Epidermal Growth Factor Receptor Isoforms Research Articles

Therapeutic application of extracellular vesicular EGFR isoform D as a co-drug to target squamous cell cancers with tyrosine kinase inhibitors

Targeting wild-type epidermal growth factor receptor (EGFR) using tyrosine kinase inhibitors (TKIs) never achieved its purported success in cancers such as head and neck squamous cell carcinoma, which are largely EGFR-dependent. We had previously shown that exceptional responders to TKIs have a genetic aberration that results in overexpression of an EGFR splice variant, isoform D (IsoD). IsoD lacks an integral transmembrane and kinase domain and is secreted in extracellular vesicles (EVs) in TKI-sensitive patient-derived cultures. Remarkably, the exquisite sensitivity to TKIs could be transferred to TKI-resistant tumor cells, and IsoD protein in the EV is necessary and sufficient to transfer the phenotype invitro and invivo across multiple models and drugs. This drug response requires an intact endocytic mechanism, binding to full-length EGFR, and signaling through Src-phosphorylation within the endosomal compartment. We propose a therapeutic strategy using EVs containing EGFR IsoD as a co-drug to expand the use of TKI therapy to EGFR-driven cancers.

Super enhancer loci of EGFR regulate EGFR variant 8 through enhancer RNA and strongly associate with survival in HNSCCs.

Epidermal growth factor receptor (EGFR) has been shown to be overexpressed in human cancers due to mutation, amplification, and epigenetic hyperactivity, which leads to deregulated transcriptional mechanism. Among the eight different EGFR isoforms, the mechanism of regulation of full-length variant 1 is well-known, no studies have examined the function & factors regulating the expression of variant 8. This study aimed to understand the function of EGFR super-enhancer loci and its associated transcription factors regulating the expression of EGFR variant 8. Our study shows that overexpression of variant 8 and its transcription was more prevalent than variant 1 in many cancers and positively correlated with the EGFR-AS1 expression in oral cancer and HNSCC. Notably, individuals overexpressing variant 8 showed shorter overall survival and had a greater connection with other clinical traits than patients with overexpression of variant 1. In this study, TCGA enhancer RNA profiling on the constituent enhancer (CE1 and CE2) region revealed that the multiple enhancer RNAs formed from CE2 by employing CE1 as a promoter. Our bioinformatic analysis further supports the enrichment of enhancer RNA specific chromatin marks H3K27ac, H3K4me1, POL2 and H2AZ on CE2. GeneHancer and 3D chromatin capture analysis showed clustered interactions between CE1, CE2 loci and this interaction may regulates expression of both EGFR-eRNA and variant 8. Moreover, increased expression of SNAI2 and its close relationship to EGFR-AS1 and variant 8 suggest that SNAI2 could regulates variant 8 overexpression by building a MegaTrans complex with both EGFR-eRNA and EGFR-AS1. Our findings show that EGFR variant 8 and its transcriptional regulation & chromatin modification by eRNAs may provide a rationale for targeting RNA splicing in combination with targeted EGFR therapies in cancer.

Expression of EGFR isoform D is regulated by HER receptor activators in breast cancer cells

Human epidermal growth factor receptor isoform D (EGFR; isoform D) is a soluble protein from a 3 kb alternate mRNA transcript that arises from the human EGFR gene. Several studies have identified this circulating isoform of EGFR as a potential diagnostic biomarker for the detection of early stage of cancers. While the expression of the full-length EGFR (isoform A) is regulated by its cognate ligand, EGF, as well as by phorbol myristate acetate (PMA), no studies have examined the factors regulating the expression of EGFR isoform D. In this study, using breast cancer cell lines, we show that the HER receptor ligands, EGF and neuregulin (NRG-1β), as well as the phorbol ester, PMA, can increase the expression of EGFR isoform D, as well as isoform A. Our results, based on measurement of mRNA levels, suggest that EGF induced expression of both isoform A and isoform D occur through a mitogen activated protein kinase (MAPK)-dependent mechanism, and also suggest that protein kinase C is involved in PMA-induced regulation of both isoforms. We also demonstrate that NRG-1β increases isoform A and isoform D expression via the MAPK-dependent pathway, but this regulation occurs independently of phosphatidylinositol 3-kinase/Akt activation. These results suggest that regulation of EGFR isoform A and isoform D expression occur using similar mechanisms. Despite commonalities in the transcriptional regulation of these two EGFR isoforms, the half-lives of these two transcripts is quite different. Moreover, EGFR isoform D, unlike isoform A, is not post-transcriptionally modulated by EGFR activators in the breast cancer cell line MDA-MB-468.

