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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 Sixteen h after plating, cells were cultured under normoxic (−) or hypoxic (+, 1% O2) conditions for 24 h (MDA-MB-231 and Hs578T) or 6 h (shCtrl-3 and shA8-20 clones). WCEs were subjected to WB for ADAM8 (LSBio antibody). Representative blots are shown ( n = 3).B ADAM8 expression in mouse mammary tumors derived from shCtrl-3 or shA8-20 cells was analyzed by immunohistochemistry. H&E staining was performed in parallel. Representative panels are shown ( n = 7/group). Bar: 100 μm.C Angiogenesis was evaluated by CD31 immunohistochemical staining of tumor sections from shCtrl-3 and shA8-20 groups ( n = 7/group). Vessel density for each mouse is given as the average number of vessels in 2 slides/tumor (3 peritumoral hot spots/slide). P: Peritumoral area, T: Tumor. Bar: 100 μm. * P = 0.01, Student's t-test.D Pearson's pairwise correlation plot shows a significant positive correlation between ADAM8 and CD31 (PECAM1) mRNA expression in tumors from patients with basal-like breast cancer (GenExMiner microarray database). r: correlation ratio. P < 0.0001, Student's t-test.E, F HUVECs were subjected to tube formation assays in the presence of conditioned medium from the indicated shA8 and shCtrl clones, or obtained in absence of tumor cells (−). Values for branch points and closed networks (polygons) are given as averages of nine fields ± s.d. Branch point: * P = 1.3E-6 versus shCtrl-3, * P = 3.2E-9 versus shCtrl-5, ** P = 6.9E-29; Polygons: * P = 5.2E-6 versus shCtrl-3, * P = 1.3E-6 versus shCtrl-5, ** P = 6.2E-24; Student's t-test; n = 4 (E). Representative images from 4 independent experiments are shown. Bar: 30 μm (F).G Conditioned media from two shCtrl and two shA8 clones were subjected to a Human Angiogenesis antibody array. Expression levels of the detected proteins were quantified using ImageJ and the angiogenesis mediators significantly downregulated by more than 2-fold in shA8 clones are presented as mean of the two clones ± s.d. Fold change (F.C.) and P-values are given, Student's t-test.H Conditioned serum-free medium from shCtrl and shA8 clones was analyzed by WB for VEGF-A. A representative blot is shown ( n = 3).I VEGF-A in the conditioned serum-free medium from shCtrl-3 or shA8-20 clones transfected with the indicated ADAM8 forms or empty vector (EV) DNA was assessed by WB (lower panel). The quantification of average levels from 3 experiments is presented as percent relative to the shCtrl set to 100%.
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fig05: A Sixteen h after plating, cells were cultured under normoxic (−) or hypoxic (+, 1% O2) conditions for 24 h (MDA-MB-231 and Hs578T) or 6 h (shCtrl-3 and shA8-20 clones). WCEs were subjected to WB for ADAM8 (LSBio antibody). Representative blots are shown ( n = 3).B ADAM8 expression in mouse mammary tumors derived from shCtrl-3 or shA8-20 cells was analyzed by immunohistochemistry. H&E staining was performed in parallel. Representative panels are shown ( n = 7/group). Bar: 100 μm.C Angiogenesis was evaluated by CD31 immunohistochemical staining of tumor sections from shCtrl-3 and shA8-20 groups ( n = 7/group). Vessel density for each mouse is given as the average number of vessels in 2 slides/tumor (3 peritumoral hot spots/slide). P: Peritumoral area, T: Tumor. Bar: 100 μm. * P = 0.01, Student's t-test.D Pearson's pairwise correlation plot shows a significant positive correlation between ADAM8 and CD31 (PECAM1) mRNA expression in tumors from patients with basal-like breast cancer (GenExMiner microarray database). r: correlation ratio. P < 0.0001, Student's t-test.E, F HUVECs were subjected to tube formation assays in the presence of conditioned medium from the indicated shA8 and shCtrl clones, or obtained in absence of tumor cells (−). Values for branch points and closed networks (polygons) are given as averages of nine fields ± s.d. Branch point: * P = 1.3E-6 versus shCtrl-3, * P = 3.2E-9 versus shCtrl-5, ** P = 6.9E-29; Polygons: * P = 5.2E-6 versus shCtrl-3, * P = 1.3E-6 versus shCtrl-5, ** P = 6.2E-24; Student's t-test; n = 4 (E). Representative images from 4 independent experiments are shown. Bar: 30 μm (F).G Conditioned media from two shCtrl and two shA8 clones were subjected to a Human Angiogenesis antibody array. Expression levels of the detected proteins were quantified using ImageJ and the angiogenesis mediators significantly downregulated by more than 2-fold in shA8 clones are presented as mean of the two clones ± s.d. Fold change (F.C.) and P-values are given, Student's t-test.H Conditioned serum-free medium from shCtrl and shA8 clones was analyzed by WB for VEGF-A. A representative blot is shown ( n = 3).I VEGF-A in the conditioned serum-free medium from shCtrl-3 or shA8-20 clones transfected with the indicated ADAM8 forms or empty vector (EV) DNA was assessed by WB (lower panel). The quantification of average levels from 3 experiments is presented as percent relative to the shCtrl set to 100%.

