STAT3 activation confers trastuzumab-emtansine (T-DM1) resistance in HER2-positive breast cancer

Abstract

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate that has been approved for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. Despite the remarkable efficacy of T-DM1 in many patients, resistance to this therapeutic has emerged as a significant clinical problem. In the current study, we used BT-474/KR cells, a T-DM1-resistant cell line established from HER2-positive BT-474 breast cancer cells, as a model to investigate mechanisms of T-DM1 resistance and explore effective therapeutic regimens. We show here for the first time that activation of signal transducer and activator of transcription 3 (STAT3) mediated by leukemia inhibitory factor receptor (LIFR) overexpression confers resistance to T-DM1. Moreover, secreted factors induced by activated STAT3 in resistant cells limit the responsiveness of cells that were originally sensitive to T-DM1. Importantly, STAT3 inhibition sensitizes resistant cells to T-DM1, both in vitro and in vivo, suggesting that the combination T-DM1 with STAT3-targeted therapy is a potential treatment for T-DM1-refractory patients.

Keywords: STAT3; drug resistance; leukemia inhibitory factor receptor; secreted factor; trastuzumab-emtansine.

Figure 1

BT‐474/KR cells are resistant to trastuzumab‐emtansine (T‐DM1) both in vitro and in vivo. A, BT‐474 and BT‐474/KR cells were treated with different concentrations of T‐DM1 for 120 h, and cell survival was measured using sulforhodamine B assay. Data represent mean ± SD of 3 independent experiments. B, Nude mice bearing BT‐474 or BT‐474/KR xenograft tumors were treated with vehicle or 5 mg/kg T‐DM1 weekly for 21 days. Tumor volume was measured on the indicated days, and tumor growth inhibition (TGI) was calculated. IC₅₀, 50% inhibitory concentration

Figure 2

Trastuzumab‐emtansine (T‐DM1) trafficking, microtubule dynamics, and drug efflux are not significantly different between BT‐474 and BT‐474/KR cells. A, Human epidermal growth factor receptor 2 (HER2) status. Western blotting of HER2 in BT‐474 and BT‐474/KR cells. B, T‐DM1 binding. BT‐474 and BT‐474/KR cells were incubated with DyLight 488 NHS‐ester‐labeled T‐DM1 (1 μg/mL) on ice for 1 h, and binding of T‐DM1 to cells was analyzed on flow cytometry. C, T‐DM1 endocytosis. BT‐474 and BT‐474/KR cells were incubated with DyLight 488 NHS‐ester‐linked T‐DM1 (1 μg/mL) at 37°C for the indicated times, and surface fluorescence was quenched using stripping buffer. T‐DM1 endocytosis was analyzed on flow cytometry and indicated as mean fluorescence intensity (MFI). D, Co‐localization of T‐DM1 (green) with lysosomes (red). BT‐474 and BT‐474/KR cells were incubated with DyLight 488 NHS‐ester‐labeled T‐DM1 (1 μg/mL), and lysosomes were labeled with Lyso‐Tracker Red. Samples were analyzed on confocal microscopy. E, Microtubule polymerization. BT‐474 and BT‐474/KR cells were treated with the indicated concentrations of T‐DM1 for 48 h, and polymeric tubulin was measured on western blotting. F, P‐glycoprotein (P‐gp) expression on western blotting

Figure 3

Trastuzumab‐emtansine (T‐DM1) arrested the cell cycle at M‐phase in BT‐474/KR cells, but did not induce apoptosis. A, BT‐474 and BT‐474/KR cells were treated with T‐DM1, trastuzumab or mertansine (DM1) for 48 h, and cell cycle distribution was analyzed on flow cytometry. Upper, representative images; lower, data from 3 separate experiments expressed as mean ± SD. B, BT‐474 and BT‐474/KR cells were treated with 1 μg/mL T‐DM1 for 48 h. Phospho‐histone H3 and cleaved caspase‐3 were measured on western blotting

