Discovered in 1965 by Cohen [1], the epidermal growth factor receptor (EGFR) is a member of the transmembrane tyrosine kinase human EGFR (HER)/ErbB family. The HER family of receptors are potent drivers of growth, proliferation, and migration [2] and consist of EGFR, ErbB2, ErbB3, and ErbB4. While essential for the development and maintenance of normal epithelial tissue, changes to EGFR biology including overexpression/amplification [3, 4], mutation [5, 6], and altered trafficking [7, 8], allow aberrant signaling and functions leading to cancer and other diseases. As a transmembrane tyrosine kinase, therapeutic interventions for EGFR-driven cancers have largely focused on targeting via monoclonal antibodies and tyrosine kinase inhibitors (TKIs). These therapies have shown some success, as in EGFR overexpressing head and neck [9], and lung adenocarcinoma [10]. Although these treatments are initially effective, the majority of tumors eventually acquire resistance via several mechanisms [11].

In TNBC, EGFR is frequently found amplified and overexpressed, but not mutated. In these tumors, it is observed in intracellular locations in addition to the plasma membrane [45]. Several protein interactions and regulatory events are required for altered intracellular localization of EGFR to occur, including the complexing of EGFR and MUC1 (model of trafficking; Figure 1). MUC1 is comprised of a C-terminal cytoplasmic tail, including a transmembrane and small extracellular domain, that is bound through cysteine bonds to the heavily O-glycosylated N-terminal extracellular domain [46]. In healthy epithelium, MUC1 is localized to the apical domain and acts as a protective barrier against infection and inflammation [47]. However, during cancer progression MUC1 regulation is altered, resulting in its overexpression and gain of several oncogenic functions [48]. Concurrent with MUC1 overexpression during tumorigenesis, a loss of cellular polarity across the epithelium occurs. Loss of polarity allows traditionally apical and basolateral localized proteins to interact, as is the case with EGFR and MUC1. The interaction of MUC1 and EGFR at the plasma membrane inhibits the ubiquitylation of EGFR [49]. This interaction increases the rate of receptor internalization into the early endosome (EE) and is paired with a loss in degradation. Under these conditions, EGFR is found to interact with the SNX, specifically SNX1 which directly binds EGFR and regulates its intracellular trafficking [50] (Figure 1).

The sorting nexin family is a diverse group of peripheral membrane proteins that are classified based on the SNX-phox homology (PX) domain [51]. The PX domain of the SNX family is a phosphatidylinositol 3-phosphate (PI3P) binding motif, with minimal affinity for PI2P, that allows recruitment to membranes throughout the endosomal network and plasma membrane [52]. Initial studies suggested that SNX1 enhanced the degradation of EGFR when expressed in cells that did not normally express SNX1 [50]. This is likely due to the ability of SNX to elongate tubule vesicles out of domains such as the EE [53]. SNX, including SNX1, can associate their cargo with the retromer complex, a heterotrimer of proteins required for sorting (Vps26, Vps29, and Vps35). This complex is involved in cargo recognition at the endosomal network and sorting for retrograde trafficking. Unlike the SNX which directly binds the membrane via its PX domains, the retromer has no membrane binding domains. Instead of directly binding the membrane, the retromer complex binds sorting proteins like Rab7 or the SNX, as they are recruited to late endosomes [54] (Figure 1). Together, SNX proteins recognize and bind cargo proteins such as EGFR, induce the formation of vesicles, associate with the retromer complex, and route cargo for retrograde trafficking.

EGFR has several roles in the nucleus that can be either kinase dependent or more often kinase independent (Figure 2). While EGFR has no DNA binding domain, it can act as a co-transcriptional regulator for a growing list of oncogenes through associations with several nuclear proteins [55] (Figure 2). Nuclear EGFR (nEGFR) has been shown to interact with the transcription factors E2F1, STAT3, and STAT5 as well as RNA helicase A [56–59]. nEGFR can induce the expression of cyclinD1 [60], nitric oxide synthase (iNOS) [57], B-Myb [56], Aurora kinase A (Aurora-A) [58], cyclooxygenase-2 (COX-2) [61], c-Myc [62], and the breast cancer resistance protein, breast cancer resistance protein (BCRP) [63].

nEGFR has also been linked to resistance to radiotherapy through the activation of DNA repair pathways [64]. Infrared (IR) radiation can induce nuclear localization and interaction with DNA-PK and induction of DNA repair [65] and DNA repair can be eliminated by blocking retrograde trafficking and the nuclear accumulation of EGFR [66] (Figure 2). In another study, EGFR was shown to be recruited to the nucleus and aid in DNA repair in response to cisplatin treatment [67]. nEGFR has also been shown to modulate the epigenetic landscape by phosphorylating histone 4Y72 [68] (Figure 2). This phosphorylation increases the recruitment of the histone methyltransferases SET8 and SUV 4-20H leading to an increase in DNA transcription and repair.

