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Contributed by Maria I. New, November 11, 2014 (sent for review October 2, 2014; reviewed by Wafik El-Deiry and H. Michael Shepard)
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Figures
- Fig. 1.
Bisphosphonates inhibit HER-driven tumor growth in prevention and treatment protocols. (A) Colony formation assays performed with HCC827, MB231, and SW620 cells to study the effect of zoledronic acid (ZA; mean colony counts ± SEM; three experiments with two or three replicate wells pooled). (B and C) Tumor volume was measured sequentially following transplant of HCC827 (lung), MB231 (breast), or SW620 (colon) cancer cells into BALB/c nu/nu mice. Drugs were begun daily by oral gavage once tumors became palpable (treatment; B) or at the time of graft (prevention; C). HCC827 and MB231 tumors showed evidence of reduced growth with Ris (1.42 µg/kg, daily, gavage) or ZA (1.36 µg/kg, daily, gavage), whereas HERlow SW620 cells did not. Change (Δ) in tumor volume plotted for single mice or as group means ± SEM; statistics: ANOVA with Bonferroni’s correction; bisphosphonate- vs. vehicle-treated mice; *P < 0.05; number of mice used for the analysis corresponds to the number of animals shown in the plot for individual tumor volumes, e.g., n = 12 mice in B, Upper, control.
- Fig. 2.
Combinatorial binding of bisphosphonates and TKIs. (A) Docking of ZA in the HER1 kinase crystal structure that was cocrystallized with erlotinib (Ert) (PDB ID code 1M17). The phosphate backbone of ZA interacts with Mg2+, the removal of which prevents ZA docking. ZA also interacts with the NH group between the two aromatic rings in Ert either by itself (as a deprotonated tautomer) or via a structural water (WAT) (as a protonated tautomer). Ert associates with T790 via WAT. Fluid phase: binding mode of ZA and Ert observed from the solvent. Tiludronate (Til) does not have an imidazole ring to make an H-bond with Ert. It instead contains a p-chlorophenyl that results in severe steric clashes (green sphere) with Ert. ZA docked together with gefitinib (Gef) (PDB ID code 2ITY) and binds in a similar orientation to that observed in the ZA–Ert complex, as a deprotonated (pink dashed line) or protonated (orange dashed line) tautomer. Minodronic acid (MA) can likewise bind with Ert as a deprotonated or protonated tautomer. (B) Molecular docking reveals that the HER1L858R mutant conformation (cyan) is similar to the active state of HER1wt (yellow) in presence of Ert and ZA. However, the simultaneous binding of Ert and ZA inhibits the kinase by preventing the downstream phosphorylation because of the absence of a hydrolysable ɤ-phosphate. ANM of the HER1L858R mutant with Ert and ZA. Eigenvectors highlighting conformational fluctuations in the Cα-helix and activation loop (A loop) are shown. Of note is that, despite the presence of Ert and ZA, the Cα-helix is in a collapsed conformation. MD confirms ANM findings showing that, in presence of Ert and ZA, the interaction between R858 and Y891 locks the activation loop allowing the Cα-helix to collapse.
- Fig. 3.
Combination of bisphosphonate and TKI causes tumor regression. (A) Effect of relotinib (Ert), zoledronic acid (ZA), or Ert plus ZA on tumor volume in BALB/c nu/nu mice grafted with HCC827 cells [Waterfall plot or mean change (Δ) in tumor volume in mouse groups, versus DMSO]. Whereas Ert and ZA prevented tumor growth, the two drugs in combination caused tumors to regress. (B) Apoptotic cells stained with TUNEL (green) are shown in representative sections (Upper) and as cell number (Lower; percent of total cells; Box plots with upper and lower quartiles and range). Statistics: Two-tailed Student t test with Bonferroni’s correction; *P < 0.05, **P < 0.01; n = 8 mice per group. (C and D) Immunolabeling for pHER1, pAKT, and pERK (counterstained with hematoxylin/eosin) (D) confirmed by Western blotting for phosphorylated (p) and total (t) ERK1/2 and AKT (on representative tumor tissue displaying median volume) (C). Note: only relevant bands from Western blots are shown, with gaps introduced where irrelevant lanes are excised (SI Methods). (Magnification: B and D, 20×.)
- Fig. 4.
HER1 mutation T790M abolishes erlotinib but not bisphosphonate sensitivity. (A) Influence of the gatekeeper T790M mutation on the conformation of the HER1wt and HER1L858R. The HER1T790M mutation (PDB ID code 2JIU) allows only a partial collapse of the Cα-helix as mutated M790 acts like a wedge to obstruct the position of M766, a highly conserved residue throughout the kinase family. In HER1wt (orange), M766 and T790 are far apart. In HERT790M (blue), although the tilt angle of Cα-helix is ∼15°, the position of M766 is still tolerated by M790. Thus, erlotinib can bind to HER1T790M. In the HER1L858R mutant (cyan), the observed tilt angle is ∼23°; this positions M766 too close to T790. In the double-mutant HER1L858R/T790M, we therefore predict that the tilt caused by the HER1L858R activating mutation will be obstructed by M790, preventing total Cα-helix collapse. MD on HER1L858R/T790M in complex with ATP confirms that movement of the mutated activation loop allows the Cα-helix to assume an activated conformation, but prevents complete collapse of the helix over the binding site. However, the T790M mutation does not affect the binding mode of ZA’s imidazole ring by preserving the water molecule (WAT) that bridges ZA to T790 and T854. (B) Although Ert and tiludronate (Til) do not inhibit colony formation in HER1L858R/T790M (H1975) cells, ZA displays a strong inhibitory effect (mean colony counts per well ± SEM; two tailed Student t test with Bonferroni’s correction, versus zero dose; *P < 0.05, **P < 0.01; repeated three times, each in duplicate, data pooled). Furthermore, ZA inhibits H1975 cell viability (MTT assay). In contrast, Ert neither itself inhibits nor enhances the inhibitory action of ZA (unlike its effect in HER1L857R cells) (triplicate wells, done three times, data pooled; mean ± SEM; ANOVA with Bonferroni’s Correction, versus zero-dose; *P < 0.05, **P < 0.01; or combined treatment versus Ert; ^^P < 0.01). Western blots (biological quadruplicates) showing the inhibitory effect of alendronate (Aln) on EGF-induced phosphorylation of HER1L858R/T790M (pHER1) (β-actin and tHER1 as controls; versus without Aln; statistics by two-tailed Student t test; **P < 0.01, n = 4). Flow cytometry showing cell-cycle profile of H1975 cells in response to ZA, which stimulates apoptosis (repeated three times). Western blots showing the effect of ZA on PARP, pAKT, cyclin D1, cyclin B1, and PCNA (GAPDH: loading control; repeated three times).
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