Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3

Abstract

Oncogenic tyrosine kinases have proved to be promising targets for the development of highly effective anticancer drugs. However, tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (HER) family show only limited activity against HER2-driven breast cancers, despite effective inhibition of epidermal growth factor receptor (EGFR) and HER2 in vivo. The reasons for this are unclear. Signalling in trans is a key feature of this multimember family and the critically important phosphatidylinositol-3-OH kinase (PI(3)K)/Akt pathway is driven predominantly through transphosphorylation of the kinase-inactive HER3 (refs 9, 10). Here we show that HER3 and consequently PI(3)K/Akt signalling evade inhibition by current HER-family TKIs in vitro and in tumours in vivo. This is due to a compensatory shift in the HER3 phosphorylation-dephosphorylation equilibrium, driven by increased membrane HER3 expression driving the phosphorylation reaction and by reduced HER3 phosphatase activity impeding the dephosphorylation reaction. These compensatory changes are driven by Akt-mediated negative-feedback signalling. Although HER3 is not a direct target of TKIs, HER3 substrate resistance undermines their efficacy and has thus far gone undetected. The experimental abrogation of HER3 resistance by small interfering RNA knockdown restores potent pro-apoptotic activity to otherwise cytostatic HER TKIs, re-affirming the oncogene-addicted nature of HER2-driven tumours and the therapeutic promise of this oncoprotein target. However, because HER3 signalling is buffered against an incomplete inhibition of HER2 kinase, much more potent TKIs or combination strategies are required to silence oncogenic HER2 signalling effectively. The biologic marker with which to assess the efficacy of HER TKIs should be the transphosphorylation of HER3 rather than autophosphorylation.

Figure 1. HER TK inhibitors fail to induce sustained inhibition of HER3 signaling in HER2-driven breast cancer cells

(A) BT474 cells were treated with 5 μM gefitinib for the indicated times and assayed for expression and phosphorylation of the indicated proteins. Lane 0* is an IgG immunoprecipitation control. (B) Data of interest shown from the identical experiment done in SkBr3 cells. (C) SKBr3 cells were treated with 5 μM erlotinib for the indicated times and lysates immunoblotted with the indicated antibodies. (D) SKBr3 cells were treated with 20 μM AG825 for the indicated times with drug being replenished 1 hr before the 72 hour harvest. (E) Mice bearing HCC1569 human breast cancer xenografts were treated with gefitinib at 150 mg/kg once daily. Animals were sacrificed at 2 hours after the first dose and 2 hours after the 4^th dose (96 hours total treatment) and tumors rapidly harvested for immunoblotting with the indicated antibodies. The six lanes corresponding to each timepoint represent six different animals.

Figure 2. Forward shift in HER3 phosphorylation-dephosphorylation equilibrium following extended HER TKI treatment

(A) BT474 cells were transfected with anti-HER2 (H) or control (C) siRNA and harvested 64 hours after transfection (lanes 1,2). Additional arms were treated with 48 hours of gefitinib untransfected (lanes 3,4) or following siRNA transfection (lanes 5,6). (B) SKBr3 cells were treated with 5μM gefitinib for 0,1, or 48 hours. Arm W was treated for 48 hours, washed, and incubated in drug-free media for one more hour. (C) SKBr3 cells were treated with the indicated concentrations of gefitinib for one hour (left side). Additional arms were treated with 5 μM gefitinib for 48 hours and subsequently treated with the indicated concentrations of gefitinib for one additional hour (right side). (D) SKBr3 cells were treated with the indicated concentrations and durations of PD168393. (E) SKBr3 cells were treated with 2 μM PD168393 for the indicated times. (F) SKBr3 cells were transfected with anti-HER3 (H) or control (C) siRNA and harvested 4 days after transfection (lanes 1,2). Additional arms were treated with gefitinib or control in untransfected cells (lanes 3,4) or following siRNA transfection (lanes 5,6). (G) In parallel with figure F, SkBr3 cells were either left untransfected, or transfected with anti-HER3 or control siRNA followed by 5uM gefitinib or control for 48 hours. Apoptotic cells were identified by their subG1 DNA content. (H) SKBr3 cells were treated as indicated for 48 hours. Apoptotic cells were identified by Annexin V expression.

Figure 3. Mechanism of HER3 reactivation following extended HER TKI treatment

(A) SKBr3 cells were treated with 5 μM gefitinib or control for 48h and stained with anti-HER2 or anti-HER3 antibodies for immunofluorescence microscopy. (B) In addition, cell surface proteins in control or 48 hour pre-treated cells were biotinylated, precipitated, and immunoblotted as indicated. (C) Control or gefitinib pre-treated (5 μM, 48h) SKBr3 cells were treated with 20 μM monensin for the final 6h and analyzed by western blotting. (D) SkBr3 cells were treated with gefitinib for 48 hrs and with 20uM monensin for the final 6 hours. Membrane and total HER3 was immunoblotted from cell surface proteome pulldowns (above) or total lysates (below). (E) The dephosphorylation rate of p-HER3 following 48hrs of gefitinib treatment was determined immediately after initiation of fully inactivating concentrations of the irreversible HER TK inhibitor PD168393 (2 μM). (F) Reactive oxygen species were quantified in control (A) or gefitinib pre-treated (B) (5 μM, 48h)SKBr3 cells as described in methods. (G) SKBr3 cells were treated with 5 uM gefitinib or in combination with the indicated concentrations of the anti-oxidants Tempol or α-lipoic acid for 48 hours.

Figure 4. Akt regulates HER3 signaling via negative feedback signaling

(A) 2×10⁶ SKBr3 cells were transiently transfected with 5 μg of pcDNA3-myr-Akt plasmid or control pcDNA3 plasmid. The following day 5 μM gefitinib was added for an additional 48h where indicated. Lysates were immunoblotted as indicated. (B) SKBr3 cells were treated with 5 or 20 μM LY294002 for 8h and lysates immunoblotted as indicated.