EGFR signaling and autophagy dependence for growth, survival, and therapy resistance

Abstract

The epidermal growth factor receptor (EGFR) is amplified or mutated in various human epithelial tumors. Its expression and activation leads to cell proliferation, differentiation, and survival. Consistently, EGFR amplification or expression of EGFR variant 3 (EGFRvIII) is associated with resistance to conventional cancer therapy through activation of pro-survival signaling and DNA-repair mechanisms. EGFR targeting has successfully been exploited as strategy to increase treatment efficacy. Nevertheless, these targeting strategies have only been proven effective in a limited percentage of human tumors.

 

Recent knowledge indicates that EGFR deregulated tumors display differences in autophagy and dependence on autophagy for growth and survival and the use of autophagy to increase resistance to EGFR-targeting drugs. In this review the dependency on autophagy and its role in mediating resistance to EGFR-targeting agents will be discussed. Considering the current knowledge, autophagy inhibition could provide a novel strategy to enhance therapy efficacy in treatment of EGFR deregulated tumors.

Introduction

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase within the ErbB family consisting of 4 members; EGFR (ErbB1, HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). ErbBs are typical receptor tyrosine kinases that were implicated in cancer in the early 1980s, when the avian erythroblastosis tumor virus was found to encode an aberrant form of the human epidermal growth factor receptor.¹ In many different cancer cell types, the ErbB pathway becomes hyperactivated by a range of mechanisms, including overproduction of ligands, overproduction of receptors, or constitutive activation of receptors.² In general, EGFR signaling is triggered by ligand binding to the extracellular ligand binding domain. This initiates receptor homo-/hetero-dimerization and autophosphorylation through the intracellular kinase domain, resulting in receptor activation. Following activation, cytoplasmic substrates are phosphorylated and initiate a signaling cascade that drives multiple cellular responses, including changes in gene expression, cytoskeletal rearrangement, apoptosis inhibition, and increased cell proliferation.^3,4 In cancer, EGFR signaling is often deregulated, leading to treatment resistance of the tumor and poor survival of patients. This deregulation is often mediated by overexpression (e.g., through gene amplification) and numerous mutations that lead to uncontrolled and sustained EGFR-signaling. Several EGFR targeting therapies have been developed (e.g., tyrosine kinase inhibitors (TKI) that inhibit EGFR signaling and antibodies that prevent EGFR expression and dimerization). Unfortunately, these therapies have only been proven effective in a limited percentage of cancer patients despite the presence of EGFR in many of the targeted tumors.⁵ Novel strategies that, potentially combined with earlier EGFR-targeting agents, lead to enhanced cell killing are therefore still desired.

Current research has indicated that EGFR-deregulated cells and tumors display alterations in their autophagic response, a pro-survival mechanism that allows cells to recycle nutrients for energy- and macromolecule production.⁶ Importantly: (1) EGFR-deregulated cells seem to be more dependent on autophagy for growth and survival; and (2) resistance to EGFR-targeting agents can be reduced through autophagy inhibition, providing a potential novel modality to target these tumors. In this review we highlight current knowledge that may provide insights as to why EGFR-deregulated cells display differences in autophagic responses and dependency on autophagy for survival and provide rationale for combining autophagy inhibition with conventional cancer therapy.

Gene Amplification and Overexpression

One of the most investigated alterations in the EGFR function is activation of signaling through increased gene copy number arising from amplification or polysomy.^7-9 Elevated EGFR expression is a strong prognostic indicator in head and neck, ovarian, cervical, bladder, and esophageal cancer. In gastric, breast endometrial, and colorectal cancers (CRC) EGFR expression is a modest predictor. This in contrast to non-small cell lung carcinoma (NSCLC), where increased EGFR expression rarely has a prognostic value.¹⁰

EGFR mutations often determine the responsiveness of tumors to EGFR inhibitors; this is often related to the dependency of cancer on continued oncogenic signaling (oncogene addiction). For a number of different oncogenes, data supporting addiction in tumors have been gathered.^11,12 For EGFR in particular, positive results in clinical trials with different antagonists have been considered as clinical evidence of oncogene addiction, even though the clinical benefits from the use of either monoclonal antibodies (mAbs) or TKIs have been limited.⁵ Only a small portion (9–20%) of tumors with hyperactive EGFR signaling is exquisitely sensitive to such specific inhibitors.^13-15 This percentage is much higher (88–94.1%) when sensitizing mutations (e.g., L858R) in the EGFR gene are present.^16,17

