Doxycycline-Inducible Expression of SPARC/ Osteonectin/ BM40 in MDA-MB-231 Human Breast Cancer Cells Results in Growth Inhibition

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.

Overexpression of SPARC gene in human gastric carcinoma and its clinic–pathologic significance

“…Two recent in vitro studies delineate the potential of SPARC as a suppressor of tumorigenic potential in human melanoma and breast cancer cells via the addition of either antisense RNA or the transfection of SPARC (Ledda et al, 1997;Dhanesuan et al, 2002). Finally, our study demonstrates that the further investigation of SPARC is warranted due to its status as a potential prognostic and therapeutic agent.…”

Common deleted genes in the 5q− syndrome: thrombocytopenia and reduced erythroid colony formation in SPARC null mice

“…26 Other investigators showed that forced expression of SPARC inhibited growth of human ovarian cancer cells in immunodeficient mice and in breast cancer cells in vitro. 27 SPARC-null mice exhibited increased proliferation of mesangial cells, 28 as well as, increased insulin growth factor signaling. 29 The protein also suppressed the growth of pancreatic cancer cells and glioma cells.…”

The role of MMP-9 in the anti-angiogenic effect of secreted protein acidic and rich in cysteine

“…Similarly, the tumour growth of glioma cells overexpressing SPARC was delayed in vivo (Schultz et al, 2002). An inhibitory effect of SPARC on proliferation and migration has been found in breast and ovarian carcinoma cells (Dhanesuan et al, 2002). Infection of MDA-231 breast carcinoma cells with osteonectin decreased the in vitro invasion of these cells through Matrigel (Koblinski et al, 2005).…”