A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer

Abstract

The development of acquired resistance to ErbB2 tyrosine kinase inhibitors limits the clinical efficacy of this class of cancer therapeutics. Little is known about the mechanism(s) of acquired resistance to these agents. Here we establish a model of acquired resistance to N-{3-chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2 (methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (lapatinib), an inhibitor of ErbB2 and ErbB1 tyrosine kinases by chronically exposing lapatinib-sensitive ErbB2-overexpressing breast cancer cells to lapatinib, simulating the clinic where lapatinib is administered on a daily chronic basis. Analysis of baseline gene expression in acquired lapatinib-resistant and parental cells indicates estrogen receptor (ER) signaling involvement in the development of resistance. Using gene interference, we confirm that acquired resistance to lapatinib is mediated by a switch in cell survival dependence and regulation of a key antiapoptotic mediator from ErbB2 alone to codependence upon ER and ErbB2 rather than loss of ErbB2 expression or insensitivity of ErbB2 signaling to lapatinib. Increased ER signaling in response to lapatinib is enhanced by the activation of factors facilitating the transcriptional activity of ER, notably FOXO3a and caveolin-1. Importantly, we confirm that lapatinib induces ER signaling in tumor biopsies from patients with ErbB2-overexpressing breast cancers receiving lapatinib therapy. These findings provided the rationale for preventing the development of acquired resistance by simultaneously inhibiting both ER and ErbB2 signaling pathways. Establishing clinically relevant models of acquired resistance to ErbB2 kinase inhibitors will enhance therapeutic strategies to improve clinical outcomes for patients with ErbB2-overexpressing breast cancers.

Conflict of interest statement

Conflict of interest statement: W.X., S.B., P.H., I.H., J.S., L. Liu, G.P., J. Harris, and N.L.S. are employees of GlaxoSmithKline.

Figures

Fig. 1.

FOXO3a and ER-regulated gene products are induced by lapatinib. Steady-state protein levels of FOXO3a, PR, and bcl-2 were assessed in BT474 cells treated with lapatinib (1 μM) for 24 h. Cells treated with vehicle (DMSO) served as controls. Steady-state actin protein levels served as controls for equal loading of protein. Results are representative of three independent experiments.

Fig. 2.

Lapatinib modulates the expression and cell localization of molecules that promote transcription of ER-regulated genes. Western blot was performed on equal amounts of protein from nuclear, cytoplasmic, and whole-cell extracts. Steady-state protein levels of AIB1, caveolin-1, and FOXO3a were assessed in vehicle-treated BT474 cells (lanes 1, 4, and 7); BT474 cells treated with lapatinib (1 μM) for 24 h (lanes 2, 5, and 8); and B5 cells cultured in the continuous presence of lapatinib (5 μM; lanes 3, 6, and 9). Oct-1 and IκΒα were used to verify the integrity of the nuclear and cytoplasmic extracts, respectively (data not shown). Results were confirmed in three independent experiments.

Fig. 3.

Acquired resistance is not mediated by loss of target expression or insensitivity of the ErbB2-mitogen-activated protein kinase–PI3K pathways to lapatinib. (A) ErbB2, p-ErbB2, and ErbB3 steady-state protein levels in parental and rBT474. Actin steady-state protein levels served as a control to ensure equal loading of protein. (B) Steady-state levels of the indicated proteins in untreated parental (P) BT474 cells and in B5, C5, and F8 cells (see Materials and Methods).

Fig. 4.

Lapatinib therapy enhances the expression of ER-regulated gene products in patients with ErbB2-overexpressing/ER+ breast cancers. Sequential tumor biopsies obtained before (day 0) and after 14 days of lapatinib therapy (1,500 mg per day, day 14) were analyzed by IHC for expression of ErbB2, ER, PR, bcl-2, and FOXO3a proteins.

Fig. 5.

Regulation of cell survival and survivin switches from ErbB2 to ER during the development of acquired resistance to lapatinib. (A) siRNA targeted knockdown of ER but not ErbB2 reduces survivin steady-state protein levels in resistant cells. (B) Survivin, but not ErbB2 knockdown, induces apoptosis in rBT474 cells. (C) ER knockdown in the B5 cells induces marked tumor cell apoptosis. The data are representative of three independent experiments.

Fig. 6.

Therapeutic strategies combining lapatinib with antiestrogens delay or prevent the development of lapatinib resistance in ErbB2-overexpressing/ER+ breast cancer cells. (A) Parental BT474 cells were treated with: (a) vehicle (DMSO); (b) ICI 182.780 (10 nM); (c) lapatinib (500 nM); (d) tamoxifen (1 μM); (e) lapatinib plus tamoxifen (1 μM); (f) ICI 182.780 (10 nM) plus lapatinib; (g) estrogen deprivation (charcoal-stripped serum/phenol red-free medium) alone; (h) estrogen deprivation plus lapatinib. After 21 days, viable cells were assessed by methylene blue staining. Results were confirmed in three independent experiments. (B) Exponentially growing parental BT474 cells were treated according to conditions described in A. After 6 weeks, viable cell colonies were visualized by methylene blue staining. Conditions were repeated in triplicate (rows).