The epidermal growth factor receptor (EGFR) is one of the most well-studied signaling pathways in cancer progression. As a result, numerous therapeutics including small-molecule inhibitors and monoclonal antibodies have been developed to target this critical oncogenic driver. Several of these EGFR inhibitors (EGFRi) have been evaluated in metastatic breast cancer, as high-level EGFR expression in primary tumors correlates with the highly aggressive basal-like phenotype and predicts for poor patient prognosis. Surprisingly, these trials have been unanimously unsuccessful at improving patient outcomes. Numerous factors, such as lack of proper patient selection may have contributed to the failure of these trials. However, recent findings suggest that there are fundamental changes in EGFR signaling that take place during primary tumor invasion, dissemination and ultimate metastasis of breast cancer cells. Herein, we review the outcomes of EGFR-targeted clinical trials in breast cancer and explore our current understanding of EGFR signaling within primary mammary tumors and how these events are altered in the metastatic setting. Overall, we put forth the hypothesis that fundamental changes in EGFR signaling between primary and metastatic tumors, a process we term the ‘EGFR paradox,’ contribute to the clinically observed inherent resistance to EGFRi. Furthermore, this hypothesis introduces the possibility of utilizing EGFR agonism as a potential therapeutic approach for the treatment of metastatic breast cancer.
Epidermal growth factor receptor (EGFR) was the first discovered of the ErbB family of receptor tyrosine kinases which includes a total of four members: Erbb1/EGFR, ErbB2/Her2, ErbB3 and ErbB4. 1 ErbB members form homo- and heterodimeric cell-surface receptors with unique extracellular domains yielding ligand-binding specificity. Downstream signaling from these receptors proceeds via tyrosine phosphorylation. 2 Since its discovery, EGFR has been characterized as a mediator of a wide variety of signal transduction events that control cell proliferation, migration and survival. Overexpression of EGFR transforms NIH3T3 fibroblasts in an EGF-dependent manner. 3 Aberrant EGFR activation in tumor cells can result from increased transcriptional expression and/or gene amplification. Increased EGFR protein and transcript levels correlate with poor prognosis in various epithelial cancers, such as colorectal cancer (CRC), 4 non-small cell lung cancer (NSCLC), 5 endometrial cancer, 6 and squamous-cell carcinoma of the head and neck (SCCHN). 7 Another mode of EGFR activation in cancer is activating somatic mutations that result in constitutive kinase activity, and these are particularly prevalent in NSCLC (reviewed in Morgensztern et al. 8 ). These findings have led to the development of numerous FDA-approved EGFR inhibitors for many of these cancers (Figure 1). Gefitinib is a small molecule EGFR kinase inhibitor that received accelerated approval from the FDA in 2003 but was pulled from the market due to lack of efficacy. These findings were the result of not selecting patients whose tumors contain EGFR activating mutations. Since then, it has been recognized that only NSCLC patients with activating mutations in EGFR respond to gefitinib. This led to the 2015 approval of gefitinib as a first-line therapy for NSCLC specifically in patients that test positive for activating EGFR mutations. The addiction of these tumors to EGFR signaling is further demonstrated by the emergence of the secondary activating T790M mutation as a major cause of tumor resistance to gefitinib. This has resulted in the recent formulation and FDA approval of osimertinib, a compound capable of inhibiting T790M mutant EGFR. 9 These lessons in NSCLC have served as a critical example of the need for biomarkers to drive application of kinase inhibitors to EGFR. Although activating mutations in EGFR are prevalent in NSCLC patients, inhibition of wild-type EGFR has shown success in pancreatic cancer, 10 head and neck cancer 11 and colorectal cancer. 12 Ultimately, these studies have led to the FDA approval of EGFR ligand blocking antibodies (cetuximab and panitumumab) for the treatment of colorectal and head and neck cancers. However, studies are still ongoing to determine other biomarkers that might improve patient selection for these cancers. 13
Targeting EGFR in metastatic breast cancer
Breast cancer (BC) is the most commonly diagnosed and the second most lethal cancer in American women. 14 Metastasis is invariably responsible for patient death in BC. The triple negative BC subtype (TNBC) is characterized by metastatic progression, poor patient prognosis, and is identified by the absence of bio-molecules that form the basis for targeted therapies for the other BC subtypes, namely estrogen receptor, progesterone receptor, and Her2 amplification. 15 Thus, there are currently no FDA approved targeted therapies for TNBC. TNBC is initially highly sensitive to chemotherapy, but many TNBC patients rapidly develop resistance, at which point metastatic disease is highly lethal. 16 Although activating mutations and gene amplification of EGFR are rare in BC, EGFR expression can be enhanced by increased gene copy number due to polysomy, and enhanced expression of EGFR in the primary tumor is associated with increased metastasis and decreased survival of TNBC patients. 17,18 Concomitant with these clinical findings, studies from the Condeelis lab established a paracrine signaling loop in which macrophage-produced EGF supported tumor cell invasion and dissemination from the primary tumor. 19,20 Experimental findings such as these prompted the initiation of several clinical trials to assess the effectiveness of EGFR inhibition (EGFRi) in metastatic TNBC. The EGFR kinase inhibitor erlotinib was evaluated in a phase II trial of unselected patients with advanced BC having had previously received chemotherapy. 21 In addition, erlotinib was evaluated in combination with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab. 22 Both of these studies determined that erlotinib did not provide clinical benefit to BC patients and erlotinib responsiveness was not predicted by EGFR expression levels in the primary tumor.
