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.

Distinct E-cadherin-based complexes regulate cell behaviour through miRNA processing or Src and p120 catenin activity

Nature Cell Biology volume 17, pages 1145–1157 (2015)Cite this article

Subjects

Abstract

E-cadherin and p120 catenin (p120) are essential for epithelial homeostasis, but can also exert pro-tumorigenic activities. Here, we resolve this apparent paradox by identifying two spatially and functionally distinct junctional complexes in non-transformed polarized epithelial cells: one growth suppressing at the apical zonula adherens (ZA), defined by the p120 partner PLEKHA7 and a non-nuclear subset of the core microprocessor components DROSHA and DGCR8, and one growth promoting at basolateral areas of cell–cell contact containing tyrosine-phosphorylated p120 and active Src. Recruitment of DROSHA and DGCR8 to the ZA is PLEKHA7 dependent. The PLEKHA7–microprocessor complex co-precipitates with primary microRNAs (pri-miRNAs) and possesses pri-miRNA processing activity. PLEKHA7 regulates the levels of select miRNAs, in particular processing of miR-30b, to suppress expression of cell transforming markers promoted by the basolateral complex, including SNAI1, MYC and CCND1. Our work identifies a mechanism through which adhesion complexes regulate cellular behaviour and reveals their surprising association with the microprocessor.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Polarized epithelial cells show distinct p120-associated populations at the junctions.
Figure 2: Biochemical separation of two distinct junctional complexes by proteomics.
Figure 3: PLEKHA7 suppresses anchorage-independent growth and expression of transformation-related markers.
Figure 4: The basolateral junctional complex promotes anchorage-independent growth and expression of transformation-related markers.
Figure 5: PLEKHA7 suppresses protein expression through miRNAs.
Figure 6: PLEKHA7 associates with DROSHA and DGCR8 at the ZA.
Figure 7: Localization of DROSHA and DGCR8 at the ZA is PLEKHA7 dependent.
Figure 8: PLEKHA7 regulates pri-miR-30b processing at the junctions.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Reynolds, A. B., Roesel, D. J., Kanner, S. B. & Parsons, J. T. Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene. Mol. Cell. Biol. 9, 629–638 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Ireton, R. C. et al. A novel role for p120 catenin in E-cadherin function. J. Cell Biol. 159, 465–476 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Ishiyama, N. et al. Dynamic and static interactions between p120 catenin and E-cadherin regulate the stability of cell–cell adhesion. Cell 141, 117–128 (2010).

    CAS  PubMed  Google Scholar 

  4. Yap, A. S., Niessen, C. M. & Gumbiner, B. M. The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn. J. Cell Biol. 141, 779–789 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Davis, M. A., Ireton, R. C. & Reynolds, A. B. A core function for p120-catenin in cadherin turnover. J. Cell Biol. 163, 525–534 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Anastasiadis, P. Z. p120-ctn: A nexus for contextual signaling via Rho GTPases. Biochim. Biophys. Acta 1773, 34–46 (2007).

    CAS  PubMed  Google Scholar 

  7. Wildenberg, G. A. et al. p120-catenin and p190RhoGAP regulate cell–cell adhesion by coordinating antagonism between Rac and Rho. Cell 127, 1027–1039 (2006).

    CAS  PubMed  Google Scholar 

  8. Smith, A. L., Dohn, M. R., Brown, M. V. & Reynolds, A. B. Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin. Mol. Biol. Cell 23, 99–110 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Schackmann, R. C. et al. Cytosolic p120-catenin regulates growth of metastatic lobular carcinoma through Rock1-mediated anoikis resistance. J. Clin. Invest. 121, 3176–3188 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Stairs, D. B. et al. Deletion of p120-catenin results in a tumor microenvironment with inflammation and cancer that establishes it as a tumor suppressor gene. Cancer Cell 19, 470–483 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Mariner, D. J., Davis, M. A. & Reynolds, A. B. EGFR signaling to p120-catenin through phosphorylation at Y228. J. Cell Sci. 117, 1339–1350 (2004).

