EGFR-targeted Cancer Research Articles

Functional loss of ERBB receptor feedback inhibitor 1 (MIG6) promotes glioblastoma tumorigenesis by aberrantactivation of epidermal growth factor receptor (EGFR).

Dysregulation of epidermal growth factor receptor (EGFR) is one of the most common mechanisms associated with the pathogenesis of various cancers. Mitogen-inducible gene 6 [MIG6; also known as ERBB receptor feedback inhibitor 1 (ERRFI1)], identified as a feedback inhibitor of EGFR, negatively regulates EGFR by directly inhibiting its kinase activity and facilitating its internalization, subsequently leading to degradation. Despite its proposed role as an EGFR-dependent tumor suppressor, the functional consequences and clinical relevance in cancer etiology remain incompletely understood. Here, we identify that the stoichiometric balance between MIG6 and EGFR is crucial in promoting EGFR-dependent oncogenic growth in various experimental model systems. In addition, a subset of ERRFI1 (the official gene symbol of MIG6) mutations exhibit impaired ability to suppress the enzymatic activation of EGFR at multiple levels. In summary, our data suggest that decreased or loss of MIG6 activity can lead to abnormal activation of EGFR, potentially contributing to cellular transformation. We propose that the mutation status of ERRFI1 and the expression levels of MIG6 can serve as additional biomarkers for guiding EGFR-targeted cancer therapies, including glioblastoma.

New Directions for Advanced Targeting Strategies of EGFR Signaling in Cancer.

Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling studies paved the way for a basic understanding of growth factor and oncogene signaling pathways and the development of tyrosine kinase inhibitors (TKIs). Due to resistance mutations and the activation of alternative pathways when cancer cells escape TKIs, highly diverse cell populations form in recurrent tumors through mechanisms that have not yet been fully elucidated. In this review, we summarize recent advances in EGFR basic research on signaling networks and intracellular trafficking that may clarify the novel mechanisms of inhibitor resistance, discuss recent clinical developments in EGFR-targeted cancer therapy, and offer novel strategies for cancer drug development.

TARGETING EPIDERMAL GROWTH FACTOR RECEPTOR FOR CANCER TREATMENT

The Epidermal Growth Factor Receptor (EGFR) is a pivotal player in cellular signalling pathways and its dysregulation is implicated in various cancers. Targeting EGFR has emerged as a promising strategy for cancer treatment. This abstract provides Comprehensive analysis of EGFR-targeted therapies, including small molecule tyrosine kinase inhibitors and monoclonal antibodies, reveals their efficacy in suppressing aberrant signalling cascades associated with tumor growth. Mechanisms of resistance, biomarkers for patient stratification, and ongoing clinical trials are discussed to highlight the evolving landscape of EGFR-targeted cancer therapies. The multifaceted nature of EGFR signaling necessitates a personalized approach, emphasizing the need for precision medicine to optimize therapeutic outcomes. This abstract encapsulates the significance and challenges of targeting EGFR for cancer treatment, providing a foundation for further research and clinical advancements in the field. KEYWORDS: Epidermal Growth Factor Receptor (EGFR), Cancer Treatment, Tyrosine Kinase Inhibitors, Monoclonal Antibodies, Biomarkers, Precision Medicine

Targeted Inhibitors of EGFR: Structure, Biology, Biomarkers, and Clinical Applications.

Members of the EGFR family of tyrosine kinase receptors are major regulators of cellular proliferation, differentiation, and survival. In humans, abnormal activation of EGFR is associated with the development and progression of many cancer types, which makes it an attractive target for molecular-guided therapy. Two classes of EGFR-targeted cancer therapeutics include monoclonal antibodies (mAbs), which bind to the extracellular domain of EGFR, and tyrosine kinase inhibitors (TKIs), which mostly target the intracellular part of EGFR and inhibit its activity in molecular signaling. While EGFR-specific mAbs and three generations of TKIs have demonstrated clinical efficacy in various settings, molecular evolution of tumors leads to apparent and sometimes inevitable resistance to current therapeutics, which highlights the need for deeper research in this field. Here, we tried to provide a comprehensive and systematic overview of the rationale, molecular mechanisms, and clinical significance of the current EGFR-targeting drugs, highlighting potential candidate molecules in development. We summarized the underlying mechanisms of resistance and available personalized predictive approaches that may lead to improved efficacy of EGFR-targeted therapies. We also discuss recent developments and the use of specific therapeutic strategies, such as multi-targeting agents and combination therapies, for overcoming cancer resistance to EGFR-specific drugs.