Inhibition of Epidermal Growth Factor Receptor Signaling by Antisense Oligonucleotides as a Novel Approach to Epidermal Growth Factor Receptor Inhibition.

We report a novel method to inhibit epidermal growth factor receptor (EGFR) signaling using custom morpholino antisense oligonucleotides (ASOs) to drive expression of dominant negative mRNA isoforms of EGFR by ASO-induced exon skipping within the transmembrane (16) or tyrosine kinase domains (18 and 21). In vivo ASO formulations induced >95% exon skipping in several models of nonsmall cell lung cancer (NSCLC) and were comparable in efficacy to erlotinib in reducing colony formation, cell viability, and migration in EGFR mutant NSCLC (PC9). However, unlike erlotinib, ASOs maintained their efficacy in both erlotinib-resistant subclones (PC9-GR) and wild-type overexpressing EGFR models (H292), in which erlotinib had no significant effect. The most dramatic ASO-induced phenotype resulted from targeting the EGFR kinase domain directly, which resulted in maximal inhibition of phosphorylation of EGFR, Akt, and Erk in both PC9 and PC9GR cells. Phosphoproteomic mass spectrometry confirmed highly congruent impacts of exon 16-, 18-, and 21-directed ASOs compared with erlotinib on PC9 genome-wide cell signaling. Furthermore, EGFR-directed ASOs had no impact in EGFR-independent NSCLC models, confirming an EGFR-specific therapeutic mechanism. Further exploration of synergy of ASOs with existing tyrosine kinase inhibitors may offer novel clinical models to improve EGFR-targeted therapies for both mutant and wild-type NSCLC patients.

Secondary Structure Analysis by SHAPE‐MaP of the EGFR pre‐mRNA Transcript to Identify Target Regions for RNA Anti‐sense Therapies

Glioblastoma Multiforme (GBM) is the most common primary brain malignancy with a median survival of 10–14 months. A common aberration in GBM is the overexpression of epidermal growth factor receptor (EGFR), dysregulated in 57% of all GBM. Standard care increases survival to only 14–15 months indicating a necessity to generate novel therapeutics. Our strategy is to deliver a gene directly to the CNS using an adeno‐associated virus gene transfer vector encoding antisense RNA therapeutics. This therapy targets critical splicing elements surrounding Intron 10 of the EGFR pre‐mRNA transcript. Cryptic poly‐a signals within intron 10 have the potential to activate the expression of a therapeutic isoform of EGFR. Alternative splicing and polyadenylation is regulated by secondary structure of the pre‐mRNA transcript. To improve our therapeutic strategy, we have analyzed the EGFR secondary structure using selective 2′ hydroxyl acylation and primer extension followed by mutational profiling (SHAPE‐MaP). The SHAPE reagent 1M7 reacts with the 2′ hydroxyl of RNA molecules when the RNA molecule is in a conformationally flexible position creating a 2′ O‐adduct. The modified RNA is reverse transcribed, incorporating mismatches at the acylated positions; a comparison of unmodified to modified RNA allows us to determine RNA nucleotides that are involved in secondary structure, part of RNA‐binding‐protein complexes, or single stranded. The EGFR DNA sequence of Exon 10, Intron 10, Exon 11 was cloned into a plasmid. The plasmid was PCR amplified with a forward T7 promoter primer and reverse primer corresponding to exon 11, this generated the template for T7 RNA transcription. RNA was T7 transcribed and purified by RNA gel electrophoresis. RNA was treated with 1M7 (+) or DMSO (−), reverse transcribed under SHAPE conditions with Superscript II then converted to cDNA. Reverse transcription conditions for SHAPE experiments incorporate Mn++ as the divalent cation, which allows the enzyme to incorporate nucleotide mismatches. 3.5 million reads generated using Nanopore MinION sequencing corresponded to (+) and (−) conditions with a mean read length of 936 nucleotides and a quality score of 9.7. Sequencing output was run through the ShapeMapper2 pipeline. The median read depth at each nucleotide was at least 92K for all conditions after elimination of reads under quality control score of 15. SHAPE‐MaP reactivity profiles were generated using RNAfold. Splicing elements within the targeted region were identified and compared to reactivity profiles. SRp40 binding motifs show high accessibility upstream of the 5′ splice site. The exon‐intron junction of the 5′ splice site is structurally available while the 3′ intron‐exon junction shows moderate accessibility. We identified a putative poly‐a signal exists within intron 10 and the literature shows expression of this shortened EGFR isoform in various cell types. Importantly, this cryptic poly‐a signal shows no reactivity indicating high secondary structures blocking recognition by the poly‐a machinery. Based on these structural profiles, we generated RNA antisense molecules to unravel the hidden poly‐a signal to activate expression of the short isoform. In conjunction, we used eCLIP to identify target specific RNA‐binding proteins.

