Epidermal Growth Factor Receptor Nuclear Translocation Research Articles

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Ginsenoside Rg3 Sensitizes Nasopharyngeal Carcinoma Cells to Radiation by Suppressing Epithelial Mesenchymal Transition.

Radioresistance restrains the therapeutic effect of nasopharyngeal carcinoma (NPC). Ginsenoside Rg3 (Rg3), an active pharmaceutical component extracted from ginseng, shows antitumor effects in various cancers. In this study, we aimed to determine whether Rg3 sensitized NPC cells to radiation and to explore the possible mechanisms. Our results revealed that Rg3 increased radiosensitivity in both HNE1 and CNE2 cell lines. Radiation-induced epithelial mesenchymal transition (EMT) in NPC cells and Rg3 blocked this effect. In addition, Rg3 attenuated radiation-induced epidermal growth factor receptor (EGFR) nuclear transport and DNA-dependent protein kinase expression. What's more, Rg3 significantly accelerated the apoptosis rates in irradiated NPC cells. In summary, our data suggested that Rg3 sensitized NPC cells to radiation and suppressed radiation-induced EMT. This effect is mediated through restrained EGFR nuclear translocation and increased cell apoptosis. Thus, Rg3 may be a potential radiation sensitizing agent for NPC.

In Situ Visualization of Epidermal Growth Factor Receptor Nuclear Translocation with Circular Bivalent Aptamer.

Epidermal growth factor receptor (EGFR) nuclear translocation correlates with the abnormal proliferation, migration, and anti-apoptosis of tumor cells. Monitoring EGFR nuclear translocation provides insights into the molecular mechanisms underlying cancers. EGFR nuclear translocation includes two processes, EGFR phosphorylation and phosphorylated EGFR translocation to the nucleus. With the help of aptamers, probes that can achieve the first step of anchoring phosphorylated EGFR have been developed. However, the EGFR nuclear translocation can last for hours, posing a challenge to monitor the entire nuclear translocation in living cells. Herein, we designed a circular bivalent aptamer-functionalized optical probe with greatly enhanced stability for long-term visualization of EGFR nuclear translocation in situ. The results of cell experiments show that the probe could monitor the entire nuclear translocation of EGFR. The findings of tissue and in vivo experiments demonstrate that the probe can evaluate the development and progression of tumors by imaging EGFR nuclear translocation in situ. The proposed approach allows us to monitor EGFR nuclear translocation in the long term, indicating its great potential in investigating the mechanisms of cancers and guiding for tumor treatment.

Downregulation of annexin A3 promotes ionizing radiation-induced EGFR activation and nuclear translocation and confers radioresistance in nasopharyngeal carcinoma

Radioresistance currently poses a significant challenge to successful disease control of nasopharyngeal carcinoma (NPC). We previously uncovered that annexin A3 (ANXA3), a calcium-dependent phospholipid binding protein, is underexpressed in radioresistant NPC cells and mouse xenografts. This study aims to further unravel the mechanistic basis underlying ANXA3-mediated radioresistance in NPC. We show that either innate ANXA3 downregulation or short hairpin RNA(shRNA)-based knockdown of ANXA3 confers resistance to ionizing radiation (IR) in NPC both in vitro and in mouse xenograft models in vivo, whereas radiosensitization was observed when ANXA3 was ectopically expressed. Mechanistically, ANXA3 knockdown dramatically enhances IR-induced epidermal growth factor receptor (EGFR) phosphorylation and nuclear translocation, leading to increased post-IR phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) concomitant with markedly accelerated DNA DSB repair. In addition, pretreatment with cetuximab efficiently abrogated the radioresistant phenotype of ANXA3-low cells as well as the ANXA3 knockdown-induced post-IR EGFR nuclear accumulation, suggesting that EGFR is an essential mediator for ANXA3 depletion-mediated radioprotection in NPC. Collectively, this work reveals for the first time a critical role of ANXA3 in radiation survival and DNA repair mechanism of NPC and provides mechanistic evidence to support ANXA3 as a potential therapeutic target to improve radiocurability for NPC.

