small intestine. The small intestine samples were stained with αSMA (α-Smooth Muscle Actin: green) to visualize the basement membrane. The basement membrane of APCMIN:CRAD+/− is disrupted/discontinued tumor cell infiltration. Arrow indicates invasive tumor cell and yellow dashed lines mark the border of the basement membrane. (g), No EMT in tumour region of APCMIN:CRAD+/− mice. EMT marker analysis in APCMIN and APCMIN:CRAD+/− small intestine samples. Markers of mesenchymal cell (Vimentin: green; N-cadherin: red) were not detected in tumors of both strains. Representative images of three independent experiments; Scale bars indicate 20 μm; Error bars: mean +/− S.D.; Two-sided unpaired t-test.

Supplementary Figure 8 Accelerated intestinal tumorigenesis by CRAD KO.

(a and b), IHC analysis of the organoids derived from CRAD WT and KO mouse intestine. Compared to WT, CRAD KO-derived cystic spheroids showed the increased cell proliferation (Ki67; a) and the increase of β-catenin (b). Quantification was performed using 10 cystic spheroids. n = 10 organoids from three different experiments. (c), Increased Mucin expression in CRAD KO. The small intestine samples from CRAD WT (2mo) and CRAD KO mice (from different ages as indicated) were examined by qRT-PCR for mMUC1, mMUC2, mMUC4, and mMUC5AC. n = 3 independent experiments. (d), Increased goblet cell in CRAD KO mice. After fixation and paraffin embedding, each sample was stained with PAS and quantified. n = 10 villi. (e), Increased mucin deposition in CRAD KO-induced tumors. qRT-PCR of MUC1 and MUC2 from the small intestine samples of each mice. n = 3 independent experiments. (f), The increase of TOP-1 expression in CRAD KO mouse. Tumor of APCMIN (4mo) and CRAD KO (3, 4, 6, 8, and 12mo) were immunostained with a TOP-1 antibody. (g), Upregulation of TOP-1 in CRAD KO tumors. CRAD WT (2mo) intestine and CRAD KO tumors (from different age were examined by qRT-PCR of mTOP-1. n = 3 independent experiments. (h), Mutual exclusive expression of CRAD and MUCs. Oncomine analysis of TCGA datasets; 10% gene rank; P < 0.0001; fold change >2; compared with normal cells. Representative images of three independent experiments; Scale bars indicate 20 μm; Error bars: mean +/− S.D.; NS: not significant (P > 0.05); Two-sided unpaired t-test.

Supplementary Figure 9 Unprocessed blots.

Figures 1e-3j Fig. 3n-S6c.

Supplementary information

Supplementary Information

Supplementary Figures 1–9, Supplementary Table legends and Supplementary References.

Reporting Summary

Supplementary Table 1

Analysis of CRC tumour microarray for CRAD expression.

Supplementary Table 2

CRAD-interacting proteins identified by tandem affinity purification and mass spectrometry.

Supplementary Table 3

Information of MC microarray for CRAD expression.

Supplementary Table 4

Primer information.

Supplementary Table 5

Antibody information.

Supplementary Table 6

Statistics Source Data.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jung, YS., Wang, W., Jun, S. et al. Deregulation of CRAD-controlled cytoskeleton initiates mucinous colorectal cancer via β-catenin. Nat Cell Biol 20, 1303–1314 (2018). https://doi.org/10.1038/s41556-018-0215-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-018-0215-z

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer