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Maisel et al. J Cancer Metastasis Treat 2019;5:7 I http://dx.doi.org/10.20517/2394-4722.2018.82 Page 5 of 14

in organ morphogenesis, maintenance and repair pathways, tumor progression, and metastasis through
[65]
upregulated EGFR signaling . EGFR signaling is potentiated by phosphorylation of tyrosines found in the
cytoplasmic domain, whose activation is determined by ligand and dimerization partner. Phosphorylated
tyrosines act as docking sites for many proteins and interactors, such as Src homology 2 or phosphotyrosine
binding domain-containing proteins, including growth factor receptor-bound protein 2, Src-homology
and collagen, PLCγ, PI3K subunit p85, GTPase activating protein, Cbl, and adaptor protein-2 (AP-2). EGFR
phosphorylation remains active in endosomal vesicles, where EGFR is hyperphosphorylated, allowing for
continued signal transduction [66-70] . Signaling through PLC has been shown to be preferentially activated
[71]
when at the cell surface, while MAPK signaling continues to occur while in intracellular vesicles .
[66]
PI3K signaling is also preferentially activated in endosomes, as Vieira et al. demonstrated PI3K activation
is unnecessary for internalization and instead plays a role in endosomal trafficking of EGFR. Similarly,
[67]
Garay et al. indicated preferential AKT signaling when EGFR is internalized in clathrin-coated vesicles
rather than at the plasma membrane through siRNA against clathrin.

Vesicular EGFR alternatively trafficked away from the lysosome (demonstrated with a SNX1-directed
[72]
siRNA) results in augmented AKT signaling in non-small-cell lung cancer , and we have previously
demonstrated that endosomally retained and mislocalized EGFR is capable of upregulating AKT activity,
resulting in increased metastatic potential, as well increased nuclear localization of transcription factor TAZ
(transcription co-activator with a PDZ-binding domain), an event associated with cancer stem cells [42,73] .
AKT is involved in driving EMT through disassembly of cell-cell junctions and upregulation of SNAIL,
[74]
confirmed by loss of EMT phenotype in prostate cancer cells when treated with AKT inhibitors . AKT1
kinase activity and AKT2 overexpression are associated with ovarian, breast, and thyroid cancers - three
cancer types commonly hallmarked by high EGFR expression [75-81] . While AKT activation is strongly
associated with the inhibition of apoptosis, at least one study has found endocytosed EGFR that does not
[82]
traffic to the lysosome correlates with an induction of apoptosis . More work to determine the mechanisms
of these events will be required to fully understand these processes.

Though EGFR is primarily targeted to the lysosome to prevent constitutively activated signal transduction,
when stimulated by EGF, it has been shown that a fraction of EGFR will be transported to the Golgi and the
ER, carried via COP-I vesicles en route to trafficking to the nucleus [83,84] . Studies have shown that after 20
min of EGF stimulation, 10 percent of EGFR colocalizes to the Golgi and remains phosphorylated, even in
the presence of protein synthesis inhibitors, indicating that the retrograde trafficking observed is not due to
[85]
post-translational modifications .

Upon translocation to the Golgi, the amino-terminal domain of EGFR sits within the lumen of the Golgi,
[84]
leaving the carboxy-terminal domain exposed in the cytoplasm to interact with importinβ-1 via the NLS .
All ErbB family members can enter the nucleus, with full-length, phosphorylated EGFR entering via the
Sec61 translocon and interacting with importin α-β1 complexes [43,86,87] . EGFR is capable of transactivational
[88]
activity, but lacks a DNA-binding domain, providing a need for transcription co-factors . Upon entry
into the nucleus, EGFR can associate with transcription co-factors such as signal transducer and activator
of transcription 3 (STAT3) to co-regulate inducible nitric oxide synthase expression and E2F1 to promote
cell cycle progression, as well as the proliferation marker Ki-67 [88-90] . Nuclear EGFR interactions with
proliferating cell nuclear antigen (PCNA) also promote cell cycle progression, while increasing PCNA
phosphorylation and the DNA damage response [91,92] . Nuclear EGFR has been found to be associated with
more than 40 percent of breast cancer tumors and 35 percent of esophageal squamous cell cancers, correlated
to shorter overall survival in patients and increased metastatic potential, respectively [93,94] . Nuclear EGFR
also contributes to cetuximab and gefitinib resistance, likely due to the restricted access of the therapeutics
to the target protein [41,85] . Finally, the role of retrotranslocated EGFR as a transcriptional co-factor further
exemplifies its kinase-independent function, highlighting the need for therapeutically targeting this process.