Abstract LB096: EGFR-dependent inflammation as a driver of breast cancer

Abstract Triple Negative Breast Cancer (TNBC) is one of the most aggressive and metastatic subtypes of hormone receptor negative breast cancers. Evidence supports that most patients with TNBC overexpress the Epidermal Growth Factor Receptor (EGFR), yet its role in driving the disease remains unclear. Therapies have been developed to target EGFR, but EGFR-targeted antibodies and tyrosine kinase inhibitors fail to work effectively in breast cancer patients. A possible explanation is that in addition to its canonical function at the membrane, EGFR has a non-canonical function where it can undergo retrograde trafficking to the nucleus, making it resistant to antibody-based therapeutics. Once in the nucleus, EGFR is known to function as a transcriptional cofactor for STAT3, STAT5, and E2F1, thereby regulating the expression of genes that promote inflammation and cell proliferation. Via RNA-Seq analysis, we demonstrate that EGFR can regulate different subsets of genes depending on its canonical signaling activity or nuclear role. Our goal is to understand and distinguish this difference in regulation to assess how EGFR affects inflammation. We hypothesize that EGFR is modifying inflammation differently depending on its canonical or non-canonical role, thereby driving disease progression in multiple ways. We have found that canonical EGFR can induce interleukin 8 while nuclear EGFR can repress interleukin 6 through the use of either kinase inhibitors (IL8) or blocking nuclear translocation of EGFR (IL6). Correlated with these changes are alterations in growth and inflammatory profiles in both a triple negative inflammatory mouse model and an EGFR-driven breast cancer mouse model. We are now investigating the role of nuclear-EGFR-dependent inflammation in driving tumor progression. Understanding the different mechanisms by which EGFR modifies inflammation and the tumor microenvironment would allow us to develop more specific targets for patients with TNBC. Citation Format: Angelica Escoto. EGFR-dependent inflammation as a driver of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB096.

Primaquine Inhibits the Endosomal Trafficking and Nuclear Localization of EGFR and Induces the Apoptosis of Breast Cancer Cells by Nuclear EGFR/Stat3-Mediated c-Myc Downregulation.

Triple-negative breast cancer (TNBC) cells overexpress the epidermal growth factor receptor (EGFR). Nuclear EGFR (nEGFR) drives resistance to anti-EGFR therapy and is correlated with poor survival in breast cancer. Inhibition of EGFR nuclear translocation may be a reasonable approach for the treatment of TNBC. The anti-malarial drugs chloroquine and primaquine have been shown to promote an anticancer effect. The aim of the present study was to investigate the effect and mechanism of chloroquine- and primaquine-induced apoptosis of breast cancer cells. We showed that primaquine, a malaria drug, inhibits the growth, migration, and colony formation of breast cancer cells in vitro, and inhibits tumor growth in vivo. Primaquine induces damage to early endosomes and inhibits the nuclear translocation of EGFR. Primaquine inhibits the interaction of Stat3 and nEGFR and reduces the transcript and protein levels of c-Myc. Moreover, primaquine and chloroquine induce the apoptosis of breast cancer cells through c-Myc/Bcl-2 downregulation, induce early endosome damage and reduce nEGFR levels, and induce apoptosis in breast cancer through nEGFR/Stat3-dependent c-Myc downregulation. Our study of primaquine and chloroquine provides a rationale for targeting EGFR signaling components in the treatment of breast cancer.

