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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 inhibition or knockdown decreases activation of HER receptors and cell viability in trastuzumab resistant cell linesAll resistant cells were continuously treated with 40μg/ml trastuzumab unless otherwise stated. (A) Resistant SKBR3 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) for 24h in serum-free media or (B) were transfected with 20nM of siRNAs against ADAM10 for 72h before western blotting and quantification of three blots is shown next to the respective blot (phosphorylated proteins relative to the respective total proteins). (C) For FACS analysis, resistant BT474 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) in serum-reduced media for 5 days. Camptothecin (6μM) was used as positive control. Cells were stained for Annexin V and percentage of positive cells analysed by FACS. (D) For the clonogenic assay, resistant SKBR3 cells were treated as in (C) and re-plated in duplicate for colony-formation for 12 days. (E) For cell counting studies, trastuzumab was withdrawn overnight from resistant SKBR3 cell lines. For the “withdrawal group”, trastuzumab remained withdrawn whereas the “continuous group” was co-treated with 40μg/ml trastuzumab. Both groups were treated in as in (C), or were transfected with 20nM of specific siRNAs against ADAM10 for 5 days (right panel). (F) BT474 and SKBR3 cells were treated in triplicate with 40μg/ml trastuzumab, 5μM of the ADAM10 inhibitor INCB8765 (ADAM10i), the ADAM17 inhibitor INCB4298 (ADAM17i), the ADAM10/17 inhibitor INCB3619 (ADAM10/17i), or their combination for 5 days before cell counting. (G) BT474 resistant cells were treated as in (F) with continuous 40μg/ml trastuzumab for 5 days before cell counting. Graphs represent data from three independent experiments and show means ± SD.
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Figure 6: ADAM10 inhibition or knockdown decreases activation of HER receptors and cell viability in trastuzumab resistant cell linesAll resistant cells were continuously treated with 40μg/ml trastuzumab unless otherwise stated. (A) Resistant SKBR3 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) for 24h in serum-free media or (B) were transfected with 20nM of siRNAs against ADAM10 for 72h before western blotting and quantification of three blots is shown next to the respective blot (phosphorylated proteins relative to the respective total proteins). (C) For FACS analysis, resistant BT474 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) in serum-reduced media for 5 days. Camptothecin (6μM) was used as positive control. Cells were stained for Annexin V and percentage of positive cells analysed by FACS. (D) For the clonogenic assay, resistant SKBR3 cells were treated as in (C) and re-plated in duplicate for colony-formation for 12 days. (E) For cell counting studies, trastuzumab was withdrawn overnight from resistant SKBR3 cell lines. For the “withdrawal group”, trastuzumab remained withdrawn whereas the “continuous group” was co-treated with 40μg/ml trastuzumab. Both groups were treated in as in (C), or were transfected with 20nM of specific siRNAs against ADAM10 for 5 days (right panel). (F) BT474 and SKBR3 cells were treated in triplicate with 40μg/ml trastuzumab, 5μM of the ADAM10 inhibitor INCB8765 (ADAM10i), the ADAM17 inhibitor INCB4298 (ADAM17i), the ADAM10/17 inhibitor INCB3619 (ADAM10/17i), or their combination for 5 days before cell counting. (G) BT474 resistant cells were treated as in (F) with continuous 40μg/ml trastuzumab for 5 days before cell counting. Graphs represent data from three independent experiments and show means ± SD.

Mentions: In view of the upregulation of ADAM10 during trastuzumab treatment in naïve cells, we hypothesized that ADAM10 might be implicated in acquired trastuzumab resistance. ADAM10 mRNA and protein levels were increased in the resistant cells compared to parental cells (figure 5A, each n=3, p≤0.05). Moreover, betacellulin levels in the media of resistant cells were also increased (figure 5B, n=3, p≤0.05). ADAM10 knockdown decreased the level of betacellulin in the media of SKBR3 resistant cells (figure 5C). In addition, 24h treatment with ADAM10i (figures 6A; S3A) and ADAM10 knockdown (figures 6B; S3B) decreased HER member phosphorylation and downstream pathway activation in trastuzumab resistant cells. ADAM10 inhibition increased the percentage of apoptotic cells (figure 6C, n=3, p ≤ 0.01) and decreased the number of colonies of trastuzumab resistant cells (figure 6D, n=3, p≤0.01) in comparison to control. However, the effect of ADAM10 knockdown could be counteracted by the addition of exogenous betacellulin in the resistant cells, similar to the result in naïve cells (figure S3C). Although withdrawal of trastuzumab from the resistant cells led to an enhanced proliferation (n=3, p≤0.01), ADAM10 inhibition or knockdown reduced cell numbers compared to control and this effect was independent of trastuzumab treatment in both resistant cell lines (figure 6E; S3D, left and right panels).


