Abstract
IntroductionSrc tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. To identify the mechanism by which Src overexpression sustains this resistance, we tested a panel of breast cancer cell lines either sensitive or resistant to lapatinib.MethodsTo determine the role of Src in lapatinib resistance, we evaluated the effects of Src inhibition/silencing in vitro on survival, migration, and invasion of lapatinib-resistant cells. In vivo experiments were performed in JIMT-1 lapatinib-resistant cells orthotopically implanted in nude mice. We used artificial metastasis assays to evaluate the effect of Src inhibition on the invasiveness of lapatinib-resistant cells. Src-dependent signal transduction was investigated with Western blot and ELISA analyses.ResultsSrc activation was higher in lapatinib-resistant than in lapatinib-sensitive cells. The selective small-molecule Src inhibitor saracatinib combined with lapatinib synergistically inhibited the proliferation, migration, and invasion of lapatinib-resistant cells. Saracatinib combined with lapatinib significantly prolonged survival of JIMT-1-xenografted mice compared with saracatinib alone, and impaired the formation of lung metastases. Unexpectedly, in lapatinib-resistant cells, Src preferentially interacted with epidermal growth factor receptor (EGFR) rather than with HER2. Moreover, EGFR targeting and lapatinib synergistically inhibited survival, migration, and invasion of resistant cells, thereby counteracting Src-mediated resistance. These findings demonstrate that Src activation in lapatinib-resistant cells depends on EGFR-dependent rather than on HER2-dependent signaling.ConclusionsComplete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer.
Highlights
Src tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer
Saracatinib combined with lapatinib synergistically inhibits survival of lapatinib-resistant human breast cancer cell lines We first evaluated the effect of lapatinib in a panel of HER2-positive cells: MDA-MB-361, SKBR-3, BT474, KPL4, and JIMT-1
(See figure on previous page.) Figure 1 Saracatinib combined with lapatinib synergistically inhibits survival of human breast cancer cell lines. (A) Percentage of survival of MDA-MB-361, SKBR-3, BT474, KPL-4, MDA-MB-361-LR, and JIMT-1 breast cancer cell lines grown in complete medium and treated with saracatinib, lapatinib, or both, measured by MTT assay
Summary
Src tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor tyrosine kinase (RTK) and a member of the HER family that includes HER1, known as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 3 (HER3), and human epidermal growth factor receptor 4 (HER4) It controls growth, differentiation, and cell survival through dimerization with other HER receptors, most notably HER3 and EGFR. HER2 amplification occurs in approximately 25% of breast cancers and correlates with a poor prognosis and resistance to conventional antitumor therapies [2,3] It is an important target for antiHER2 drugs, namely, monoclonal antibodies that target the extracellular domain of the receptor, such as trastuzumab and pertuzumab, small-molecule adenosine triphosphate (ATP) competitors able to block tyrosine kinase (TK) activity within the intracellular domain of HER2, such as lapatinib, and antibody-drug conjugates such as trastuzumab emtansine [4,5]. Some patients are constitutively resistant to lapatinib treatment, and, even in responders, the disease often progresses because of the selection of tumor cells that have acquired resistance to the drug
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