In NSCLC and CRC, increased EGFR gene copy number has been associated with increased clinical efficacy of EGFR antagonists erlotinib and cetuximab.¹⁸ Both drugs have shown clinical promise, and the anti-EGFR antibody cetuximab is used in treatment of head and neck squamous cell cancer (HNSCC) and CRC. Despite clinical gain, both intrinsic resistance and the development of acquired resistance have been observed.¹⁹

The Tyrosine Kinase Domain

Both mutations associated with drug resistance and sensitivity have been described within the tyrosine kinase (TK) domain of EGFR in subsets of NSCLC, rare cases in HNSCC, CRC, small cell lung carcinomas (SCLC), ovarian, esophageal, and pancreatic cancers.²⁰ Distribution of mutations is not random and may be related to cancer etiology. For instance, in NSCLC the incidence of EGFR mutations among clinical responders to gefitinib or erlotinib is 77%, compared with only 7% in NSCLC cases that are refractory to tyrosine kinase inhibitor (TKI) treatment.²⁰

Multiple studies have shown differences in treatment outcome associated with EGFR mutations. For example, mutations in exon 18 (nucleotide-binding loop), accounting for 5% of the mutations, are usually amino acid substitutions that contribute to drug sensitivity. Mutations in exon 19 are characterized by small in-frame deletions and account for 45% of EGFR mutations, making it the most prominent EGFR kinase domain mutation in NSCLC. These tumors are, in general, sensitive to TKIs like gefitinib and erlotinib.²⁰ The L858R substitution in exon 21, within the activation loop of EGFR, comprises approximately 40–45% of EGFR mutations. Tumors harboring the L858R mutation are, in general, sensitive to TKIs, although some clinical studies have shown that these tumors are not as responsive in comparison to tumors with deletion mutations in exon 19.²⁰

EGFR exon 20 mutations, typically located after the C-helix of the tyrosine kinase domain, may account for up to 4% of all EGFR mutations, with the T790M substitution as the most prominent one (up to 50% of all mutations in exon 20). This T790M mutation is considered an acquired mutation and converts TKI-sensitive tumors into (reversible) TKI-resistant tumors.²¹ Like the T790M mutation, other exon 20 mutated proteins are resistant to clinically achievable doses of reversible (gefitinib, erlotinib) and irreversible (neratinib, afatinib, PF00299804) TKIs in preclinical models.²² Growing clinical experience with tumors harboring EGFR exon 20 insertions correspond with the preclinical data; only few patients have shown responsiveness to EGFR TKIs.²²

EGFRvIII

In a significant proportion of tumors, amplification of the EGFR gene is accompanied by rearrangements, although amplification is not mandatory for gene rearrangement.²³ The most abundant rearrangement is a deletion variant that lacks exon 2–7 of the extracellular domain, yielding a constitutively active receptor, EGFRvIII or Δ2–7.^24-26 This mutation is most prevalent in malignant gliomas (20–30% in unselected patients with a glioblastoma multiforme [GBM] and 50–60% in patients whose tumors show amplification of wild-type EGFR).²⁷ Recent studies identified EGFRvIII in head and neck squamous cell carcinomas (~21%),²⁸ squamous cell carcinomas of the lung (~5%),^29,30 and breast (~5%),³¹ suggesting broader implications to human cancer.³²

EGFRvIII is known to contribute to radio resistance of tumor cells³³ at least in part through enhanced repair of DNA double-strand breaks.³⁴ Additionally, EGFRvIII expression is associated with resistance to gefitinib and leads to sustained EGFR signaling and AKT activity.³⁵ Furthermore, the tumor microenvironment, and in particular tumor hypoxia, significantly contributes to therapy resistance.^36-38 Expression of EGFRvIII provides cells with a survival advantage when exposed to stresses such as hypoxia and nutrient starvation.³⁹