    CAS  PubMed  Google Scholar 

  12. Johnson, E. et al. HER2/ErbB2-induced breast cancer cell migration and invasion require p120 catenin activation of Rac1 and Cdc42. J. Biol. Chem. 285, 29491–29501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Dohn, M. R., Brown, M. V. & Reynolds, A. B. An essential role for p120-catenin in Src- and Rac1-mediated anchorage-independent cell growth. J. Cell Biol. 184, 437–450 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Yanagisawa, M. & Anastasiadis, P. Z. p120 catenin is essential for mesenchymal cadherin-mediated regulation of cell motility and invasiveness. J. Cell Biol. 174, 1087–1096 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Soto, E. et al. p120 catenin induces opposing effects on tumor cell growth depending on E-cadherin expression. J. Cell Biol. 183, 737–749 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Silvera, D. et al. Essential role for eIF4GI overexpression in the pathogenesis of inflammatory breast cancer. Nat. Cell Biol. 11, 903–908 (2009).

    CAS  PubMed  Google Scholar 

  17. Liu, W. F., Nelson, C. M., Pirone, D. M. & Chen, C. S. E-cadherin engagement stimulates proliferation via Rac1. J. Cell Biol. 173, 431–441 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lewis-Tuffin, L. J. et al. Misregulated E-cadherin expression associated with an aggressive brain tumor phenotype. PLoS ONE 5, e13665 (2010).

    PubMed  PubMed Central  Google Scholar 

  19. Rodriguez, F. J., Lewis-Tuffin, L. J. & Anastasiadis, P. Z. E-cadherin’s dark side: Possible role in tumor progression. Biochim. Biophys. Acta 1826, 23–31 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kuphal, S. & Bosserhoff, A. K. E-cadherin cell–cell communication in melanogenesis and during development of malignant melanoma. Arch. Biochem. Biophys. 524, 43–47 (2012).

    CAS  PubMed  Google Scholar 

  21. Meng, W., Mushika, Y., Ichii, T. & Takeichi, M. Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell–cell contacts. Cell 135, 948–959 (2008).

    CAS  PubMed  Google Scholar 

  22. Pulimeno, P., Bauer, C., Stutz, J. & Citi, S. PLEKHA7 is an adherens junction protein with a tissue distribution and subcellular localization distinct from ZO-1 and E-cadherin. PLoS ONE 5, e12207 (2010).

    PubMed  PubMed Central  Google Scholar 

  23. Miyoshi, J. & Takai, Y. Structural and functional associations of apical junctions with cytoskeleton. Biochim. Biophys. Acta 1778, 670–691 (2008).

    CAS  PubMed  Google Scholar 

  24. Smutny, M. et al. Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens. Nat. Cell Biol. 12, 696–702 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Mariner, D. J. et al. Identification of Src phosphorylation sites in the catenin p120ctn. J. Biol. Chem. 276, 28006-28013 (2001).

    Google Scholar 

  26. Ma, L. W., Zhou, Z. T., He, Q. B. & Jiang, W. W. Phosphorylated p120-catenin expression has predictive value for oral cancer progression. J. Clin. Pathol. 65, 315–319 (2012).

    PubMed  Google Scholar 

  27. Kourtidis, A., Ngok, S. P. & Anastasiadis, P. Z. p120 catenin: an essential regulator of cadherin stability, adhesion-induced signaling, and cancer progression. Prog. Mol. Biol. Transl. Sci. 116, 409–432 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Tikhmyanova, N. & Golemis, E. A. NEDD9 and BCAR1 negatively regulate E-cadherin membrane localization, and promote E-cadherin degradation. PLoS ONE 6, e22102 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Chang, J. H., Gill, S., Settleman, J. & Parsons, S. J. c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation. J. Cell Biol. 130, 355–368 (1995).