Gefitinib is another EGFR-specific kinase inhibitor that has been evaluated in metastatic BC in multiple trials. A multicenter phase II study examined the outcomes of gefitinib treatment in unselected metastatic BC patients that had previously received standard chemotherapies. In all, 98.3% of these patients were non-responders and as above there was no correlation between EGFR expression and response to gefitinib. 23 Similarly, gefitinib as a monotherpay in metastatic estrogen receptor alpha (ER-α) negative BC patients did not provide clinical benefit in another phase II clinical trial. 24 Engebraaten et al. tested the efficacy of combining gefitinib with docetaxel in metastatic BC as compared with docetaxel alone. In this study, the combination was associated with lower partial response rate and higher toxicity than chemotherapy alone. 25 In addition to kinase inhibitors, clinical trials have also evaluated the addition of the ligand blocking monoclonal antibody cetuximab to the DNA-alkylating agent carboplatin. 26 Similarly, this study found that fewer than 20% of metastatic TNBC patients responded to cetuximab plus carboplatin. In subsequent studies, the combination of cetuximab with antimicrotubule agents or topoisomerase inhibitors did not increase patient overall survival as compared with these chemotherapies alone, leading to premature trial termination. 27,28 These findings have been confirmed in more recent trials examining the efficacy of panitumumab, another ligand-blocking anti-EGFR monoclonal antibody, in the treatment of TNBC. As with other EGFRi, panitumumab did not improve progression-free survival over chemotherapy alone when used in metastatic TNBC. 29 In contrast to these adjuvant trials in metastatic disease, use of panitumumab in combination with chemotherapy did appear efficacious as a neoadjuvant therapy for operable stage II–III TNBC. 30 Overall, despite strong pre-clinical data linking high levels of EGFR to increased metastatic progression and decreased patient survival, TNBC in the metastatic setting appears to be unresponsive to EGFRi (Table 1). The mechanisms of inherent resistance of metastatic BC to EGFRi remain to be fully established.
The ‘EGFR paradox’ during the metastatic progression of breast cancer
Recently, our lab reported findings that demonstrate a switch in EGFR function between primary and metastatic tumors. 31 In this study, EGF treatment of EGFR-amplified primary tumor cells resulted in increased proliferation, and these cells were particularly sensitive to EGFR inhibition. Conversely, after epithelial–mesenchymal transition (EMT)-driven in vivo metastasis, cells derived from pulmonary metastases are inherently resistant to EGFRi and undergo robust growth inhibition in response to EGF. 31 This idea that growth factors have context dependent dual effects on cell growth has long been proposed. 32 Indeed, growth factors such as interleukin 6 (IL-6) and platelet-derived growth factor (PDGF) are known to paradoxically inhibit the growth of some cell types. 33,34 Furthermore, the recognized growth-promoting roles of estrogen in BC are coupled with accounts of estrogen-induced apoptosis, termed ‘the estrogen paradox’ nicely reviewed in Jordan and Ford. 35 Another well-established shift in function in BC is that of transforming growth factor-beta (TGF-β) where it functions as a powerful tumor suppressor in primary tumors but drives disease progression in the metastatic setting. 36 Further understanding of this shift in EGFR signaling will likely serve to explain the failure of EGFRi in the treatment of metastatic BC. Furthermore, these findings also present the opportunity to exploit the antimetastatic function of EGFR agonism as a therapeutic approach. Below we review some of the established findings that support the existence of the EGFR paradox during BC growth, dissemination and metastasis.
Potential mechanisms of inherent resistance to EGFRi in metastatic breast cancer
Diminution of EGFR expression with metastatic progression
As mentioned above, our lab recently developed a model in which overexpression of WT EGFR transforms normal murine mammary gland (NMuMG) cells.
EGFR enhanced nuclear transport after metastasis
EGFR is primarily localized to the plasma membrane, but numerous studies have demonstrated nuclear localization of EGFR where it can undergo several poorly understood functions that are both dependent and independent of kinase activity.
