Autocrine CSF-1R activation promotes Src-dependent disruption of mammary epithelial architecture

Carolyn N Wrobel, Jayanta Debnath, Eva Lin, Sean Beausoleil, Martine F Roussel, Joan S Brugge, Carolyn N Wrobel, Jayanta Debnath, Eva Lin, Sean Beausoleil, Martine F Roussel, Joan S Brugge

Abstract

Elevated coexpression of colony-stimulating factor receptor (CSF-1R) and its ligand, CSF-1, correlates with invasiveness and poor prognosis of a variety of epithelial tumors (Kacinski, B.M. 1995. Ann. Med. 27:79-85). Apart from recruitment of macrophages to the tumor site, the mechanisms by which CSF-1 may potentiate invasion are poorly understood. We show that autocrine CSF-1R activation induces hyperproliferation and a profound, progressive disruption of junctional integrity in acinar structures formed by human mammary epithelial cells in three-dimensional culture. Acini coexpressing receptor and ligand exhibit a dramatic relocalization of E-cadherin from the plasma membrane to punctate intracellular vesicles, accompanied by its loss from the Triton-insoluble fraction. Interfering with Src kinase activity, either by pharmacological inhibition or mutation of the Y561 docking site on CSF-1R, prevents E-cadherin translocation, suggesting that CSF-1R disrupts cell adhesion by uncoupling adherens junction complexes from the cytoskeleton and promoting cadherin internalization through a Src-dependent mechanism. These findings provide a mechanistic basis whereby CSF-1R could contribute to invasive progression in epithelial cancers.

