Toni M. Brand

Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the HER family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the PI3K/AKT, RAS/RAF/MEK/ERK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. Increased activation by gene amplification, protein overexpression or mutations of the EGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NSCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the EGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five EGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKIs) and two monoclonal antibodies have gained FDA approval for use in oncology. Both approaches to targeting the EGFR have shown clinical promise and the anti-EGFR antibody cetuximab is used to treat HNSCC and CRC. Despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. In this review we focus on the biology of the EGFR, the role of EGFR in human cancer, the development of antibody-based anti-EGFR therapies and a summary of their clinical successes. Further, we provide an in depth discussion of described molecular mechanisms of resistance to cetuximab and potential strategies to circumvent this resistance.

Nuclear EGFR as a Molecular Target in Cancer

The epidermal growth factor receptor (EGFR) has been one of the most targeted receptors in the field of oncology. While anti-EGFR inhibitors have demonstrated clinical success in specific cancers, most patients demonstrate either intrinsic or acquired resistance within one year of treatment. Many mechanisms of resistance to EGFR inhibitors have been identified, one of these being attributed to alternatively localized EGFR from the cell membrane into the cells nucleus. Inside the nucleus, EGFR functions as a co-transcription factor for several genes involved in cell proliferation and angiogenesis, and as a tyrosine kinase to activate and stabilize proliferating cell nuclear antigen and DNA dependent protein kinase. Nuclear localized EGFR is highly associated with disease progression, worse overall survival in numerous cancers, and enhanced resistance to radiation, chemotherapy, and the anti-EGFR therapies gefitinib and cetuximab. In this review the current knowledge of how nuclear EGFR enhances resistance to cancer therapeutics is discussed, in addition to highlighting ways to target nuclear EGFR as an anti-cancer strategy in the future.

AXL Mediates Resistance to Cetuximab Therapy

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. In this study, we show that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated, and tightly associated with EGFR expression in cells resistant to cetuximab (Ctx(R) cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in Ctx(R) cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in Ctx(R) cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft models, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers. Cancer Res; 74(18); 5152-64. ©2014 AACR.

Yes and Lyn play a role in nuclear translocation of the Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR antibody that has been approved for use in oncology. Previously we investigated mechanisms of resistance to cetuximab using a model derived from the non-small cell lung cancer line NCI-H226. We demonstrated that cetuximab-resistant clones (CtxR) had increased nuclear localization of the EGFR. This process was mediated by Src family kinases (SFKs), and nuclear EGFR had a role in resistance to cetuximab. To better understand SFK-mediated nuclear translocation of EGFR, we investigated which SFK member(s) controlled this process as well as the EGFR tyrosine residues that are involved. Analyses of mRNA and protein expression indicated upregulation of the SFK members Yes (v-Yes-1 yamaguchi sarcoma viral oncogene) and Lyn (v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog) in all CtxR clones. Further, immunoprecipitation analysis revealed that EGFR interacts with Yes and Lyn in CtxR clones, but not in cetuximab-sensitive (CtxS) parental cells. Using RNAi interference, we found that knockdown of either Yes or Lyn led to loss of EGFR translocation to the nucleus. Conversely, overexpression of Yes or Lyn in low nuclear EGFR-expressing CtxS parental cells led to increased nuclear EGFR. Chromatin immunoprecipitation (ChIP) assays confirmed nuclear EGFR complexes associated with the promoter of the known EGFR target genes B-Myb and iNOS. Further, all CtxR clones exhibited upregulation of B-Myb and iNOS at the mRNA and protein levels. siRNAs directed at Yes or Lyn led to decreased binding of EGFR complexes to the B-Myb and iNOS promoters based on ChIP analyses. SFKs have been shown to phosphorylate EGFR on tyrosines 845 and 1101 (Y845 and Y1101), and mutation of Y1101, but not Y845, impaired nuclear entry of the EGFR. Taken together, our findings demonstrate that Yes and Lyn phosphorylate EGFR at Y1101, which influences EGFR nuclear translocation in this model of cetuximab resistance.

Nuclear EGFR protein expression predicts poor survival in early stage non-small cell lung cancer