Estrogen receptor β and treatment with a phytoestrogen are associated with inhibition of nuclear translocation of EGFR in the prostate

Knockout of ERβ in the mouse leads to nuclear expression of epidermal growth factor receptor (EGFR) in the prostate. To examine whether ERβ plays a similar role in the human prostate, we used four cohorts of men: 1) a Swedish cohort of normal prostates and PCa (prostate cancer) of different Gleason grades; 2) men with benign prostatic hyperplasia (BPH) treated with the 5α-reductase inhibitor, finasteride, and finasteride together with the ERβ agonists, soy isoflavones; 3) men with PCa above Gleason grade 4 (GG4), treated with ADT (androgen deprivation therapy) and abiraterone (AA), the blocker of androgen synthesis for different durations; and 4) men with GG4 PCa on ADT or ADT with the AR (androgen receptor) blocker, enzalutamide, for 4 mo to 6 mo. In men with BPH, finasteride treatment induced EGFR nuclear expression, but, when finasteride was combined with isoflavones, EGFR remained on the cell membrane. In GG4 patients, blocking of AR for 4 mo to 6 mo resulted in loss of ERβ and PTEN expression and increase in patients with nuclear EGFR from 10 to 40%. In the men with GG4 PCa, blocking of adrenal synthesis of testosterone for 2 mo to 7 mo had the beneficial effect of increasing ERβ expression, but, on treatment longer than 8 mo, ERβ was lost and EGFR moved to the nucleus. Since nuclear EGFR is a predictor of poor outcome in PCa, addition of ERβ agonists together with abiraterone should be considered as a treatment that might sustain expression of ERβ and offer some benefit to patients.

Valproic Acid Synergizes With Cisplatin and Cetuximab in vitro and in vivo in Head and Neck Cancer by Targeting the Mechanisms of Resistance.

Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. The combination of cisplatin (CDDP) plus cetuximab (CX) is one of the standard first-line treatments in this disease. However, this therapeutic regimen is often associated with high toxicity and resistance, suggesting that new combinatorial strategies are needed to improve its therapeutic index. In our study, we evaluated the antitumor effects of valproic acid (VPA), a well-known antiepileptic agent with histone deacetylase inhibitory activity, in combination with CDDP/CX doublet in head and neck squamous cell carcinoma (HNSCC) models. We demonstrated, in HNSCC cell lines, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX resulted in a clear synergistic antiproliferative and pro-apoptotic effects. The synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, suggesting the ability of the combined approach to affect also the cancer stem cells compartment. Mechanistically, VPA enhanced DNA damage in combination treatment by reducing the mRNA expression of ERCC Excision Repair 1, a critical player in DNA repair, and by increasing CDDP intracellular concentration via upregulation at transcriptional level of CDDP influx channel copper transporter 1 and downregulation of the ATPAse ATP7B involved in CDDP-export. Valproic acid also induced a dose-dependent downregulation of epidermal growth factor receptor (EGFR) expression and of MAPK and AKT downstream signaling pathways and prevent CDDP- and/or CX-induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of genes induced by non-canonical activity of nuclear EGFR, such as cyclin D1 and thymidylate synthase. Finally, we confirmed the synergistic antitumor effect also in vivo in both heterotopic and orthotopic models, demonstrating that the combined treatment completely blocked HNSCC xenograft tumors growth in nude mice. Overall, the introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. A phase II clinical trial exploring the combination of VPA and CDDP/CX in R/M HNSCC patients is currently ongoing in our institute.

Epidermal growth factor receptor-mediated regulation of matrix metalloproteinase-2 and matrix metalloproteinase-9 in MCF-7 breast cancer cells