Nuclear Delivery of Radiosensitizing EGFR-Targeted Nanoparticles Is Abrogated in Isogenic Cervical Cancer Cell Lines Following Mutagenesis of the Ligand Binding Domain

The development of targeted treatment regimens for cervical cancer remains a critically unexplored area of research. One such possible target is epidermal growth factor receptor (EGFR). Our laboratory has previously synthesized radiosensitizing nanoparticles (NPs) that specifically target cells that express EGFR. Here, we test the hypothesis that nuclear localization following treatment of EGFR-targeted NPs is preferentially achieved in cervical cancer cell lines with high levels of EGFR expression and is dependent upon an intact ligand binding domain. A panel of cervical cancer cell lines (HeLa, SiHa, CaSki, ME180, and C33A) with varying levels of endogenous and genetically manipulated EGFR expression were cultured and treated with Fe3O4@TiO2 NPs conjugated to EGF “mimic” (B-loop) peptide (EGFR-targeted NPs) or its scrambled version (scrambled non-targeted NPs). Transient overexpression and knockdown of EGFR was achieved with transfection of EGFR DNA or small interfering RNA (siRNA). Isogenic cell lines were expanded from single cell clones of the ME-180 cervical cancer cell line with heterozygous or homozygous mutations, introduced by CRISPR-Cas9 technology, of the critical amino acids Leucine-14 (Leu-14) and Tyrosine-45 (Tyr-45) located within the ligand binding domain of the EGFR protein. Immunocytochemistry with confocal microscopy and flow cytometry was utilized to assess EGF-binding ability of isogenic cell lines as well ascellular delivery, subcellular distribution, and receptor binding ability of EGFR-targeted NPs and scrambled non-targeted NPs in this panel of cell lines. All tested cervical cancer cell lines endogenously produced both full-length EGFR mRNA variant 1 (v1), as well as shorter mRNA variants 3 and 4 which encode soluble isoforms of EGFR, while expression of full-length EGFR (v1) varied dramatically across cell lines. Cell lines with high levels of endogenous or genetically upregulated EGFR expression experienced increased nuclear uptake of EGFR-targeted NPs compared to cell lines with low levels of endogenous EGFR expression or following siRNA-mediated knockdown, while uptake of scrambled non-targeted NPs was similar across all cell lines. Isogenic clones of the ME-180 cervical cancer cell line demonstrated impaired EGF-binding as well as nuclear localization of EGFR-targeted NPs but scrambled non-targeted NPs. Nuclear accumulation of EGFR-targeted radiosensitizing NPs, but not scrambled non-targeted NPs, occurred preferentially in cervical cancer cell lines with high expression of EGFR and was abrogated following mutation of the ligand binding domain. Further studies are required to demonstrate in vivo radiosensitization of cervical cancer following treatment with EGFR-targeted NPs.

Prediction of regulatory targets of alternative isoforms of the epidermal growth factor receptor in a glioblastoma cell line

BackgroundThe epidermal growth factor receptor (EGFR) is a major regulator of proliferation in tumor cells. Elevated expression levels of EGFR are associated with prognosis and clinical outcomes of patients in a variety of tumor types. There are at least four splice variants of the mRNA encoding four protein isoforms of EGFR in humans, named I through IV. EGFR isoform I is the full-length protein, whereas isoforms II-IV are shorter protein isoforms. Nevertheless, all EGFR isoforms bind the epidermal growth factor (EGF). Although EGFR is an essential target of long-established and successful tumor therapeutics, the exact function and biomarker potential of alternative EGFR isoforms II-IV are unclear, motivating more in-depth analyses. Hence, we analyzed transcriptome data from glioblastoma cell line SF767 to predict target genes regulated by EGFR isoforms II-IV, but not by EGFR isoform I nor other receptors such as HER2, HER3, or HER4.ResultsWe analyzed the differential expression of potential target genes in a glioblastoma cell line in two nested RNAi experimental conditions and one negative control, contrasting expression with EGF stimulation against expression without EGF stimulation. In one RNAi experiment, we selectively knocked down EGFR splice variant I, while in the other we knocked down all four EGFR splice variants, so the associated effects of EGFR II-IV knock-down can only be inferred indirectly. For this type of nested experimental design, we developed a two-step bioinformatics approach based on the Bayesian Information Criterion for predicting putative target genes of EGFR isoforms II-IV. Finally, we experimentally validated a set of six putative target genes, and we found that qPCR validations confirmed the predictions in all cases.ConclusionsBy performing RNAi experiments for three poorly investigated EGFR isoforms, we were able to successfully predict 1140 putative target genes specifically regulated by EGFR isoforms II-IV using the developed Bayesian Gene Selection Criterion (BGSC) approach. This approach is easily utilizable for the analysis of data of other nested experimental designs, and we provide an implementation in R that is easily adaptable to similar data or experimental designs together with all raw datasets used in this study in the BGSC repository, https://github.com/GrosseLab/BGSC.