The growth-inhibitory function of EGFR
The first observation that EGF inhibits cancer-cell growth at concentrations that are stimulatory to other cells was reported for the rat pituitary GH4CI tumor cell line and the human epidermoid carcinoma A431 cell line.
The impact of co-expressed receptors on resistance to EGFR kinase inhibitors
The above mechanisms of inherent resistance to EGFRi dictate the emergence of alternative signaling pathways that sustain tumor cell survival and metastatic growth. In esophageal cancer, resistance to EGFRi is associated with fibroblast growth factor receptor 2 (FGFR2) amplification and overexpression.
In NSCLC, the amplification of hepatocyte growth factor receptor (c-MET) has been implicated in resistance to EGFR kinase inhibition by reactivating ErbB signaling through the ErbB3 receptor.
Studies in NSCLC also suggest the Axl receptor can facilitate resistance to erlotinib.
Similarly, Axl has been found to interact with and transactivate signaling from EGFR and other receptors independent of ligand engagement in TNBC cells.
Clearly this mechanism would contribute to resistance to ligand-blocking EGFR antibodies. In our EGFR-driven metastatic BC model, the diminution and functional switch of EGFR in metastatic lesions is associated with an increase in fibroblast growth factor receptor 1 splice variant β (FGFR1-β) expression.
Indeed, recent studies from our lab and others highlight the value of FGFR targeting therapies in TNBC and lapatinib-resistant BC models.
Discussion and conclusion
EGFR is a critical signaling molecule involved in a myriad of biological processes and carcinogenic events. In various cancers, aberrant EGFR activation contributes to the initial oncogenic transformation of cells and their subsequent invasion and exit from the primary tumor. These canonical signaling events generated from plasma membrane localized receptors drive the well-established oncogenic effects of EGFR. In contrast, progression of disseminated BC cells into macrometastases is associated with downregulation of EGFR, shunting of EGFR away from the cell surface, and non-canonical pro-apoptotic signaling through STAT1. Although a precise mechanism that unifies these observations remains unknown, EGFR induction of apoptosis has previously been demonstrated to result from intracellular signaling from endosomes. 66,67 Thus, it is tempting to speculate a model where cell surface EGFR signaling has a crucial role for the initial invasion of BC. 37 However, following systemic dissemination, tumor cells will increase EGFR internalization and/or decrease its expression as part of an adaptation response to the metastatic microenvironment. Thus, established macrometastases evolve to become EGFR independent and are therefore inherently resistant to targeted inhibition of EGFR (Figure 2).
Future challenges of EGFR therapy in BC
Inhibition of EGFR signaling via kinase inhibitors and monoclonal antibodies has resulted in fundamental changes in patient care for some tumor types. However, numerous attempts to apply these therapies to metastatic BC patients have been unsuccessful. We believe the literature as a whole supports a paradoxical shift in EGFR function during BC metastasis to a STAT1-dominated pro-apoptotic signaling mechanism. Other STAT1-activating cytokines are heavily used therapeutically, yet EGFR–STAT1 signaling is virtually unexplored in cancer treatment. We conclude that EGFR agonism could be pursued as a potential adjuvant therapy for metastatic BC. Despite the potential utility of EGFR agonism as a therapeutic approach in metastatic BC, predicting patient groups that might benefit from EGFR agonists versus inhibitors faces many challenges. Paramount to these challenges includes the design of effective biomarkers to predict the pro- versus anti-tumorigenic effect of EGFR. Although EGFR expression and cellular localization can be assessed in primary mammary tumor biopsies, these types of analyses would need to be standardized into reproducible diagnostics that could be introduced to the clinic. Furthermore, these detection methods may not be feasible on metastatic BC tissues.
However, using the estrogen paradox as a model, estrogen treatment has demonstrated growth inhibitory effects on BC cells in culture and in mouse models. 83,84 Similarly, patients pretreated and resistant to endocrine inhibition therapies do show antitumor responses when switched to high-dose estrogen. 85,86 Therefore, one potential course of therapy for patients who present with metastatic lesions and display EGFR expression in their primary tumor would be to initiate EGFRi treatment, and at the point of disease progression, abruptly switch to a high-dose EGFR agonist. Indeed, a recent study using the A431 model of EGF-induced growth inhibition has established proof-of-concept for in vivo tumor inhibition upon systemic administration of supraphysiologic levels of recombinant EGF. 87 Overall, more thorough preclinical and clinical studies will establish if we will be able to harness the power of the EGFR paradox for the therapeutic benefit of metastatic BC patients.
We thank the Members of the Wendt Laboratory for critical reading of the manuscript. This work was supported in part by the National Institutes of Health (R00CA166140) and the METavivor Foundation.
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