    CAS  PubMed  Google Scholar 

  30. Wang, Y. et al. Synergistic effect of cyclin D1 and c-Myc leads to more aggressive and invasive mammary tumors in severe combined immunodeficient mice. Cancer Res. 67, 3698–3707 (2007).

    CAS  PubMed  Google Scholar 

  31. Eiseler, T. et al. Protein kinase D1 mediates anchorage-dependent and -independent growth of tumor cells via the zinc finger transcription factor Snail1. J. Biol. Chem. 287, 32367–32380 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Thoreson, M. A. et al. Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion. J. Cell Biol. 148, 189-202 (2000).

    PubMed  PubMed Central  Google Scholar 

  33. Ozawa, M. & Ohkubo, T. Tyrosine phosphorylation of p120(ctn) in v-Src transfected L cells depends on its association with E-cadherin and reduces adhesion activity. J. Cell Sci. 114, 503–512 (2001).

    CAS  PubMed  Google Scholar 

  34. Zhou, B. P. et al. Dual regulation of Snail by GSK-3β-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat. Cell Biol. 6, 931–940 (2004).

    CAS  PubMed  Google Scholar 

  35. Yook, J. I. et al. A Wnt-Axin2-GSK3β cascade regulates Snail1 activity in breast cancer cells. Nat. Cell Biol. 8, 1398-1406 (2006).

    Google Scholar 

  36. Fabian, M. R., Sonenberg, N. & Filipowicz, W. Regulation of mRNA translation and stability by microRNAs. Annu. Rev. Biochem. 79, 351–379 (2010).

    CAS  PubMed  Google Scholar 

  37. Krol, J., Loedige, I. & Filipowicz, W. The widespread regulation of microRNA biogenesis, function and decay. Nat. Rev. Genet. 11, 597–610 (2010).

    CAS  PubMed  Google Scholar 

  38. Meijer, H. A. et al. Translational repression and eIF4A2 activity are critical for microRNA-mediated gene regulation. Science 340, 82–85 (2013).

    CAS  PubMed  Google Scholar 

  39. Pillai, R. S. et al. Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309, 1573–1576 (2005).

    CAS  PubMed  Google Scholar 

  40. Croce, C. M. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genet. 10, 704–714 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Adams, B. D., Kasinski, A. L. & Slack, F. J. Aberrant regulation and function of MicroRNAs in cancer. Curr. Biol. 24, R762-R776 (2014).

    PubMed Central  Google Scholar 

  42. Joglekar, M. V. et al. The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells. Islets 1, 137–147 (2009).

    PubMed  Google Scholar 

  43. Watanabe, S. et al. HMGA2 maintains oncogenic RAS-induced epithelial-mesenchymal transition in human pancreatic cancer cells. Am. J. Pathol. 174, 854–868 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Zhang, J. et al. miR-30 inhibits TGF-β1-induced epithelial-to-mesenchymal transition in hepatocyte by targeting Snail1. Biochem. Biophys. Res. Commun. 417, 1100–1105 (2012).

    CAS  PubMed  Google Scholar 

  45. Buechner, J. et al. Tumour-suppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma. Br. J. Cancer 105, 296–303 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Lan, F. F. et al. Hsa-let-7g inhibits proliferation of hepatocellular carcinoma cells by downregulation of c-Myc and upregulation of p16(INK4A). Int. J. Cancer 128, 319–331 (2011).

    CAS  PubMed  Google Scholar 

  47. Lal, A. et al. miR-24 Inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to ”seedless” 3′UTR microRNA recognition elements. Mol. Cell 35, 610–625 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Ha, M. & Kim, V. N. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell. Biol. 15, 509–524 (2014).

    CAS  PubMed  Google Scholar 

  49. Gregory, R. I. et al. The microprocessor complex mediates the genesis of microRNAs. Nature 432, 235–240 (2004).

    CAS  PubMed  Google Scholar 

  50. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003).

