Abstract
Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer, leading to improved overall survival. However, acquired resistance inevitably occurs. We aimed to identify, quantify, and assess the mechanisms of acquired resistance to trastuzumab. We established an acquired trastuzumab-resistant model in vitro from BT-474, a trastuzumab-sensitive, HER2-amplified breast-cancer cell line. A multi-omic strategy was implemented to obtain gene, proteome, and phosphoproteome signatures associated with acquired resistance to trastuzumab in HER2-positive breast cancer, followed by validation in human clinical samples. YAP1 dephosphorylation and TEAD2 overexpression were detected as significant alterations in the Hippo pathway in trastuzumab-resistant breast cancer. Because of the emerging role of these proteins as mediators of normal growth and tumorigenesis, we assessed the exogenous modulation of their activity, either by in vitro gene silencing or by pharmacological inhibition of the YAP1/TEAD complexes, both in vitro and in vivo. Moreover, we identified increased signaling through the Hippo pathway in human samples after progression following trastuzumab treatment. Finally, YAP1/TAZ nuclear accumulation in malignant cells in HER2 breast tumor was significantly associated with worse progression-free and overall survival in metastatic HER2-positive breast-cancer patients. Our results suggest the involvement of Hippo signaling in acquired trastuzumab resistance in breast cancer. Additionally, we provide novel evidence for a potential breast-cancer treatment strategy based on dual targeting of HER2 and Hippo pathway effectors, which may improve the antitumor activity of trastuzumab and help overcome resistance.
Highlights
Among the molecular markers employed in routine clinical practice for prognosis and predictive aims, human epidermal growth factor receptor 2 (HER2; ErbB2) defines a discrete category of breast tumors with specific characteristics
We verified a switch in the phenotype to epithelial-mesenchymal transition (EMT), which may be indicative of a more aggressive behavior, in accordance with the other cellular findings associated with the acquired resistance (Figure 1B): we observed, in resistant BT-474.r2T cells, a downregulation of epithelial markers (E-cadherin, occludin), upregulation of mesenchymal markers (N-cadherin, fibronectin, vimentin), as well as an increase in the level of CD24
HER2-positive breast cancer, a global phosphoproteome analysis based on discovery SILAC approach for was performed in 12 samples: lysates from BT-474 and BT-474.r2T cells, which were either untreated or exposed to 15 μg/mL trastuzumab, and collected at three different times: 12 min, to capture rapid changes in phosphorylation patterns; 24 h, to identify sustained variations related to the acquisition of resistance; and 7 days, to measure the response of the cells to treatment with trastuzumab
Summary
Among the molecular markers employed in routine clinical practice for prognosis and predictive aims, human epidermal growth factor receptor 2 (HER2; ErbB2) defines a discrete category of breast tumors with specific characteristics. The development of the monoclonal anti-HER2 antibody trastuzumab has been shown to have a substantial clinical benefit, inhibiting tumor progression by targeting the HER2 receptor in early and metastatic breast-cancer patients [3,4]. There is a pressing need to further our knowledge of the molecular bases that limit the response to trastuzumab in order to discover predictive biomarkers, overcome resistance to standard anti-HER2 treatment by developing alternative therapies, and eventually enhance treatment response and survival for HER2-positive breast-cancer patients [3,5]
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