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.

176 Hair eruption initiates adverse events of EGFR-targeted cancer therapy

176 Hair eruption initiates adverse events of EGFR-targeted cancer therapy

EGFR-Induced and c-Src-Mediated CD47 Phosphorylation Inhibits TRIM21-Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion.

Tumor cells often overexpress immune checkpoint proteins, including CD47, for immune evasion. However, whether or how oncogenic activation of receptor tyrosine kinases, which are crucial drivers in tumor development, regulates CD47 expression is unknown. Here, it is demonstrated that epidermal growth factor receptor (EGFR) activation induces CD47 expression by increasing the binding of c-Src to CD47, leading to c-Src-mediated CD47 Y288 phosphorylation. This phosphorylation inhibits the interaction between the ubiquitin E3 ligase TRIM21 and CD47, thereby abrogating TRIM21-mediated CD47 K99/102 polyubiquitylation and CD47 degradation. Knock-in expression of CD47 Y288F reduces CD47 expression, increases macrophage phagocytosis of tumor cells, and inhibits brain tumor growth in mice. In contrast, knock-in expression of CD47 K99/102R elicits the opposite effects compared to CD47 Y288F expression. Importantly, CD47-SIRPα blockade with an anti-CD47 antibody treatment significantly enhances EGFR-targeted cancer therapy. In addition, CD47 expression levels in human glioblastoma (GBM) specimens correlate with EGFR and c-Src activation and aggravation of human GBM. These findings elucidate a novel mechanism underlying CD47 upregulation in EGFR-activated tumor cells and underscore the role of the EGFR-c-Src-TRIM21-CD47 signaling axis in tumor evasion and the potential to improve the current cancer therapy with a combination of CD47 blockade with EGFR-targeted remedy.

Effective inhibition of cancer cells by recombinant adenovirus expressing EGFR-targeting artificial microRNA and reversed-caspase-3.

The EGFR-targeting cancer therapies are commonly facing drug resistance, mostly due to mutations. Gene therapy with artificial microRNA targeting EGFR conserved sequence may avoid such problem. In this study, we constructed a recombinant adenovirus expressing EGFR-targeting artificial microRNA and active revCASP3 (Ad-EC), under the control of tumor-specific SLPI promoter, and evaluated its inhibitory effect on HEP-2 cancer cells both in vitro and in vivo. MTT assay showed that cell growth inhibition rate at 72h was 44.0% in Ad-EC group at MOI 50, while the rate was 7.7% in the control virus Ad-GFP group and 3.6% in Cetuximab (500 μg/ml) group respectively. Flow cytometry analysis revealed the late apoptotic cells rate was 36.1% in Ad-EC group, significantly higher than 6.5% of Ad-GFP group (p < 0.001). When Ad-EC (MOI 50) was combined with CDDP (0.25 μg/ml), late apoptotic cells rate increased to 61.2%, significantly higher than each monotherapy group (P < 0.001). The real-time xCELLigence system recorded an effective cell growth inhibition in Ad-EC and CDDP groups, and more enhanced effect in Ad-EC plus CDDP group. Western blot revealed that Ad-EC could inhibit the activation of AKT pathway and ERK1/2 pathway, while Cetuximab had the AKT pathway over-activated. In vivo experiments with HEP-2 xenograft in nude mice confirmed the tumor inhibition in Ad-EC, CDDP and Ad-EC plus CDDP groups compared with PBS group (P < 0.01). Collectively, these data support the effective inhibition of cancer cells by this novel gene therapy strategy.

