Abstract
SPARC (secreted protein acidic and rich in cysteine)/BM40/Osteonectin is a matricellular protein with multiple effects on cell behaviour. In vitro, its major known functions are anti-adhesive and anti-proliferative, and it is associated with tissue remodelling and cancer in vivo. SPARC is overexpressed in many cancers, including breast cancer, and the effects of SPARC seem to be cell type-specific. To study the effects of SPARC on breast cancer, we transfected SPARC into the MDA-MB-231 BAG, human breast cancer cell line using the Tet-On inducible system. By western analysis, we found low background levels in the MDA-MB-231 BAG and clone X parental cells, and prominent induction of SPARC protein expression after doxycycline treatment in SPARC transfected clones X5, X21, X24 and X75. Induction of SPARC expression did not affect cell morphology or adhesiveness to collagens type I and IV, but it slowed the rate of proliferation in adherent cultures. Cell cycle analysis showed that SPARC slowed the progression to S phase. Doxycycline induction of SPARC also slowed the rate of monolayer wound closure in the cultured wound healing assay. Thymidine inhibition of proliferation abrogated this effect, confirming that it was due to anti-proliferation rather than inhibition of migration. Consistent with this, we were unable to detect any differences in migration and Matrigel outgrowth analysis of doxycycline-stimulated cells. We conclude that SPARC is inhibitory to human breast cancer cell proliferation, and does not stimulate migration, in contrast to its stimulatory effects reported for melanoma (proliferation and migration) and glioma (migration) cells. Similar growth repression by SPARC has been reported for ovarian cancer cells, and this may be a common feature among carcinomas.
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References
Mann K, Deutzmann R, Paulsson M, Timpl R: Solubilization of protein BM-40 from a basement membrane tumor with chelating agents and evidence for its identity with osteonectin and SPARC. FEBS Lett 218(1): 167–172, 1987
Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML, Martin GR: Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26(1 Pt 1): 99–105, 1981
Yan Q, Sage EH: SPARC, a matricellular glycoprotein with important biological functions. J Histochem Cytochem 47(12): 1495–1506, 1999
Brekken RA, Sage EH: SPARC, a matricellular protein: at the crossroads of cell-matrix. Matrix Biol 19(7): 569–580, 2000
Sage H, Vernon RB, Funk SE, Everitt EA, Angello J: SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca +2-dependent binding to the extracellular matrix. J Cell Biol 109(1): 341–356, 1989
Funk SE, Sage EH: The Ca 2 +)-binding glycoprotein SPARC modulates cell cycle progression in bovine aortic endothelial cells. Proc Natl Acad Sci USA 88(7): 2648–2652, 1991
Ledda MF, Adris S, Bravo AI, Kairiyama C, Bover L, Chernajovsky Y, Mordoh J, Podhajcer OL: Suppression of SPARC expression by antisense RNA abrogates the tumorigenicity of human melanoma cells. Nat Med 3(2): 171–176, 1997
Jacob K, Webber M, Benayahu D, Kleinman HK: Osteonectin promotes prostate cancer cell migration and invasion: a possible mechanism for metastasis to bone. Cancer Res 59(17): 4453–4457, 1999
Mok SC, Chan WY, Wong KK, Muto MG, Berkowitz RS: SPARC, an extracellular matrix protein with tumor-suppressing activity in human ovarian epithelial cells. Oncogene 12(9): 1895–1901, 1996
Mason IJ, Taylor A, Williams JG, Sage H, Hogan BL: Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell 'culture shock' glycoprotein of Mr 43,000. EMBO J 5(7): 1465–1472, 1986
Vial E, Castellazzi M: Down-regulation of the extracellular matrix protein SPARC in vSrc-and vJun-transformed chick embryo fibroblasts contributes to tumor formation in vivo. Oncogene 19(14): 1772–1782, 2000
Reed MJ, Puolakkainen P, Lane TF, Dickerson D, Bornstein P, Sage EH: Differential expression of SPARC and thrombospondin 1 in wound repair: immunolocalization and in situ hybridization. J Histochem Cytochem 41(10): 1467–1477, 1993.