The non-canonical trafficking and activities of EGFR may drive resistance to conventional therapies in several ways. The presence of EGFR within intracellular endosomes or the nucleus would prevent cell impermeable antibodies from reaching the receptor. Further, antibodies like cetuximab which act by competitively inhibiting the ligand binding pocket would not be effective as the ligand is already bound, and receptor activated and internalized [12]. Antibodies, like nimotuzumab, that act by recruiting immune cells would also be ineffective as they need to be on the cell surface to recruit immune cells [18]. TKIs on the other hand are freely cell permeable and inhibit EGFR activation through competitive inhibition of the ligand binding pocket. However, the noncanonical roles of nEGFR also evade TKI therapies as many of these roles may not involve EGFR’s kinase function. Instead, nEGFR can directly bind transcription factors within the nucleus and alter their DNA binding profiles to induce a novel suite of oncogenic protein expression. The shortcomings of conventional therapies to target intracellular and nEGFR highlight the need for new and different therapies that can inhibit the nuclear roles of EGFR in TNBC.

The nuclear accumulation of EGFR represents an ideal target for therapeutic intervention as it appears to rarely occur outside the context of cancer, yet drives tumor progression, therapeutic resistance, and correlates with worse patient outcomes [69–71]. nEGFR has been shown to drive a growing list of oncogenes when associated with transcription factors in the nucleus, as well as modulate the epigenetic landscape to favor tumor progression [56–58, 60–62, 65, 66, 68]. The nuclear localization of EGFR has been shown to correlate with worse prognosis in an ever growing list of cancer including, breast [71], colorectal [70], ovarian [72], lung [73], oropharyngeal [74] and laryngeal [75]. Importantly, these studies demonstrate that nEGFR levels are a better predictor of outcomes than surface or whole cell EGFR levels. This presents an opportunity to develop therapeutics to block the nuclear functions of EGFR, to make clinical impact in TNBC and other tumors where EGFR undergoes retrograde trafficking.

Several therapeutic agents have been developed to target the retrograde trafficking of EGFR, primarily by blocking one of several protein interactions which drive this trafficking. At the plasma membrane in unpolarized epithelia during tumor progression, EGFR forms a complex with MUC1. This complex initiates a series of events that drive EGFR retrograde trafficking and has been targeted with several peptide-based inhibitors. These therapeutic peptides typically mimic some portion of the binding interface to act as a competitive inhibitor once introduced to the cell. However, therapeutic peptides are typically too large and charged or polar to be readily cell permeable. To allow cell penetration, a protein transduction domain is added to these peptides, allowing them to freely enter the cell [76].

The Schroeder lab and others have previously produced therapeutic cell penetrating peptides that target the interaction of EGFR and MUC1. The first peptide generated, PTD4-MUC1-inhibitory-peptide (PMIP), based on the cytoplasmic domain of MUC1 which interacts with EGFR, initiating its retrograde trafficking [77] (Table 1). PMIP was shown to reduce EGFR levels in cell culture and in the mouse mammary tumor virus-driven polyoma middle T-antigen (MMTV-PyV MT) mouse model of breast cancer. Along with the reduction of EGFR levels, a reduction in tumor growth and an induction of caspase3 cleavage was observed, suggesting these tumors were undergoing apoptotic cell death. Clinical translation of this peptide was not possible, as it proved to be unstable in high concentrations (data not shown). Another series of peptides were generated to target the cytoplasmic domain of MUC1 called GO-201 [78], GO-202 [78], GO-203 [79] (Table 1). These peptides mimicked regions of the intracellular domain and induced tumor regression in several xenograph models of breast, prostate cancer [80], and NSCLC [81]. The GO-203 peptide was then reformulated into a nanoparticle containing several 203 peptides called GO-203-NP which increases efficacy of GO-203 by increasing the cellular uptake of GO-203 [82]. Phase 1/2 clinical trial results demonstrate efficacy for GO-203-2C, a peptide that blocks MUC1 dimerization, in combination therapy with decitabine in acute myeloid leukemia (AML) patients [83].