    CAS  PubMed  Google Scholar 

  51. Han, J. et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. He, L. et al. A microRNA polycistron as a potential human oncogene. Nature 435, 828–833 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Olive, V. et al. miR-19 is a key oncogenic component of mir-17-92. Genes Dev. 23, 2839–2849 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Pulimeno, P., Paschoud, S. & Citi, S. A role for ZO-1 and PLEKHA7 in recruiting paracingulin to tight and adherens junctions of epithelial cells. J. Biol. Chem. 286, 16743–16750 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Kurita, S., Yamada, T., Rikitsu, E., Ikeda, W. & Takai, Y. Binding between the junctional proteins afadin and PLEKHA7 and implication in the formation of adherens junction in epithelial cells. J. Biol. Chem. 288, 29356–29368 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Paschoud, S., Jond, L., Guerrera, D. & Citi, S. PLEKHA7 modulates epithelial tight junction barrier function. Tissue Barriers 2, e28755 (2014).

    PubMed  PubMed Central  Google Scholar 

  57. Smith, A. L., Friedman, D. B., Yu, H., Carnahan, R. H. & Reynolds, A. B. ReCLIP (reversible cross-link immuno-precipitation): an efficient method for interrogation of labile protein complexes. PLoS ONE 6, e16206 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Smoot, M. E., Ono, K., Ruscheinski, J., Wang, P. L. & Ideker, T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 27, 431–432 (2011).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH R01 CA100467, R01 NS069753, P50 CA116201 (P.Z.A.); NIH R01 GM086435, Florida Department of Health, Bankhead-Coley 10BG11 (P.S.); NIH/NCI R01CA104505, R01CA136665 (J.A.C.); BCRF (E.A.P.); the Swiss Cancer League (S.C., Project KLS-2878-02-2012). A.K. is supported by the Jay and Deanie Stein Career Development Award for Cancer Research at Mayo Clinic. We thank Mayo Clinic’s Proteomics Core and B. Madden for assistance with mass spectrometry, B. Edenfield for immunohistochemistry, M. Takeichi, D. Radisky and M. Cichon for constructs, and D. Radisky, L. Lewis-Tuffin, J. C. Dachsel, B. Necela and the late G. Hayes for suggestions and comments.

Author information

Author notes

  1. Siu P. Ngok & Pamela Pulimeno

    Present address: Present addresses: Division of Hematology, Oncology, Stem Cell Transplantation and Cancer Biology, Department of Pediatrics, Stanford School of Medicine, Stanford, California 94305-5457, USA (S.P.N.); Department of Genetics and Complex Diseases, Harvard T. Chan School of Public Health, 665, Huntington Avenue, Boston, Massachusetts 02115, USA (P.P.).,

Authors and Affiliations

  1. Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, 4500 San Pablo Road, Jacksonville, Florida 32224, USA

    Antonis Kourtidis, Siu P. Ngok, Ryan W. Feathers, Lomeli R. Carpio, Tiffany R. Baker, Jennifer M. Carr, Irene K. Yan, Sahra Borges, Peter Storz, John A. Copland, Tushar Patel, E. Aubrey Thompson & Panos Z. Anastasiadis

  2. Department of Molecular Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211, Geneva 4, Switzerland

    Pamela Pulimeno

  3. Division of Hematology/Oncology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, Florida 32224, USA

    Edith A. Perez

  4. Department of Cell Biology and Institute of Genetics and Genomics of Geneva, University of Geneva, 30 quai Ernest-Ansermet, CH-1211, Geneva 4, Switzerland

