Validation of a Phosphoprotein Array Assay for Characterization of Human Tyrosine Kinase Receptor Downstream Signaling in Breast Cancer

Chergui, Fadila; Chrétien, Anne-Sophie; Bouali, Sanae; Ramacci, Carole; Rouyer, Marie; Bastogne, Thierry; Genin, Pascal; Leroux, Agnès; Merlin, Jean‐Louis

doi:10.1373/clinchem.2008.116632

Cited by 18 publications

(22 citation statements)

References 29 publications

(30 reference statements)

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“…Bio-Plex ® phosphoprotein arrays do not provide subcellular localization data or take into account the heterogeneity of the tumor tissue (27). Tumor heterogeneity is a major source of variability in results.…”

Section: Discussionmentioning

confidence: 99%

“…The expression levels of p-AKT, phosphorylated-ribosomal protein S6 kinase B1 (p-P70S6K), phosphorylated-mitogen-activated protein kinase kinase 1 (p-MEK1) and phosphorylated-extracellular signal regulated kinase 1/2 (p-ERK1/2) were quantitatively assessed using a multiplex bead immunoassay, as previously described (27). Briefly, proteins were extracted from cell cultures and tumor specimens using cell lysis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

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Trastuzumab as a preoperative monotherapy does not inhibit HER2 downstream signaling in HER2-positive breast cancer

Lion¹,

Harlé²,

Salleron³

et al. 2016

Oncology Letters

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Abstract. Human epidermal growth factor 2 (HER2) is overexpressed in 15-20% of breast carcinomas. The overexpression of HER2 was previously associated with a poor prognosis until the development of the first anti-HER2 therapy, trastuzumab, which drastically improves the prognosis of HER2-overexpressing breast cancers. However, its mechanism of action remains not fully understood. Several studies have proposed that the behavior and mechanism of action of trastuzumab may be drastically altered in vitro and in vivo. The present study assesses the ability of trastuzumab to inhibit the phosphorylation of the key-proteins of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin and Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways in vitro, in breast cancer cell lines and in tumor biopsies obtained from patients treated with trastuzumab preoperative monotherapy as part of the Unicancer GEP04 RADHER phase II clinical trial. HER2-positive SKBR3 and HER2-negative MCF-7 cell lines were exposed to trastuzumab for 72 h. In total, 41 patients received trastuzumab alone for 6 weeks of preoperative treatment. Biopsies were collected at the baseline and at surgery. A total of 19 pairs of associated baseline and surgery tumor specimens were eligible for protein extraction and comparative phosphoprotein expression analysis, prior to and subsequent to treatment. The expression of phosphoproteins was quantitatively assessed using a multiplex immunoassay. In the SKBR3 cell line, a statistically significant decrease of the expression level of phosphorylated (p-)AKT, p-ribosomal protein S6 kinase B1, p-extracellular signal regulated kinase 1/2 and p-mitogen-activated protein kinase kinase 1 was observed after exposure to trastuzumab. In contrast, no statistically significant variations for levels expression of these phosphoproteins were observed in patients following treatment. The lack of downregulation of PI3K and MAPK pathways could probably be explained by the implementation of a predominant immunological mechanism of action for trastuzumab, a type of antibody-dependent cell-mediated toxicity, which has previously been reported in preoperative monotherapy settings. The present study confirms that trastuzumab involves various modes of action when assayed in vitro and used clinically.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Trastuzumab as a preoperative monotherapy does not inhibit HER2 downstream signaling in HER2-positive breast cancer

Lion¹,

Harlé²,

Salleron³

et al. 2016

Oncology Letters

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“…The expression of the signaling phosphoproteins was analyzed using multiplex bead immunoassay as described and validated previously (26). Briefly, protein extracts were transferred into 96-well dishes and diluted with 25 µl buffered solution.…”

Section: Multiplex Bead Immunoassaymentioning

confidence: 99%

“…We first investigated the correlation of their expression with ER and HER2 expression, and subsequently evaluated the correlation with expression levels of MAPK and PI3K signaling phosphorylated proteins such as p-AKT, p-GSK3β, p-S6 kinase, p-MEK1 and p-ERK1/2 quantitatively determined using multiplex bead immunoassay as previously described and validated in breast cancer (26).…”

Section: Introductionmentioning

confidence: 99%

Expression and activation of P38 MAP kinase in invasive ductal breast cancers: Correlation with expression of the estrogen receptor, HER2 and downstream signaling phosphorylated proteins

Merlin¹,

Harlé²,

Lion³

et al. 2013

Oncology Reports

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Abstract. MAP kinase signaling proteins have major implications in the molecular oncogenesis of breast cancers and have been extensively investigated as putative targets for therapy. This study reports the investigation of the expression of P38 MAPK and its phosphorylated form (p-P38 MAPK) in clinical specimens of invasive breast carcinomas and their correlation with estrogen receptor (ER) and HER2 expression, as well as MAPK and PI3 kinase-AKT pathway signaling phosphorylated proteins. Expression levels of P38 MAPK and p-P38 MAPK as well as p-AKT, p-GSK3β, p-S6 kinase, p-MEK1 and p-ERK1/2 were quantitatively assessed using multiplex bead immunoassay in frozen specimens from 45 invasive ductal breast cancers. Twenty-nine specimens were ER + , 15 were HER2 + and 10 were triple-negative breast cancers (TNBCs). P38 MAPK was found to be expressed in all tumor specimens and was significantly (P= 0.002) overexpressed in ER + tumors. P38 MAPK expression was lower in TNBCs than in all of the other tumors.

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“…Tot-ERK1/2 was present on all plates and was used for normalization of the data set before log 2 -transformation. The suitability of the technology for phosphoprotein analysis of human tumor samples has been reported recently (24).…”

Section: Analysis Of Phospho-proteins By Western Blot and Bio-plex Anmentioning

confidence: 99%

Pathway Analysis of Glioblastoma Tissue after Preoperative Treatment with the EGFR Tyrosine Kinase Inhibitor Gefitinib—A Phase II Trial

Hegi

Diserens

Bady

et al. 2011

Molecular Cancer Therapeutics

169

109

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Amplification of the epidermal growth factor receptor (EGFR) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we aimed at investigating the molecular effects of the tyrosine kinase inhibitor gefitinib on the EGFR signaling pathway in human glioblastoma. Twenty-two patients selected for reoperation of recurrent glioblastoma were treated within a phase II trial for 5 days with 500 mg gefitinib before surgery followed by postoperative gefitinib until recurrence. Resected glioblastoma tissues exhibited high concentrations of gefitinib (median, 4

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Validation of a Phosphoprotein Array Assay for Characterization of Human Tyrosine Kinase Receptor Downstream Signaling in Breast Cancer

Cited by 18 publications

References 29 publications

Trastuzumab as a preoperative monotherapy does not inhibit HER2 downstream signaling in HER2-positive breast cancer

Trastuzumab as a preoperative monotherapy does not inhibit HER2 downstream signaling in HER2-positive breast cancer

Expression and activation of P38 MAP kinase in invasive ductal breast cancers: Correlation with expression of the estrogen receptor, HER2 and downstream signaling phosphorylated proteins

Pathway Analysis of Glioblastoma Tissue after Preoperative Treatment with the EGFR Tyrosine Kinase Inhibitor Gefitinib—A Phase II Trial

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