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Generation, characterization, and maintenance of trastuzumab-resistant HER2+ breast cancer cell lines.

ABSTRACT: Trastuzumab became the therapy of choice for patients with HER2-positive breast cancer in 1998, and it has provided clinical benefit ever since. However, a significant percentage of patients show primary resistance to trastuzumab at diagnosis, and most patients with metastatic disease that initially respond to trastuzumab eventually progress (acquired resistance). Consequently, there is an urgent need to improve our knowledge of the mechanisms governing resistance, so that specific therapeutic strategies can be developed to provide improved efficacy. We generated new cell lines derived from BCCL through extended exposure to trastuzumab. Drug-conditioned populations were authenticated for their molecular profile and their resistance rate was determined. Heterogeneous HER2 amplification was observed across most of the BCCLs, ranging from cells without HER2 amplification to elevated HER2 gene copy numbers in others. Using a phospho-antibody array we analyzed the status of kinase receptors and effectors from different cellular pathways. This revealed that HER2, AKT, and S6RP presented high phosphorylation levels with specific variations between sensitive and resistant populations. In addition, differences in phosphorylation levels for several of those pathways targets were found between sensitive and resistant lines. Furthermore, a biochemical study characterized patterns of molecular alterations similar to those commonly described in breast cancer. Finally, a subcutaneous xenograft murine model confirmed the resistance to trastuzumab of the established cell line. We conclude that these resistant BCCLs can be a valuable tool to gain insight into the mechanisms of acquisition of trastuzumab resistance.

SUBMITTER: Zazo S

PROVIDER: S-EPMC5126281 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Generation, characterization, and maintenance of trastuzumab-resistant HER2+ breast cancer cell lines.

Zazo Sandra S González-Alonso Paula P Martín-Aparicio Ester E Chamizo Cristina C Cristóbal Ion I Arpí Oriol O Rovira Ana A Albanell Joan J Eroles Pilar P Lluch Ana A Madoz-Gúrpide Juan J Rojo Federico F

American journal of cancer research 20161101 11

Trastuzumab became the therapy of choice for patients with HER2-positive breast cancer in 1998, and it has provided clinical benefit ever since. However, a significant percentage of patients show primary resistance to trastuzumab at diagnosis, and most patients with metastatic disease that initially respond to trastuzumab eventually progress (acquired resistance). Consequently, there is an urgent need to improve our knowledge of the mechanisms governing resistance, so that specific therapeutic s ...[more]

PMID: 27904779

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Project description:HER2-positive breast cancer is one of the most prevalent forms of cancer among women worldwide. Generally, the molecular characteristics of this breast cancer include activation of human epidermal growth factor receptor-2 (HER2) and hormone receptor activation. HER2-positive is associated with a higher death rate, which led to the development of a monoclonal antibody called trastuzumab, specifically targeting HER2. The success rate of HER2-positive breast cancer treatment has been increased; however, drug resistance remains a challenge. This fact motivated us to explore the underlying molecular mechanisms of trastuzumab resistance. For this purpose, a two-fold approach was taken by considering well-known breast cancer cell lines SKBR3 and BT474. In the first fold, trastuzumab treatment doses were optimized separately for both cell lines. This was done based on the proliferation rate of cells in response to a wide variety of medication dosages. Thereafter, each cell line was cultivated with a steady dosage of herceptin for several months. During this period, six time points were selected for further in vitro analysis, ranging from the untreated cell line at the beginning to a fully resistant cell line at the end of the experiment. In the second fold, nucleic acids were extracted for further high throughput-based microarray experiments of gene and microRNA expression. Such expression data were further analyzed in order to infer the molecular mechanisms involved in the underlying development of trastuzumab resistance. In the list of differentially expressed genes and miRNAs, multiple genes (e.g., BIRC5, E2F1, TFRC, and USP1) and miRNAs (e.g., hsa miR 574 3p, hsa miR 4530, and hsa miR 197 3p) responsible for trastuzumab resistance were found. Downstream analysis showed that TFRC, E2F1, and USP1 were also targeted by hsa-miR-8485. Moreover, it indicated that miR-4701-5p was highly expressed as compared to TFRC in the SKBR3 cell line. These results unveil key genes and miRNAs as molecular regulators for trastuzumab resistance.

Project description:BackgroundTrastuzumab improves survival in the adjuvant treatment of HER-positive breast cancer, although combined therapy with anthracycline-based regimens has been associated with cardiac toxicity. We wanted to evaluate the efficacy and safety of a new nonanthracycline regimen with trastuzumab.MethodsWe randomly assigned 3222 women with HER2-positive early-stage breast cancer to receive doxorubicin and cyclophosphamide followed by docetaxel every 3 weeks (AC-T), the same regimen plus 52 weeks of trastuzumab (AC-T plus trastuzumab), or docetaxel and carboplatin plus 52 weeks of trastuzumab (TCH). The primary study end point was disease-free survival. Secondary end points were overall survival and safety.ResultsAt a median follow-up of 65 months, 656 events triggered this protocol-specified analysis. The estimated disease-free survival rates at 5 years were 75% among patients receiving AC-T, 84% among those receiving AC-T plus trastuzumab, and 81% among those receiving TCH. Estimated rates of overall survival were 87%, 92%, and 91%, respectively. No significant differences in efficacy (disease-free or overall survival) were found between the two trastuzumab regimens, whereas both were superior to AC-T. The rates of congestive heart failure and cardiac dysfunction were significantly higher in the group receiving AC-T plus trastuzumab than in the TCH group (P<0.001). Eight cases of acute leukemia were reported: seven in the groups receiving the anthracycline-based regimens and one in the TCH group subsequent to receiving an anthracycline outside the study.ConclusionsThe addition of 1 year of adjuvant trastuzumab significantly improved disease-free and overall survival among women with HER2-positive breast cancer. The risk-benefit ratio favored the nonanthracycline TCH regimen over AC-T plus trastuzumab, given its similar efficacy, fewer acute toxic effects, and lower risks of cardiotoxicity and leukemia. (Funded by Sanofi-Aventis and Genentech; BCIRG-006 ClinicalTrials.gov number, NCT00021255.).

Dataset Information

Generation, characterization, and maintenance of trastuzumab-resistant HER2+ breast cancer cell lines.

Publications

Generation, characterization, and maintenance of trastuzumab-resistant HER2+ breast cancer cell lines.

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