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ADAM10 mediates trastuzumab resistance and is correlated with survival in HER2 positive breast cancer.

Feldinger K, Generali D, Kramer-Marek G, Gijsen M, Ng TB, Wong JH, Strina C, Cappelletti M, Andreis D, Li JL, Bridges E, Turley H, Leek R, Roxanis I, Capala J, Murphy G, Harris AL, Kong A - Oncotarget (2014)

Bottom Line: However, resistance remains a challenge.We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment.Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding.

View Article: PubMed Central - PubMed

Affiliation: Human Epidermal Growth Factor Group, Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.

ABSTRACT
Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p ≤ 0.001 in BT474; p ≤ 0.01 in SKBR3) and in vivo (p ≤ 0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p ≤ 0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p ≤ 0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p ≤ 0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.

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ADAM10 knockdown decreases cell viability and enhances trastuzumab response by inhibiting the activation of HER receptors(A) In SKBR3 cells, ADAM10 was knocked down using two siRNAs (20nM) or their combination before western blotting after 72h. Quantification of three blots is shown (phosphorylated proteins relative to the respective total proteins). (B) BT474 cells were transfected with 20nM of siRNA against ADAM10 for 72h and camptothecin (6μM) was used as positive control. Cells were stained for Annexin V and the percentage of positive cells was assessed by FACS analysis. (C) SKBR3 cells were transfected as in (B) before cells were re-plated in duplicate and left for colony-formation for 12 days. (D) In cell counting assays, SKBR3 cells transfected as in (B) were seeded in triplicate and treated the next day with 40μg/ml trastuzumab as indicated for 5 days. (E) In SKBR3 cells, ADAM10 was knocked down and the cells were co-stimulated with 50ng/ml betacellulin as indicated for 5 days before cell counting experiments (left) or for 72h before western blot analysis for the indicated proteins (F). Graphs show means ± SD.
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Figure 4: ADAM10 knockdown decreases cell viability and enhances trastuzumab response by inhibiting the activation of HER receptors(A) In SKBR3 cells, ADAM10 was knocked down using two siRNAs (20nM) or their combination before western blotting after 72h. Quantification of three blots is shown (phosphorylated proteins relative to the respective total proteins). (B) BT474 cells were transfected with 20nM of siRNA against ADAM10 for 72h and camptothecin (6μM) was used as positive control. Cells were stained for Annexin V and the percentage of positive cells was assessed by FACS analysis. (C) SKBR3 cells were transfected as in (B) before cells were re-plated in duplicate and left for colony-formation for 12 days. (D) In cell counting assays, SKBR3 cells transfected as in (B) were seeded in triplicate and treated the next day with 40μg/ml trastuzumab as indicated for 5 days. (E) In SKBR3 cells, ADAM10 was knocked down and the cells were co-stimulated with 50ng/ml betacellulin as indicated for 5 days before cell counting experiments (left) or for 72h before western blot analysis for the indicated proteins (F). Graphs show means ± SD.

Mentions: To further prove the role of ADAM10, we silenced ADAM10 using two different siRNAs (and a combination). The knockdown was first optimized and ADAM17 levels were not affected (figure S2A and B). ADAM10 knockdown decreased the phosphorylation of all HER receptors and the downstream pathways in comparison to control (figures 4A, left and right panels, n=3, S2C, 4E), fortifying the ADAM10 inhibitor results above. It also enhanced apoptosis (figure 4B, n=3, p≤0.001) and decreased colony formation (figure 4C, n=3, p≤0.0001) in comparison to control. Furthermore, ADAM10 knockdown enhanced response to trastuzumab treatment in both cell lines (figures 4D, n=3, p≤0.01, S2D). However, the addition of exogenous betacellulin reversed the inhibitory effect of ADAM10 knockdown on the activation of HER receptors (EGFR, HER2, and HER4) and the downstream pathways, which correlated with an increase in cell proliferation (measured by cell number) compared to ADAM10 knockdown alone (figure 4E). This is not surprising since ADAM10 knockdown effect on the endogenous release of HER ligands and should not have an effect on the exogenous ligand stimulation. HER3 phosphorylation was not recovered by the addition of exogenous betacellulin since it is not a HER3 ligand. These results confirm the role of ADAM10 in the shedding of endogenous HER ligands in HER2 positive breast cancer cells.