Mentions: When solid tumors reach a few millimeters in diameter, nutrients and oxygen become insufficient and hypoxic stress is induced (Majmundar et al, 2010). This leads to cell death by necrosis as well as to the release of factors that promote endothelial cell recruitment and angiogenesis that are needed to support continued tumor growth (Bergers & Benjamin, 2003; Naumov et al, 2006). ADAM8 levels were induced under hypoxia in pancreatic cancer cells (Valkovskaya et al, 2007). Thus, the effects of growth under reduced oxygen on ADAM8 expression in breast cancer cells were studied. Incubation of MDA-MB-231 and Hs578T cells in 1% oxygen led to substantial increases in ADAM8 protein levels (Fig 5A, left panels). As seen with 3D-culture of Hs578T cells, levels of the ADAM8 proform and active form increased, and the proform now co-migrated with the band observed in MDA-MB-231 cells. Similar to the MDA-MB-231 parental cell line, ADAM8 expression was increased in shCtrl-3 cells under hypoxic conditions; whereas no induction was seen in the shA8-20 clone (Fig 5A, right panels). The typical hypoxia-inducible factor 1-alpha (HIF-1α) induction was essentially comparable in these cells (supplementary Fig S5), suggesting that the hypoxic pathway is not impaired in the clones. Consistently, strong ADAM8 staining was detected around the necrotic areas in shCtrl-3 tumors, but absent in tumors derived from shA8-20 cells (Fig 5B), even though the extent of necrosis was similar in both tumor populations.


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 Sixteen h after plating, cells were cultured under normoxic (−) or hypoxic (+, 1% O2) conditions for 24 h (MDA-MB-231 and Hs578T) or 6 h (shCtrl-3 and shA8-20 clones). WCEs were subjected to WB for ADAM8 (LSBio antibody). Representative blots are shown ( n = 3).B ADAM8 expression in mouse mammary tumors derived from shCtrl-3 or shA8-20 cells was analyzed by immunohistochemistry. H&E staining was performed in parallel. Representative panels are shown ( n = 7/group). Bar: 100 μm.C Angiogenesis was evaluated by CD31 immunohistochemical staining of tumor sections from shCtrl-3 and shA8-20 groups ( n = 7/group). Vessel density for each mouse is given as the average number of vessels in 2 slides/tumor (3 peritumoral hot spots/slide). P: Peritumoral area, T: Tumor. Bar: 100 μm. * P = 0.01, Student's t-test.D Pearson's pairwise correlation plot shows a significant positive correlation between ADAM8 and CD31 (PECAM1) mRNA expression in tumors from patients with basal-like breast cancer (GenExMiner microarray database). r: correlation ratio. P < 0.0001, Student's t-test.E, F HUVECs were subjected to tube formation assays in the presence of conditioned medium from the indicated shA8 and shCtrl clones, or obtained in absence of tumor cells (−). Values for branch points and closed networks (polygons) are given as averages of nine fields ± s.d. Branch point: * P = 1.3E-6 versus shCtrl-3, * P = 3.2E-9 versus shCtrl-5, ** P = 6.9E-29; Polygons: * P = 5.2E-6 versus shCtrl-3, * P = 1.3E-6 versus shCtrl-5, ** P = 6.2E-24; Student's t-test; n = 4 (E). Representative images from 4 independent experiments are shown. Bar: 30 μm (F).G Conditioned media from two shCtrl and two shA8 clones were subjected to a Human Angiogenesis antibody array. Expression levels of the detected proteins were quantified using ImageJ and the angiogenesis mediators significantly downregulated by more than 2-fold in shA8 clones are presented as mean of the two clones ± s.d. Fold change (F.C.) and P-values are given, Student's t-test.H Conditioned serum-free medium from shCtrl and shA8 clones was analyzed by WB for VEGF-A. A representative blot is shown ( n = 3).I VEGF-A in the conditioned serum-free medium from shCtrl-3 or shA8-20 clones transfected with the indicated ADAM8 forms or empty vector (EV) DNA was assessed by WB (lower panel). The quantification of average levels from 3 experiments is presented as percent relative to the shCtrl set to 100%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: A Sixteen h after plating, cells were cultured under normoxic (−) or hypoxic (+, 1% O2) conditions for 24 h (MDA-MB-231 and Hs578T) or 6 h (shCtrl-3 and shA8-20 clones). WCEs were subjected to WB for ADAM8 (LSBio antibody). Representative blots are shown ( n = 3).B ADAM8 expression in mouse mammary tumors derived from shCtrl-3 or shA8-20 cells was analyzed by immunohistochemistry. H&E staining was performed in parallel. Representative panels are shown ( n = 7/group). Bar: 100 μm.C Angiogenesis was evaluated by CD31 immunohistochemical staining of tumor sections from shCtrl-3 and shA8-20 groups ( n = 7/group). Vessel density for each mouse is given as the average number of vessels in 2 slides/tumor (3 peritumoral hot spots/slide). P: Peritumoral area, T: Tumor. Bar: 100 μm. * P = 0.01, Student's t-test.D Pearson's pairwise correlation plot shows a significant positive correlation between ADAM8 and CD31 (PECAM1) mRNA expression in tumors from patients with basal-like breast cancer (GenExMiner microarray database). r: correlation ratio. P < 0.0001, Student's t-test.E, F HUVECs were subjected to tube formation assays in the presence of conditioned medium from the indicated shA8 and shCtrl clones, or obtained in absence of tumor cells (−). Values for branch points and closed networks (polygons) are given as averages of nine fields ± s.d. Branch point: * P = 1.3E-6 versus shCtrl-3, * P = 3.2E-9 versus shCtrl-5, ** P = 6.9E-29; Polygons: * P = 5.2E-6 versus shCtrl-3, * P = 1.3E-6 versus shCtrl-5, ** P = 6.2E-24; Student's t-test; n = 4 (E). Representative images from 4 independent experiments are shown. Bar: 30 μm (F).G Conditioned media from two shCtrl and two shA8 clones were subjected to a Human Angiogenesis antibody array. Expression levels of the detected proteins were quantified using ImageJ and the angiogenesis mediators significantly downregulated by more than 2-fold in shA8 clones are presented as mean of the two clones ± s.d. Fold change (F.C.) and P-values are given, Student's t-test.H Conditioned serum-free medium from shCtrl and shA8 clones was analyzed by WB for VEGF-A. A representative blot is shown ( n = 3).I VEGF-A in the conditioned serum-free medium from shCtrl-3 or shA8-20 clones transfected with the indicated ADAM8 forms or empty vector (EV) DNA was assessed by WB (lower panel). The quantification of average levels from 3 experiments is presented as percent relative to the shCtrl set to 100%.
Mentions: When solid tumors reach a few millimeters in diameter, nutrients and oxygen become insufficient and hypoxic stress is induced (Majmundar et al, 2010). This leads to cell death by necrosis as well as to the release of factors that promote endothelial cell recruitment and angiogenesis that are needed to support continued tumor growth (Bergers & Benjamin, 2003; Naumov et al, 2006). ADAM8 levels were induced under hypoxia in pancreatic cancer cells (Valkovskaya et al, 2007). Thus, the effects of growth under reduced oxygen on ADAM8 expression in breast cancer cells were studied. Incubation of MDA-MB-231 and Hs578T cells in 1% oxygen led to substantial increases in ADAM8 protein levels (Fig 5A, left panels). As seen with 3D-culture of Hs578T cells, levels of the ADAM8 proform and active form increased, and the proform now co-migrated with the band observed in MDA-MB-231 cells. Similar to the MDA-MB-231 parental cell line, ADAM8 expression was increased in shCtrl-3 cells under hypoxic conditions; whereas no induction was seen in the shA8-20 clone (Fig 5A, right panels). The typical hypoxia-inducible factor 1-alpha (HIF-1α) induction was essentially comparable in these cells (supplementary Fig S5), suggesting that the hypoxic pathway is not impaired in the clones. Consistently, strong ADAM8 staining was detected around the necrotic areas in shCtrl-3 tumors, but absent in tumors derived from shA8-20 cells (Fig 5B), even though the extent of necrosis was similar in both tumor populations.

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