Figure 4

Signal transducer and activator of transcription 3 (STAT3) activation mediates trastuzumab‐emtansine (T‐DM1) resistance in BT‐474/KR cells. A, Growth inhibitory effects of T‐DM1, alone or in combination with 0.3 μmol/L napabucasin, on BT‐474/KR cells. Cell growth was measured using the sulforhodamine B assay. Data shown represent mean ± SD of 3 independent experiments. B, BT‐474/KR cells were treated with napabucasin for 24 h. STAT3 activation was detected on western blotting. C, Phosphorylated and total STAT1, STAT3, and STAT5 in BT‐474 and BT‐474/KR cells on western blotting. D, BT‐474/KR cells transfected with either non‐targeting control (NTC) or STAT3 small interfering RNA (siRNA) for 48 h were exposed to T‐DM1 (1 μg/mL) for an additional 72 h. Human epidermal growth factor receptor 2 (HER2), STAT3 and cleaved caspase 3 were detected on western blotting. IC₅₀, 50% inhibitory concentration

Figure 5

Leukemia inhibitory factor receptor (LIFR) overexpression leads to signal transducer and activator of transcription 3 (STAT3) activation and limits the anticancer effect of trastuzumab‐emtansine (T‐DM1). A, Phosphorylation of growth factor receptors and expression of cytokine receptors in BT‐474 and BT‐474/KR cells on western blotting. B, mRNA of LIFR and LIF in BT‐474 and BT‐474/KR cells on quantitative reverse transcription‐polymerase chain reaction. C, BT‐474/KR cells were transfected with either non‐targeting control (NTC) small interfering RNA (siRNA) or LIFR siRNA for 48 h. LIFR knockdown efficiency and STAT3 activation were measured on western blotting. D, BT‐474/KR cells were transfected with the indicated siRNA for 48 h, followed by treatment with T‐DM1 (0.1 μg/mL) for an additional 96 h. Cell survival was analyzed on sulforhodamine B assay. Error bars represent mean ± SD from triplicates. **P < .01, ***P < .001. GM‐CSFR, granulocyte‐macrophage colony‐stimulating factor receptor; IL‐6R, interleukin‐6 receptor; pEGFR, phospho‐EGFR; pFGFR1, phospho‐FGFR1; pHER3, phospho‐HER3

Figure 6

Factors secreted by BT‐474/KR cells confer resistance on BT‐474 cells. A, BT‐474 and BT‐474/KR cells were cultured for 96 h. Interleukin‐6 (IL‐6) level in the supernatants was analyzed on ELISA. B, mRNA level of IL‐6 in BT‐474 and BT‐474/KR cells was analyzed on quantitative reverse transcription‐polymerase chain reaction. C, BT‐474 cells were incubated with conditioned media (CM) from BT‐474 or BT‐474/KR cells for 24 h. Signal transducer and activator of transcription 3 (STAT3) activation was measured on western blotting. D, BT‐474 and BT‐474/KR cells were cultured separately, and lysates were mixed at a 1:1 ratio, or BT‐474 and BT‐474/KR cells were co‐cultured at a 1:1 ratio. STAT3 activation was measured on western blotting. E, BT‐474 cells were incubated in CM from BT‐474 or BT‐474/KR cells, with or without trastuzumab‐emtansine (T‐DM1; 30 ng/mL). Colonies were stained with crystal violet. F, The number of colonies formed in (E) was quantified; percent inhibition is expressed relative to colonies formed following incubation in CM from BT‐474 cells without T‐DM1 exposure. Error bars represent mean ± SD from triplicates. **P < .01

Figure 7

Signal transducer and activator of transcription 3 (STAT3) inhibition overcomes trastuzumab‐emtansine (T‐DM1) resistance in vivo. A, Leukemia inhibitory factor receptor (LIFR) expression was detected on immunohistochemistry. Scale bar, 50 μm. B, BT‐474 and BT‐474/KR xenograft tumors were lysed. C, BT‐474/KR xenograft tumors and an equal amount of BT‐474/KR cells were lysed. D, Mice bearing BT‐474/KR xenograft tumors were treated with vehicle or napabucasin. Tumors were isolated and lysed. B‐D, Phosphorylated (activated) STAT3 (pSTAT3) was measured on western blotting. E, Nude mice bearing BT‐474/KR xenograft tumors were treated with T‐DM1, napabucasin, or a combination of T‐DM1 and napabucasin for 17 days. Tumor volume was measured on the indicated days. Error bars represent mean ± SD. n = 8. *P < .05, **P < .01. F, Proposed model of T‐DM1 resistance in human epidermal growth factor receptor 2 (HER2)‐positive breast cancer