Although EGFRvIII expression is frequently observed in GBM, culturing GBM cells in vitro will lead to a rapid loss of EGFRvIII expression,^40,41 and thus complicates assessment of EGFRvIII-targeting strategies in GBM. Researchers therefore often use cell lines that artificially express EGFRvIII. Although informative, these cell lines have their limitations as, unlike in primary GBM (a range of 1–100% EGFRvIII positive cells in GBM is observed),²³ all cells will express EGFRvIII. Furthermore, the heterogeneous expression levels observed in GBM are difficult to mimic due to the use of artificial promoters; in addition, the cells were established without EGFRvIII and are thus not dependent on EGFRvIII for growth and survival. To maintain EGFRvIII expression in vitro, cells could be cultured under stem cell culture conditions.⁴² Alternatively, EGFRvIII expression is also maintained when primary tumors are xenografted subcutaneously on mice⁴² and should be considered in validating results obtained in transgenic models.

For GBM patients, EGFR overexpression is a significant prognostic value for predicting survival, and the expression of EGFRvIII with EGFR amplification plays an important role in enhanced tumorigenicity. EGFRvIII overexpression in the presence of EGFR amplification is the strongest indicator for poor survival prognosis in 2 large cohorts of patients. Shinojima and colleagues found in a cohort of 87 patients that EGFRvIII expression, assessed by immunohistochemistry (IHC), was not a predictor for overall survival (OS). However, in patients with EGFR amplification, multivariate analysis revealed that EGFRvIII expression was an independent, significant, poor prognostic factor for OS (P = 0.0044, HR = 2.71).²³ These findings were endorsed by Pelloski et al.,⁴³ who observed that the median survival of a patient group with EGFRvIII expression (n = 36, assessed by IHC) was reduced from 85 to 47 wk compared with EGFRvIII-negative patient group (n = 81). In contrast, Montano et al.⁴⁴ showed, in a cohort of 73 patients, that EGFRvIII (assessed by reverse transcription-PCR) is a molecular predictor of improved overall survival (P = 0.0023, HR = 2.59) in GBM patients treated with surgery followed by adjuvant radiotherapy and temozolomide (TMZ). This discrepancy could potentially be explained by the EGFRvIII detection method. Montano used the more sensitive RT-PCR, whereas Pelloski and Shinojima used IHC and may have missed very low levels of EGFRvIII expression. Another possible explanation for the differences could be the uniformness of the patient group. Montano used patients that all underwent surgery, radiotherapy, and TMZ treatment, whereas the other cohorts were treated more heterogeneously. Furthermore, all patients in Pelloski’s study were wild-type for YKL-40 (a Ras activator), were Montano does not discriminate between Ras activator status, and the Karnofsky performance status (KPS score) of the patients in Pelloski’s and Shinojima’s cohort was much higher.^23,43,44 Taken together, more and lager cohorts with uniform treatment are required to gain additional insight in the clinical relevance of EGFRvIII.

Cancer Stem Cells

Recent data showed that EGFR and EGFRvIII signaling are involved in maintaining a cancer stem cell (CSC) phenotype. In glioblastoma, both the EGFR^pos and EGFR^neg tumor-initiating cells (TICs) derived from primary GBM can give rise to experimental tumors. However, the EGFR^pos TICs displayed enhanced tumorigenic potential and highly invasive behavior.⁴⁵ Conversely, EGFR^neg TICs formed tumors with low efficiency and needed to re-upregulate their EGFR expression to become tumorigenic. These “potential” CSCs might be kept in a dormancy-like state by EGFR downregulation and be reactivated when exposed to stimuli of the in vivo tumor microenvironment.⁴⁵ Indeed, GBMs that were EGFR^neg in origin expressed EGFR on recurrence.⁴⁶ The idea of EGFR^pos and EGFR^neg CSC is further supported by the finding that CSC propagation is possible in the absence of exogenous growth factors (like EGF), suggesting that EGF signaling is not critical for GBM CSC maintenance.⁴⁷ In contrast, EGFR signaling is required for GMB CSC proliferation,^48,49 and gefitinib treatment decreases CSC number in nasopharyngeal carcinoma models.⁵⁰ In this study, cisplatin-treated tumor cells regrew rapidly upon re-implantation, whereas regrowth of gefitinib-treated tumor cells was severely diminished.⁵⁰ Furthermore, Clark et al.⁵¹ showed that GBM CSC lines displayed tumor-initiating capacity after EGF withdrawal or cetuximab treatment by compensatory activation of ErbB2 and ErbB3, suggesting a resistance mechanism for EGFR-targeted therapy. Lapatinib, a dual EGFR/ErbB2 inhibitor, treatment inhibited CSCs proliferation, indicating that a simultaneous blockade of multiple ErbB family members could be needed for more efficient GBM treatment.