Cho WJ, Kim EJ, Lee SJ, Kim HD, Shin HJ, Lim WK: Involvement of SPARC in in vitro differentiation of skeletal myoblasts. Biochem Biophys Res Commun 271(3): 630–634, 2000
Gilmour DT, Lyon GJ, Carlton MB, Sanes JR, Cunningham JM, Anderson JR, Hogan BL, Evans MJ, Colledge WH: Mice deficient for the secreted glycoprotein SPARC/osteonectin/ BM40 develop normally but show severe age-onset cataract formation and disruption of the lens. Embo J 17(7): 1860–1870, 1998
Bradshaw AD, Francki A, Motamed K, Howe C, Sage EH: Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation. Mol Biol Cell 10(5): 1569–1579, 1999
Kato Y, Sakai N, Baba M, Kaneko S, Kondo K, Kubota Y, Yao M, Shuin T, Saito S, Koshika S, Kawase T, Miyagi Y, Aoki I, Nagashima Y: Stimulation of motility of human renal cell carcinoma by SPARC/Osteonectin/BM-40 associated with type IV collagen. Invasion Metastasis 18(2): 105–114, 1998
Gilles C, Bassuk JA, Pulyaeva H, Sage EH, Foidart JM, Thompson EW: SPARC/Osteonectin induces MMP-2-activation in human breast cancer cell lines. Cancer Res 58: 5529–5536, 1998
Gilles C, Thompson EW: The epithelial to mesenchymal transition and metastatic progression in carcinoma. Breast J 2: 83–96, 1996
Brunner N, Thompson EW, Spang-Thomsen M, Rygaard J, Dano K, Zwiebel JA: LacZ transduced human breast cancer xenografts as an in vivo model for the study of invasion and metastasis. Eur J Cancer 28A(12): 1989–1995, 1992
Price JT, Tiganis T, Agarwal A, Djakiew D, Thompson EW: Epidermal growth factor promotes MDA-MB-231 breast cancer cell migration through a phosphatidylinositol 3-kinase and phospholipase C-dependent mechanism. Cancer Res 59(21): 5475–5478, 1999
Nischt R, Pottgiesser J, Krieg T, Mayer U, Aumailley M, Timpl R: Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40. Eur J Biochem 200(2): 529–536, 1991
Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H: Transcriptional activation by tetracyclines in mammalian cells. Science 268(5218): 1766–1769, 1995
Resnitzky D, Gossen M, Bujard H, Reed SI: Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol 14(3): 1669–1679, 1994
Young MF, Day AA, Dominquez P, McQuillan CI, Fisher LW, Termine JD: Structure and expression of osteonectin mRNA in human tissue. Connect Tissue Res 24(1): 17–28, 1990
Miller DL, el-Ashry D, Cheville AL, Liu Y, McLeskey SW, Kern FG: Emergence of MCF-7 cells overexpressing a transfected epidermal growth factor receptor (EGFR) under estrogen-depleted conditions: evidence for a role of EGFR in breast cancer growth and progression. Cell Growth Differ 5(12): 1263–1274, 1994
Price JT, Thompson EW: Models for studying cellular invasion of basement membranes. Meth Mol Biol 129: 231–249, 1999
Golembieski WA, Ge S, Nelson K, Mikkelsen T, Rempel SA: Increased SPARC expression promotes U87 glioblastoma invasion in vitro. Int J Dev Neurosci 17(5–6): 463–472, 1999
Porter PL, Sage EH, Lane TF, Funk SE, Gown AM: Distribution of SPARC in normal and neoplastic human tissue. J Histochem Cytochem 43(8): 791–800, 1995
Stenner DD, Tracy RP, Riggs BL, Mann KG: Human platelets contain and secrete osteonectin, a major protein of mineralized bone. Proc Natl Acad Sci USA 83(18): 6892–6896, 1986
Frizell E, Liu SL, Abraham A, Ozaki I, Eghbali M, Sage EH, Zern MA: Expression of SPARC in normal and fibrotic livers. Hepatology 21(3): 847–854, 1995
Maillard C, Malaval L, Delmas PD: Immunological screening of SPARC/Osteonectin in nonmineralized tissues. Bone 13(3): 257–264, 1992
Ledda F, Bravo AI, Adris S, Bover L, Mordoh J, Podhajcer OL: The expression of the secreted protein acidic and rich in cysteine (SPARC) is associated with the neoplastic progression of human melanoma. J Invest Dermatol 108(2): 210–214, 1997
Rempel SA, Ge S and Gutierrez JA, SPARC: a potential diagnostic marker of invasive meningiomas. Clin Cancer Res 5(2): 237–241, 1999
