Bisphosphonates inactivate human EGFRs to exert antitumor actions

Abstract

Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers.

Keywords: Her2/neu; drug repurposing; osteoporosis; receptor tyrosine kinase; tyrosine kinase inhibitor.

Fig. 1.

Bisphosphonates bind to the kinase domain of the HER family. (A) Protein thermal shift uses Sypro Orange to capture hydrophobic domains during heat-induced protein unfolding to yield a Tm. The Tm increases when ligand binds to protein resulting in a thermal shift (ΔTm = Tm_ligand – Tm_veh)._. Risedronate (Ris) or erlotinib (Ert) binding to recombinant HER1^wt or constitutively active HER1^L858R protein causes a significant thermal shift, whereas tiludronate (Til) does not. HER1^{K745A/N842A/D855A}, in which the predicted interacting amino acids are mutated, does not display a bisphosphonate-induced thermal shift: this constitutes biophysical proof for the binding of bisphosphonates to the HER1 kinase domain. With recombinant HER2^wt protein, a second peak is induced upon bisphosphonate binding, again indicative of direct protein–ligand interaction. Tm (°C) is shown on representative traces; ΔTm, Tm, P values (determined by pairwise comparisons using two-tailed Student t test), and number of replicate traces (in parentheses) are shown in the table (Inset). (B) Molecular docking using the X-ray crystal structure of HER1^wt (PDB ID code 2GS7) confirms binding of Ris, but not Til to the kinase pocket. Bisphosphonates containing one or more N-atoms interact with T790 (gatekeeper residue) via a water (WAT) molecule, whereas Til, which has a p-chlorophenyl group, does not (also see Fig. S2). Similarly, the N6 atom of the adenine group of AMP-PNP interacts with T790 via WAT. Superimposition of crystallized AMP-PNP and docked Ris thus shows an overlap of the purine ring of adenine and the pyridine ring of Ris. Activated HER1^L858R (PDB ID code 2ITV) resembles the active state of HER1 (PDB ID code 2GS6) and binds Ris, Ert, and AMP-PNP. Docking also confirms loss of bisphosphonate binding to HER1^{K745A/N842A/D855A}, where substituted Ala residues are unable to coordinate with Mg²⁺. Similar interactions between Ris and zoledronic acid (ZA) occur with HER2; here WAT bridges the bisphosphonates to the gatekeeper residue T798. (C) Ris causes a concentration-dependent inhibition of the kinase activity (in triplicate) of recombinant HER1 and HER2. The inhibition is similar to that with recombinant FGFR, considerably less marked with VEGFR, and virtually absent with the INSR. Correspondingly, the structure of HER1 and FGFR kinase domains superimpose well and residues that coordinate Mg²⁺ are well positioned for bisphosphonate binding. In contrast, the VEGFR structure is in a more open conformation, where D1046 and N1033 are positioned further away from the binding pocket. This shifts Mg²⁺ coordination outwards (arrow) and reduces the bisphosphonate fit. The INSR kinase domain is structurally distinct from that of HER1 and is unable to bind bisphosphonates.

Fig. 2.

Bisphosphonates inhibit cancer cell viability through a HER-dependent mechanism. Effects of bisphosphonates or erlotinib (Ert) (as shown) on cell viability assessed by the MTT assay (mean % ± SEM) in HER1^ΔE746-A750 (HCC827), HER1^wt (H1666 and H1703), and HER1^wt:RAS^G12S (A549) lung cancer cells, as well as in HER1^wt breast cancer (MB231) and HER^low colon cancer cells (SW620) (triplicate wells; repeated three times; statistics by ANOVA with Bonferroni’s Correction against zero-dose; P values versus zero-dose; mean ± SEM; *P < 0.05, **P < 0.01). qPCR was used to determine mRNA expression of all four HER isoforms HER1 to -4 (transcript copy number per cell ± SEM; three biological replicates, with three technical replicates each; estimated using 2,500 copies of β-actin per cell). To study whether the bisphosphonate effect was mediated by one or more HER isoforms, siRNAs for each of the four isoforms (siHER) were applied all at once, with scrambled siRNAs (siScr) as controls. Knockdown of individual HER proteins was confirmed by Western blotting using isoform-specific antibodies SI Methods). MTT assay assessed the viability of siRNA- or siScr-transfected cells in response to zoledronic acid (ZA, 40 µM). The extent of reduction in HER corresponded to the extent of reversal of the ZA effect on cell viability (four biological replicates transfected separately; expressed as mean percent reduction with bisphosphonate ± SEM; statistics: two-tailed Student t test; P values from siScr vs. siHER transfectants; *P < 0.05, **P < 0.01). Of note is that siHER itself reduced viability in all but SW620 cells; results are therefore expressed as a percentage of vehicle-treated siScr and siHER transfectants, respectively. A partial down-regulation of HER1 in MB231 cells corresponds with a partial attenuation of bisphosphonate action. With HCC827 cells, the oncogenic stimulation is because of mutated HER1^ΔE746-A750; hence, knockdown almost abolishes bisphosphonate action, despite a relatively lower extent of HER2 knockdown. Note: Only relevant bands from Western blots are shown, with gaps introduced where irrelevant lanes are excised.

Fig. 3.

Bisphosphonates inhibit global HER signaling to cause cell-cycle arrest and apoptosis. (A) Flow cytometry showing the effect of the bisphosphonate zoledronic acid (ZA) on the cell-cycle profile of HER1^wt (H1666), HER1^ΔE746-A750 (HCC827), HER1^L858R (H3255), and HER1^wt:RAS^G12S (A549) expressing lung cancer cells, as well as HER1^wt-expressing breast cancer (MB231) and HER^low colon cancer (SW620) cells (repeated three times on single samples; colored bars include mean ± SEM). Representative Western blots show PARP cleavage and cyclin D1, cyclin B1, and PCNA protein expression. (B) Downstream signaling pathways triggered by HER1 activation, of which the ones shown in red were examined. ZA inhibited EGF-induced phosphorylation of Y845, Y992 (Western blots), Y1045, Y1068, and Y1173 (ELISA) (two-tailed Student t test; in duplicate; mean fold-change over no-EGF ± SD; *P < 0.05, **P < 0.01). Western blots also show reduced pAKT and pERK in whole cell extracts of HER1^wt-expressing H1666 cells, whereas there was no inhibition in HER^low SW620 cells. Western blots of cytosolic (C) and nuclear (N) subfractions of H1666 and A549 cells show a marked reduction by ZA of pSTAT3, pSTAT5, and p50 in the nuclear subcompartment (normalized to histone-H3). Note: Only relevant bands from Western blots are shown, with gaps introduced where irrelevant lanes are excised.