In relation to EGFRvIII in CSC, a population of the cells derived from pediatric diffuse intrinsic pontine gliomas (DIPG) neurospheres displayed co-expression of the CSC marker CD133 and EGFRvIII.⁵² In another study, EGFRvIII expression on invasive breast cancer carcinomas resulted in increased expression of genes related to self-renewal and epithelial–mesenchymal transition, along with a higher percentage of CSC-like cells.³¹ Furthermore, Liu et al.⁵³ showed that the CD133+ fraction of GBM exclusively expressed EGFRvIII, whereas wild-type EGFR was not detected. These data indicate a role for EGFRvIII in the propagation of CSC that could explain the relative therapy resistance of EGFRvIII tumors.

EGFR Signaling Pathways Implicated in Autophagy

After ligand binding by EGFR or constitutive signaling by EGFRvIII the activation of several parallel pathways has been described. These include: (1) activation of the PI3K-AKT-mTOR pathway; (2) increased Ras and (3) STAT3 signaling; and (4) Beclin1 (Fig. 1).⁵⁴ All pathways involved in autophagy regulation.

Figure 1. EGFR- and EGFRvIII-signaling pathways associated with autophagy regulation. Both receptors signal through all 4 pathways; nevertheless, EGFR preferentially signals via the RAS pathway, whereas EGFRvIII predominantly uses mTOR signaling.

Autophagy is a catabolic process that allows cells to recycle cellular components through degradation by the lysosomal machinery.^55,56 Autophagy is an evolutionarily conserved process that results in the targeting of cellular proteins and organelles to lysosomes for degradation. Autophagy serves to regulate normal organelle turnover and the removal of those with compromised function to maintain cellular homeostasis. Additionally, autophagy is a survival mechanism during periods of metabolic stress, where self-digestion provides an alternative energy source and facilitates the disposal of unfolded proteins.^57-60

Previously, we and others showed that cells with deregulated EGFR signaling display differences in autophagic response.^61-63 Interestingly, EGFR expression represses autophagy activity. For example, EGFR reduction by siRNA treatment leads to an induction of autophagy activity in prostate cancer cells.⁶³ Furthermore, induction in autophagy was observed after targeting with TKIs or cetuximab.⁶⁴ Recently, in a panel of HNSSC xenografts, we observed a correlation between EGFR and expression of the autophagy marker Lc3b, suggesting a close interplay between EGFR signaling and autophagy. This correlation is most likely mediated through controlling Lc3b protein production, as this correlation was also observed on the mRNA level.⁶¹ This was further confirmed in a panel of cell lines, where EGFR expression negatively correlated with autophagic flux, as determined by Lc3b-turnover. Interestingly, the suppressive activity of EGFR in these cells can be independent of its kinase activity⁶⁵ and mediated through maintaining high glucose levels through association with sodium/glucose cotransporter 1 (SGLT1).⁶³ In addition, EGFR can suppress autophagy dependent on its kinase domain through maintaining high activation of the PI3K/Akt/mTOR pathway.⁶⁶ Furthermore, EGFR activity results in inhibition of autophagy through inhibition of beclin1,⁶² a potent inducer of autophagy. Together these data indicate that the expression of EGFR is closely related to expression of autophagic markers and autophagic activity of cells.

Although the effect of EGFR seems to be mostly autophagy-suppressive, in constitutive EGFR-signaling cells the effect on autophagy activity is less pronounced during normal conditions and seems to be stimulatory during metabolic stresses. For example, in stably transduced glioblastoma cell lines and prostate cancer cells that express EGFRvIII, a faster and more pronounced autophagic response during starvation or severe hypoxia is observed (unpublished data). The enhanced autophagic response provides these cells with survival and growth advantage.

The suppressive action of EGFR on autophagy activity and the opposing action of EGFRvIII during stressful conditions could result from signaling via different signal-transduction pathways. For example, Wolf-Yadlin et al.⁶⁷ showed that EGFR predominantly signals via Erk1, Erk2, and STAT3, whereas EGFRvIII favors signaling via the PI3K and STAT3 pathway.^68,69 This difference in signaling preference of these pathways associated with autophagy activity is likely to result in differences between EGFR and EGFRvIII.