    Sandra Citi

Authors
  1. Antonis Kourtidis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Siu P. Ngok
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pamela Pulimeno
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ryan W. Feathers
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lomeli R. Carpio
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tiffany R. Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jennifer M. Carr
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Irene K. Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sahra Borges
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Edith A. Perez
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Peter Storz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. John A. Copland
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Tushar Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. E. Aubrey Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Sandra Citi
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Panos Z. Anastasiadis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.K. designed the study, conceived and designed experiments, carried out all experiments except those described below, analysed the data and wrote the manuscript. S.P.N. made constructs. P.P. and S.C. developed antibodies and constructs. R.W.F. provided technical support. L.R.C. assisted with IF. T.R.B., J.M.C. and E.A.T. carried out the NanoString experiment and assisted with qRT–PCRs. I.K.Y. and T.P. assisted with the ISH assay. E.A.P., P.S., S.B. and J.A.C. developed and provided tissue micro-arrays. P.Z.A. conceived and designed the study, conceived and designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Panos Z. Anastasiadis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Distinct complexes exist at the junctions of epithelial cells.

Caco2 or PLEKHA7-GFP transfected MDCK cells were grown to polarize, subjected to IF for PLEKHA7 or GFP respectively and co-stained for (a) p120; (b,c) E-cadherin (Ecad); (d) Afadin, Actin (phalloidin), Myosin IIA; (e) phosphorylated p120: Y228; (f) phosphorylated p120: Y96; (g) phosphorylated p120: T310; (h,i) Rac1; (j) RhoA. Stained cells were imaged by confocal microscopy and image stacks were acquired, as in Fig. 1. Representative x–y image stacks are shown and/or the merged composite x–z images. Scale bars for x–y images: 20 μM; for x–z images: 5 μM.

Supplementary Figure 2 PLEKHA7 localization to the junctions is E-cadherin- and p120-dependent.

(a) Western blot of the lysates from the separated apical and basolateral fractions shown in Fig. 2 for E-cadherin (Ecad), α-catenin, and β-catenin. (b) Caco2 control (NT) or E-cadherin knockdown (shEcad) cells, stained by IF for E-cadherin and PLEKHA7. (c) p120 and PLEKHA7 IF stainings of Caco2 control (NT) cells, p120 knockdown (shp120) cells, and of p120 knockdown cells transfected either with the full length murine mp120-1A isoform (shp120 + 1A) or the murine mp120-4A isoform that lacks the N-terminal PLEKHA7-binding domain (shp120 + 4A). All scale bars: 20 μM.

Supplementary Figure 3 PLEKHA7 loss from the junctions results in increased anchorage-independent growth and related signalling.

(a) Demonstration of the PLEKHA7 mRNA knockdown by qRT–PCR after infection of Caco2 cells with two PLEKHA7 shRNAs (shPLEKHA7 no. 8 and no. 10) or non-target control shRNA (NT) (mean ± s.d. from n = 3 independent experiments; ∗P < 0.0001, Student’s two-tailed t-test). Source data are provided in Supplementary Table 2. (b) Caco2 control (NT) and PLEKHA7 shRNA knockdown (shPLEKHA7) cells were stained and imaged for PLEKHA7 and p120. (c) Caco2 control (NT) or PLEKHA7 knockdown cells (shPLEKHA7 #8, #10) grown on soft agar and imaged for colony formation (images are in 2× magnification; see Fig. 3c for quantitation). (d) MDCK cells were infected with either control (NT) or PLEKHA7 shRNA (shPLEKHA7#8) and subjected to western blot for the markers shown. Phosphorylation sites are denoted by p-. Actin is the loading control. (e) Soft agar assay of Caco2 cells infected with either vector control (adGFP) or a SNAI1-expressing construct (adSNAI1) (images are in 2× magnification; see Fig. 3h for quantitation). (f) Western blot of control (NT) or NEZHA knockdown (shNEZHA#72 and #73 shRNAs) Caco2 cells for the markers shown; Actin is the loading control. (g) IF of control (NT) or NEZHA knockdown (shNEZHA) Caco2 cells for PLEKHA7 and NEZHA. (h) IF of Caco2 cells transfected with either wild type murine mp120-1A (1A) or the murine mp120-ΔARM1 (ΔARM1) construct that cannot bind E-cadherin, stained with the murine-specific p120 antibody 8D11 (mp120) and co-stained with PLEKHA7. (i) Western blot of pcDNA (vector control), mp120-ΔARM1, or mp120-1A transfected cells, for the markers shown; Actin is the loading control. Single and double stars on the p120 blot indicate the 1A and ΔARM1 bands, respectively, right above the endogenous p120 bands. Scale bars for x–y images: 20 μM; for x–z images: 2.5 μM; for panels c and e: 2 mm.