ADAM10 mediates trastuzumab resistance and is correlated with survival in HER2 positive breast cancer.

Feldinger K, Generali D, Kramer-Marek G, Gijsen M, Ng TB, Wong JH, Strina C, Cappelletti M, Andreis D, Li JL, Bridges E, Turley H, Leek R, Roxanis I, Capala J, Murphy G, Harris AL, Kong A - Oncotarget (2014)

ADAM10 knockdown decreases cell viability and enhances trastuzumab response by inhibiting the activation of HER receptors(A) In SKBR3 cells, ADAM10 was knocked down using two siRNAs (20nM) or their combination before western blotting after 72h. Quantification of three blots is shown (phosphorylated proteins relative to the respective total proteins). (B) BT474 cells were transfected with 20nM of siRNA against ADAM10 for 72h and camptothecin (6μM) was used as positive control. Cells were stained for Annexin V and the percentage of positive cells was assessed by FACS analysis. (C) SKBR3 cells were transfected as in (B) before cells were re-plated in duplicate and left for colony-formation for 12 days. (D) In cell counting assays, SKBR3 cells transfected as in (B) were seeded in triplicate and treated the next day with 40μg/ml trastuzumab as indicated for 5 days. (E) In SKBR3 cells, ADAM10 was knocked down and the cells were co-stimulated with 50ng/ml betacellulin as indicated for 5 days before cell counting experiments (left) or for 72h before western blot analysis for the indicated proteins (F). Graphs show means ± SD.
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Figure 4: ADAM10 knockdown decreases cell viability and enhances trastuzumab response by inhibiting the activation of HER receptors(A) In SKBR3 cells, ADAM10 was knocked down using two siRNAs (20nM) or their combination before western blotting after 72h. Quantification of three blots is shown (phosphorylated proteins relative to the respective total proteins). (B) BT474 cells were transfected with 20nM of siRNA against ADAM10 for 72h and camptothecin (6μM) was used as positive control. Cells were stained for Annexin V and the percentage of positive cells was assessed by FACS analysis. (C) SKBR3 cells were transfected as in (B) before cells were re-plated in duplicate and left for colony-formation for 12 days. (D) In cell counting assays, SKBR3 cells transfected as in (B) were seeded in triplicate and treated the next day with 40μg/ml trastuzumab as indicated for 5 days. (E) In SKBR3 cells, ADAM10 was knocked down and the cells were co-stimulated with 50ng/ml betacellulin as indicated for 5 days before cell counting experiments (left) or for 72h before western blot analysis for the indicated proteins (F). Graphs show means ± SD.
Mentions: To further prove the role of ADAM10, we silenced ADAM10 using two different siRNAs (and a combination). The knockdown was first optimized and ADAM17 levels were not affected (figure S2A and B). ADAM10 knockdown decreased the phosphorylation of all HER receptors and the downstream pathways in comparison to control (figures 4A, left and right panels, n=3, S2C, 4E), fortifying the ADAM10 inhibitor results above. It also enhanced apoptosis (figure 4B, n=3, p≤0.001) and decreased colony formation (figure 4C, n=3, p≤0.0001) in comparison to control. Furthermore, ADAM10 knockdown enhanced response to trastuzumab treatment in both cell lines (figures 4D, n=3, p≤0.01, S2D). However, the addition of exogenous betacellulin reversed the inhibitory effect of ADAM10 knockdown on the activation of HER receptors (EGFR, HER2, and HER4) and the downstream pathways, which correlated with an increase in cell proliferation (measured by cell number) compared to ADAM10 knockdown alone (figure 4E). This is not surprising since ADAM10 knockdown effect on the endogenous release of HER ligands and should not have an effect on the exogenous ligand stimulation. HER3 phosphorylation was not recovered by the addition of exogenous betacellulin since it is not a HER3 ligand. These results confirm the role of ADAM10 in the shedding of endogenous HER ligands in HER2 positive breast cancer cells.

Bottom Line: However, resistance remains a challenge.We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment.Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding.

View Article: PubMed Central - PubMed

Affiliation: Human Epidermal Growth Factor Group, Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.

ABSTRACT
Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p ≤ 0.001 in BT474; p ≤ 0.01 in SKBR3) and in vivo (p ≤ 0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p ≤ 0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p ≤ 0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p ≤ 0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.

Show MeSH
Related in: MedlinePlus