INTRODUCTION Nuclear EGFR (nEGFR) has been identified in various human tumor tissues, including cancers of the breast, ovary, oropharynx, and esophagus, and has predicted poor patient outcomes. We sought to determine if protein expression of nEGFR is prognostic in early stage non-small cell lung cancer (NSCLC). METHODS Resected stages I and II NSCLC specimens were evaluated for nEGFR protein expression using immunohistochemistry (IHC). Cases with at least one replicate core containing ≥5% of tumor cells demonstrating strong dot-like nucleolar EGFR expression were scored as nEGFR positive. RESULTS Twenty-three (26.1% of the population) of 88 resected specimens stained positively for nEGFR. Nuclear EGFR protein expression was associated with higher disease stage (45.5% of stage II vs. 14.5% of stage I; p = 0.023), histology (41.7% in squamous cell carcinoma vs. 17.1% in adenocarcinoma; p = 0.028), shorter progression-free survival (PFS) (median PFS 8.7 months [95% CI 5.1-10.7 mo] for nEGFR positive vs. 14.5 months [95% CI 9.5-17.4 mo] for nEGFR negative; hazard ratio (HR) of 1.89 [95% CI 1.15-3.10]; p = 0.011), and shorter overall survival (OS) (median OS 14.1 months [95% CI 10.3-22.7 mo] for nEGFR positive vs. 23.4 months [95% CI 20.1-29.4 mo] for nEGFR negative; HR of 1.83 [95% CI 1.12-2.99]; p = 0.014). CONCLUSIONS Expression of nEGFR protein was associated with higher stage and squamous cell histology, and predicted shorter PFS and OS, in this patient cohort. Nuclear EGFR serves as a useful independent prognostic variable and as a potential therapeutic target in NSCLC.

Targeting AKT with the allosteric AKT inhibitor MK-2206 in non-small cell lung cancer cells with acquired resistance to cetuximab

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (CtxR) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in CtxR clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all CtxR clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of CtxR cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting.

AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma

Purpose: Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy worldwide. Standard-of-care treatments for patients with HNSCC include surgery, radiation, and chemotherapy. In addition, the anti-EGFR monoclonal antibody cetuximab is often used in combination with these treatment modalities. Despite clinical success with these therapeutics, HNSCC remains a difficult malignancy to treat. Thus, identification of new molecular targets is critical. Experimental Design: In the current study, the receptor tyrosine kinase AXL was investigated as a molecular target in HNSCC using established cell lines, HNSCC patient-derived xenografts (PDX), and human tumors. HNSCC dependency on AXL was evaluated with both anti-AXL siRNAs and the small-molecule AXL inhibitor R428. Furthermore, AXL inhibition was evaluated with standard-of-care treatment regimens used in HNSCC. Results: AXL was found to be highly overexpressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases, and shorter relapse-free survival in patients with HNSCC. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration, and invasion. In addition, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab, and radiation. Moreover, radiation-resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance. Conclusions: This study provides evidence for the role of AXL in HNSCC pathogenesis and supports further preclinical and clinical evaluation of anti-AXL therapeutics for the treatment of patients with HNSCC. Clin Cancer Res; 21(11); 2601–12. ©2015 AACR.

Nuclear Epidermal Growth Factor Receptor Is a Functional Molecular Target in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a subclass of breast cancers (i.e., estrogen receptor–negative, progesterone receptor–negative, and HER2-negative) that have poor prognosis and very few identified molecular targets. Strikingly, a high percentage of TNBCs overexpresses the EGF receptor (EGFR), yet EGFR inhibition has yielded little clinical benefit. Over the last decade, advances in EGFR biology have established that EGFR functions in two distinct signaling pathways: (i) classical membrane-bound signaling and (ii) nuclear signaling. Previous studies have demonstrated that nuclear EGFR (nEGFR) can enhance resistance to anti-EGFR therapies and is correlated with poor overall survival in breast cancer. On the basis of these findings, we hypothesized that nEGFR may promote intrinsic resistance to cetuximab in TNBC. To examine this question, a battery of TNBC cell lines and human tumors were screened and found to express nEGFR. Knockdown of EGFR expression demonstrated that TNBC cell lines retained dependency on EGFR for proliferation, yet all cell lines were resistant to cetuximab. Furthermore, Src Family Kinases (SFKs) influenced nEGFR translocation in TNBC cell lines and in vivo tumor models, where inhibition of SFK activity led to potent reductions in nEGFR expression. Inhibition of nEGFR translocation led to a subsequent accumulation of EGFR on the plasma membrane, which greatly enhanced sensitivity of TNBC cells to cetuximab. Collectively, these data suggest that targeting both the nEGFR signaling pathway, through the inhibition of its nuclear transport, and the classical EGFR signaling pathway with cetuximab may be a viable approach for the treatment of patients with TNBC. Mol Cancer Ther; 13(5); 1356–68. ©2014 AACR.

Mapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3

Nuclear localized HER family receptor tyrosine kinases (RTKs) have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3) have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs) of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain) contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B1) and 27 (B2) amino acids in length that conferred the majority of HER3’s transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B1 and B2 regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B1 and B2 regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs.

Paxillin mutations affect focal adhesions and lead to altered mitochondrial dynamics: Relevance to lung cancer

Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified, and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages, and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L, and K506R) in a GFP-tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, and P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1), and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets.