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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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Treatment with trastuzumab leads to an increase of ADAM10 levels in vitro and in vivo(A) BT474 and SKBR3 cells were treated for 24h with the indicated doses of trastuzumab before ADAM10 mRNA and protein levels were assessed. The relative protein levels from the semi-quantification of three western blots are shown. (B) BT474 and SKBR3 cells were treated with 40μg/ml trastuzumab in serum-free media and betacellulin levels in the media were assessed in triplicate using ELISA after 24h. (C) Paraffin-embedded tumor slides from xenograft mice bearing BT474 tumors treated with trastuzumab (50 mg/kg loading dose and 25 mg/kg maintenance dose administered intraperitoneally twice a week) or saline (control) for a total of 5 doses [30] were stained for ADAM10 expression by IHC before being scored using IRS. Graphs show means ± SD.
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Figure 1: Treatment with trastuzumab leads to an increase of ADAM10 levels in vitro and in vivo(A) BT474 and SKBR3 cells were treated for 24h with the indicated doses of trastuzumab before ADAM10 mRNA and protein levels were assessed. The relative protein levels from the semi-quantification of three western blots are shown. (B) BT474 and SKBR3 cells were treated with 40μg/ml trastuzumab in serum-free media and betacellulin levels in the media were assessed in triplicate using ELISA after 24h. (C) Paraffin-embedded tumor slides from xenograft mice bearing BT474 tumors treated with trastuzumab (50 mg/kg loading dose and 25 mg/kg maintenance dose administered intraperitoneally twice a week) or saline (control) for a total of 5 doses [30] were stained for ADAM10 expression by IHC before being scored using IRS. Graphs show means ± SD.

Mentions: To assess the effect of trastuzumab treatment on ADAM10 expression, BT474 and SKBR3 cells were treated with two doses of trastuzumab for 24 hours. ADAM10 mRNA levels were increased in a dose-dependent manner: a 3.6–fold increase in BT474 cells and a 2.5-fold in SKBR3 after 40μg/ml trastuzumab treatment, compared to untreated control (figure 1A, left upper and lower panels, both cell lines, n=3, p≤0.01). ADAM10 protein levels were increased by 7–fold in BT474 and 5–fold in SKBR3 cells (figure 1A, middle and right panels, n=3 each, p≤0.01). The upregulation of ADAM10 coincided with an increase of the ligand betacellulin, which is shed by this protease, in the media of trastuzumab treated cells in comparison to control (figure 1B, n=3, p≤0.05).


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)

Treatment with trastuzumab leads to an increase of ADAM10 levels in vitro and in vivo(A) BT474 and SKBR3 cells were treated for 24h with the indicated doses of trastuzumab before ADAM10 mRNA and protein levels were assessed. The relative protein levels from the semi-quantification of three western blots are shown. (B) BT474 and SKBR3 cells were treated with 40μg/ml trastuzumab in serum-free media and betacellulin levels in the media were assessed in triplicate using ELISA after 24h. (C) Paraffin-embedded tumor slides from xenograft mice bearing BT474 tumors treated with trastuzumab (50 mg/kg loading dose and 25 mg/kg maintenance dose administered intraperitoneally twice a week) or saline (control) for a total of 5 doses [30] were stained for ADAM10 expression by IHC before being scored using IRS. Graphs show means ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196152&req=5

Figure 1: Treatment with trastuzumab leads to an increase of ADAM10 levels in vitro and in vivo(A) BT474 and SKBR3 cells were treated for 24h with the indicated doses of trastuzumab before ADAM10 mRNA and protein levels were assessed. The relative protein levels from the semi-quantification of three western blots are shown. (B) BT474 and SKBR3 cells were treated with 40μg/ml trastuzumab in serum-free media and betacellulin levels in the media were assessed in triplicate using ELISA after 24h. (C) Paraffin-embedded tumor slides from xenograft mice bearing BT474 tumors treated with trastuzumab (50 mg/kg loading dose and 25 mg/kg maintenance dose administered intraperitoneally twice a week) or saline (control) for a total of 5 doses [30] were stained for ADAM10 expression by IHC before being scored using IRS. Graphs show means ± SD.
Mentions: To assess the effect of trastuzumab treatment on ADAM10 expression, BT474 and SKBR3 cells were treated with two doses of trastuzumab for 24 hours. ADAM10 mRNA levels were increased in a dose-dependent manner: a 3.6–fold increase in BT474 cells and a 2.5-fold in SKBR3 after 40μg/ml trastuzumab treatment, compared to untreated control (figure 1A, left upper and lower panels, both cell lines, n=3, p≤0.01). ADAM10 protein levels were increased by 7–fold in BT474 and 5–fold in SKBR3 cells (figure 1A, middle and right panels, n=3 each, p≤0.01). The upregulation of ADAM10 coincided with an increase of the ligand betacellulin, which is shed by this protease, in the media of trastuzumab treated cells in comparison to control (figure 1B, n=3, p≤0.05).

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