EGF family hormones stimulate distinct biological responses: Implications for tumorigenesis and cancer treatment

Kristy J Wilson, Purdue University

Abstract

Breast, prostate, pancreatic, colorectal, lung, and head and neck cancers exploit deregulated signaling by ErbB family receptors. ErbB family receptors are activated by the fifteen peptide hormones that comprise the EGF family. The fundamental question is why are there so many EGF family hormones. One possible explanation is varied affinities for the ErbB receptors. But this fails to account for ligands that have similar affinities but still differentially stimulate biological responses. We hypothesize that EGF family ligands differ in intrinsic activity. Intrinsic activity is the maximal biological effect stimulated by a ligand. So, we propose to compare the intrinsic activity of EGF family hormones and to understand the mechanism underlying differences in intrinsic activity. Numerous EGF family peptides activate EGF receptor (EGFR). We examined these peptides in 32D/EGFR myeloid and MCF10A breast cells and found two distinct classes of ligands. Amphiregulin (AR), transforming growth factor alpha, neuregulin 2 beta (NRG2β), and epigen stimulate greater EGFR coupling to cell proliferation and DNA synthesis than do EGF, betacellulin, heparin-binding EGF-like growth factor, and epiregulin. Thus, EGF family ligands stimulate different biological responses through the same receptor. We hypothesized that agonists with lower intrinsic activity would antagonize agonists with higher intrinsic activity. Indeed, EGF competitively antagonizes AR, indicating that their functional differences reflect dissimilar intrinsic activity at EGFR. We hypothesized that the mechanism for differences in intrinsic activity is changes in the pattern of receptor phosphorylation and activation of signaling effectors. The literature indicates that EGFR Tyr992 is phosphorylated following AR and EGF stimulation. We hypothesized that phosphorylation of Tyr992 and the subsequent binding and activation of PLCγ would be necessary for AR stimulated proliferation. Indeed, the EGFR Y992F mutation and the PLCγ inhibitor U73122 preclude AR stimulation of cell proliferation. But neither the EGFR Y992F nor the PLCγ inhibitor affects EGF stimulation. These data suggest that PLCγ signaling is necessary for AR stimulated proliferation. Phosphorylation of Tyr1045 creates a binding site for the ubiquitin ligase c-cbl, leading to EGFR ubiquitation and degradation. The literature indicates that EGF stimulates greater Tyr1045 phosphorylation than AR. Thus, we hypothesized that Tyr1045 and c-cbl are in part responsible for the differences in intrinsic activity of EGFR agonists. The EGFR Y1045F mutation allowed EGF to stimulate proliferation in 32D/EGFR cells. Likewise, expression of z-cbl (dominant negative mutant of c-cbl) allows EGF to stimulate proliferation. These data suggest that EGFR degradation and signaling duration of PLCγ regulate EGFR ligand intrinsic activity. Thus, divergence in the sets of EGFR tyrosine residues that are phosphorylated upon stimulation with different ligands appears to underlie differences in intrinsic activities. The differences in the intrinsic activity of EGF family hormones at EGFR are not atypical. The NRG2 gene encodes multiple splicing isoforms that differ only in the carboxyl-terminus. NRG2β stimulates ErbB4 coupling to proliferation; in contrast, the NRG2α fails to stimulate ErbB4 coupling. As previously demonstrated, the NRG2α K45F mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to proliferation. Here we investigate the regulation of NRG2 intrinsic activity. The NRG2β Q43L mutant potently stimulates ErbB4 phosphorylation but not ErbB4 coupling to proliferation. In contrast, the NRG2α L43Q/K45F mutant stimulates proliferation, even though it does not have greater affinity for ErbB4 than does NRG2α/K45F. Collectively, these data indicate that Gln43 of NRG2β is both necessary and sufficient for NRG2 stimulation of ErbB4 coupling to proliferation. We hypothesized that NRG2α/K45F and NRG2β/Q43L would antagonize NRG2β stimulated proliferation because these point mutants bind to the receptor but fail to stimulate biological effect. Indeed, here we demonstrate that both NRG2α/K45F and NRG2β/Q43L antagonize NRG2β stimulated proliferation. We demonstrate that increased concentrations of the agonist NRG2β can overcome the effects of NRG2β/Q43L and NRG2α/K45F. This indicates that these mutants competitively inhibit agonist stimulation of ErbB4 coupling to cell proliferation. We hypothesized that the mechanism underlying the functional differences of NRG2 isoforms and mutants is changes in the activation of signaling effectors. The antagonists NRG2α/K45F and NRG2β/Q43L stimulate less Akt phosphorylation than do the agonists NRG2β and NRG2α/L43Q/K45F. This indicates that the ability to activate Akt may differentiate between agonists and antagonists. The ErbB4 Ct-b isoform lacks a WW binding motif for the transcription regulator YAP and does not allow NRG2β to stimulate proliferation. Thus, the antagonistic activity of NRG2β/Q43L and NRG2α/K45F mutants may be due to their reduced stimulation of the PI3 kinase/Akt signaling pathway and of the ErbB4 effector YAP. NRG2β is also an agonist for EGFR and ErbB3; thus, we hypothesized that NRG2β/Q43L would fail to stimulate receptor coupling to proliferation. Indeed, NRG2β/Q43L does not stimulate EGFR or ErbB3/ErbB2 coupling to proliferation. Moreover, NRG2β/Q43L competitively antagonizes agonist-induced coupling of EGFR or ErbB3 to cell survival or proliferation. These data are the first evidence that mutants of EGF family ligands may antagonize agonist-induced ErbB receptor signaling. Thus, these data suggest a novel paradigm for cancer drug discovery targeted to ErbB receptors.

Degree

Ph.D.

Advisors

Riese, Purdue University.

Subject Area

Molecular biology|Pharmacology

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