Abstract 2643: TNFAIP2 interacts with EGFR to modulate EGF-induced EGFR auto-phosphorylation and internalization and sequential ERK1/2 activation in nasopharyngeal carcinoma cells

Abstract TNFAIP2 (TNF-α-inducible protein 2) is identified as an angiogenic and proinflammatory factor and is essential for formation of tunneling nanotubes connecting between remote cells. Cancer-associated TNFAIP2 overexpression is first demonstrated in nasopharyngeal carcinoma (NPC) and is significantly correlated with poor survival of patients. We previously revealed that TNFAIP2 is associated with actin filaments, inducing actin remodeling and membrane protrusion and promotes cell motility of NPC cells. To elucidate functional roles of TNFAIP2 in cancer progression, we exploited co-immunoprecipitation (co-IP) coupled with mass spectrometry (MS)-based proteomic analysis to identify interacting proteins of TNFAIP2 in NPC cells. Epidermal growth factor (EGF) receptor (EGFR) is one of the identified candidates and is frequently overexpressed in various cancer types. We validated the interaction between TNFAIP2 and EGFR in two types of NPC cell lines; nevertheless, the interaction of TNFAIP2 with EGFR was reduced along with EGF stimulation. Reciprocal domain mapping analyses and immunofluorescence staining further demonstrated that the C-terminal amino acid residues 461-520 of TNFAIP2, which are dispensable for its association with the plasma membrane, are required for TNFAIP2 interaction with EGFR. On the other hand, the intracellular juxtamembrane domain (amino acids 645-689) and kinase domain (amino acids 690-945) of EGFR are needed for its interaction with TNFAIP2. Furthermore, EGF-triggered internalization of EGFR was significantly interfered in TNFAIP2-knockout NPC cells generated using the CRISPR-Cas9 system, accompanied by alleviated auto-phosphorylation of EGFR on the 1068 and 1173 tyrosine residues and subsequent activation of downstream ERK1/2. This impairment of liganded EGFR-transduced signaling in TNFAIP2-knockout NPC cells was correlated with a decrease in cell migration as revealed by transwell migration assays. These data collectively reveal a novel role of TNFAIP2 in modulation of EGF-triggered EGFR activation and sequential signaling transduction through protein-protein interactions. Efforts are made to pursue if the aforementioned events involve modulation of EGFR oligomerization or recruitment of internalized activated EGFR into endosomes. The results could shed light on how TNFAIP2 promotes cancer progression via coordinating with EGFR and the impact of TNFAIP2 on EGFR-targeted cancer therapy. Citation Format: Hao-Ping Liu, Chih-Ching Wu. TNFAIP2 interacts with EGFR to modulate EGF-induced EGFR auto-phosphorylation and internalization and sequential ERK1/2 activation in nasopharyngeal carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2643.

SCD1 is required for EGFR-targeting cancer therapy of lung cancer via re-activation of EGFR/PI3K/AKT signals

BackgroundCancer cells are characterized by aberrant activation of lipid biosynthesis, producing saturated fatty acids and monounsaturated fatty acids via stearoyl-CoA desaturases (SCD) for regulating metabolic and signaling platforms. SCD1 overexpression functions as an oncogene in lung cancer and predicts a poor clinical outcome. This study aimed to investigate the role of SCD1 inhibition by EGFR inhibitor (Gefitinib)-based anti-tumor therapy of lung cancer both in vitro and in vivo.MethodsCCK-8 assay was performed to determine cell viability. The SCD1 mRNA level was detected by qPCR. The protein levels were assessed by Western blotting. E-cadherin and N-cadherin levels were determined by immunofluorescence. Apoptosis detection was conducted by flow cytometry. Cell migration or invasion was evaluated by transwell assay. The tumor sizes and tumor volumes were calculated in nude mice by subcutaneous injection of A549 cells transfected with vector of pcDNA3.1-SCD1 or negative control. Expression of Ki-67 was detected by immunohistochemistry.ResultSCD1 up-regulated expression was observed in lung cancer cell lines. Cells with overexpressed SCD1 had high IC50 values for Gefitinib in A549 and H1573 cell lines. Overexpression of SCD1 inhibited Gefitinib-induced apoptosis, decreased cell vitality and impaired ability of migration and invasion, while these effects were counteracted by A939572. Mechanistically, SCD1 promoted the activation of proliferation and metastasis-related EGFR/PI3K/AKT signaling, and up-regulated epithelial to mesenchymal transition (EMT) phenotype in the two cell lines, which was restored by SCD1 inhibition. Furthermore, in spite of EGFR inhibition, overexpression of SCD1 in vivo significantly promoted tumor growth by activating EGFR/PI3K/AKT signaling in tumor tissues, but A939572 treatment restricted SCD1-induced tumor progression and inhibited EMT phenotype of cancer cells in vivo.ConclusionThese findings indicated that inhibition of oncogene SCD1 is required for targeting EGFR therapy in lung cancer.