Abstract 2591: Role of EGFR-AS1 lncRNA in growth factor addiction and treatment response in squamous cell cancers

Abstract Squamous-cell cancers (SCCs) represent one of the commonest lethal malignancy worldwide. Despite evidence demonstrating that majority are addicted to Epidermal Growth Factor Receptor (EGFR)-signaling, targeting with monoclonal antibodies or tyrosine-kinase inhibitors (TKIs) has been met with limited success. We recently demonstrated that TKI-sensitivity is modulated by a long non-coding RNA (lncRNA) situated in the antisense strand of EGFR (EGFR-AS1). ‘Synonymous' alterations in EGFR-AS1 affected TKI-response in patient-derived models, and could be used to predict response in a small patient cohort. The lncRNA likely functions in cis by altering EGFR splicing, favouring expression of a truncated EGFR isoform (isoform D), with previously unknown function. Importantly, mediation of this effect requires EGFR ligand derived from paracrine or autocrine sources. Remarkably, even modest EGFR-AS1 knockdown showed profound effects on TKI sensitivity and tumor growth in vivo; the latter suggesting lncRNA-addiction. TKI-resistance models were established from patient-derived lines, through serial passaging with sub-lethal drug doses. Detailed analyses of four of these models demonstrate alterations that affect each of the steps involved in this addiction pathway: ligand binding, EGFR-AS1 expression/amplification and isoform D expression. Based on our mechanistic insights, we propose that patient-response to EGFR inhibitors is contingent on establishing a comprehensive suite of inter-related biomarkers as delineated here. This data also supports a novel therapeutic approach that involves modulating a lncRNA and/or splice variants of a well-established oncogenic driver. Citation Format: Daniel SW Tan, Fui-Teen Chong, Hui-Sun Leong, Shen Yon Toh, Dawn P. Lau, Xue Lin Kwang, Xiaoqian Zhang, Gek San Tan, Anders J. Skanderup, Tony KH Lim, Ramanuj Dasgupta, N Gopalakrishna Iyer. Role of EGFR-AS1 lncRNA in growth factor addiction and treatment response in squamous cell cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2591.

Characterization of two splice variants of EGFR and their effects on the growth of the razor clam

The epidermal growth factor receptor (EGFR) plays a vital role in cell growth, proliferation, and body growth. In this study, two EGFR isoforms (Sc-EGFR-1a and Sc-EGFR-1b) were obtained from Sinonovacula constricta. Sequence analysis of the coding region of Sc-EGFRs, revealed the 2 isoforms were generated from a common gene by alternative splicing and that one form possessed an extra 81 bp that corresponded to an insertion of 27 amino acids in the C-terminus. Expression of the Sc-EGFR isoforms were detectable in the early embryo stages and increased in the middle-late stages. Both isoforms were widely expressed in tissues, but the highest levels were detected in the siphon. Although both isoforms had a typical extracellular region, transmembrane region, and intracellular region, the insertion/deletion of 27 aa in the C-terminus changed the phosphorylation sites and may have influenced downstream kinase activities. In vitro, the proliferating cell nuclear antigen indicator and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that HEK293T cells transfected with Sc-EGFR-1a and Sc-EGFR-1b exhibited different proliferation patterns. Sc-EGFR-1a caused more rapid proliferation than Sc-EGFR-1b and the EGFP-N1 control. In vivo, Sc-EGFRs were successfully knocked-down for at least 15 days by injecting the clams with the dsRNA and was associated with a significant reduction in shell length. Our results reveal for the first time in mollusks the existence of alternative splicing of EGFR and provide the basis for further studies to establish the role of EGFR in growth and development of S. constricta.

