EGFR transactivates RON to drive oncogenic crosstalk

Abstract

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation – arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.

Pervasive aggregation and depletion of host and viral proteins in response to cysteine-reactive electrophilic compounds

“…More broadly, we observe formation of stress granules in response to nearly every cysteine-reactive compound evaluated, including the drugs afatinib and auranofin, with the latter recently implicated in increasing nuclear H 2 O 2 levels 147 . Notably, afatinib- induced stress granules were only observed to occur at 10 µM, which, while a dosage that is reported in a number of prior studies 158–163 , is >50-fold above 175 the dose require to effect full covalent modification of EGFR 84 . The parallel recruitment of proteasome subunits into stress granules, which aligns with prior reports of stress-induced phase separation of the proteasome 167 , allows us to put forth a model whereby covalent modifiers cause widespread protein aggregation and subsequent proteasome-mediated degradation, driven by elevated local concentrations of hyper-active proteasomes.…”

“…Thus, we further broadened the scope of compounds assessed to include RA190, which, like other cysteinereactive electrophiles, increased proteasome activity in our proteasome activation assays (Figure S12A), bardoxolone, a widely utilized NRF2 activator 154 , sulforaphane, a covalent modulator of the ubiquitin-proteasome and autophagic pathways with anticancer activity 155 , as well as chemotherapeutic agents such as FDA-approved ibrutinib and afatinib, which are covalent kinase inhibitors, auranofin, which targets TXNRD1/2 156,157 and was recently reported to increase the activity of the DNA damage checkpoint kinase CHK1 via oxidation of an allosteric cysteine residue 147 . We subjected cells to compound dosing consistent with prior reports-for afatinib and ibrutinib we chose to treat with 10 µM compound, which, while a dosage used widely [158][159][160][161][162][163] are in substantial excess of those required to achieve full target engagement 84 . With the exception of ibrutinib, all cysteine-reactive compounds induced G3BP puncta consistent with SG formation (Figure 6B, S31A).…”

Crosstalk of growth factor receptors at plasma membrane clathrin-coated sites

“…The fact that drugs against either receptor influenced the behavior of the other point to direct signaling through binding or phosphorylation. Interestingly, past work has shown a direct interaction between EGFR and another RTK RON ( 16 ). Clathrin-coated pits are relatively small structure of ~100 nm in diameter.…”

“…While receptors have been mostly studied in isolation, it is becoming clear that different members of the RTK family can crosstalk with other receptors (14)(15)(16). For example, EGFR inhibitors were identified in a screen for drugs that improve outcomes in cancers driven by the fibroblast growth factor receptor (FGFR) (17).…”

Overexpression of ErbB-1 (EGFR) Protein in Eutopic Endome-Trium of Infertile Women with Severe Ovarian Endometriosis during the ‘Implantation Window’ of Menstrual Cycle

“…6). Such expression pattern of ErbB-1 induces higher proliferative capacity, increased vessel density, cellular atypias, high mitotic activity, and distinctive infiltrative phenotype in both types of tissues, and these changes may bring forth their oncogenic potential [57,[60][61][62][63][64][65]. The observed cytoplasmic and nuclear distribution of ErbBs, especially for ErbB-1 and ErbB-3, besides their membranous localization in the eutopic endometrium during endometriosis may trigger pathogenic potential of eutopic endometrium [66][67][68][69][70].…”