In breast cancer, increased epidermal growth factor receptor (EGFR) expression and phosphorylation have been correlated with increased invasive potential and poor prognosis. Interaction of EGFR with its ligand epidermal growth factor (EGF) activates cellular signalling cascades promoting tumour invasion. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are upregulated in most cancers and play crucial roles in modulating invasion and metastasis. EGFR-mediated regulation of MMP-2 and MMP-9 in breast cancer was investigated using metastatic human breast ductal carcinoma cell line MCF-7. Culture of MCF-7 cells on 1µg/ml EGF-coated culture dishes for 24h led to appreciable increase in MMP-2 and MMP-9 expression and activity. Expression of membrane type-1 matrix metalloproteinase (MT1-MMP) and focal adhesion kinase (FAK), phosphorylation of EGFR and phosphatidylinositol 3' kinase (PI3K), and nuclear translocation of EGFR and cellular migration were also appreciably increased. Targeting EGFR-EGF interactions by treatment of MCF-7 cells with anti-EGFR monoclonal antibodies prior to culture on EGF prevented appreciable upregulation of MMP-2 and MMP-9 expression and activity. Increased expression of MT1-MMP and FAK, phosphorylation of EGFR and PI3K and enhanced cell migration were also inhibited. Treatment of cells with PI3K inhibitor LY294002 prevented upregulation of MMP-2 and MMP-9 indicating that EGFR-mediated signalling for MMP regulation occurs through PI3K. As increased EGFR activity has been observed in highly invasive breast cancers, targeting EGFR-EGF interactions might render such cancers less invasive by inhibiting EGFR-mediated upregulation of MMP-2 and MMP-9 and therefore could be of importance in their clinical management.

PGE2 mediates EGFR internalization and nuclear translocation via caveolin endocytosis promoting its transcriptional activity and proliferation in human NSCLC cells.

Prostaglandin E2 (PGE2) contributes to tumor progression by promoting cancer cell growth, invasion and by creating a favorable pro-tumor microenvironment. PGE2 has been reported to transactivate and internalize into the nucleus receptor tyrosine kinases such as Epidermal growth factor receptor (EGFR), thereby supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, PGE2 induces EGFR nuclear translocation via different dynamin-dependent endocytic pathways, promotes the formation of an EGFR-STAT3 complex, affects nuclear EGFR target gene expression and mediates tumor cell proliferation. Indeed, we find that PGE2 induces EGFR internalization and consequent nuclear import through Clathrin- and Caveolin-mediated endocytosis and through the interaction of EGFR with Importin β1. Within the nucleus, EGFR forms a complex with STAT3, an event blocked by ablation of Clathrin Heavy Chain or Caveolin-1. The combination of EGF and PGE2 prolongs nuclear EGFR transcriptional activity manifested by the upregulation of CCND1, PTGS2, MYC and NOS2 mRNA levels and potentiates nuclear EGFR-induced NSCLC cell proliferation. Additionally, NSCLC patients with high expression of a nuclear EGFR gene signature display shorter survival times than those with low expression, thus showing a putative correlation between nuclear EGFR and poor prognosis in NSCLC. Together, our findings indicate a complex mechanism underlying PGE2-induced EGF/EGFR signaling and transcriptional control, which plays a key role in cancer progression.

Abstract 3333: The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor

Abstract The epidermal growth factor receptor (EGFR) is an important therapeutic target in patients with various cancers. Unfortunately, resistance to anti-EGFR therapeutics is common. Previous studies in our laboratory identified two mechanisms of resistance to the EGFR monoclonal antibody cetuximab: First, the nuclear translocation of EGFR bypasses the inhibitory effects of cetuximab; and second, the receptor tyrosine kinase AXL mediates cetuximab resistance by maintaining EGFR activation and downstream signaling. On the basis of these findings, we hypothesized that AXL could mediate the nuclear translocation of EGFR in the setting of cetuximab resistance. We found that NSCLC cetuximab-resistant clones and patient-derived xenografts (PDXs) had increased abundance of nuclear EGFR (nEGFR) and AXL. Cellular fractionation techniques, super resolution microscopy, and electron microcopy revealed that genetic loss of AXL diminished the nuclear translocation and accumulation of EGFR. Building on previous studies indicating that SRC family kinases (SFKs) and HER family ligands mediate the nuclear translocation of EGFR, we found that AXL knockdown down-regulated the expression of the SFKs YES and LYN, and the ligand neuregulin-1 (NRG1). Furthermore, AXL knockdown decreased the interaction between EGFR and HER3 and the nuclear abundance of HER3. Nuclear localization of EGFR could be rescued only upon simultaneous overexpression of Lyn and NRG1 in cells depleted of AXL. Collectively, these data uncover a previously unrecognized role for AXL in regulating the nuclear translocation of EGFR and suggest that AXL-mediated SFK activation and NRG1 expression are necessary and sufficient to regulate this process. Citation Format: Toni M. Brand, Mari Iida, Kelsey L. Corrigan, Cara M. Braverman, John P. Coan, Bailey G. Flanigan, Andrew P. Stein, Ravi Salgia, Jana Rolff, Randall J. Kimple, Deric L. Wheeler. The receptor tyrosine kinase AXL mediates nuclear translocation of the epidermal growth factor receptor [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 3333. doi:10.1158/1538-7445.AM2017-3333