Abstract 1232: Development of novel therapeutic splice-switching oligonucleotides targeting oncogenic RNA isoforms driving race-related aggressive prostate cancer

Abstract Background: Age-adjusted incidence and mortality rates for prostate cancer (PCa) among African American (AA) men are 1.6- and 2.4-fold greater, respectively, than among white men. More aggressive characteristics of AA PCa account for a significant component of the PCa disparity, in addition to social determinants of health. Previous work from our laboratory and others have identified deregulation of the androgen receptor (AR) and epidermal growth factor receptor (EGFR) pathways in AA versus white PCa. Thus, there is an urgent need to develop a novel therapeutic strategy capable of inhibiting oncogenic AR and EGFR RNA isoforms enriched for in AA PCa. Methods: We have designed and synthesized novel chemically modified splice switching oligonucleotides (SSOs) to: 1) correct aberrant splicing leading to production of AR-V7 (AR-V7 SSO), an AR variant that lacks the ligand binding domain, is constitutively active and associates with castration-resistant PCa, poorer clinical outcomes and resistance to androgen ablation/AR inhibition therapies, 2) drive production of AR45 (AR45 SSO), an AR variant that has a unique, non-functional transactivation domain and acts in a dominant-negative manner and 3) drive production of inhibitory or dominant-negative EGFR isoforms (EGFR-TM or EGFR-TK SSOs), lacking the transmembrane or tyrosine kinase domain, respectively. Results: Transfection of a panel of PCa cell lines derived from AA and white patients with AR-V7 SSO decreases AR-V7 RNA and protein in a dose-dependent manner. Preliminary data suggests this biochemical response correlates with a biologically significant phenotype, as AR-V7 SSO also decreases proliferation, proliferation in the presence of enzalutamide and colony forming ability of PCa cells. Following transfection of PCa cell lines with AR45 SSO, an increase in AR45 RNA is seen and simultaneous decreases in wild type AR RNA and AR-V7 RNA are seen. In addition, modulation of AR signaling is detected, with decreases in RNAs corresponding to AR-induced target genes and increases in RNAs corresponding to AR-repressed target genes. Transfection of PCa cell lines with EGFR-TM or EGFR-TK SSOs increase RNA corresponding to these isoforms in a dose-dependent manner. Furthermore, a decrease in phosphorylated EGFR protein is detected. Further studies to examine the effects of these SSOs on transactivation activity, signaling and PCa cell biology and the therapeutic efficacy of these SSOs in AA and white PCa patient-derived explants are underway. Conclusions: These studies suggest that SSOs can be developed to modulate AR and EGFR signaling. Such SSOs could represent a novel therapeutic strategy with the potential to reduce PCa disparities for AA men and improve outcomes for men of all races with aggressive disease driven by these mechanisms. Citation Format: Jennifer A. Freedman, Timothy Robinson, Bonnie LaCroix, Brendon Patierno, Daniel George, Bruce Sullenger, Steven Patierno. Development of novel therapeutic splice-switching oligonucleotides targeting oncogenic RNA isoforms driving race-related aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1232. doi:10.1158/1538-7445.AM2017-1232

Epidermal growth factor receptor as an adverse survival predictor in squamous cell carcinoma of the penis

Penile carcinoma (PC) is more frequent in underdeveloped countries, generally is diagnosed at an advanced stage when therapeutic options are restricted, and thus is associated with high morbidity/mortality rates. Recent studies have demonstrated clinical benefits with epidermal growth factor receptor (EGFR)-targeted therapy in patients with PC, although there is no test that provides accurate patient selection. The aim of the present study was to evaluate the prognostic value of EGFR gene and protein status in tumor samples from patients with primary penile squamous cell carcinoma. We assessed the expression of wild-type and 2 mutant EGFR isoforms (delA746-E750 and mL858R) by immunohistochemistry in 139 samples, of which 49 were also evaluated for EGFR copy number by fluorescence in situ hybridization (FISH). Positive immunohistochemical staining of wild-type and mutant EGFR was evidenced by complete and strong membranous staining. For FISH analysis, cases were considered unaltered, polysomic, or amplified, as determined by signals of the EGFR gene and chromosome 7. An independent cohort of 107 PC samples was evaluated for mutations in EGFR, KRAS, and BRAF. Protein overexpression was noted in nearly half of the cases and was associated with cancer recurrence (P=.004) and perineural invasion (P=.005). Expression of the 2 mutated EGFR isoforms was not observed. The FISH status was not associated with protein expression. Altered FISH (polysomy and gene amplification) was an independent risk factor for dying of cancer. Only 1 patient of 107 presented KRAS mutations, and no mutations of EGFR or BRAF were observed.

