The approach described here is a departure from traditional methods used to influence HER receptor signaling. Small molecule inhibitors target kinases, whereas antibody-based approaches target receptor ectodomains and ligands (5
). Small molecule approaches also have risks of off-target toxicity, whereas antibody-based approaches are limited by affinity maxima, high dosing requirements, and relatively large molecular weights (~150 kDa) (58
). Therapeutic options are further limited for HER3 as it lacks significant kinase activity; hence, we focused our attention on this important target and exploited this deficiency. Although it is arguably counterintuitive to silence a pathway by engaging the receptor with ligand, it is possible that, compared with function-blocking antibodies, bivalent ligands (particularly NN) may exhibit some therapeutic advantage; e.g.
by engaging the receptors in more favorable steric associations for inhibiting interactions with other partners, altering internalization rates, or through improved tumor-penetrating properties.
Previous attempts to force hetero- and homodimerization of EGFR and HER2 showed altered proliferation and migration depending on the type of receptor dimer formed (59
). However, that approach relied on expression of modified receptors with rapamycin binding domains to force particular receptor pairings. The approach we describe alters receptor interactions and biases signaling with an exogenous method that does not require genetic intervention. Another report describes exogenous application of a surface-bound dimeric EGF ligand for selective expansion of neural stem cells (60
). In this study we demonstrate enhanced versatility of bivalent ligands by incorporating NRG moieties and provide a conceptually new application, inhibition of signaling. Furthermore, we establish the potential to selectively bias signaling in a particular cell type, based on its HER-family receptor expression profile, and to potentially apply bivalent ligands in a therapeutic context.
There are several possible mechanistic interpretations for how NN, which binds to HER3 (and HER4), inhibits receptor activation, downstream signaling, and phenotypic behaviors. A bias toward HER3 homotypic interactions by NN is arguably anticipated. Several lines of evidence support the idea that HER3 exists in a closely clustered state before ligand binding, possibly in association with HER4 (9
), and all cell lines used in this study exhibited low levels of HER4 compared with HER3 (supplemental Fig. S2
). Hence, bivalent NN might be able to trap HER3 receptors, either linking them as dimers or possibly resulting in a concatenation of cross-linked dimers as shown in B
. This interpretation is supported by two consistent observations in our studies, which is that biasing and/or inhibitory effects of NN increased with dose and that low doses of NN produced similar results to corresponding doses of native, monovalent NRG. At low NN doses, where HER3 receptors may not be saturated and/or complete concatenation may not occur, HER3 may still be available for interaction with HER2, as would be the case with stimulation by native NRG. Upon increasing the dose of NN, if the proposed hypothesis is true, complete concatenation or saturation of HER3 by NN would prevent interaction with HER2, and thus result in the differential signaling and phenotypic outcomes from NRG that were observed in this study. In this case, the efficacy of NN in biasing signaling would be dependent on the relative numbers and locations of HER receptors, disparities in which may explain the different effects observed in different cell lines with similar gross HER receptor profiles in this study. It is also notable that ligand-induced dimerization/homotypic association of HER3 has rarely been reported, especially in a cellular context (39
). Ligand binding is believed to unlock HER3 from the clustered, inhibited state, allowing it to find and associate with HER2 (or other partners). When HER3 partners with HER2, activation of signaling likely requires tetramers or higher order oligomers to overcome the kinase deficiency of HER3, although detailed structures and mechanisms of this activation process are still not clear (37
). It is, therefore, possible that bivalent NN could link two HER3 receptors facing each other, leaving the dimerization face available for interaction with HER2 or other partners (i.e.
instead of the concatenation with HER3, as shown in B
). If such “solo, facing-each-other” dimers are indeed formed, they are likely constrained from proper (activating) interactions with HER2, as evidenced by the lack of HER3-mediated signaling observed in the presence of NN.
In contrast to the near-complete inhibition of receptor activation and signaling observed with NN, our more limited study of bivalent EE revealed that it activated cognate receptor EGFR and only partially biased activation away from HER2 (E
). These results are consistent with a mechanistic model in which EE may opportunistically bind to a variety of pre-formed EGFR states (homodimers, oligomers, and heterodimers) that have been reported by others to exist in dynamic equilibrium on the cell surface (9
). Thus, EE may serve to stabilize dimers or concatamers (D
) upon binding to homotypically pre-associated EGFR via both EGF moieties and also may act as a monomeric ligand upon binding to EGFR that is not homotypically pre-associated. It is also possible that EE induces a solo, facing-each-other dimer that leaves the dimerization face available for interaction with HER2.
Potential mechanistic interpretations notwithstanding, these studies suggest that a bivalent NN ligand could have therapeutic applications. Recent findings point to HER3 interactions with c-MET as a mechanism for therapeutic resistance after treatment with various chemotherapeutic agents (19
). Furthermore, HER3- and/or HER4-mediated autocrine signaling may play an important role in certain types of ovarian cancer (64
). In that regard inhibition of proliferation of H1975 cells by NN is particularly interesting, as H1975 express HER4 at detectable levels (55
) and would thus presumably bind NN through both HER3 and HER4. HER4 responses can promote proliferation of breast (48
) and lung (56
) cancers; thus, the ability of NN to inhibit malignant phenotypes in cancer cells that co-express HER3 and HER4 is an encouraging observation. The therapeutic implications of the EGF-containing bivalent ligands are less clear. HER2 is implicated as an important partner in the pathogenesis of certain EGFR-overexpressing tumors (65
), as HER2-EGFR dimers more strongly activate malignant signaling pathways compared with EGFR homodimers (66
). As opposed to NN, EE and EN may bias signaling to alternative productive pathways, making them less promising to pursue as possible chemotherapeutics but of interest for future studies on phenotypes relevant for regenerative medicine. More broadly, bivalent ligands may be deployed as a new tool for dissecting the complex systems biology of the EGFR family in developmental biology, tissue regeneration, wound healing, and other physiological and pathophysiological processes (67