Alternatively, an EJ-1 was generated to interfere directly with the nuclear localization sequence of EGFR [84] (Table 1). The JXM, region of EGFR, is a significant regulator of EGFR biology as it contains a nuclear localization sequence [87], calmodulin-binding domain [88], dimerization domain [89], and basolateral targeting domain [90]. Not only is this region critical for EGFR regulation, but it is also structured as an alpha-helix, and the resulting EJ-1 peptide was far more stable than PMIP. To increase the stability further, this peptide was stapled by substituting 2 residues with non-natural amino acids that were covalently linked through a hydrocarbon linker arm [85]. The resulting stapled peptide, SAH5, had greater stability and was more potent than EJ-1 (Table 1). SAH5 significantly reduced the growth of SUM149, an inflammatory TNBC cell line, xenographs in severe combined immunodeficiency (SCID) mice. However, this peptide was shown to have toxic side effects that included injection site irritation, constriction of veins upon injection, and the induction of seizures in animals shortly after injection (data not shown). Cell culture analysis demonstrated that this peptide induced the opening of calcium channels and altered subsequent calcium signaling (data not shown). These off-target effects increased the toxicity to a level that prevented clinical translation of this peptide.

Recently, the Schroeder lab targeted the endosomal trafficking protein SNX1 to generate a peptide that mimics the interaction domain between EGFR and SNX1, called cSNX1.3 (Figure 1, Table 1) [86]. This end-capped peptide directly binds EGFR with a similar binding efficiency as SNX1 and results in a significant reduction of nEGFR in TNBC cells with amplified EGFR. cSNX1.3 treatment induces an EGFR-dependent impact on oncogenic phenotypes including proliferation, survival, migration, and mammosphere formation while having no impact on immortalized breast epithelial cells. In the transgenic mouse model whey acidic protein-transforming growth factor α (WAP-TGFα), which develops EGFR-dependent mammary carcinomas, 1/3 of animals showed partial regression, 1/3 no progression and 1/3 demonstrated a complete regression with no observable tumor upon necropsy. Further analysis demonstrated no toxicity and circulation of cSNX1.3 was found up to 24 h after injection, indicating this may be a viable therapeutic (data not shown).

Of note, in cell culture experiments, cSNX1.3 had no effect on EGFR signal transduction, internalization, or degradation. This indicates that results observed were not due to an effect on canonical activities of EGFR. Rather, cSNX1.3 seemed to only effect the retrograde trafficking of EGFR, further highlighting the importance of this pathway for the understanding and treatment of cancer. These and other peptide-based therapeutics that block protein-protein interactions hold promise for future clinical therapeutics.

EGFR is a complex protein that plays roles in many aspects of tumor growth and metastasis. Many drugs have been designed to target well characterized aspects of EGFR biology like ligand binding and kinase signaling. TKIs and antibody-based therapies have significantly improved many patient outcomes over several decades. However, tumors often develop resistance to these drugs either through activation of other signal transduction pathways or through additional mutations. In cases where there is an abundance of nEGFR these drugs don’t often work. nEGFR is a major driver of tumor progression, metastasis, and resistance to anti-EGFR therapies and has been suggested to be a better biomarker than total cell EGFR levels.

The tumor specificity of nEGFR makes it a potentially valuable therapeutic target as nEGFR is rarely seen outside the context of cancer. This tumor specificity is in stark contrast to the roles of EGFR’s kinase domain which are critical in a majority of human tissues at nearly all developmental stages. The reliance on EGFR to develop and maintain human tissues, including the skin and gastrointestinal (GI) tract, explains many side effects seen with anti-EGFR therapies including rash, nausea, and diarrhea.

As has been demonstrated with several peptide inhibitors of EGFR retrograde trafficking, nEGFR is a druggable target. The trafficking of EGFR to the nucleus requires several protein interactions that alter the posttranslational modification seen on EGFR, leading to a switch from lysosomal degradation to nuclear trafficking. The interactions of EGFR with MUC1 and SNX1 have both been targeted and shown efficacy in animal mouse tumor models. The interaction of EGFR and MUC1 is an upstream step in the nuclear trafficking of EGFR and might induce the degradation of EGFR in addition to the loss off nuclear localization making it an ideal anti-nEGFR therapy. In contrast targeting a nEGFR trafficking protein, like SNX1, is further downstream and is unlikely to induce EGFR degradation. However, targeting SNX1 might have additional benefits that have not been fully characterized. SNX1 binds EGFR on the kinase domain and induces the retrograde trafficking of EGFR. SNX1 also binds several other RTKs that have also been shown to drive oncogenesis with their nuclear activities. The peptide cSNX1.3 which was designed to block the interaction of EGFR and SNX1 inhibited 2 dimensional (2D) cell migration driven by EGFR. cSNX1.3 also inhibited migration driven by HER3 and c-Met, which are both RTKs shown to interact with SNX1. They undergo nuclear translocation and drive oncogenesis from within the nucleus suggesting the nuclear localization of RTKs more broadly could be a target for therapeutic intervention and targeting sorting proteins could be an avenue to a broad acting anti-RTK retrograde trafficking therapy.