Figures

Figure 1.
Figure 1.
Constitutive CSF-1R activation alters MCF-10As monolayer morphology and acinar morphogenesis. (A) MCF-10A cells expressing wild-type or active CSF-1R were lysed in RIPA buffer and probed for receptor levels. Receptor activation was detected with a phospho-specific antibody to the Y723 autophosphorylation site. Cells were starved overnight in assay media, and where noted were treated with 10 ng/ml CSF-1 for 5 min before lysis. (B) Phase-contrast images of MCF-10A cells expressing wild-type or active CSF-1R cultured to confluence in assay media with growth factors noted in parentheses. (C) MCF-10A cells expressing wild-type or active CSF-1R were cultured in MatrigelTM, and assay media with growth factors was replaced every 4 d. Brightfield images of day 5 and day 15 acini are shown. (D) The average size of MCF-10A acini was determined by microscopic area measurements using MetaMorph software (n = 30).
Figure 2.
Figure 2.
Autocrine or chronic activation can mimic the active CSF-1R phenotype. (A) MCF-10As expressing CSF-1R and CSF-1 were cultured in MatrigelTM in assay media. Brightfield images of day 5 and day 19 acini are shown. (B) 3D cultures of cells expressing CSF-1R were stimulated daily with 10 ng/ml CSF-1. Phase-contrast images of day 5 and day 13 acini are shown.
Figure 3.
Figure 3.
CSF-1 coexpression causes hyperproliferation and architectural disruption. MCF-10A cells expressing CSF-1R and vector control (CSF-1R/Vector) or CSF-1 (CSF-1R/CSF-1) were cultured in MatrigelTM in assay media with 10 ng/ml CSF-1 where noted (CSF-1). Confocal images of day 6 and day 17 acinar cultures are shown. Structures were stained with DAPI (blue) along with Ki67 (green) and E-cadherin (red) (A and C) or cleaved caspase-3 (green) and laminin 5 (red) (B and D).
Figure 4.
Figure 4.
Autocrine CSF-1R activation alters wound healing dynamics in MCF-10As. Confluent monolayers of MCF-10As expressing CSF-1R and vector control (CSF-1R/Vector) or CSF-1 (CSF-1R/CSF-1) were starved overnight in assay media. A wound was introduced into each monolayer with a pipette tip, and cells were incubated in assay media with 10 ng/ml CSF-1 where noted (CSF-1) for 48 h. Phase-contrast images of wound clo-sure are representative of four separate experiments.
Figure 5.
Figure 5.
Coexpression of CSF-1 with CSF-1R elicits relocalization of E-cadherin from the membrane to intracellular vesicles. (A) MCF-10As expressing CSF-1R and vector control (CSF-1R/Vector) or CSF-1 (CSF-1R/CSF-1) were fixed for E-cadherin immunofluorescence 8 h after wounding. (B) Confocal images of day 21 acini expressing wild-type or active CSF-1R, stained with DAPI (blue) and E-cadherin (green). (C) Total levels of E-cadherin from RIPA lysates of MCF-10As expressing CSF-1R and vector control (Vector) or CSF-1 (CSF-1). (D) MCF-10As expressing CSF-1R and vector control (lanes 1, 3, 5, and 7) or CSF-1 (lanes 2, 4, 6, and 8) were harvested by treatment with EDTA (1–2 and 5–6) or trypsin (3–4 and 7–8) and placed in suspension for 6 h before lysis in RIPA buffer and E-cadherin immunoblotting.
Figure 6.
Figure 6.
Autocrine CSF-1R activation prevents adhesive compaction. MCF-10As expressing CSF-1R and vector control (CSF-1R/Vector) or CSF-1 (CSF-1R/CSF-1) were placed in suspension in assay media with 10 ng/ml CSF-1 where noted (CSF-1) for 6 h before harvest. Cells were resuspended in 0.5% agarose and spotted onto glass slides for DIC imaging.
Figure 7.
Figure 7.
Assembly of the cadherin–catenin complex is unaltered in CSF-1 coexpressing cells, but association with the cytoskeleton is disrupted. Parental MCF-10As and cells expressing CSF-1R and vector control (CSF-1R/Vector) or CSF-1 (CSF-1R/CSF-1) were grown to confluence in assay media before lysis. Media was supplemented with 5 ng/ml EGF for parental cells and 10 ng/ml CSF-1 for CSF-1R/Vector cells. (A) RIPA lysates were probed for total levels of E-cadherin and catenin proteins. (B) E-cadherin immunoprecipitates from Triton lysates were probed for associated catenins. (C) Levels of E-cadherin in the Triton-soluble (TS) and Triton-insoluble (TI) lysate fractions were assayed. Actin is shown as a control for equal protein loading.
Figure 8.
Figure 8.
The ability of CSF-1R to promote junctional disruption is Src dependent. (A) MCF-10A cells expressing active CSF-1R (right and center), or active CSF-1R including a point mutation at the Src binding site (Y561F, left), were cultured in MatrigelTM. Cultures were treated every 48 h with Src inhibitor SU6656 (2 μm) where noted. Brightfield images of day 15 acini are shown. (B) CSF-1R immunoprecipitates from MCF-10As expressing active CSF-1R or active CSF-1R with the Y561F mutation were probed for Src association with the receptor. (C) MCF-10As expressing CSF-1R and vector control (CSF-1R/Vector) or CSF-1 (CSF-1R/CSF-1), active CSF-1R, or active CSF-1R with the Y561F mutation were starved overnight in assay media. Cells were treated with 10 ng/ml CSF-1 before lysis where noted. Lysates were probed for Src tyrosine 419 phosphorylation and total Src levels. (D) MCF-10As coexpressing CSF-1R and CSF-1 along with vector control or DN-Src (K295R) were cultured in MatrigelTM. Phase-contrast images of day 16 acini are shown. (E) Levels of E-cadherin in the Triton-soluble (TS) and Triton-insoluble (TI) lysate fractions of MCF-10As expressing active CSF-1R or active CSF-1R with the Y561F mutation were assayed. Actin is shown as a control for equal protein loading.
Figure 9.
Figure 9.
Autocrine stimulation of c-Met does not persistently disrupt acinar adhesion despite comparable activation of Src. (A) MCF-10A cells expressing c-Met and vector control (c-Met/Vector) or HGF (c-Met/HGF) were cultured in MatrigelTM in assay media with 40 ng/ml HGF where noted (HGF). Brightfield images of day 5 and day 17 acini are shown. (B) 3D cultures of MCF-10As expressing c-Met and HGF were stained with DAPI (blue) and E-cadherin (green). Confocal image of day 19 structure is shown. (C) MCF-10As expressing CSF-1R and vector control or CSF-1, or c-Met and vector control or HGF were starved overnight in assay media. Cells were treated with 10 ng/ml CSF-1 or 40 ng/ml HGF before lysis where noted. Lysates were probed for Src tyrosine 419 phosphorylation and total Src levels.