Abstract 3327: Sortilin controls the EGFR nuclear translocation

Abstract The epidermal growth factor receptor (EGFR) deregulations play a central role in cancer progression. EGFR generates extra- and intracellular signals through various pathways, including its release in the tumor microenvironment through extracellular vesicles, as well as the nuclear EGFR signaling. That ultimately results in oncogenic signals promoting the tumor aggressiveness. Our previous report showed that sortilin, a member of the VPS10P sorting proteins, is associated with EGFR to allow its secretion through exosomes in non-small cell lung cancer (NSCLC) cells. Furthermore, sortilin is mainly present in the Golgi which is an essential compartment involved in the EGFR bypass towards the nucleus. Therefore, our aim is to investigate the potential role of sortilin in EGFR nuclear translocation. EGFR and sortilin translocation into the nucleus has been validated by proximity ligation assay and confocal microscopy analysis. Moreover, to study the sortilin function in the EGFR nuclear translocation, we resorted to the NSCLC cell line A549 in which the sortilin expression was silenced using a shRNA delivery. Subcellular fractionations and nuclear protein extractions were performed to study the impact of the sortilin loss on the EGF-induced EGFR nuclear translocation. EGF stimulation highly increases the nuclear translocation of the EGFR and sortilin complex. Interestingly, sortilin depletion significantly decreases the EGF-induced EGFR nuclear translocation in the A549 cells. Strikingly, sortilin-depleted cells exhibit an overexpression of EGFR target genes (MYC, ABCG2, AURKA and CCND1) compared to the control cells. These results suggest that the overexpression of the EGFR target genes is independent of EGFR nuclear translocation in sortilin-depleted cells. Thus, we analyze the activation of the known transcription factors directly involved in the control of EGFR up-regulated genes to determine whether they are activated in sortilin-depleted cells. Moreover, to better understand the sortilin-dependent EGFR nuclear translocation, we compare functional properties (proliferation, migration and angiogenesis assays) of sortilin-depleted and control cells. Altogether, our findings suggest a key role of sortilin in the regulation of the EGFR nuclear transport, towards the control of target genes that are widely involved in cancer progression. Citation Format: Hussein Al Akhrass, Thomas Naves, Marie-Odile Jauberteau, François Vincent, Fabrice Lalloué. Sortilin controls the EGFR nuclear translocation [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 3327. doi:10.1158/1538-7445.AM2017-3327

Inhibition of EGFR nuclear translocation attenuate radioresistance through decreased p-DNA-PK expression in cervical cancer cells.

