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ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis.

Romagnoli M, Mineva ND, Polmear M, Conrad C, Srinivasan S, Loussouarn D, Barillé-Nion S, Georgakoudi I, Dagg Á, McDermott EW, Duffy MJ, McGowan PM, Schlomann U, Parsons M, Bartsch JW, Sonenshein GE - EMBO Mol Med (2013)

Bottom Line: Circulating tumor cells and brain metastases were also significantly reduced.Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model.As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, USA.

ABSTRACT
The transmembrane metalloprotease-disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple-negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF-A and transendothelial cell migration via β1-integrin activation. In vivo, treatment with an anti-ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.

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A–D Cells were transfected with siRNAs as in Fig 2D and E for 24 h, and tested for colony formation in soft agar (A), cell migration (B), invasion (C), and 3D-Matrigel outgrowth (D). For soft agar assays, cells were grown for 8-12 days and colonies >20 μm diameter in 3 wells/condition were counted with ImageJ software. A representative of two experiments with similar results is shown. * P = 0.002, ** P = 1.9E-6, #P = 1.3E-5, ##P = 1.0E-7, Student's t-test (A). Migration (B) and invasion (C) assays were performed for 24 h using Transwells without or with precoating of Matrigel, respectively. Control condition (siCtrl) is set to 100% (mean ± s.d. from three independent experiments). * P = 0.004, ** P = 5.3E-4, #P = 1.8E-4, ##P = 5.5E-5, Student's t-test (B). * P = 0.001, ** P = 9.3E-4, #P = 8.7E-6, ##P = 4.6E-5, Student's t-test (C). For Matrigel outgrowth assays, colonies formed after 5-7 days were photographed at 20× magnification. Experiments were done twice with similar results. Bars: 100 μm (D).E WCEs from MDA-MB-231 cells and in vivo derived LM1 and LM2 lines, either untransfected (left panel) or transfected with siCtrl or siA8 for 48 h (right panels), were subjected to WB for ADAM8 (LSBio antibody). A representative blot is shown ( n = 3).F Twenty-four h after siRNA transfection, LM1 and LM2 cells were subjected to a 3D-Matrigel outgrowth assay, as in (D), except that colonies were photographed after 4 days. Experiments were done twice with similar results. Bars: 100 μm.
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fig03: A–D Cells were transfected with siRNAs as in Fig 2D and E for 24 h, and tested for colony formation in soft agar (A), cell migration (B), invasion (C), and 3D-Matrigel outgrowth (D). For soft agar assays, cells were grown for 8-12 days and colonies >20 μm diameter in 3 wells/condition were counted with ImageJ software. A representative of two experiments with similar results is shown. * P = 0.002, ** P = 1.9E-6, #P = 1.3E-5, ##P = 1.0E-7, Student's t-test (A). Migration (B) and invasion (C) assays were performed for 24 h using Transwells without or with precoating of Matrigel, respectively. Control condition (siCtrl) is set to 100% (mean ± s.d. from three independent experiments). * P = 0.004, ** P = 5.3E-4, #P = 1.8E-4, ##P = 5.5E-5, Student's t-test (B). * P = 0.001, ** P = 9.3E-4, #P = 8.7E-6, ##P = 4.6E-5, Student's t-test (C). For Matrigel outgrowth assays, colonies formed after 5-7 days were photographed at 20× magnification. Experiments were done twice with similar results. Bars: 100 μm (D).E WCEs from MDA-MB-231 cells and in vivo derived LM1 and LM2 lines, either untransfected (left panel) or transfected with siCtrl or siA8 for 48 h (right panels), were subjected to WB for ADAM8 (LSBio antibody). A representative blot is shown ( n = 3).F Twenty-four h after siRNA transfection, LM1 and LM2 cells were subjected to a 3D-Matrigel outgrowth assay, as in (D), except that colonies were photographed after 4 days. Experiments were done twice with similar results. Bars: 100 μm.

Mentions: To test the role of ADAM8 in the aggressive phenotype of MDA-MB-231 and Hs578T cells in vitro, the effects of its KD were measured on growth in soft agar, migration and invasion through Matrigel, assays that are performed in 3D-culture conditions. Decreased ADAM8 expression in both lines led to reduced abilities to grow under anchorage-independent conditions (Fig 3A), to migrate (Fig 3B) or invade through Matrigel (Fig 3C). Consistently, the capacity of these aggressive cells to form characteristic branching colonies in a Matrigel 3D-outgrowth assay was robustly inhibited upon ADAM8 KD (Fig 3D). The MDA-MB-231-derived cells LM1 and LM2, which were selected in vivo for their ability to metastasize to the lung (Minn et al, 2005), expressed higher levels of ADAM8 than the parental line (Fig 3E), and formed large colonies more rapidly in Matrigel (3–4 versus 6–7 days for parental cells) (Fig 3F). The ability of LM1 and LM2 cells to form these highly invasive colonies in Matrigel was strikingly reduced by ADAM8 KD (Fig 3E and F). Thus, ADAM8 is required to maintain the migratory and invasive phenotype of TNBC cells in culture.


ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis.