Affibody-Based PET Imaging to Guide EGFR-Targeted Cancer Therapy in Head and Neck Squamous Cell Cancer Models.

In head and neck squamous cell cancer, the human epidermal growth factor receptor 1 (EGFR) is the dominant signaling molecule among all members of the family. So far, cetuximab is the only approved anti-EGFR monoclonal antibody used for the treatment of head and neck squamous cell cancer, but despite the benefits of adding it to standard treatment regimens, attempts to define a predictive biomarker to stratify patients for cetuximab treatment have been unsuccessful. We hypothesized that imaging with EGFR-specific radioligands may facilitate noninvasive measurement of EGFR expression across the entire tumor burden and allow for dynamic monitoring of cetuximab-mediated changes in receptor expression. Methods: EGFR-specific Affibody molecule (ZEGFR:03115) was radiolabeled with 89Zr and 18F. The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression. The protein dose for imaging studies was assessed by injecting 89Zr-deferoxamine-ZEGFR:03115 (2.4–3.6 MBq, 2 μg) either together with or 30 min after increasing amounts of unlabeled ZEGFR:03115 (1, 5, 10, 15, and 20 μg). PET images were acquired at 3, 24, and 48 h after injection, and the image quantification data were correlated with the biodistribution results. The EGFR expression and biodistribution of the tracer were assessed ex vivo by immunohistochemistry, Western blot, and autoradiography. To downregulate the EGFR level, treatment with cetuximab was performed, and 18F-aluminium fluoride-NOTA-ZEGFR:03115 (12 μg, 1.5–2 MBq/mouse) was used to monitor receptor changes. Results: In vivo studies demonstrated that coinjecting 10 μg of nonlabeled molecules with 89Zr-deferoxamine-ZEGFR:03115 allows for clear tumor visualization 3 h after injection. The radioconjugate tumor accumulation was EGFR-specific, and PET imaging data showed a clear differentiation between xenografts with varying EGFR expression levels. A strong correlation was observed between PET analysis, ex vivo estimates of tracer concentration, and receptor expression in tumor tissues. Additionally, 18F-aluminium fluoride-NOTA-ZEGFR:03115 could measure receptor downregulation in response to EGFR inhibition. Conclusion: ZEGFR:03115-based radioconjugates can assess different levels of EGFR level in vivo and measure receptor expression changes in response to cetuximab, indicating a potential for assessment of adequate treatment dosing with anti-EGFR antibodies.

EGFR-targeting, β-defensin-tailored fusion protein exhibits high therapeutic efficacy against EGFR-expressed human carcinoma via mitochondria-mediated apoptosis.

Defensins play an essential role in innate immunity. In this study, a novel recombinant β-defensin that targets the epidermal growth factor receptor (EGFR) was designed and prepared. The EGFR-targeting β-defensin consists of an EGF-derived oligopeptide (Ec), a β-defensin-1 peptide (hBD1) and a lidamycin-derived apoprotein (LDP), which serves as the "scaffold" for the fusion protein (Ec-LDP-hBD1). Ec-LDP-hBD1 effectively bound to EGFR highly expressed human epidermoid carcinoma A431 cells. The cytotoxicity of Ec-LDP-hBD1 to EGFR highly expressed A431 cells was more potent than that to EGFR low-expressed human lung carcinoma A549 and H460 cells (the IC50 values in A431, A549, and H460 cells were 1.8 ± 0.55, 11.9 ± 0.51, and 5.19 ± 1.21 μmol/L, respectively); in addition, the cytotoxicity of Ec-LDP-hBD1 was much stronger than that of Ec-LDP and hBD1. Moreover, Ec-LDP-hBD1 suppressed cancer cell proliferation and induced mitochondria-mediated apoptosis. Its in vivo anticancer action was evaluated in athymic mice with A431 and H460 xenografts. The mice were administered Ec-LDP-hBD1 (5, 10 mg/kg, i.v.) two times with a weekly interval. Administration of Ec-LDP-hBD1 markedly inhibited the tumor growth without significant body weight changes. The in vivo imaging further revealed that Ec-LDP-hBD1 had a tumor-specific distribution with a clear image of localization. The results demonstrate that the novel recombinant EGFR-targeting β-defensin Ec-LDP-hBD1 displays both selectivity and enhanced cytotoxicity against relevant cancer cells by inducing mitochondria-mediated apoptosis and exhibits high therapeutic efficacy against the EGFR-expressed carcinoma xenograft. This novel format of β-defensin, which induces mitochondrial-mediated apoptosis, may play an active role in EGFR-targeting cancer therapy.

Interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in human epithelial cancer cells

Epidermal growth factor receptor (EGFR) is an important regulator of epithelial cell growth and survival in normal and cancerous tissues and is a principal therapeutic target for cancer treatment. EGFR is associated in epithelial cells with the heavily glycosylated transmembrane mucin protein MUC1, a natural ligand of galectin-3 that is overexpressed in cancer. This study reveals that the expression of cell surface MUC1 is a critical enhancer of EGF-induced EGFR activation in human breast and colon cancer cells. Both the MUC1 extracellular and intracellular domains are involved in EGFR activation but the predominant influence comes from its extracellular domain. Binding of galectin-3 to the MUC1 extracellular domain induces MUC1 cell surface polarization and increases MUC1–EGFR association. This leads to a rapid increase of EGFR homo-/hetero-dimerization and subsequently increased, and also prolonged, EGFR activation and signalling. This effect requires both the galectin-3 C-terminal carbohydrate recognition domain and its N-terminal ligand multi-merization domain. Thus, interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in epithelial cancer cells. As MUC1 and galectin-3 are both commonly overexpressed in most types of epithelial cancers, their interaction and impact on EGFR activation likely makes important contribution to EGFR-associated tumorigenesis and cancer progression and may also influence the effectiveness of EGFR-targeted cancer therapy.

Near-infrared photothermal therapy using EGFR-targeted gold nanoparticles increases autophagic cell death in breast cancer

Although triple negative breast cancer (TNBC) is a small percentage of all breast cancers, to date, TNBC is one of the most challenging types of breast cancer for basic and clinic research because TNBC patients display a high risk of relapse, shorter overall survival and limited therapeutic options after completion of conventional chemotherapy compared with patients with other breast cancer subtypes. The epidermal growth factor receptor (EGFR) is a promising target for TNBC treatment. Although near infrared-photothermal therapy (NIR-PTT) using anti-EGFR antibody-conjugated gold nanorods (anti-EGFR-GNs), has attracted considerable interest for non-invasive and targeted TNBC treatment through an activation of apoptotic pathway, it is unclear whether anti-EGFR-GNs-combined NIR-PTT modulates the induction of autophagy contributing to cell death. Therefore, we investigated the autophagic cell death in cultured TNBC cells and mouse xenograft tumors during anti-EGFR-GNs-combined NIR-PTT. We here found that the cytotoxicity induced by anti-EGFR-GNs-combined NIR-PTT was rescued by treatment with autophagy inhibitor, 3-methyladenine (3-MA). Anti-EGFR-GNs-combined NIR-PTT induced remarkable levels of autophagy activity as evidenced by a large number of autophagic vesicles and a significant increase in autophagy-specific proteins; microtubule-associated protein light chain 3 (LC3), p62, beclin-1, and autophagy-related gene5 (Atg5), accompanying the inhibition of AKT-mTOR signaling pathway responsible for inducing autophagy. Moreover, in mouse xenograft tumors, anti-EGFR-GNs-combined NIR-PTT also increased LC3 and beclin-1 levels. Our findings, for the first time, demonstrate that anti-EGFR-GNs-combined NIR-PTT remarkably induces autophagy leading to EGFR-targeted cancer cell death.