e17011 Background: The commonly used treatment for cervical cancer is radiotherapy. However, the resistance to irradiation and metastasis at the advanced stage is a common reason for the poor prognosis and high mortality. This study was designed to elucidate the role of epidermal growth factor receptor (EGFR) nuclear translocation in radioresistance, and its correlation with DNA damage repair pathway in the cervical cancer cells. Methods: The dynamic expression of EGFR, DNA-dependent protein kinase (DNA-PK), PDK-1, PKN1 and their phosphorylation level in irradiated cervical cancer cell line CaSki at 0 10 20 40 minutes was determined by western blotting. Besides, nuclear localization signal (NLS) peptide inhibitor and control peptides was synthesized and treated cells before irradiation to elucidate the correlation between EGFR nuclear translocation and DNA damage repair after irradiation. Results: Expression of EGFR, protein kinase N1 (PKN1), and DNA-PK in nucleus was increased after irradiation in CaSki cells. Irradiation also enhanced the phosphorylation level of EGFR at Thr654, PKN1 at T774 and DNA-PK at T2609. Inhibition of EGFR nuclear translocation by NLS peptide decreased the expression level of EGFR and DNA-PK in the nucleus, and attenuated their phosphorylation process. Conclusions: EGFR nuclear translocation riggered by irradiation promoted DNA damage repair in irradiated cervical cancer cells. This work facilitated us to understand the possible molecular mechanism of the resistance to irradiation in the treatment of cervical cancer, providing a potentially potent clinical method to cancer therapy.

Knockdown of CAVEOLIN-1 Sensitizes Human Basal-Like Triple-Negative Breast Cancer Cells to Radiation

Background/Aims: Triple-negative breast cancer (TNBC) is a high-risk breast cancer phenotype without specific targeted therapy options and is significantly associated with increased local recurrence in patients treated with radiotherapy. CAVEOLIN-1 (CAV-1)-mediated epidermal growth factor receptor (EGFR) nuclear translocation following irradiation promotes DNA repair and thus induces radiation resistance. In this study, we aimed to determine whether knockdown of CAV-1 enhances the radiosensitivity of basal-like TNBC cell lines and to explore the possible mechanisms. Methods: Western blotting was used to compare protein expression in a panel of breast cancer cell lines. Nuclear accumulation of EGFR as well as DNA repair and damage at multiple time points following irradiation with or without CAV-1 siRNA pretreatment were investigated using western blotting and confocal microscopy. The radiosensitizing effect of CAV-1 siRNA was evaluated using a clonogenic assay. Flowcytometry was performed to analyse cell apoptosis and cell cycle alteration. Results: We found that CAV-1 is over-expressed in basal-like TNBC cell lines and barely expressed in HER-2-positive cells; additionally, we observed that HER-2-positive cell lines are more sensitive to irradiation than basal-like TNBC cells. Our findings revealed that radiation-induced EGFR nuclear translocation was impaired by knockdown of CAV-1. In parallel, radiation-induced elevation of DNA repair proteins was also hampered by pretreatment with CAV-1 siRNA before irradiation. Silencing of CAV-1 also promoted DNA damage 24 h after irradiation. Colony formation assays verified that cells could be radiosensitized after knockdown of CAV-1. Furthermore, G₂/M cell cycle arrest and apoptosis enhancement may also contribute to the radiosensitizing effect of CAV-1 siRNA. Conclusion: Our results support the hypothesis that CAV-1 knockdown by siRNA causes increased radiosensitivity in basal-like TNBC cells. The mechanisms associated with this effect are reduced DNA repair through delayed CAV-1-associated EGFR nuclear accumulation and induction of G₂/M arrest and apoptosis through the combined effects of CAV-1 siRNA and radiation.

Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells.

IL-17E synergizes with EGF and confers in vitro resistance to EGFR-targeted therapies in TNBC cells.

