HSP90 Inhibitor, 17-DMAG, Alone and in Combination with Lapatinib Attenuates Acquired Lapatinib-Resistance in ER-positive, HER2-Overexpressing Breast Cancer Cell Line

Abstract

Lapatinib, a Human Epidermal growth factor Receptor 2 (HER2)-targeting therapy in HER2-overexpressing breast cancer, has been widely used clinically, but the prognosis is still poor because most patients acquire resistance. Therefore, we investigated mechanisms related to lapatinib resistance to evaluate new therapeutic targets that may overcome resistance. Lapatinib-resistant cell lines were established using SKBR3 and BT474 cells. We evaluated cell viability and cell signal changes, gene expression and protein changes. In the xenograft mouse model, anti-tumor effects were evaluated using drugs. Analysis of the protein interaction network in two resistant cell lines with different lapatinib resistance mechanisms showed that HSP90 protein was commonly increased. When Heat Shock Protein 90 (HSP90) inhibitors were administered alone to both resistant cell lines, cell proliferation and protein expression were effectively inhibited. However, inhibition of cell proliferation and protein expression with a combination of lapatinib and HSP90 inhibitors showed a more synergistic effect in the LR-BT474 cell line than the LR-SKBR3 cell line, and the same result was exhibited with the xenograft model. These results suggest that HSP90 inhibitors in patients with lapatinib-resistant Estrogen Receptor (ER) (+) HER2 (+) breast cancer are promising therapeutics for future clinical trials.

Keywords: ER (+) HER2 (+); HSP90; breast cancer; lapatinib resistance.

Figure 1

Establishment of lapatinib-acquired-resistant Human Epidermal growth factor Receptor 2 (HER2) (+) cell lines and biologic changes in HER2 downstream signaling. (A) Lapatinib was administered for 48 and 72 h at different concentrations in SKBR3, BT474, LR-SKBR3, and LR-BT474 cell lines and measured using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) analysis. Results are expressed as percentage of viable cells from three independent experiments (mean ± SD) (***, p < 0.001). (B) Parental and lapatinib-resistant cell lines were treated with lapatinib at 20, 50, or 100 nM concentrations for 24 h. Western blot was performed using same amounts of protein. Protein activation was analyzed by evaluating phosphorylation status using corresponding p-HER2, p-EGFR, p-Akt, and p-Erk antibodies. Results were obtained from two independent experiments.

Figure 2

Gene set enrichment analysis (GSEA) enrichment plot in lapatinib-resistant cell lines. (A) GSEA plot shows G2M checkpoint and E2F target in LR-SKBR3 and SKBR3 cells. (B) GSEA plot shows early and late estrogen responses in LR-BT474 and BT474 cells. (C) GSEA analysis represents gene expression enrichment in LR-SKBR3 cell compared with LR-BT474 cell. (D) GSEA analysis represents gene expression enrichment in LR-BT474 cell compared with LR-SKBR3 cell.

Figure 3

Phosphoproteomic profiling of lapatinib-resistant and sensitive cell lines. (A) Experimental schematic outline of SILAC experiment. (B) Enriched cellular pathways in LR-SKBR3. (C) Enriched cellular pathways in LR-BT474. (D) Major cluster by GLay clustering in LR-SKBR3. (E) Major cluster by GLay clustering in LR-BT474.

Figure 4

17DMAG induces inhibition of cell proliferation and affects HER2 downstream signaling in lapatinib-resistant cell lines. (A) 17DMAG was administered for 48 and 72 h at different concentrations in parent and lapatinib-resistant cell lines and measured using MTT analysis. Results are expressed as percentage of viable cells from three independent experiments (mean ± SD) (***, p < 0.001). (B) Parental and lapatinib-resistant cell lines were treated with 17DMAG at 20, 50, or 100 nM concentrations for 24 h. Western blot was performed using same amounts of protein. Protein activation was analyzed using corresponding p-HER2, p-EGFR, p-Akt, and p-ERK antibodies. Results represent 2 independent experiments.

Figure 5

Combination of lapatinib and 17DMAG in lapatinib-resistant cell lines. (A) Isobologram plot shows combination treatment of lapatinib and 17-DMAG in LR-SKBR3 and LR-BT474 cell lines. (B) All cell lines were treated with lapatinib, 17DMAG, or combination lapatinib and 17DMAG for 24 h, lysed, and analyzed using western blot with indicated antibodies.

Figure 6

Antitumor efficacy of 17DMAG and lapatinib combination in BT474 and LR-BT474 xenograft models. (A,B) Mice were injected with BT474 and LR-BT474 cells on the flanks. After tumor formation, randomly grouped nude mice were treated with control, lapatinib (75 mg/kg), 17DMAG (5 mg/kg), or combination (lapatinib ,17DMAG) for 5 weeks. Tumor measurements were obtained twice a week. Average tumor volumes in control, lapatinib, 17-DMAG, and combination treatment groups are presented as mean ± SEM (*, p < 0.05; **, p < 0.01). Scale bars, 1 cm. (C) Representative image of immunohistochemical staining in BT474 and LR-BT474 tumors of xenograft models. Tumor sections from vehicle and combination groups were stained for HER2 and ER. Scale bars, 100 μm. Scatter plot of ER Allred scores in BT474 and LR-BT474 xenograft tumors measured by IHC. Data are presented as mean ± SEM (n = 4 in each group).