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 inhibition or knockdown decreases activation of HER receptors and cell viability in trastuzumab resistant cell linesAll resistant cells were continuously treated with 40μg/ml trastuzumab unless otherwise stated. (A) Resistant SKBR3 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) for 24h in serum-free media or (B) were transfected with 20nM of siRNAs against ADAM10 for 72h before western blotting and quantification of three blots is shown next to the respective blot (phosphorylated proteins relative to the respective total proteins). (C) For FACS analysis, resistant BT474 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) in serum-reduced media for 5 days. Camptothecin (6μM) was used as positive control. Cells were stained for Annexin V and percentage of positive cells analysed by FACS. (D) For the clonogenic assay, resistant SKBR3 cells were treated as in (C) and re-plated in duplicate for colony-formation for 12 days. (E) For cell counting studies, trastuzumab was withdrawn overnight from resistant SKBR3 cell lines. For the “withdrawal group”, trastuzumab remained withdrawn whereas the “continuous group” was co-treated with 40μg/ml trastuzumab. Both groups were treated in as in (C), or were transfected with 20nM of specific siRNAs against ADAM10 for 5 days (right panel). (F) BT474 and SKBR3 cells were treated in triplicate with 40μg/ml trastuzumab, 5μM of the ADAM10 inhibitor INCB8765 (ADAM10i), the ADAM17 inhibitor INCB4298 (ADAM17i), the ADAM10/17 inhibitor INCB3619 (ADAM10/17i), or their combination for 5 days before cell counting. (G) BT474 resistant cells were treated as in (F) with continuous 40μg/ml trastuzumab for 5 days before cell counting. Graphs represent data from three independent experiments and show means ± SD.
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Figure 6: ADAM10 inhibition or knockdown decreases activation of HER receptors and cell viability in trastuzumab resistant cell linesAll resistant cells were continuously treated with 40μg/ml trastuzumab unless otherwise stated. (A) Resistant SKBR3 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) for 24h in serum-free media or (B) were transfected with 20nM of siRNAs against ADAM10 for 72h before western blotting and quantification of three blots is shown next to the respective blot (phosphorylated proteins relative to the respective total proteins). (C) For FACS analysis, resistant BT474 cells were treated with 5μM ADAM10 inhibitor INCB8765 (ADAM10i) in serum-reduced media for 5 days. Camptothecin (6μM) was used as positive control. Cells were stained for Annexin V and percentage of positive cells analysed by FACS. (D) For the clonogenic assay, resistant SKBR3 cells were treated as in (C) and re-plated in duplicate for colony-formation for 12 days. (E) For cell counting studies, trastuzumab was withdrawn overnight from resistant SKBR3 cell lines. For the “withdrawal group”, trastuzumab remained withdrawn whereas the “continuous group” was co-treated with 40μg/ml trastuzumab. Both groups were treated in as in (C), or were transfected with 20nM of specific siRNAs against ADAM10 for 5 days (right panel). (F) BT474 and SKBR3 cells were treated in triplicate with 40μg/ml trastuzumab, 5μM of the ADAM10 inhibitor INCB8765 (ADAM10i), the ADAM17 inhibitor INCB4298 (ADAM17i), the ADAM10/17 inhibitor INCB3619 (ADAM10/17i), or their combination for 5 days before cell counting. (G) BT474 resistant cells were treated as in (F) with continuous 40μg/ml trastuzumab for 5 days before cell counting. Graphs represent data from three independent experiments and show means ± SD.
Mentions: In view of the upregulation of ADAM10 during trastuzumab treatment in naïve cells, we hypothesized that ADAM10 might be implicated in acquired trastuzumab resistance. ADAM10 mRNA and protein levels were increased in the resistant cells compared to parental cells (figure 5A, each n=3, p≤0.05). Moreover, betacellulin levels in the media of resistant cells were also increased (figure 5B, n=3, p≤0.05). ADAM10 knockdown decreased the level of betacellulin in the media of SKBR3 resistant cells (figure 5C). In addition, 24h treatment with ADAM10i (figures 6A; S3A) and ADAM10 knockdown (figures 6B; S3B) decreased HER member phosphorylation and downstream pathway activation in trastuzumab resistant cells. ADAM10 inhibition increased the percentage of apoptotic cells (figure 6C, n=3, p ≤ 0.01) and decreased the number of colonies of trastuzumab resistant cells (figure 6D, n=3, p≤0.01) in comparison to control. However, the effect of ADAM10 knockdown could be counteracted by the addition of exogenous betacellulin in the resistant cells, similar to the result in naïve cells (figure S3C). Although withdrawal of trastuzumab from the resistant cells led to an enhanced proliferation (n=3, p≤0.01), ADAM10 inhibition or knockdown reduced cell numbers compared to control and this effect was independent of trastuzumab treatment in both resistant cell lines (figure 6E; S3D, left and right panels).

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