Generation and Characterization of Rat Monoclonal Antibodies Against Epidermal Growth Factor Receptor.

Overexpression of the epidermal growth factor receptor (EGFR) gene and dysregulation of EGFR signaling are observed in various cancer cells, and EGFR is a validated target for cancer therapy. In the present study, we report on the generation of two rat anti-EGFR antibodies (clones 2C2D3 and 4H7F4) by using the rat lymph node method. Flow cytometric analysis and immunofluorescence showed that both antibodies specifically bound to EGFR on the surface of cancer cells. Competitive analysis demonstrated that the epitope of each antibody had no overlap with that of the therapeutic anti-EGFR antibody cetuximab. These results suggest that 2C2D3 and 4H7F4 are potentially useful in EGFR-targeted cancer therapy.

Oncogenic miR-9 is a target of erlotinib in NSCLCs.

EGFR-targeted cancer therapy is a breakthrough in non-small cell carcinoma. miRNAs have been proved to play important roles in cancer. Currently, for the role of miRNAs in EGFR-targeted cancer therapy is unclear. In this study, first we found that erlotinib reduced the expression of miR-9. MiR-9 expression was increased in human lung cancer tissues compared with peripheral normal tissues, and miR-9 promoted the growth of NSCLC cells. Overexpression of miR-9 decreased the growth inhibitory effect of erlotinib. Second, miR-9 decreased FoxO1 expression by directly inhibition of its mRNA translation. Adenovirus-mediated overexpression of FoxO1 or siRNA-mediated downregulation of FoxO1 negatively regulated cell growth. And exogenous overexpression FoxO1 reduced the pro-growth effect of miR-9. Finally, we found that erlotinib upregulated FoxO1 protein expression. Moreover, overexpression of miR-9 decreased erlotinib-induced FoxO1 expression, and overexpression of FoxO1 enhanced the growth inhibitory effects of erlotinib. Additionally, we found that erlotinib downregulates miR-9 expression through suppressing the transcrption of miR-9-1 and enhanced DNA methylation maybe involved. These findings suggest that oncogenic miR-9 targeted FoxO1 to promote cell growth, and downregulation of this axis was involved in erlotinib’s growth inhibitory effects. Clarifying the regulation of miRNAs by erlotinib may indicate novel strategies for enhancing EGFR-targeted cancer therapy.

Abstract 121: The EGFR gatekeeper mutation T790M is present in selected patients with early breast cancer

Abstract Introduction. The epidermal growth factor receptor (EGFR) is one of the major oncogenes in a variety of human cancers including breast cancer (BC). While EGFR overexpression and/or amplification has been shown to occur frequently in human breast cancer, EGFR-activating mutations are suggested to be rare if not absent. Thus, the aim of our study was to examine the presence of somatic EGFR gene mutations in a group of norwegian patients diagnosed with early breast cancer. Patients. We investigated tumor biopsies obtained from 132 patients with early breast cancer treated at the Akershus University Hospital (University of Oslo) in 2007/2008. The majority of patients (n = 82) belonged to the luminal- A/B subtypes of BC. In addition, 33 patients were classified as HER-2 positive patients (IHC 3+ or amplification verified by FISH) and 17 as triple-negative breast cancer (TNBC) patients. Methods. Briefly, DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tumor tissue using QIAsymphony DSP DNA Mini Kit (Qiagen Inc.). The extraction of DNA was performed by fully automated purification using the QIAsymphony SP/AS system. EGFR-mutations were detected using a Therascreen EGFR RGQ PCR kit according to the manufacturer´s protocol. We tested for point mutations in exon 18 (G719A/C/S), in-frame deletions in exon 19 (ΔE746-A750), in-frame insertions as well as point mutations in exon 20 (S768I, T790M) and a point mutation in exon 21 (L858R). Results. In the present study we detected three individuals with an EGFR-T790M mutation. Two of the patients were diagnosed with TNBC, while the disease of the third patient was classified as a luminal-A type breast cancer. Interestingly, the T790M-mutation was present in the biopsies obtained during surgery before adjuvant therapy was initiated. No anti-cancer therapy has been given prior to surgery. According to our best knowledge, the T790M-”TKI-resistance-mutation” has not been detected in early breast cancer previously. This finding contrasts the observations made in lung cancer patients where the T790M mutation in general is a classical second mutation causing drug resistance during ongoing TKI-therapy. No other EGFR-mutations were detected in our patients. Conclusion: To our best knowledge we provide first evidence for the presence of the EGFR-T790M-mutation in human early breast cancer. Thus, treatment with novel EGFR-targeting cancer drugs (like “pan-HER-inhibitors”) affecting also T790M-mutated-EGFR may be worthwhile to be tested in highly selected breast cancer patients. Citation Format: Vahid Bemanian, Torill Sauer, Joel Touma, Bjørn A. Lindstedt, Ying Chen, Hilde P. Ødegård, Ida R. Bukholm, Jürgen Geisler. The EGFR gatekeeper mutation T790M is present in selected patients with early breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 121. doi:10.1158/1538-7445.AM2015-121