Estrogen receptor-, progesterone receptor- and HER2-negative breast cancers, also known as triple-negative breast cancers (TNBCs), have poor prognoses and are refractory to current therapeutic agents, including epidermal growth factor receptor (EGFR) inhibitors. Resistance to anti-EGFR therapeutic agents is often associated with sustained kinase phosphorylation, which promotes EGFR activation and translocation to the nucleus and prevents these agents from acting on their targets. The mechanisms underlying this resistance have not been fully elucidated. In addition, the IL-17E receptor is overexpressed in TNBC tumors and is associated with a poor prognosis. We have previously reported that IL-17E promotes TNBC resistance to anti-mitotic therapies. Here, we investigated whether IL-17E promotes TNBC resistance to anti-EGFR therapeutic agents by exploring the link between the IL-17E/IL-17E receptor axis and EGF signaling. We found that IL-17E, similarly to EGF, activates the EGFR in TNBC cells that are resistant to EGFR inhibitors. It also activates the PYK-2, Src and STAT3 kinases, which are essential for EGFR activation and nuclear translocation. IL-17E binds its specific receptor, IL-17RA/IL17RB, on these TNBC cells and synergizes with the EGF signaling pathway, thereby inducing Src-dependent EGFR transactivation and pSTAT3 and pEGFR translocation to the nucleus. Collectively, our data indicate that the IL-17E/IL-17E receptor axis may underlie TNBC resistance to EGFR inhibitors and suggest that inhibiting IL-17E or its receptor in combination with EGFR inhibitor administration may improve TNBC management.

Abstract 247: Understanding the role of tyrosine 1101 in the nuclear translocation of the epidermal growth factor receptor

Abstract Triple-negative breast cancer (TNBC) is a subclass of breast cancers that are negative for the estrogen receptor, the progesterone receptor, and HER2. Currently, TNBCs have poor prognosis and very few proven molecular targets. One promising target in TNBC has been the epidermal growth factor receptor (EGFR), as it is overexpressed in as many as 80% of TNBCs. Strikingly, however, antibody-based therapy directed against the EGFR (cetuximab) in TNBCs has had modest clinical impact. Over the last decade, advances in EGFR biology have established that the EGFR functions in two distinct signaling pathways: classical membrane-bound signaling and the nuclear EGFR signaling pathway. We previously demonstrated that EGFR nuclear translocation is dependent on Src Family Kinases (SFKs) and phosphorylation of tyrosine 1101 (Y1101) on the cytoplasmic tail of the EGFR. In these studies, translocation of the EGFR to the nucleus led to activation of the nuclear EGFR signaling pathway, which resulted in cetuximab resistance. Resistance could be reversed by simultaneously targeting both the nuclear EGFR signaling pathway and the classical EGFR signaling pathway through the inhibition of its nuclear transport (blockade of SFKs) and with cetuximab, respectively. Despite the identification of nuclear EGFR as a functional molecular target in TNBC, the role of Y1101 in EGFR translocation has yet to be identified. In this study, we seek to better understand the role of Y1101 in the nuclear translocation of EGFR in TNBC. Using a battery of TNBC cell lines, we have created isogenic pairs that overexpress either wild type EGFR (EGFR-WT) or EGFR with a mutant Y1101 (EGFR-Y110F). Using electron and super-resolution confocal microscopy as well as cellular fractionation, we have shown that EGFR-Y1101 undergoes normal endocytosis upon EGF stimulation in multiple cell lines, but unlike EGFR-WT, EGFR-Y1101F deposits around the perinuclear region. These data suggest that EGFR-Y1101F is able to undergo normal translocation to the perinuclear space, but that amino acid Y1101 is necessary for direct nuclear entry, potentially due to its interactions with nucleoporins. Current work is focused on the identification of nucleoporins with which EGFR-Y1101 may interact. In addition, continued work focuses on allelic mutation using CrisprCas9. Ultimately, the long-term goal of this project is to understand how Y1101 mediates EGFR nuclear translocation. With this information, we will then aim to develop allosteric inhibitors of Y1101 that may abrogate EGFR nuclear translocation and lead to a novel therapeutic which targets the nuclear EGFR signaling pathway in TNBC. Citation Format: Bailey G. Flanigan, Mari Iida, Toni M. Brand, Deric L. Wheeler. Understanding the role of tyrosine 1101 in the nuclear translocation of the epidermal growth factor receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 247.