References

    1. Adams, C.L., and W.J. Nelson. 1998. Cytomechanics of cadherin-mediated cell-cell adhesion. Curr. Opin. Cell Biol. 10:572–577.
    1. Adams, C.L., Y.T. Chen, S.J. Smith, and W.J. Nelson. 1998. Mechanisms of epithelial cell–cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin–green fluorescent protein. J. Cell Biol. 142:1105–1119.
    1. Alford, D., and J. Taylor-Papadimitriou. 1996. Cell adhesion molecules in the normal and cancerous mammary gland. J. Mammary Gland Biol. Neoplasia. 1:207–218.
    1. Alonso, G., M. Koegl, N. Mazurenko, and S.A. Courtneidge. 1995. Sequence requirements for binding of Src family tyrosine kinases to activated growth factor receptors. J. Biol. Chem. 270:9840–9848.
    1. Barcellos-Hoff, M.H., J. Aggeler, T.G. Ram, and M.J. Bissell. 1989. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development. 105:223–235.
    1. Behrens, J., L. Vakaet, R. Friis, E. Winterhager, F. Van Roy, M.M. Mareel, and W. Birchmeier. 1993. Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/β-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. J. Cell Biol. 120:757–766.
    1. Blake, R.A., P. Garcia-Paramio, P.J. Parker, and S.A. Courtneidge. 1999. Src promotes PKCdelta degradation. Cell Growth Differ. 10:231–241.
    1. Blake, R.A., M.A. Broome, X. Liu, J. Wu, M. Gishizky, L. Sun, and S.A. Courtneidge. 2000. SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling. Mol. Cell. Biol. 20:9018–9027.
    1. Boterberg, T., K.M. Vennekens, M. Thienpont, M.M. Mareel, and M.E. Bracke. 2000. Internalization of the E-cadherin/catenin complex and scattering of human mammary carcinoma cells MCF-7/AZ after treatment with conditioned medium from human skin squamous carcinoma cells COLO 16. Cell Adhes. Commun. 7:299–310.
    1. Braga, V.M., L.M. Machesky, A. Hall, and N.A. Hotchin. 1997. The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell–cell contacts. J. Cell Biol. 137:1421–1431.
    1. Conacci-Sorrell, M., J. Zhurinsky, and A. Ben-Ze'ev. 2002. The cadherin-catenin adhesion system in signaling and cancer. J. Clin. Invest. 109:987–991.
    1. Dai, X.M., G.R. Ryan, A.J. Hapel, M.G. Dominguez, R.G. Russell, S. Kapp, V. Sylvestre, and E.R. Stanley. 2002. Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects. Blood. 99:111–120.
    1. Debnath, J., K.R. Mills, N.L. Collins, M.J. Reginato, S.K. Muthuswamy, and J.S. Brugge. 2002. The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell. 111:29–40.
    1. Debnath, J., S.K. Muthuswamy, and J.S. Brugge. 2003. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods. 30:256–268.
    1. Downing, J.R., and A.B. Reynolds. 1991. PDGF, CSF-1, and EGF induce tyrosine phosphorylation of p120, a pp60src transformation-associated substrate. Oncogene. 6:607–613.
    1. Flick, M.B., E. Sapi, and B.M. Kacinski. 2002. Hormonal regulation of the c-fms proto-oncogene in breast cancer cells is mediated by a composite glucocorticoid response element. J. Cell. Biochem. 85:10–23.
    1. Fujita, Y., G. Krause, M. Scheffner, D. Zechner, H.E. Leddy, J. Behrens, T. Sommer, and W. Birchmeier. 2002. Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat. Cell Biol. 4:222–231.
    1. Herrenknecht, K., M. Ozawa, C. Eckerskorn, F. Lottspeich, M. Lenter, and R. Kemler. 1991. The uvomorulin-anchorage protein alpha catenin is a vinculin homologue. Proc. Natl. Acad. Sci. USA. 88:9156–9160.
    1. Ide, H., D.B. Seligson, S. Memarzadeh, L. Xin, S. Horvath, P. Dubey, M.B. Flick, B.M. Kacinski, A. Palotie, and O.N. Witte. 2002. Expression of colony-stimulating factor 1 receptor during prostate development and prostate cancer progression. Proc. Natl. Acad. Sci. USA. 99:14404–14409.