Abstract B18: Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent

Abstract African American (AA) men exhibit a nearly 2-fold higher incidence and 3-fold higher mortality rate from prostate cancer compared to Caucasian American (CA) men and disparities in tumor aggressiveness remain after controlling for social determinants of health. Previous work from our laboratory and others has revealed differences in gene expression that contribute to prostate cancer health disparities among AAs. Oncogenic signaling pathways exhibiting up-regulation more often in AA prostate cancer include androgen receptor (AR) and epidermal growth factor receptor (EGFR). Thus, novel therapeutic strategies capable of driving production of inhibitory AR and EGFR isoforms and capable of limiting aberrant constitutively active AR isoforms are urgently needed. Such strategies will increase our understanding of these molecular mechanisms underlying prostate cancer in AA men. In addition, such strategies have the potential to ultimately result in novel specific approaches for treatment that will help reduce prostate cancer disparities for AAs and will improve treatment of advanced stage disease in all men with aggressive disease driven by these mechanisms. Splice-switching oligonucleotides (SSOs) represent a novel therapeutic strategy to combat prostate cancer. Unlike the strategy of using RNA interference to inhibit the expression of a gene, SSOs simultaneously limit the production of pathogenic proteins and induce the expression of protein variants with therapeutic value. SSOs modulate pre-mRNA splicing by binding to target pre-mRNAs and blocking access of the splicing machinery to a particular splice site, and can be used to produce novel splice variants or correct aberrant splicing. Herein we have designed and synthesized novel SSOs targeting AR and EGFR pre-mRNAs to produce inhibitory splice variants and correct aberrant splicing as follows. We have synthesized a SSO to correct the aberrant splicing that leads to production of the constitutively active AR-V7 variant leading to restoration of ligand dependency. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the cryptic exon included in AR-V7, decreases AR-V7 RNA. In addition, we have synthesized a SSO to drive production of a naturally occurring dominant-negative AR variant (AR45) leading to interference with wild type AR transactivation activity. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the first exon of wild type AR and leads to inclusion of an alternative first exon encoding the unique N-terminal extension characterizing AR45, increases AR45 RNA. Furthermore, we have synthesized SSOs targeting exons in the transmembrane domain and the tyrosine kinase domain of EGFR to drive production of a soluble, naturally expressed inhibitory EGFR isoform and a dominant-negative EGFR isoform, respectively. Transfection of prostate cancer cells with these chemically modified SSOs increase RNA corresponding to these isoforms. Studies are underway to examine the effects of these SSOs on AR and EGFR signaling, respectively, and the effects of these SSOs on prostate tumor cell biology. These studies suggest that SSOs can be developed to modulate AR and EGFR signaling. Such SSOs have the potential to further our understanding of the contribution of the targeted molecular mechanisms to AA prostate cancer and have the potential to yield novel therapeutic modalities to combat prostate cancer in AA men as well as men of all races with aggressive disease driven by these mechanisms. Citation Format: Jennifer A. Freedman, Timothy J. Robinson, Bonnie LaCroix, Brendon M. Patierno, Daniel J. George, Bruce A. Sullenger, Steven R. Patierno. Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B18.

Preliminary Evidence on the Diagnostic and Molecular Role of Circulating Soluble EGFR in Non-Small Cell Lung Cancer.

Assessment of biological diagnostic factors providing clinically-relevant information to guide physician decision-making are still needed for diseases with poor outcomes, such as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a promising molecule in the clinical management of NSCLC. While the EGFR transmembrane form has been extensively investigated in large clinical trials, the soluble, circulating EGFR isoform (sEGFR), which may have a potential clinical use, has rarely been considered. This study investigates the use of sEGFR as a potential diagnostic biomarker for NSCLC and also characterizes the biological function of sEGFR to clarify the molecular mechanisms involved in the course of action of this protein. Plasma sEGFR levels from a heterogeneous cohort of 37 non-advanced NSCLC patients and 54 healthy subjects were analyzed by using an enzyme-linked immunosorbent assay. The biological function of sEGFR was analyzed in vitro using NSCLC cell lines, investigating effects on cell proliferation and migration. We found that plasma sEGFR was significantly decreased in the NSCLC patient group as compared to the control group (median value: 48.6 vs. 55.6 ng/mL respectively; p = 0.0002). Moreover, we demonstrated that sEGFR inhibits growth and migration of NSCLC cells in vitro through molecular mechanisms that included perturbation of EGF/EGFR cell signaling and holoreceptor internalization. These data show that sEGFR is a potential circulating biomarker with a physiological protective role, providing a first approach to the functional role of the soluble isoform of EGFR. However, the impact of these data on daily clinical practice needs to be further investigated in larger prospective studies.

