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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 Stable ADAM8 KD (shA8) clones were characterized in vitro. ADAM8 expression in WCEs from two shA8 clones (shA8-17 and shA8-20) were compared with two shCtrl clones (shCtrl-3 and shCtrl-5) using WB (LSBio antibody) (A). Clones were grown in 2D-cultures for 48 h and subjected to an ATP assay. NS: non significant, Student's t-test, n = 3 (B). Migration assays were performed as described in Fig 3B. * P = 6.8E-14, Student's t-test, n = 3 (C). Matrigel outgrowth assays were performed as in Fig 3D. Experiments were done twice with similar results. Bar: 100 μm (D).E–H MDA-MB-231 derived shCtrl-3 and shA8-20 cells were injected into the MFP of female mice ( n = 7/group). Tumor volume was measured twice a week (mean ± s.e.m.). * P = 1.4E-6, ** P = 9.8E-8, #P = 3.0E-5, ##P = 9.7E-7, †P = 1.7E-5, ‡P = 5.1E-6, §P = 2.0E-7, Student's t-test (E). At the end of the experiment, tumors were photographed and weighed (mean ± s.e.m.). * P = 5.6E-8, Student's t-test. Bar: 1 cm (F). Blood was collected by cardiac puncture and GFP-positive CTCs were detected by flow cytometry. Average CTC count ± s.d. is given per μl of blood. * P = 0.006, Student's t-test (G). Presence of brain metastases was examined by fluorescent microscopy ( n = 6/group). Representative photographs are shown. Bars: 1 mm (H).I ADAM8 expression in distant metastases of breast cancer patients ( n = 56) was analyzed by immunohistochemistry. Representative pictures and percentages of ADAM8-positive samples are presented.
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fig04: A–D Stable ADAM8 KD (shA8) clones were characterized in vitro. ADAM8 expression in WCEs from two shA8 clones (shA8-17 and shA8-20) were compared with two shCtrl clones (shCtrl-3 and shCtrl-5) using WB (LSBio antibody) (A). Clones were grown in 2D-cultures for 48 h and subjected to an ATP assay. NS: non significant, Student's t-test, n = 3 (B). Migration assays were performed as described in Fig 3B. * P = 6.8E-14, Student's t-test, n = 3 (C). Matrigel outgrowth assays were performed as in Fig 3D. Experiments were done twice with similar results. Bar: 100 μm (D).E–H MDA-MB-231 derived shCtrl-3 and shA8-20 cells were injected into the MFP of female mice ( n = 7/group). Tumor volume was measured twice a week (mean ± s.e.m.). * P = 1.4E-6, ** P = 9.8E-8, #P = 3.0E-5, ##P = 9.7E-7, †P = 1.7E-5, ‡P = 5.1E-6, §P = 2.0E-7, Student's t-test (E). At the end of the experiment, tumors were photographed and weighed (mean ± s.e.m.). * P = 5.6E-8, Student's t-test. Bar: 1 cm (F). Blood was collected by cardiac puncture and GFP-positive CTCs were detected by flow cytometry. Average CTC count ± s.d. is given per μl of blood. * P = 0.006, Student's t-test (G). Presence of brain metastases was examined by fluorescent microscopy ( n = 6/group). Representative photographs are shown. Bars: 1 mm (H).I ADAM8 expression in distant metastases of breast cancer patients ( n = 56) was analyzed by immunohistochemistry. Representative pictures and percentages of ADAM8-positive samples are presented.

Mentions: Next, we tested the effects of stable ADAM8 KD in vivo using a mouse mammary fat pad (MFP) xenograft model. Clones of MDA-MB-231 cells expressing a specific ADAM8 shRNA (shA8-17 and shA8-20) or Control shRNA (shCtrl-3 and shCtrl-5) were isolated. Effective KD of ADAM8 was confirmed by WB (Fig 4A). Reduction of ADAM8 had no detectable effect on 2D-growth as assessed by an ATP assay (Fig 4B). The two shA8 MDA-MB-231 clones showed significantly reduced ability both to migrate and to invade through Matrigel compared to the shCtrl clones (Fig 4C and D). Rescue of these phenotypes by ectopic ADAM8 expression in the shA8-20 clone confirmed the role of ADAM8 KD in these cells (supplementary Fig S3).


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 Stable ADAM8 KD (shA8) clones were characterized in vitro. ADAM8 expression in WCEs from two shA8 clones (shA8-17 and shA8-20) were compared with two shCtrl clones (shCtrl-3 and shCtrl-5) using WB (LSBio antibody) (A). Clones were grown in 2D-cultures for 48 h and subjected to an ATP assay. NS: non significant, Student's t-test, n = 3 (B). Migration assays were performed as described in Fig 3B. * P = 6.8E-14, Student's t-test, n = 3 (C). Matrigel outgrowth assays were performed as in Fig 3D. Experiments were done twice with similar results. Bar: 100 μm (D).E–H MDA-MB-231 derived shCtrl-3 and shA8-20 cells were injected into the MFP of female mice ( n = 7/group). Tumor volume was measured twice a week (mean ± s.e.m.). * P = 1.4E-6, ** P = 9.8E-8, #P = 3.0E-5, ##P = 9.7E-7, †P = 1.7E-5, ‡P = 5.1E-6, §P = 2.0E-7, Student's t-test (E). At the end of the experiment, tumors were photographed and weighed (mean ± s.e.m.). * P = 5.6E-8, Student's t-test. Bar: 1 cm (F). Blood was collected by cardiac puncture and GFP-positive CTCs were detected by flow cytometry. Average CTC count ± s.d. is given per μl of blood. * P = 0.006, Student's t-test (G). Presence of brain metastases was examined by fluorescent microscopy ( n = 6/group). Representative photographs are shown. Bars: 1 mm (H).I ADAM8 expression in distant metastases of breast cancer patients ( n = 56) was analyzed by immunohistochemistry. Representative pictures and percentages of ADAM8-positive samples are presented.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927960&req=5

fig04: A–D Stable ADAM8 KD (shA8) clones were characterized in vitro. ADAM8 expression in WCEs from two shA8 clones (shA8-17 and shA8-20) were compared with two shCtrl clones (shCtrl-3 and shCtrl-5) using WB (LSBio antibody) (A). Clones were grown in 2D-cultures for 48 h and subjected to an ATP assay. NS: non significant, Student's t-test, n = 3 (B). Migration assays were performed as described in Fig 3B. * P = 6.8E-14, Student's t-test, n = 3 (C). Matrigel outgrowth assays were performed as in Fig 3D. Experiments were done twice with similar results. Bar: 100 μm (D).E–H MDA-MB-231 derived shCtrl-3 and shA8-20 cells were injected into the MFP of female mice ( n = 7/group). Tumor volume was measured twice a week (mean ± s.e.m.). * P = 1.4E-6, ** P = 9.8E-8, #P = 3.0E-5, ##P = 9.7E-7, †P = 1.7E-5, ‡P = 5.1E-6, §P = 2.0E-7, Student's t-test (E). At the end of the experiment, tumors were photographed and weighed (mean ± s.e.m.). * P = 5.6E-8, Student's t-test. Bar: 1 cm (F). Blood was collected by cardiac puncture and GFP-positive CTCs were detected by flow cytometry. Average CTC count ± s.d. is given per μl of blood. * P = 0.006, Student's t-test (G). Presence of brain metastases was examined by fluorescent microscopy ( n = 6/group). Representative photographs are shown. Bars: 1 mm (H).I ADAM8 expression in distant metastases of breast cancer patients ( n = 56) was analyzed by immunohistochemistry. Representative pictures and percentages of ADAM8-positive samples are presented.
Mentions: Next, we tested the effects of stable ADAM8 KD in vivo using a mouse mammary fat pad (MFP) xenograft model. Clones of MDA-MB-231 cells expressing a specific ADAM8 shRNA (shA8-17 and shA8-20) or Control shRNA (shCtrl-3 and shCtrl-5) were isolated. Effective KD of ADAM8 was confirmed by WB (Fig 4A). Reduction of ADAM8 had no detectable effect on 2D-growth as assessed by an ATP assay (Fig 4B). The two shA8 MDA-MB-231 clones showed significantly reduced ability both to migrate and to invade through Matrigel compared to the shCtrl clones (Fig 4C and D). Rescue of these phenotypes by ectopic ADAM8 expression in the shA8-20 clone confirmed the role of ADAM8 KD in these cells (supplementary Fig S3).

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