Synthesis and Characterization of a Melttin Expressing Recombinant Antibody Targeting Human Laryngeal Cancer

Laryngeal carcinoma is poor prognosis and patients with laryngeal carcinoma usually present late leading to the reduced treatment efficacy and high rate of recurrence. Incidence and mortality rates of laryngeal carcinoma are equivalent,suggesting a failure of current therapies,despite the variety of combined modality approaches which have been introduced to complement surgical treatment. A successful cancer therapeutic should target tumours specifically with limited systemic toxicity. Herein,the recombinant antiEGFR / MEL protein was expressed in Escherichia coli( E. coli),refolded and purified on an immobilized Ni2 +-affinity chromatography column. In the protein,anti epidermal growth factor receptor( EGFR) single chain antibody was used to target the EGFR in the laryngeal cancer cell,and the melittin was used to mediate inhibition of cell growth.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis( SDS-PAGE) and Western blotting analysis revealed that antiEGFR / MEL was sufficiently expressed. Confocal microscopy and flow cytometry demonstrated that antiEGFR / MEL bound specifically to Hep-2 cells,as almost no binding to Jurkat cells was observed under identical time and dosage conditions. MTT assay showed that antiEGFR / MEL suppressed the growth of Hep-2cells effecitively. Collectively,these results suggest that antiEGFR / MEL is biologically active and specific toward EGFR-positive tumor cells and may represent an effective EGFR-targeted cancer therapy.

Mice with epidermal-specific knockout of epidermal growth factor receptor (Egfr) to model skin toxicity associated with EGFR-targeted cancer therapy

Mice with epidermal-specific knockout of epidermal growth factor receptor (Egfr) to model skin toxicity associated with EGFR-targeted cancer therapy

Synthesis and purification of a toxin-linked conjugate targeting epidermal growth factor receptor in Escherichia coli

Aberrant epidermal growth factor receptor (EGFR) signaling is a common feature of multiple tumor types, including glioblastoma (GBM). As such, EGFR has emerged as an attractive target for antitumor therapy. In the present study, we sought to develop an immunotoxin capable of specifically targeting EGFR-expressing cells and mediating inhibition of cell growth and cell killing. The Luffin P1 (LP1) ribosome inactivating protein was chosen to generate a fusion protein, antiEGFR/LP1, based upon its potent protein synthesis inhibition and small size (5kDa). LP1 was fused to the C-terminus of an anti-EGFR single-chain antibody (scFv). The recombinant antiEGFR/LP1 protein was expressed in Escherichia coli, and refolded and purified on an immobilized Ni2+-affinity chromatography column. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting analysis revealed that antiEGFR/LP1 was sufficiently expressed. Confocal microscopy and flow cytometry demonstrated that antiEGFR/LP1 bound specifically to EGFR-positive cells (U251), as almost no binding to EGFR-negative (Jurkat cells) was observed under identical time and dosage conditions. Finally, the MTT cell viability assay showed that antiEGFR/LP1 elicited obvious cytotoxicity toward EGFR-positive tumor cells. Collectively, these results suggest that antiEGFR/LP1 is biologically active and specific toward EGFR-positive tumor cells and may represent an effective EGFR-targeted cancer therapy.