Supplementary Figure 4 PLEKHA7 is mis-localized or lost in breast and renal tumour tissues.

Representative immunohistochemistry images of (a) breast and (b) kidney (renal), normal and cancer tissues stained for PLEKHA7, p120 and E-cadherin (Ecad) (left panels) and the percentage of tissues that exhibit presence, absence, or mis-localization of the three markers examined (right panels). BC: breast cancer; RCC: renal cell carcinoma. Scale bars: 20 μM. Number of tissues examined per cancer type/stage; BC TMA: Benign, n = 8; DCIS, n = 12; ILC ER+ Her-, n = 10; IDC ER+, Her-, n = 16; IDC Her+, n = 16; IDC Triple Negative, n = 13; RCC TMA: Matched normal, n = 119; stage 1, n = 71; stage 2, n = 20; stage 3, n = 22; stage 4, n = 6. (c) Caco2 control (NT), PLEKHA7 knockdown (shPLEKHA7), and PLEKHA7, p120 (shPLEKHA7, shp120) double knockdown cells were grown on soft agar for colony formation assay (images are shown in 2× magnification; see Fig. 4a for quantitation) and (d) examined by western blot for E-cadherin (Ecad) levels; α-tubulin is the loading control. (e) Caco2 cells treated with either vehicle (DMSO) or the Src inhibitor PP2 (10 μM) were stained for p120 and phosphorylated p120: Y228. Scale bars 20 μM; for panel c: 2 mm.

Supplementary Figure 5 PLEKHA7 acts via miRNAs but not post-translational modification mechanisms.

(a) qRT–PCR analysis for the indicated mRNAs of Caco2 control (NT) or PLEKHA7 knockdown cells (shPLEKHA7) (mean ± s.d. from n = 3 independent experiments). (b) Western blot for the markers shown (Actin: loading control) of control (NT) or PLEKHA7 knockdown (shPLEKHA7) Caco2 cells treated with 20 μg ml−1 cycloheximide (CHX) or (c) 10 μM MG-132, for the indicated time points. (d) Western blot of control (NT) or PLEKHA7 knockdown (shPLEKHA7) Caco2 cells for the markers shown; Actin is the loading control. (e,f) miR-30b and miR-19a qRT–PCR analysis of PLEKHA7-knockdown (shPLEKHA7) Caco2 cells after ectopic re-expression of PLEKHA7 (LZRS-PLEKHA7) (mean ± s.d. from n = 3 independent experiments; ∗P < 0.05, Student’s two-tailed t-test) (g) Caco2 cells transfected with the indicated anti-miRs (a-miR) were subjected to western blot for the markers shown. (h) Caco2 control (NT) and PLEKHA7 knockdown (shPLEKHA7) cells were transfected with either control or miR-30b mimic and blotted for the markers shown. (i) Caco2 cells infected with either vector control (adGFP) or a SNAI1-expressing construct (adSNAI1) were subjected to western blot for the markers shown; Actin is the loading control. Source data for panels a,e,f are provided in Supplementary Table 2.

Supplementary Figure 6 The microprocessor complex localizes at the ZA.