    1. Iyer, A., T.E. Kmiecik, M. Park, I. Daar, D. Blair, K.J. Dunn, P. Sutrave, J.N. Ihle, M. Bodescot, and G.F. Vande Woude. 1990. Structure, tissue-specific expression, and transforming activity of the mouse met protooncogene. Cell Growth Differ. 1:87–95.
    1. Kacinski, B.M. 1995. CSF-1 and its receptor in ovarian, endometrial and breast cancer. Ann. Med. 27:79–85.
    1. Kacinski, B.M., K.A. Scata, D. Carter, L.D. Yee, E. Sapi, B.L. King, S.K. Chambers, M.A. Jones, M.H. Pirro, E.R. Stanley, and L.R. Rohrschneider. 1991. FMS (CSF-1 receptor) and CSF-1 transcripts and protein are expressed by human breast carcinomas in vivo and in vitro. Oncogene. 6:941–952.
    1. Kowalski, P.J., M.A. Rubin, and C.G. Kleer. 2003. E-cadherin expression in primary carcinomas of the breast and its distant metastases. Breast Cancer Res. 5:R217–R222.
    1. Langer, S.J., D.M. Bortner, M.F. Roussel, C.J. Sherr, and M.C. Ostrowski. 1992. Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression. Mol. Cell. Biol. 12:5355–5362.
    1. Le, T.L., A.S. Yap, and J.L. Stow. 1999. Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics. J. Cell Biol. 146:219–232.
    1. Le, T.L., S.R. Joseph, A.S. Yap, and J.L. Stow. 2002. Protein kinase C regulates endocytosis and recycling of E-cadherin. Am. J. Physiol. Cell Physiol. 283:C489–C499.
    1. Lipsich, L.A., A.J. Lewis, and J.S. Brugge. 1983. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J. Virol. 48:352–360.
    1. Marks, D.C., X.F. Csar, N.J. Wilson, U. Novak, A.C. Ward, V. Kanagasundarum, B.W. Hoffmann, and J.A. Hamilton. 1999. Expression of a Y559F mutant CSF-1 receptor in M1 myeloid cells: a role for Src kinases in CSF-1 receptor-mediated differentiation. Mol. Cell Biol. Res. Commun. 1:144–152.
    1. McCrea, P.D., C.W. Turck, and B. Gumbiner. 1991. A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin. Science. 254:1359–1361.
    1. Mukhopadhyay, D., L. Tsiokas, X.M. Zhou, D. Foster, J.S. Brugge, and V.P. Sukhatme. 1995. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature. 375:577–581.
    1. Muthuswamy, S.K., D. Li, S. Lelievre, M.J. Bissell, and J.S. Brugge. 2001. ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Nat. Cell Biol. 3:785–792.
    1. Niemann, C., V. Brinkmann, E. Spitzer, G. Hartmann, M. Sachs, H. Naundorf, and W. Birchmeier. 1998. Reconstitution of mammary gland development in vitro: requirement of c-met and c-erbB2 signaling for branching and alveolar morphogenesis. J. Cell Biol. 143:533–545.
    1. Nollet, F., G. Berx, and F. van Roy. 1999. The role of the E-cadherin/catenin adhesion complex in the development and progression of cancer. Mol. Cell Biol. Res. Commun. 2:77–85.
    1. Owens, D.W., G.W. McLean, A.W. Wyke, C. Paraskeva, E.K. Parkinson, M.C. Frame, and V.G. Brunton. 2000. The catalytic activity of the Src family kinases is required to disrupt cadherin-dependent cell-cell contacts. Mol. Biol. Cell. 11:51–64.
    1. Palacios, F., L. Price, J. Schweitzer, J.G. Collard, and C. D'Souza-Schorey. 2001. An essential role for ARF6-regulated membrane traffic in adherens junction turnover and epithelial cell migration. EMBO J. 20:4973–4986.
    1. Pollack, A.L., R.B. Runyan, and K.E. Mostov. 1998. Morphogenetic mechanisms of epithelial tubulogenesis: MDCK cell polarity is transiently rearranged without loss of cell-cell contact during scatter factor/hepatocyte growth factor-induced tubulogenesis. Dev. Biol. 204:64–79.
    1. Pollard, J.W., and L. Hennighausen. 1994. Colony stimulating factor 1 is required for mammary gland development during pregnancy. Proc. Natl. Acad. Sci. USA. 91:9312–9316.
    1. Price, F.V., S.K. Chambers, J.T. Chambers, M.L. Carcangiu, P.E. Schwartz, E.I. Kohorn, E.R. Stanley, and B.M. Kacinski. 1993. Colony-stimulating factor-1 in primary ascites of ovarian cancer is a significant predictor of survival. Am. J. Obstet. Gynecol. 168:520–527.
