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Characterization of T-DM1-resistant breast cancer cells.

ABSTRACT: The development of targeted therapies has drastically improved the outcome of patients with different types of cancer. T-DM1 (trastuzumab-emtansine) is an antibody-drug conjugate used for the treatment of HER2-positive breast cancer combining the FDA approved mAb (monoclonal antibody) trastuzumab and the microtubule cytotoxic agent DM1 (emtansine). Despite clinical successes achieved by targeted therapies, a large number of patients develop resistance during treatment. To explore mechanisms of resistance to T-DM1, the MDA-MB-361 HER2-positive breast cancer cell line was exposed in vitro to T-DM1 in the absence or presence of ciclosporin A. Previously reported mechanisms of resistance such as trastuzumab-binding alterations, MDR1 upregulation, and SLC46A3 downregulation were not observed in these models. Despite a decrease in HER2 expression at the cell surface, both resistant cell lines remained sensitive to HER2 targeted therapies such as mAbs and tyrosine kinase inhibitors. In addition, sensitivity to DNA damaging agents and topoisomerase inhibitors were unchanged. Conversely resistance to anti-tubulin agents increased. Resistant cells displayed a decreased content of polymerized tubulin and a decreased content of ?III tubulin although the downregulation of ?III tubulin by siRNA in the parental cell line did not modified the sensitivity to T-DM1. Both cell lines resistant to T-DM1 also presented giant aneuploid cells. Several SLC (solute carrier) transporters were found to be differentially expressed in the resistant cells in comparison to parental cells. These results suggest that some characteristics such as increased baseline aneuploidy and altered intracellular drug trafficking might be involved in resistance to T-DM1.

SUBMITTER: Sauveur J

PROVIDER: S-EPMC7314699 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Characterization of T-DM1-resistant breast cancer cells.

Sauveur Juliette J Conilh Louise L Beaumel Sabine S Chettab Kamel K Jordheim Lars-Petter LP Matera Eva-Laure EL Dumontet Charles C

Pharmacology research & perspectives 20200801 4

The development of targeted therapies has drastically improved the outcome of patients with different types of cancer. T-DM1 (trastuzumab-emtansine) is an antibody-drug conjugate used for the treatment of HER2-positive breast cancer combining the FDA approved mAb (monoclonal antibody) trastuzumab and the microtubule cytotoxic agent DM1 (emtansine). Despite clinical successes achieved by targeted therapies, a large number of patients develop resistance during treatment. To explore mechanisms of r ...[more]

PMID: 32583565

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Project description:Aromatase inhibitors (AIs), such as letrozole, are considered as first-line treatment for estrogen receptor-positive breast cancer in postmenopausal women. Despite the successful use of letrozole, resistance to therapy, tumor relapse and metastasis remain principal causes of patient mortality. Although there is no therapy currently available for AI-resistant breast cancer, previous reports have demonstrated that AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and epithelial to mesenchymal transition (EMT). This suggests a convergence of EMT and cancer stem cells (CSCs) in endocrine resistance. The present study evaluated the contribution of mammospheres in letrozole-resistant breast cancer by characterizing mammospheres and their potential impact on cellular motility. Ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either letrozole-resistant MCF-7 cells (LTLT-Ca) or letrozole-sensitive MCF-7 cells (AC-1). Subsequently, intratumoral CSC marker expression was assessed by immunohistochemistry. The results indicated that LTLT-Ca tumors were CD44+/CD24+, while AC-1 tumors presented low CD44/CD24 expression. Since mammosphere formation depends on CSCs, both cell lines were cultured either adherently (2D) or as mammospheres (3D) to assess the CD44/CD24 protein expression profile. When 3D culturing both cell lines, higher expression levels of CD44 and CD24 were observed when compared with their adherent counterparts, with the most robust change observed in the LTLT-Ca cell line. To quantitate the breast cancer stem cell activity, mammosphere formation assays were performed, and the LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared with AC-1 cells. Additionally, targeted gene expression arrays were conducted to compare the LTLT-Ca 3D and 2D cells, revealing that LTLT-Ca 3D cells displayed decreased expression levels of genes involved in cell adhesion and tumor suppression (e. g., E-cadherin, caveolin 1 and β-catenin). To validate this finding, wound healing assays were performed, and LTLT-Ca mammospheres exhibited a 70% wound closure, whereas AC-1 mammospheres exhibited a 39% wound closure. Collectively, the present findings demonstrated a strong association between AI-resistant mammospheres and an increased propensity for migration, which may be indicative of a poor prognosis.

Project description:The humanized monoclonal antibody-drug conjugate trastuzumab emtansine (T-DM1, Kadcyla) has been approved by the U.S. FDA to treat human epidermal growth factor receptor 2 (HER-2)-positive metastatic breast cancer. Despite its effectiveness in most patients, some are initially resistant or develop resistance. No biomarker of drug resistance to T-DM1 has been identified. Antibody-drug efficacy is associated with antibody internalization in the cell; therefore, cellular sensitivity of cells to the drug may be linked to cellular vesicle trafficking systems. Caveolin-1 is a 22 KD protein required for caveolae formation and endocytic membrane transport. In this study, the relationship between caveolin-1 expression and the chemosensitivity of HER-2-positive breast cancer cells to T-DM1 was investigated. Samples from 32 human breast cancer biopsy and normal tissue specimens were evaluated immunohistochemically for caveolin-1 expression. Caveolin-1 was shown to be expressed in 68% (22/32) of the breast cancer specimens. In addition, eight (72.7%, 8/11) HER-2 positive breast cancer specimens had a higher caveolin-1 expression than normal tissues. HER-2-positive BT-474 and SKBR-3 breast cancer cells that express low and moderate levels of caveolin-1, respectively, were treated with trastuzumab or its conjugate T-DM1. Cell viability and molecular localizations of caveolin-1, antibody and its conjugate were examined. Confocal microscopy showed that T-DM1 and caveolin-1 colocalized in SKBR-3 cells, which also were five times more sensitive to the conjugate in terms of cell survival than BT-474 cells, although T-DM1 also showed improved drug efficacy in BT-474 cells than trastuzumab treatment. Caveolin-1 expression in these lines was manipulated by transfection of GFP-tagged caveolin-1 or caveolin-1 siRNA. BT-474 cells overexpressing caveolin-1 were more sensitive to T-DM1 treatment than mock-transfected cells, whereas the siRNA-transfected SKBR-3 cells had decreased sensitivity to T-DM1 than mock-transfected SKBR-3 cells. The expression of caveolin-1 could mediate endocytosis and promote the internalization of T-DM1 into HER-2 positive cancer cells. Thus, caveolin-1 protein may be an effective predictor for determining the outcome of T-DM1 treatment in breast cancer patients.

Dataset Information

Characterization of T-DM1-resistant breast cancer cells.

Publications

Characterization of T-DM1-resistant breast cancer cells.

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