Romagnoli M, Mineva ND, Polmear M, Conrad C, Srinivasan S, Loussouarn D, Barillé-Nion S, Georgakoudi I, Dagg Á, McDermott EW, Duffy MJ, McGowan PM, Schlomann U, Parsons M, Bartsch JW, Sonenshein GE - EMBO Mol Med (2013)

A–D Cells were transfected with siRNAs as in Fig 2D and E for 24 h, and tested for colony formation in soft agar (A), cell migration (B), invasion (C), and 3D-Matrigel outgrowth (D). For soft agar assays, cells were grown for 8-12 days and colonies >20 μm diameter in 3 wells/condition were counted with ImageJ software. A representative of two experiments with similar results is shown. * P = 0.002, ** P = 1.9E-6, #P = 1.3E-5, ##P = 1.0E-7, Student's t-test (A). Migration (B) and invasion (C) assays were performed for 24 h using Transwells without or with precoating of Matrigel, respectively. Control condition (siCtrl) is set to 100% (mean ± s.d. from three independent experiments). * P = 0.004, ** P = 5.3E-4, #P = 1.8E-4, ##P = 5.5E-5, Student's t-test (B). * P = 0.001, ** P = 9.3E-4, #P = 8.7E-6, ##P = 4.6E-5, Student's t-test (C). For Matrigel outgrowth assays, colonies formed after 5-7 days were photographed at 20× magnification. Experiments were done twice with similar results. Bars: 100 μm (D).E WCEs from MDA-MB-231 cells and in vivo derived LM1 and LM2 lines, either untransfected (left panel) or transfected with siCtrl or siA8 for 48 h (right panels), were subjected to WB for ADAM8 (LSBio antibody). A representative blot is shown ( n = 3).F Twenty-four h after siRNA transfection, LM1 and LM2 cells were subjected to a 3D-Matrigel outgrowth assay, as in (D), except that colonies were photographed after 4 days. Experiments were done twice with similar results. Bars: 100 μm.
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fig03: A–D Cells were transfected with siRNAs as in Fig 2D and E for 24 h, and tested for colony formation in soft agar (A), cell migration (B), invasion (C), and 3D-Matrigel outgrowth (D). For soft agar assays, cells were grown for 8-12 days and colonies >20 μm diameter in 3 wells/condition were counted with ImageJ software. A representative of two experiments with similar results is shown. * P = 0.002, ** P = 1.9E-6, #P = 1.3E-5, ##P = 1.0E-7, Student's t-test (A). Migration (B) and invasion (C) assays were performed for 24 h using Transwells without or with precoating of Matrigel, respectively. Control condition (siCtrl) is set to 100% (mean ± s.d. from three independent experiments). * P = 0.004, ** P = 5.3E-4, #P = 1.8E-4, ##P = 5.5E-5, Student's t-test (B). * P = 0.001, ** P = 9.3E-4, #P = 8.7E-6, ##P = 4.6E-5, Student's t-test (C). For Matrigel outgrowth assays, colonies formed after 5-7 days were photographed at 20× magnification. Experiments were done twice with similar results. Bars: 100 μm (D).E WCEs from MDA-MB-231 cells and in vivo derived LM1 and LM2 lines, either untransfected (left panel) or transfected with siCtrl or siA8 for 48 h (right panels), were subjected to WB for ADAM8 (LSBio antibody). A representative blot is shown ( n = 3).F Twenty-four h after siRNA transfection, LM1 and LM2 cells were subjected to a 3D-Matrigel outgrowth assay, as in (D), except that colonies were photographed after 4 days. Experiments were done twice with similar results. Bars: 100 μm.
Mentions: To test the role of ADAM8 in the aggressive phenotype of MDA-MB-231 and Hs578T cells in vitro, the effects of its KD were measured on growth in soft agar, migration and invasion through Matrigel, assays that are performed in 3D-culture conditions. Decreased ADAM8 expression in both lines led to reduced abilities to grow under anchorage-independent conditions (Fig 3A), to migrate (Fig 3B) or invade through Matrigel (Fig 3C). Consistently, the capacity of these aggressive cells to form characteristic branching colonies in a Matrigel 3D-outgrowth assay was robustly inhibited upon ADAM8 KD (Fig 3D). The MDA-MB-231-derived cells LM1 and LM2, which were selected in vivo for their ability to metastasize to the lung (Minn et al, 2005), expressed higher levels of ADAM8 than the parental line (Fig 3E), and formed large colonies more rapidly in Matrigel (3–4 versus 6–7 days for parental cells) (Fig 3F). The ability of LM1 and LM2 cells to form these highly invasive colonies in Matrigel was strikingly reduced by ADAM8 KD (Fig 3E and F). Thus, ADAM8 is required to maintain the migratory and invasive phenotype of TNBC cells in culture.

Bottom Line: Circulating tumor cells and brain metastases were also significantly reduced.Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model.As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, USA.

ABSTRACT
The transmembrane metalloprotease-disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple-negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF-A and transendothelial cell migration via β1-integrin activation. In vivo, treatment with an anti-ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.

Show MeSH
Related in: MedlinePlus