Ezrin Enhances EGFR Signaling and Modulates Erlotinib Sensitivity in Non–Small Cell Lung Cancer Cells

Ezrin is a scaffolding protein that is involved in oncogenesis by linking cytoskeletal and membrane proteins. Ezrin interacts with epidermal growth factor receptor (EGFR) in the cell membrane, but little is known about the effects of this interaction on EGFR signaling pathway. In this study, we established the biological and functional significance of ezrin-EGFR interaction in non–small cell lung cancer (NSCLC) cells. Endogenous ezrin and EGRF interaction was confirmed by co-immunoprecipitation and immunofluorescent staining. When expression of ezrin was inhibited, EGFR activity and phosphorylation levels of downstream signaling pathway proteins ERK and STAT3 were decreased. Cell fractionation experiments revealed that nuclear EGFR was significantly diminished in ezrin-knockdown cells. Consequently, mRNA levels of EGFR target genes AURKA, COX-2, cyclin D1, and iNOS were decreased in ezrin-depleted cells. A small molecule inhibitor of ezrin, NSC305787, reduced EGF-induced phosphorylation of EGFR and downstream target proteins, EGFR nuclear translocation, and mRNA levels of nuclear EGFR target genes similar to ezrin suppression. NSC305787 showed synergism with erlotinib in wild-type EGFR-expressing NSCLC cells, whereas no synergy was observed in EGFR-null cells. Phosphorylation of ezrin on Y146 was found as an enhancer of ezrin-EGFR interaction and required for increased proliferation, colony formation, and drug resistance to erlotinib. These findings suggest that ezrin-EGFR interaction augments oncogenic functions of EGFR and that targeting ezrin may provide a potential novel approach to overcome erlotinib resistance in NSCLC cells.

Epidermal Growth Factor based Therapy Promotes Intracellular Trafficking and Accumulation of its Receptor in the Nucleus of Fibroblasts from Diabetic Foot Ulcers

Objective: To gain a better understanding of the Epidermal Growth Factor (EGF) Receptor (EGFR) activation, trafficking and biological response in diabetic foot ulcers (DFU), exposed to recombinant human EGF via intra-ulcer infiltration as a healing alternative. Methods: We studied by immunoelectron microscopy the intracellular localization of the EGFR and Proliferating Cell Nuclear Antigen (PCNA) in fibroblast-like cells (FLC) from granulation tissue of DFU patients, collected before and at different time points after EGF treatment. Results: EGF therapy appears to increase EGFR immunolabeling. At early time-points, EGFR labeling is observed predominantly in the nucleus, suggesting a fast EGFR internalization and nuclear translocation. Interestingly EGFR is also detected in the mitochondrial outer membrane. PCNA expression and trafficking were also detected in a time-dependent manner after EGF infiltration. Conclusion: Differential subcellular distribution of EGFR and PCNA and accumulation in the nucleus, in a timepoint specific manner, supports the induction of an EGF-mediated activation program that is sustained for at least 24 hours after the EGF administration. These findings substantiate the therapeutic ability of EGF to restore the healing process in DFU.

The Soluble Heparin-Binding EGF-Like Growth Factor Stimulates EGF Receptor Trafficking to the Nucleus.

Most ligands of epidermal growth factor receptor (EGFR) have the ability to induce EGFR translocation into the nucleus, where EGFR acts as an important transcriptional regulator. Soluble form of heparin-binding EGF-like growth factor (sHB-EGF) is one of the ligands for EGFR in many cell types; however, there is no evidence for the ability of sHB-EGF to induce EGFR nuclear importation. Here, we demonstrated that treatment of A431 cells with sHB-EGF resulted in nuclear localization of EGFR and such translocation occurs via retrograde pathway. It was shown by confocal microscopy and co-immunoprecipitation assay that the translocation complex consisted of both ligand and receptor. The chromatin immunoprecipitation assay showed the association of sHB-EGF–EGFR complex with promoter region of cyclin D1 in the cell nucleus and this association was prevented by application of EGFR kinase inhibitor AG-1478. The obtained data suggest that sHB-EGF acts similarly to other EGFR ligands and is capable to induce EGFR nuclear translocation as a part of ligand-receptor complex in a tyrosine phosphorylation-dependent manner.