EGFR–PI3K–AKT–mTOR Pathway

Activated EGFR binds GRB2-associated binding protein 1 (GAB1) together with growth factor receptor-bound protein 2 (GRB2) to recruit phosphoinositide-3-kinase (PI3K). PI3K phosphorylates PI(4,5)P2 (phosphatidylinositol) into PI(3,4,5)P3. This process is negatively regulated by phosphatase and tensin homolog (PTEN). 3-phosphoinositide dependent protein kinase-1 (PDK1) brings v-akt murine thymoma viral oncogene homolog 1 (AKT) to the plasma membrane, where PIP3 is located, to phosphorylate and activate AKT. AKT subsequently activates mTOR (mammalian target of Rapamycin).⁵⁴

mTOR, a central growth regulator downstream of oxygen, energy, nutrient, and growth factor signaling, inhibits autophagy. Hence, insufficiency in either results in mTOR inhibition and rapid induction of autophagy in most systems. In conditions of nutrient sufficiency, high mTOR activity prevents Unc-51-like kinase (ULK1) activation by phosphorylating ULK1 Ser757 and disrupting the interaction between ULK1 and 5′ AMP-activated protein kinase (AMPK), thereby preventing ULK1 to initiate an autophagy activating complex with FIP200 and ATG13.^70,71 During periods of starvation, mTOR dissociates from the ULK1 complex, leading to less ULK1 phosphorylation, and increases ULK1 kinase activity.^72,73 Recently, a role for ULK1 activation for survival of hypoxic cells was identified.^74,75

EGFR–RAS Signaling Pathway

The RAS oncogene is a member of small GTPase family involved in the regulation of cell survival and growth and is frequently activated in cancer.⁷⁶ Next to frequently detected activating mutations in RAS, growth factor signaling, e.g., through EGFR, can lead to uncontrolled RAS signaling. After auto-phosphorylation, the adaptor protein growth factor receptor-bound protein 2 (GRB2) binds EGFR at the phosphorylated sites and activates Son of sevenless (SOS), a GTP-exchange factor for RAS. SOS then converts RAS-GDP into active RAS-GTP.

Several studies have implicated RAS activity in the induction of autophagy, as displayed by a high autophagic flux after oncogenic RAS transformation.⁷⁷ Increased autophagy in these cells is required to sustain a high metabolic rate, to prevent accumulation of damaged mitochondria, reduce oxygen consumption, and to prevent metabolic substrate depletion.^77-79 In relation, autophagy inhibition in RAS transformed cells leads to enhanced cell killing during nutrient deprivation.⁷⁷ Furthermore, it has been shown that RAS plays a role in regulating the redox state of the cell, and that constitutive production of ROS correlates with RAS-induced cell transformation^80,81 and mediates autophagy induction through activation of protein kinase 8 (JNK) and subsequent upregulation of ATG5 and ATG7.⁸⁰

EGFR–STAT3 Signaling Pathway

The third main signaling mediator downstream of activated EGFR is the signal transducer and activator of transcription (STAT3) protein. STAT3 belongs to a family of at least 7 transcription factors that share conservation in coiled-coil, SRC homology (SH2), and DNA-binding domains.⁸² STAT3 is a latent transcription factor present in the cytoplasm of cells. Phosphorylation at Y705, is mediated through activation of several transmembrane receptors, such as EGFR,⁸³ and is required for transcriptional activity or transactivation of members of the Janus kinase (JAK) protein family.⁸⁴ Phosphorylation leads to dimerization, nuclear translocation, DNA binding, and gene activation.⁸⁵ Recently, STAT3 has been recognized as a new autophagy regulator through suppression of PKR.⁸⁶ Shen et al.⁸⁶ proposed that in normal conditions, latent cytoplasmic STAT3 binds to protein kinase R (PKR), inhibiting its activity, and reduces autophagy levels through eIF2α inhibition, a signaling cascade involved in both transcriptional and translational regulation of Lc3b and ATG5 production.⁶⁰ Hence, STAT3 phosphorylation leads to homodimerization and enables the free PKR to phosphorylate eIF2α via direct interaction between STAT3 and PKR.⁸⁷ Furthermore, STAT3 controls the expression of several autophagy-associated proteins, including BCL-2, Bcl-X_L, and MCL-1,^88,89 which inhibit autophagy through sequestration of Beclin 1.⁶⁰

EGFR-Beclin 1

Beclin 1 is a coiled-coil protein involved in the regulation of autophagy in mammalian cells and is a component of the class III phosphatidylinositol-3-kinase (PI3K) complex.⁹⁰ Beclin 1 promotes autophagy, and cells with reduced Beclin 1 expression exhibit reduced autophagic activity.⁹¹ Beclin 1 is an essential gene for early embryonic development and is a haploinsufficient tumor suppressor.⁹² Intriguingly, Beclin 1 is tumor suppressive in breast cancer cells; mice that have only one functional allele of Beclin 1 display higher incidence of spontaneous tumors, and mono-allelical deletions of Beclin 1 have been described for 40–75% of human ovarian, breast, and prostate cancers.^91,93-95 Beclin 1 may also promote survival as an interacting partner of an anti-apoptotic protein Bcl-2.⁹⁶ Binding of Bcl-2 to Beclin 1 inhibits Beclin 1-dependent autophagy and Beclin 1-dependent autophagic cell death.^91,97 Recently, it was shown that EGFR phosphorylates Beclin 1 at 3 different tyrosine residues, Y229, Y233, and Y352, after activation by EGF. This tyrosine phosphorylation favors the formation of Beclin 1 dimers, which are incapable of VPS34 binding, and results in reduced autophagy activation (Fig. 1).⁶²

EGFRvIII Tumors Require Increased Metabolism

Why EGFRvIII-expressing tumors require higher activation of autophagy during metabolic stress remains unclear, but could be related to the higher proliferation rate and associated nutritional demand. For example, Guo et al.⁹⁸ showed that EGFRvIII expression induces major shifts in GBM cell metabolism. Uptake of ¹⁸FDG in EGFRvIII-expressing U87 xenografts was doubled compared with volume matched control xenografts. In relation, gene expression arrays showed upregulation of genes involved in regulation of the cell metabolism, e.g., glucose transporter 1 (GLUT1) and GLUT3, Hexokinase2 (HK2), and pyruvate dehydrogenase kinase (PDK1).⁹⁹

In general, EGFRvIII-expressing tumors require upregulation of cell metabolism proteins and require increased glucose uptake to maintain their elevated growth rate. This might explain why these tumors may display increased dependence on autophagy for their energy supply in a tumor microenvironment that is low in glucose or deprived of oxygen.

EGFR Mediates Mitochondrial Homeostasis

In relation to the involvement of EGFR in cell metabolism, Rasbach et al. showed the involvement of EGFR in mitochondrial biogenesis after oxidant injury via EGFR-dependent p38 MAPK activation of the mitochondrial biogenesis regulator PPAR-γ cofactor-1α (PGC-1),¹⁰⁰ allowing the cells to maintain high metabolism and their increased proliferation rate.

Additionally, EGFR is involved in stabilizing mitochondria and preventing apoptosis. Synergistic interaction between EGFR and c-Src via phosphorylation of EGFR at Y845 causes translocation to the mitochondria. There, it interacts with cytochrome c oxidase subunit II (COX II) and prevents apoptosis. This seems independent of EGFR kinase activity but is enhanced by EGF treatment.^101,102 Although cells did not undergo apoptosis, ATP production was drastically reduced by binding of EGFR to COX II.¹⁰² Similar mechanisms and translocation to the mitochondria to antagonize apoptosis have been observed for EGFRvIII.^103,104

COX II inhibition by EGFR leaves the cell with reduced ATP production after insults such as chemo- and radiotherapy or starvation and must revert to other sources for their ATP production. Autophagy may provide an alternative source for energy production, and could be exploited therapeutically in stressed cells with EGFR overexpression. This could also explain why EGFR-expressing cells are more dependent on autophagy for their survival.⁶¹

