Autophosphorylation of the carboxyl‐terminal domain is not required for oncogenic transformation by lung‐cancer derived EGFR mutants

Abstract

Aberrant activation of cancer‐derived mutants of the epidermal growth factor receptor (EGFR) is closely associated with cancer pathogenesis and is thought to be mediated through multiple tyrosine phosphorylations within the C‐terminal domain. Here, we examined the consequences of the loss of these C‐terminal phosphorylation sites on cellular transformation in the context of lung‐cancer‐derived L858R, exon 19 deletion and exon 20 insertion mutant EGFR. Oncogenic EGFR mutants with substitution of the 10 potential C‐terminal tyrosine autophosphorylation sites for phenylalanine (CYF10) were still able to promote anchorage‐independent growth in soft agar at levels comparable to the parental L858R or exon19 deletion or exon 20 insertion mutants with intact autophosphorylation sites. Furthermore, these CYF10 mutants retained the ability to transform Ba/F3 cells in the absence of IL‐3. Bead‐based phosphorylation and immunoprecipitation analyses demonstrated that key EGFR‐associated proteins—including Grb2 and PLC‐γ—are neither phosphorylated nor bound to CYF10 mutants in transformed cells. Taken together, we conclude that tyrosine phosphorylation is not required for oncogenic activity of lung‐cancer‐derived mutant EGFR, suggesting these mutants can lead to cellular transformation by an alternative mechanism independent of EGFR phosphorylation.

Kinase-Inactivated EGFR Is Required for the Survival of Wild-Type EGFR-Expressing Cancer Cells Treated with Tyrosine Kinase Inhibitors

“…All the results combined suggest that TKI-induced kinase-inactivated EGFR dimers have a critical function in sustaining survival of cancer cells in both intrinsic and acquired resistances. In accordance with our previous studies, the pro-survival role of TKI-induced kinase-inactivated EGFR dimers can be attributed to its scaffolding function, which is involved in interacting with and stabilizing other pro-survival proteins [4,27,28,29,30,31,32]. This provides a mechanistic explanation for the lack of therapeutic efficacy associated with TKIs as its main function is to inhibit EGFR’s kinase activity without affecting EGFR’s kinase-independent function.…”

“…Studies have shown that EGFR possesses pro-survival function, independent of its canonical kinase activity, as a scaffold protein interacting and stabilizing key survival proteins such as sodium/glucose co-transporter (SGLT1) to maintain glucose uptake of cancer cells [4], p53-upregulated modulator of apoptosis (PUMA) to repress apoptosis [27], lysosomal-associated transmembrane protein 4B (LAPTM4B) to promote pro-survival autophagy [28], fatty acid synthase (FASN) to promote de novo fatty acid synthesis [29], system x c −antiporter to maintain cysteine import [30], and mammalian target of rapamycin complex 2 (mTORC2) to inhibit mitophagy [31]. In addition, recent study has found that the C-terminus non-phosphorylatable EGFR mutant is oncogenic [32], clearly suggesting that the oncogenic potential of EGFR resides in its kinase-independent functions. Moreover, downregulation of EGFR protein causes cancer cell death through induction of mitophagy [31].…”

Defining molecular risk in ALK+ NSCLC

“…However, there are important differences. First, while in EGFR + NSCLC the oncogene variants, such as exon 19 indels, L858R, “rare” point mutations and exon 20 insertions, cause a largely similar oncogenic drive [23-25], which nevertheless translates into a different clinical course only after institution of EGFR-directed therapies due to differential TKI sensitivity [26], the unfavourable EML4-ALK V3 variant in ALK + NSCLC has a different biology per se. There is evidence that the shorter V3 oncoprotein is more stable [11, 27, 28], causes stronger ALK phosphorylation [11] and promotes cell motility and metastasis more efficiently [13, 16], resulting in a higher a priori clinical risk [29].…”

Mechanistic insights into differential requirement of receptor dimerization for oncogenic activation of mutant EGFR and its clinical perspective

“…In contrast, introduction of the same dimerizationdisruption mutations into the dimerization-independent group of EGFR mutants had little effect on their ability to induce colony formation under the same experimental condition, suggesting that this group of mutants can indeed result in cellular transformation irrespective of dimerization (25). Notably, these unexpected results are further supported by a recent report showing that C-terminal autophosphorylation of dimerization-independent mutant EGFR such as Ex20Ins is not required for oncogenic transformation (37). One proposed explanation for this result is that Gab1/2, Shc1 and Bcar1 adaptors may interact in a mutant-EGFR-specific manner and function as crucial factors in mediating constitutive oncogenic activation of various signaling pathways independently of asymmetric dimerization and C-terminal phosphorylation (37).…”

“…Notably, these unexpected results are further supported by a recent report showing that C-terminal autophosphorylation of dimerization-independent mutant EGFR such as Ex20Ins is not required for oncogenic transformation (37). One proposed explanation for this result is that Gab1/2, Shc1 and Bcar1 adaptors may interact in a mutant-EGFR-specific manner and function as crucial factors in mediating constitutive oncogenic activation of various signaling pathways independently of asymmetric dimerization and C-terminal phosphorylation (37).…”