Epidermal growth factor receptor signaling pathway involved in progestin‐resistance of human endometrial carcinoma: In a mouse model

The aim of these investigations was to study the role of gefitinib (a specific oral epidermal growth factor receptor-tyrosine kinase inhibitor) on reversing progestin-resistance in a human endometrial carcinoma xenograft model. To study the effect of gefitinib and epidermal growth factor receptor (EGFR) overexpression on tumor progestin resistance, the Ishikawa endometrial carcinoma cell line was transfected to stably express a high level of EGFR, which resulted in the progestin-resistant Ishikawa-pLWERNL subcell line. BALB/c nude mice were injected subcutaneously with the parental Ishikawa cell line and the Ishikawa-pLWERNL cell line. Therapy experiments with gefitinib alone or in combination with medroxyprogesterone acetate (MPA) were done and samples were analyzed for EGFR and progesterone receptor isoform B (PR-B) expression by Western blot and immumohistochemistry analyses. Role in blocking EGFR autophosphorylation and its downstream signaling pathway and antagonizing progestin resistance by gefitinib was investigated by Western blot analysis. EGFR expression was higher in progestin-resistant Ishikawa-pLWERNL endometrial cancer (EC) xenografts than in progestin-sensitive Ishikawa EC xenografts; in contrast, PR-B was higher in Ishikawa xenografts than in Ishikawa-pLWERNL xenografts. Higher EGFR expression reduced sensitivity to progestin and decreased PR-B expression in Ishikawa xenografts; it also abnormally activated EGFR autophosphorylation and its downstream signaling pathway. Gefitinib effectively inhibited the proliferation of EC xenografts that overexpressed EGFR, and reversed hormone resistance in progestin-resistant EC xenografts. The present study describes an in vivo model that can provide a valuable tool in studying the interaction of overexpressed EGFR and progestin resistance in EC. Gefitinib may be useful in the treatment of progestin-resistant EC.

EGFR Soluble Isoforms and Their Transcripts Are Expressed in Meningiomas

The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%.Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types.

Soluble epidermal growth factor receptor isoforms in non-small cell lung cancer tissue and in blood

Epidermal growth factor receptor (EGFR) is implicated in tumor development and is highly expressed in many human tumors. EGFR overexpression has been observed in both premalignant lesions and in malignant lung tumors, as well as in 40–80% of patients with non-small cell lung cancer (NSCLC). EGFR is a 170-kDa transmembrane glycoprotein with an extracellular ligand-binding domain and a cytoplasmic domain with intrinsic tyrosine kinase activity. Soluble forms of EGFR (sEGFR) containing the extracellular domain have been described both in conditioned media from EGFR overexpressing cells as well as in peripheral blood. However, very little is known regarding the molecular function and the biochemical properties of these circulating EGFR isoforms.This study investigates the expression of sEGFR in lung cancer cultured cells and NSCLC patients with the aim of identifying clinically relevant isoforms specifically produced by tumor cells.Proteomic approaches including OFFGEL electrophoresis and Western blotting analysis were used to assess the sEGFR expression pattern in primary lung tumor samples, normal counterparts and matched plasma.We discover that the isoelectric points of sEGFR isoforms in NSCLC biopsy tissue differ from those of the isoforms present in healthy tissue and detected in the plasma of all subjects.These results demonstrate, for the first time, the existence of sEGFR isoforms specifically produced by NSCLC tumor cells which could represent a new potential biomarker for diagnosis and therapy of lung tumors. However, our observations indicate that more highly sensitive and specific quantitative assays are needed in order to reliably detect the tumor-associated sEGFR isoforms in plasma samples.

Adult diffuse gliomas produce mRNA transcripts encoding EGFR isoforms lacking a tyrosine kinase domain

The epidermal growth factor receptor (EGFR) gene encodes four alternatively spliced mRNA, variants 1, 2, 3 and 4, respectively, encoding the whole isoform a (EGFR) and truncated isoforms b, c and d, all of which lack the receptor’s intracellular domain. In addition, a mutant EGFRvIII differs from isoform a in a truncated extracellular domain. The expression pattern of these isoforms is unknown in adult diffuse gliomas. Thus, we investigated in 47 cases: i) EGFR protein expression by immunohistochemistry using an extracellular domain-recognizing antibody (Ext-Ab) and an intracellular domain specific one (Int-Ab), ii) mRNA expression of EGFRv1, -v2, -v3, -v4 and -vIII by RT-PCR and iii) EGFR amplification by fluorescent in situ hybridization. The relation of these data with histological criteria and patient outcome was studied. The immunostaining was stronger with the Ext-Ab than with the Int-Ab. EGFRv1, -v2, -v3 and -v4 mRNA expression were highly correlated. They were expressed in all tumors, with highest levels in glioblastomas. EGFRv1 strong levels and the presence of vIII mRNAs were more closely associated with Int-Ab staining. EGFR gene amplification concerned only glioblastomas and was associated with the presence of EGFRvIII and high levels of EGFRv2, -v3 and -v4 transcripts. A pejorative outcome was associated with: histology (glioblastomas), EGFR amplification, strong Int-Ab labeling and high levels of variant mRNAs. Our results indicated that the full-length EGFR and mutant EGFRvIII are not the sole EGFR isoform expressed in diffuse gliomas. This could explain discordant immunohistochemical results reported in the literature and may have therapeutic implications.