EGFR, Treatment Resistance, and Therapeutic Potential of Autophagy Inhibition

EGFR expression or mutations contribute to tumor treatment resistance. For instance, acquired mutations in the kinase domain of EGFR (like the T790M) can abrogate the susceptibility to TKIs like gefitinib or erlotinib.²¹ Furthermore, EGFR contributes to radiotherapy resistance either through activation of the pro-survival pathway PLCγ-PKC-RAF¹⁰⁵ or through activation of DNA repair via DNA-PK.¹⁰⁶ We have also shown that expression of EGFRvIII contributes to stress resistance typical for the tumor microenvironment, including nutrient deprivation and hypoxia.³⁹ Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance,^36,37,107 and in HNSCC, the degree of hypoxia is the most significant factor explaining variability in survival.³⁷ Targeting hypoxia in pre-clinical models has been shown to sensitize tumors to therapy through various modalities.^60,108,109 Importantly, a meta-analysis in HNSCC demonstrated therapeutic benefit of hypoxia modification.¹¹⁰

Tumor cells adapt to hypoxia through multiple mechanisms, including activation of autophagy.^6,60,111-115 Genetic and pharmacological inhibition of autophagy sensitizes human tumor cells to hypoxia, reduces the fraction of viable hypoxic tumor cells, and sensitizes human tumors xenografts to irradiation (Fig. 2A).⁶⁰

Figure 2. (A) In EGFR-deregulated tumors, inhibition of autophagy leads to increased cell killing of metabolic stressed cells. (B) Resistance of tumor cells with active EGFR signaling to monoclonal antibodies (mAbs) or tyrosine kinase inhibitors (TKIs) can be reduced by autophagy inhibition.

In relation to EGFR expression, although we showed reduced autophagic flux in cells expressing EGFR, these cells were already under normal conditions dependent on autophagy for proliferation and survival.⁶¹ In general, EGFR-expressing tumors are considered highly radioresistant;¹¹⁶ also in our setting, a large dose irradiation had only a minor effect on tumor delay. Interestingly, chloroquine administration to inhibit autophagy led to a large delay in tumor growth that exceeded the effect of irradiation and, in addition, sensitized tumors to irradiation.⁶¹

Recent data showed that EGFR and EGFRvIII signaling is involved in maintaining a CSC phenotype, and recently it was shown that autophagy is important for CSC self-renewal and tumorigenic potential in breast cancer stem cells,¹¹⁷ and for regulation of energy metabolism and migration and invasion of GBM-derived stem cells.¹¹⁸ Taken together, these data suggest that autophagy and EGFR or EGFRvIII signaling are very important in CSC and could therefore be considered for dual targeted therapy for treatment of CSCs in patients. Why EGFR- and EGFRvIII-expressing cells and tumors are more sensitive to chloroquine treatment remains matter of investigation.

Clinical efficacy of anti EGFR drugs to date has been limited by both acquired and intrinsic resistance. Cancer cells adapt rapidly to EGFR inhibition treatment, resulting in only a small success rate for EGFR inhibition as mono therapy in cancer treatment^119,120 (Fig. 2B). Nevertheless, inhibition of EGFR signaling in combination with autophagy inhibition looks promising in vitro. In NSCLC cell lines with activating EGFR mutation (exon 19 deletion) erlotinib induces both apoptosis and autophagy. Inhibition of autophagy can further enhance sensitivity to erlotinib in these NSCLC cells, suggesting that autophagy serves as a protective mechanism.¹²¹ Moreover, wild-type EGFR-expressing NSCLC cells’ resistance to erlotinib can be abrogated through autophagy inhibition.¹²² Furthermore, ZD6474, a small molecule inhibitor of VEGFR, EGFR, and RET induces apoptosis in 2 glioblastoma cell lines, which can be enhanced by the inhibition of autophagy.¹²³ These findings suggest a therapeutic opportunity for the inhibition of autophagy in combination with conventional cancer therapies.

Conclusion

Over the last decades EGFR has evolved as highly investigated target in the field of anti-cancer treatment. This has led to the development of EGFR-targeting antibodies like cetuximab or panitumumab and TKIs like gefitinib, erlotinib, and lapatinib. More recently, the potential of autophagy inhibition as therapy in cancer is being evaluated. Several reports indicate that cells and tumors with amplified or overactivated EGFR are particularly sensitive to autophagy inhibition for growth, survival, and resistance to conventional therapies. Additionally, resistance to EGFR-targeting therapies can also be reduced by autophagy-inhibition. Inhibition of autophagy may therefore provide a novel treatment opportunity for EGFR-overexpressing tumors and should be pursued clinically.

10.4161/cc.27518