(a) Northern blot analysis of control (NT) or PLEKHA7 knockdown (shPLEKHA7) Caco2 cells using a miR-30b probe, indicating the pre-miR-30b and the mature miR-30b. U6 is the loading control. (b,c) IF of polarized Caco2 cells for p120 and DROSHA or DGCR8. Antibodies used here: DROSHA: Sigma and DGCR8: Sigma. The indication ∗2nd ab refers to the use here of a different antibody for DROSHA and DGCR8, compared to the antibodies used in Fig. 6d, e (for antibody details, see Supplementary Table 3). Enlarged details in boxes are shown on top of apical fields. The x–z composite image of panel b is shown in Fig. 6f and of c in Fig. 6g. (d,e) IF of polarized MDCK cells for p120 and DROSHA (antibody: Abcam) or DGCR8 (antibody: Abcam). (f,g) siRNA-mediated knockdown of DROSHA (siDROSHA) and DGCR8 (siDGCR8) in Caco2 cells, shown both by IF (left side of each panel), and western blot (right side of each panel). Non-target (NT) siRNA is the control. p120 was used as a co-stain for the IF; Actin is the loading control for the blots; molecular weights (kDa) are indicated on the left side of each blot. (h) Western blots of PLEKHA7, p120, DROSHA, and DGCR8 IPs for PLEKHA7, p120 and E-cadherin (Ecad). IgG is the negative IP control. Scale bars: 20 μM; for enlarged parts of panels b and c: 3 μM.

Supplementary Figure 7 Regulation of the microprocessor at the ZA is PLEKHA7-depended.

(a–c) IF of Caco2 cells transfected with either the wild type murine mp120-1A (1A) construct or the murine mp120-ΔARM1 (ΔARM1) construct that cannot bind E-cadherin, both stained with the murine-specific p120 antibody 8D11 (mp120) and co-stained with PLEKHA7, DROSHA, or DGCR8. Arrows indicate affected junctional staining of the indicated markers in transfected cells. (d) IF of control (NT) or p120 knockdown (shp120) Caco2 cells for DROSHA and p120. (e) qRT–PCR of control (NT) and p120 knockdown (shp120) Caco2 cells for the miRNAs shown (mean ± s.d. from n = 3 independent experiments; ∗P < 0.05, Student’s two-tailed t-test). (f) IF of control (DMSO) or Nocodazole (10 μM for 8 h) treated Caco2 cells for PLEKHA7, DROSHA, or DGCR8. (g,h) IF of control (NT) or NEZHA knockdown (shNEZHA) Caco2 cells for DROSHA and DGCR8, co-stained with NEZHA. (i) qRT–PCR of control (NT), PLEKHA7 knockdown (shPLEKHA7), PLEKHA7 + p120 (shPLEKHA7 + shp120), and PLEKHA7 + Cadherin-11 (shPLEKHA7 + shCad11) double knockdown Caco2 cells for the indicated miRNAs (individual data points and mean from n = 2 independent experiments are shown). (j,k) IF of control (NT), PLEKHA7 knockdown (shPLEKHA7), and PLEKHA7 + p120 double knockdown (shPLEKHA7 + shp120) Caco2 cells for DROSHA and DGCR8, co-stained with p120. (l,m) IF of control (NT), PLEKHA7 knockdown (shPLEKHA7), and PLEKHA7 + Cadherin-11 double knockdown (shPLEKHA7 + Cad11) Caco2 cells for DROSHA and DGCR8, co-stained with Cadherin 11 (Cad11). Non-specific cytoplasmic or nuclear background appears respectively for the two Cadherin-11 antibodies used in the IF (see Methods for antibody details). All scale bars: 20 μM. Source data for panels e and i are provided in Supplementary Table 2.

Supplementary information

Supplementary Information

Supplementary Information (PDF 4579 kb)

Supplementary Information

Supplementary Table 1 (XLSX 49 kb)

Supplementary Information

Supplementary Table 2 (XLSX 2376 kb)

Supplementary Information

Supplementary Table 3 (XLSX 42 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kourtidis, A., Ngok, S., Pulimeno, P. et al. Distinct E-cadherin-based complexes regulate cell behaviour through miRNA processing or Src and p120 catenin activity. Nat Cell Biol 17, 1145–1157 (2015). https://doi.org/10.1038/ncb3227

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3227

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing