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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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Related in: MedlinePlus

A Blood was drawn using the submandibular collection method from 4 mice/group on the indicated days after tumor cell implantation into the MFP and subjected to flow cytometry to measure GFP-positive CTCs. Average count for the 4 mice ± s.d. per μl of blood is given for each time point. * P = 0.03, #P = 0.05, †P = 0.02, Student's t-test.B Cells were incubated, in duplicate, on a HUVEC monolayer or in empty wells. Attached cells were counted in three fields per well. Mean ± s.d. from three independent experiments. NS, non significant, * P = 1.6E-6, Student's t-test.C Cells were subjected to an overnight transendothelial migration assay on HUVECs. Mean ± s.d. from three independent experiments. * P = 1.7E-5, Student's t-test.D, E Cells were plated on fibronectin-coated coverslips, stained with antibodies against active β1-integrin, vinculin or phosphotyrosine (focal adhesions markers) and phalloidin-488 (F-actin). Confocal microscopy images show adhesion protein channel (red) or merge with F-actin (green). Bar: 10 μm (D). Signal intensity for active β1-integrin and vinculin was determined using ImageJ software (arbitrary units). Mean intensity ± s.d. from >20 cells over 2 experiments. * P = 1.4E-4, NS, non significant, Student's t-test (E).F Adhesion of shCtrl-3 cells on HUVECs was assessed as in (B) with prior incubation of shCtrl-3 cells with an antagonist β1-integrin antibody (+) or a control isotype (−). Mean ± s.d. from three independent experiments. * P = 3.0E-5, Student's t-test.G, H Adhesion (G) and transendothelial migration (H) of shCtrl-3 cells on HUVECs was assessed as in B and C, respectively. Prior to the assays, shCtrl-3 cells were incubated either with monoclonal antibodies targeting the ectodomain of ADAM8 (Mab10311 or Mab1031) or an appropriate isotype control (IgG1 or IgG2B, respectively). The transmigration assay was performed for 9 h. Mean ± s.d. from three independent experiments. * P = 0.001, #P = 5.0E-4, Student's t-test (G). * P = 6.3E-7, #P = 4.0E-7, Student's t-test (H).I Active β1-integrin expression was assessed by immunohistochemistry in mouse mammary tumors (shCtrl-3 and shA8-20) and contralateral mammary glands (shCtrl-3) ( n = 7/group). P: Peritumoral area, T: Tumor. Bar: 100 μm.
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fig06: A Blood was drawn using the submandibular collection method from 4 mice/group on the indicated days after tumor cell implantation into the MFP and subjected to flow cytometry to measure GFP-positive CTCs. Average count for the 4 mice ± s.d. per μl of blood is given for each time point. * P = 0.03, #P = 0.05, †P = 0.02, Student's t-test.B Cells were incubated, in duplicate, on a HUVEC monolayer or in empty wells. Attached cells were counted in three fields per well. Mean ± s.d. from three independent experiments. NS, non significant, * P = 1.6E-6, Student's t-test.C Cells were subjected to an overnight transendothelial migration assay on HUVECs. Mean ± s.d. from three independent experiments. * P = 1.7E-5, Student's t-test.D, E Cells were plated on fibronectin-coated coverslips, stained with antibodies against active β1-integrin, vinculin or phosphotyrosine (focal adhesions markers) and phalloidin-488 (F-actin). Confocal microscopy images show adhesion protein channel (red) or merge with F-actin (green). Bar: 10 μm (D). Signal intensity for active β1-integrin and vinculin was determined using ImageJ software (arbitrary units). Mean intensity ± s.d. from >20 cells over 2 experiments. * P = 1.4E-4, NS, non significant, Student's t-test (E).F Adhesion of shCtrl-3 cells on HUVECs was assessed as in (B) with prior incubation of shCtrl-3 cells with an antagonist β1-integrin antibody (+) or a control isotype (−). Mean ± s.d. from three independent experiments. * P = 3.0E-5, Student's t-test.G, H Adhesion (G) and transendothelial migration (H) of shCtrl-3 cells on HUVECs was assessed as in B and C, respectively. Prior to the assays, shCtrl-3 cells were incubated either with monoclonal antibodies targeting the ectodomain of ADAM8 (Mab10311 or Mab1031) or an appropriate isotype control (IgG1 or IgG2B, respectively). The transmigration assay was performed for 9 h. Mean ± s.d. from three independent experiments. * P = 0.001, #P = 5.0E-4, Student's t-test (G). * P = 6.3E-7, #P = 4.0E-7, Student's t-test (H).I Active β1-integrin expression was assessed by immunohistochemistry in mouse mammary tumors (shCtrl-3 and shA8-20) and contralateral mammary glands (shCtrl-3) ( n = 7/group). P: Peritumoral area, T: Tumor. Bar: 100 μm.

Mentions: Recent studies have demonstrated CTCs can be measured prior to formation of a detectable primary tumor (Eyles et al, 2010). Thus, we investigated whether the appearance of these early CTCs requires ADAM8 using a second set of mice ( n = 4/group). Blood was collected at day 7, when tumors were barely detectable and at days 14 and 21, when a difference in size was only beginning to be measured (Fig 4E). CTCs were detected as early as 7 days after cell implantation (Fig 6A). The numbers of CTCs were significantly lower in the mice injected with shA8-20 versus shCtrl-3 cells throughout the time course (Fig 6A). This suggests a lower potential risk for developing metastases to distant organs when ADAM8 is knocked down, independent of tumor size.


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 Blood was drawn using the submandibular collection method from 4 mice/group on the indicated days after tumor cell implantation into the MFP and subjected to flow cytometry to measure GFP-positive CTCs. Average count for the 4 mice ± s.d. per μl of blood is given for each time point. * P = 0.03, #P = 0.05, †P = 0.02, Student's t-test.B Cells were incubated, in duplicate, on a HUVEC monolayer or in empty wells. Attached cells were counted in three fields per well. Mean ± s.d. from three independent experiments. NS, non significant, * P = 1.6E-6, Student's t-test.C Cells were subjected to an overnight transendothelial migration assay on HUVECs. Mean ± s.d. from three independent experiments. * P = 1.7E-5, Student's t-test.D, E Cells were plated on fibronectin-coated coverslips, stained with antibodies against active β1-integrin, vinculin or phosphotyrosine (focal adhesions markers) and phalloidin-488 (F-actin). Confocal microscopy images show adhesion protein channel (red) or merge with F-actin (green). Bar: 10 μm (D). Signal intensity for active β1-integrin and vinculin was determined using ImageJ software (arbitrary units). Mean intensity ± s.d. from >20 cells over 2 experiments. * P = 1.4E-4, NS, non significant, Student's t-test (E).F Adhesion of shCtrl-3 cells on HUVECs was assessed as in (B) with prior incubation of shCtrl-3 cells with an antagonist β1-integrin antibody (+) or a control isotype (−). Mean ± s.d. from three independent experiments. * P = 3.0E-5, Student's t-test.G, H Adhesion (G) and transendothelial migration (H) of shCtrl-3 cells on HUVECs was assessed as in B and C, respectively. Prior to the assays, shCtrl-3 cells were incubated either with monoclonal antibodies targeting the ectodomain of ADAM8 (Mab10311 or Mab1031) or an appropriate isotype control (IgG1 or IgG2B, respectively). The transmigration assay was performed for 9 h. Mean ± s.d. from three independent experiments. * P = 0.001, #P = 5.0E-4, Student's t-test (G). * P = 6.3E-7, #P = 4.0E-7, Student's t-test (H).I Active β1-integrin expression was assessed by immunohistochemistry in mouse mammary tumors (shCtrl-3 and shA8-20) and contralateral mammary glands (shCtrl-3) ( n = 7/group). P: Peritumoral area, T: Tumor. Bar: 100 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3927960&req=5

fig06: A Blood was drawn using the submandibular collection method from 4 mice/group on the indicated days after tumor cell implantation into the MFP and subjected to flow cytometry to measure GFP-positive CTCs. Average count for the 4 mice ± s.d. per μl of blood is given for each time point. * P = 0.03, #P = 0.05, †P = 0.02, Student's t-test.B Cells were incubated, in duplicate, on a HUVEC monolayer or in empty wells. Attached cells were counted in three fields per well. Mean ± s.d. from three independent experiments. NS, non significant, * P = 1.6E-6, Student's t-test.C Cells were subjected to an overnight transendothelial migration assay on HUVECs. Mean ± s.d. from three independent experiments. * P = 1.7E-5, Student's t-test.D, E Cells were plated on fibronectin-coated coverslips, stained with antibodies against active β1-integrin, vinculin or phosphotyrosine (focal adhesions markers) and phalloidin-488 (F-actin). Confocal microscopy images show adhesion protein channel (red) or merge with F-actin (green). Bar: 10 μm (D). Signal intensity for active β1-integrin and vinculin was determined using ImageJ software (arbitrary units). Mean intensity ± s.d. from >20 cells over 2 experiments. * P = 1.4E-4, NS, non significant, Student's t-test (E).F Adhesion of shCtrl-3 cells on HUVECs was assessed as in (B) with prior incubation of shCtrl-3 cells with an antagonist β1-integrin antibody (+) or a control isotype (−). Mean ± s.d. from three independent experiments. * P = 3.0E-5, Student's t-test.G, H Adhesion (G) and transendothelial migration (H) of shCtrl-3 cells on HUVECs was assessed as in B and C, respectively. Prior to the assays, shCtrl-3 cells were incubated either with monoclonal antibodies targeting the ectodomain of ADAM8 (Mab10311 or Mab1031) or an appropriate isotype control (IgG1 or IgG2B, respectively). The transmigration assay was performed for 9 h. Mean ± s.d. from three independent experiments. * P = 0.001, #P = 5.0E-4, Student's t-test (G). * P = 6.3E-7, #P = 4.0E-7, Student's t-test (H).I Active β1-integrin expression was assessed by immunohistochemistry in mouse mammary tumors (shCtrl-3 and shA8-20) and contralateral mammary glands (shCtrl-3) ( n = 7/group). P: Peritumoral area, T: Tumor. Bar: 100 μm.
Mentions: Recent studies have demonstrated CTCs can be measured prior to formation of a detectable primary tumor (Eyles et al, 2010). Thus, we investigated whether the appearance of these early CTCs requires ADAM8 using a second set of mice ( n = 4/group). Blood was collected at day 7, when tumors were barely detectable and at days 14 and 21, when a difference in size was only beginning to be measured (Fig 4E). CTCs were detected as early as 7 days after cell implantation (Fig 6A). The numbers of CTCs were significantly lower in the mice injected with shA8-20 versus shCtrl-3 cells throughout the time course (Fig 6A). This suggests a lower potential risk for developing metastases to distant organs when ADAM8 is knocked down, independent of tumor size.

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