    1. Pujuguet, P., L. Del Maestro, A. Gautreau, D. Louvard, and M. Arpin. 2003. Ezrin regulates E-cadherin–dependent adherens junction assembly through Rac1 activation. Mol. Biol. Cell. 14:2181–2191.
    1. Reynolds, A.B., D.J. Roesel, S.B. Kanner, and J.T. Parsons. 1989. 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.
    1. Reynolds, A.B., J. Daniel, P.D. McCrea, M.J. Wheelock, J. Wu, and Z. Zhang. 1994. Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes. Mol. Cell. Biol. 14:8333–8342.
    1. Roura, S., S. Miravet, J. Piedra, A. Garcia de Herreros, and M. Dunach. 1999. Regulation of E-cadherin/catenin association by tyrosine phosphorylation. J. Biol. Chem. 274:36734–36740.
    1. Roussel, M.F. 1998. Key effectors of signal transduction and G1 progression. Adv. Cancer Res. 74:1–24.
    1. Roussel, M.F., T.J. Dull, C.W. Rettenmier, P. Ralph, A. Ullrich, and C.J. Sherr. 1987. Transforming potential of the c-fms proto-oncogene (CSF-1 receptor). Nature. 325:549–552.
    1. Roussel, M.F., J.R. Downing, C.W. Rettenmier, and C.J. Sherr. 1988. A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene product) activates its transforming potential. Cell. 55:979–988.
    1. Sapi, E., M.B. Flick, S. Rodov, M. Gilmore-Hebert, M. Kelley, S. Rockwell, and B.M. Kacinski. 1996. Independent regulation of invasion and anchorage-independent growth by different autophosphorylation sites of the macrophage colony-stimulating factor 1 receptor. Cancer Res. 56:5704–5712.
    1. Sapi, E., M.B. Flick, S. Rodov, D. Carter, and B.M. Kacinski. 1998. a. Expression of CSF-I and CSF-I receptor by normal lactating mammary epithelial cells. J. Soc. Gynecol. Investig. 5:94–101.
    1. Sapi, E., M.B. Flick, S. Rodov, and B.M. Kacinski. 1998. b. Ets-2 transdominant mutant abolishes anchorage-independent growth and macrophage colony-stimulating factor-stimulated invasion by BT20 breast carcinoma cells. Cancer Res. 58:1027–1033.
    1. Schmeichel, K.L., V.M. Weaver, and M.J. Bissell. 1998. Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial cell phenotype. J. Mammary Gland Biol. Neoplasia. 3:201–213.
    1. Swift, S., J. Lorens, P. Achacoso, and G.P. Nolan. 1999. Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell-based systems. In Current Protocols in Immunology. Unit 10.28 (Suppl. 31).
    1. Takeda, H., A. Nagafuchi, S. Yonemura, S. Tsukita, J. Behrens, and W. Birchmeier. 1995. V-src kinase shifts the cadherin-based cell adhesion from the strong to the weak state and beta catenin is not required for the shift. J. Cell Biol. 131:1839–1847.
    1. Van Nguyen, A., and J.W. Pollard. 2002. Colony stimulating factor-1 is required to recruit macrophages into the mammary gland to facilitate mammary ductal outgrowth. Dev. Biol. 247:11–25.
    1. Warren, S.L., and W.J. Nelson. 1987. Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures. Mol. Cell. Biol. 7:1326–1337.
    1. Watabe, M., A. Nagafuchi, S. Tsukita, and M. Takeichi. 1994. Induction of polarized cell–cell association and retardation of growth by activation of the E-cadherin–catenin adhesion system in a dispersed carcinoma line. J. Cell Biol. 127:247–256.
    1. Wilhelmsen, K., S. Burkhalter, and P. van der Geer. 2002. C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor's carboxy-terminus. Oncogene. 21:1079–1089.
    1. Zhang, Q., J. Calafat, H. Janssen, and S. Greenberg. 1999. ARF6 is required for growth factor- and rac-mediated membrane ruffling in macrophages at a stage distal to rac membrane targeting. Mol. Cell. Biol. 19:8158–8168.

Source: PubMed

Sponsorer og samarbejdspartnere

Medicinske tilstande

Narkotikainterventioner

3
Abonner