Rempel SA, Golembieski WA, Ge S, Lemke N, Elisevich K, Mikkelsen T, Gutierrez JA: SPARC: a signal of astrocytic neo-plastic transformation and reactive response in human primary and xenograft gliomas. J Neuropathol Exp Neurol 57(12): 1112–1121, 1998
Porte H, Triboulet JP, Kotelevets L, Carrat F, Prevot S, Nord-linger B, DiGioia Y, Wurtz A, Comoglio P, Gespach C, Chastre E: Overexpression of stromelysin-3, BM-40/SPARC, and MET genes in human esophageal carcinoma: implications for prognosis. Clin Cancer Res 4(6): 1375–1382, 1998
Le Bail B, Faouzi S, Boussarie L, Guirouilh J, Blanc JF, Carles J, Bioulac-Sage P, Balabaud C, Rosenbaum J: Osteonectin/ SPARC is overexpressed in human hepatocellular carcinoma. J Pathol 189(1): 46–52, 1999
Thomas R, True LD, Bassuk JA, Lange PH, Vessella RL: Differential expression of osteonectin/SPARC during human prostate cancer progression. Clin Cancer Res 6(3): 1140–1149, 2000
Bellahcene A, Castronovo V: Increased expression of osteonectin and osteopontin, two bone matrix proteins, in human breast cancer. Am J Pathol 146(1): 95–100, 1995
Podhajcer OL, Wolf C, Lefebvre O, Segain JP, Rouyer N, Stoll I, Rio MC, Chambon P, Basset P: Comparative expression of the SPARC and stromelysin-3 genes in mammary tumours. Breast 5: 13–20, 1996
Sommers CL, Byers SW, Thompson EW, Torri JA, Gelmann EP: Differentiation state and invasiveness of human breast cancer cell lines. Breast Cancer Res Treat 31(2–3): 325–335, 1994
Pichler RH, Hugo C, Shankland SJ, Reed MJ, Bassuk JA, Andoh TF, Lombardi DM, Schwartz SM, Bennett WM, Alpers CE, Sage EH, Johnson RJ, Couser WG: SPARC is expressed in renal interstitial fibrosis and in renal vascular injury. Kidney Int 50(6): 1978–1989, 1996
Pichler RH, Bassuk JA, Hugo C, Reed MJ, Eng E, Gordon KL, Pippin J, Alpers CE, Couser WG, Sage EH, Johnson RJ: SPARC is expressed by mesangial cells in experimental mesangial proliferative nephritis and inhibits platelet-derived-growth-factor-medicated mesangial cell proliferation in vitro. Am J Pathol 148(4): 1153–1167, 1996
Lane TF, Sage EH: The biology of sparc, a protein that modulates cell-matrix interactions. FASEB J 8(2): 163–173, 1994.
Sage EH: Terms of attachment: SPARC and tumorigenesis. Nat Med 3(2): 144–146, 1997
Howe CC, Kath R, Mancianti ML, Herlyn M, Mueller S, Cristofalo V: Expression and structure of human SPARC transcripts: SPARC mRNA is expressed by human cells involved in extracellular matrix production and some of these cells show an unusual expression pattern. Exp Cell Res 188(2): 185–191, 1990
Lane TF, Iruela-Arispe ML, Sage EH: Regulation of gene expression by SPARC during angiogenesis in vitro. Changes in fibronectin, thrombospondin-1, and plasminogen activator inhibitor-1. J Biol Chem 267(23): 16736–16745, 1992
Kupprion C, Motamed K, Sage EH: SPARC (BM-40, osteonectin) inhibits the mitogenic effect of vascular endothelial growth factor on microvascular endothelial cells. J Biol Chem 273(45): 29635–29640, 1998
Bradshaw AD, Sage EH: SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J Clin Invest 107(9): 1049–1054, 2001
Meng Q, Xu J, Goldberg ID, Rosen EM, Greenwald RA, Fan S: Influence of chemically modified tetracyclines on proliferation, invasion and migration properties of MDA-MB-468 human breast cancer cells. Clin Exp Metastasis 18(2): 139–146, 2000
Graham JD, Balleine RL, Milliken JS, Bilous AM, Clarke CL: Expression of osteonectin mRNA in human breast tumours is inversely correlated with oestrogen receptor content. Eur J Cancer 33(10): 1654–1660, 1997
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Dhanesuan, N., Sharp, J.A., Blick, T. et al. Doxycycline-Inducible Expression of SPARC/ Osteonectin/ BM40 in MDA-MB-231 Human Breast Cancer Cells Results in Growth Inhibition. Breast Cancer Res Treat 75, 73–85 (2002). https://doi.org/10.1023/A:1016536725958
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DOI: https://doi.org/10.1023/A:1016536725958