Membranous Expression of Ectodomain Isoforms of the Epidermal Growth Factor Receptor Predicts Outcome after Chemoradiotherapy of Lymph Node–Negative Cervical Cancer

We compared the prognostic significance of ectodomain isoforms of the epidermal growth factor receptor (EGFR), which lack the tyrosine kinase (TK) domain, with that of the full-length receptor and its autophosphorylation status in cervical cancers treated with conventional chemoradiotherapy. Expression of EGFR isoforms was assessed by immunohistochemistry in a prospectively collected cohort of 178 patients with squamous cell cervical carcinoma, and their detection was confirmed with Western blotting and reverse transcriptase PCR. A proximity ligation immunohistochemistry assay was used to assess EGFR-specific autophosphorylation. Pathways associated with the expression of ectodomain isoforms were studied by gene expression analysis with Illumina beadarrays in 110 patients and validated in an independent cohort of 41 patients. Membranous expression of ectodomain isoforms alone, without the coexpression of the full-length receptor, showed correlations to poor clinical outcome that were highly significant for lymph node-negative patients (locoregional control, P = 0.0002; progression-free survival, P < 0.0001; disease-specific survival, P = 0.005 in the log-rank test) and independent of clinical variables. The ectodomain isoforms were primarily 60-kD products of alternative EGFR transcripts. Their membranous expression correlated with transcriptional regulation of oncogenic pathways including activation of MYC and MAX, which was significantly associated with poor outcome. This aggressive phenotype of ectodomain EGFR expressing tumors was confirmed in the independent cohort. Neither total nor full-length EGFR protein level, or autophosphorylation status, showed prognostic significance. Membranous expression of ectodomain EGFR isoforms, and not TK activation, predicts poor outcome after chemoradiotherapy for patients with lymph node-negative cervical cancer.

Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor and α5-Integrin

Proteins encoded by the epidermal growth factor receptor (EGFR/HER1/ERBB1) gene are being studied as diagnostic, prognostic, and theragnostic biomarkers for numerous human cancers. The clinical application of these tissue/tumor biomarkers has been limited, in part, by discordant results observed for epidermal growth factor receptor (EGFR) expression using different immunological reagents. Previous studies have used EGFR-directed antibodies that cannot distinguish between full-length and soluble EGFR (sEGFR) expression. We have generated and characterized an anti-sEGFR polyclonal antiserum directed against a 31-mer peptide (residues 604-634) located within the unique 78-amino acid carboxy-terminal sequence of sEGFR. Here, we use this antibody to demonstrate that sEGFR is coexpressed with EGFR in a number of carcinoma-derived cell lines. In addition, we show that a second protein of ~140 kDa (p140) also is detected by this antibody. Rigorous biochemical characterization identifies this second protein to be α5-integrin. We show that a 26-amino acid peptide in the calf domain of α5-integrin (residues 710-735) is 35% identical in sequence with a 31-mer carboxy-terminal sEGFR peptide and exhibits an approximately 5-fold lower affinity for anti-sEGFR than the homologous 31-mer sEGFR peptide does. We conclude that the carboxy terminus of sEGFR and the calf-1 domain of α5-integrin share a region of sequence identity, which results in their mutual immunological reactivity with anti-sEGFR. We also demonstrate that anti-sEGFR promotes three-dimensional tissue cohesion and compaction in vitro, further suggesting a functional link between sEGFR and α5-integrin and a role of the calf-1 domain in cell adhesion. These results have implications for the study of both EGFR and sEGFR as cancer biomarkers and also provide new insight into the mechanisms of interaction between cell surface EGFR isoforms and integrins in complex processes such as cell adhesion and survival signaling.

Abstract CN09-03: The EGFR conundrum: EGFR-based diagnostics in the era of precision medicine

Abstract CN09-03: The EGFR conundrum: EGFR-based diagnostics in the era of precision medicine