Upregulation Of Epidermal Growth Factor Receptor Research Articles

The role of systemic therapy in advanced skull base chordomas: overview of the current state and the MD Anderson protocol.

The role of systemic therapy in primary or advanced and metastatic chordoma has been traditionally limited because of the inherent resistance to cytotoxic therapies and lack of specific or effective therapeutic targets. Despite resection and adjuvant radiation therapy, local recurrence rates in clival chordoma remain high and the risk of systemic metastases is not trivial, leading to significant morbidity and mortality. Recently, molecular targeted therapies (MTTs) and immune checkpoint inhibitors (ICIs) have emerged as promising therapeutic avenues in chordoma. In recent years, preclinical studies have identified potential targets based on intrinsic genetic dependencies, epigenetic modulators, or newly identified tumor-associated cell populations driving treatment resistance and recurrence. Nonetheless, the role of systemic therapies in the neoadjuvant or adjuvant setting for primary, locally progressive, and distant metastatic chordomas is still being investigated. Herein, an overview of current and emerging systemic treatment strategies in advanced clival chordoma is provided. Furthermore, several molecular biomarkers have been recently uncovered as potential predictors of the response to specific molecular therapeutics. The authors describe the recently discovered role of 1p36 and 9p21 deletions as biomarkers capable of guiding drug selection. Then they discuss completed and ongoing clinical trials of MTTs, including several tyrosine kinase inhibitors used as monotherapy or in combination, such as imatinib, sorafenib, dasatinib, and lapatinib, among others, as well as mammalian target of rapamycin inhibitors such as everolimus and rapamycin. They present their experience and other recent studies demonstrating vast benefits in advanced chordoma from ICIs. Additionally, they provide a brief overview of novel systemic strategies such as adoptive cell transfer (CAR-T and NK cells), oncolytic viruses, epigenetic targeting (KDM6, HDAC, and EZH2 inhibitors), and several promising preclinical studies with high translational potential. Finally, the authors present their institutional multidisciplinary protocol for the incorporation of systemic therapy for both newly diagnosed and recurrent chordomas based on molecular studies including upfront enrollment in MTT trials in patients with epidermal growth factor receptor upregulation or INI-1 deficiency or enrollment in ICI clinical trials for patients with high tumor mutational burden or high PD-L1 expression on tumor cells or in the tumor microenvironment.

Abstract 2724: BCG020, a novel bispecific antibody targeting EGFR and CD70, is effective against EGFR and CD70 high expressed tumors

Abstract The epidermal growth factor receptor (EGFR) is highly expressed across a wide range of different cancers, and is a known driver of cancer growth; EGFR upregulation is frequently associated with adverse prognosis. Many patients undergoing EGFR-TKI (tyrosine kinase inhibitors) treatment will acquire resistance, which can be accompanied by increased expression of CD70 in some cancers. Simultaneous engagement of both EGFR and CD70 with a bispecific antibody (bsAb) is a potential strategy to overcome such resistance and achieve greater efficacy. To explore this strategy, we developed a novel IgG1 bsAb, BCG020, targeting EGFR and CD70. The parental EGFR antibody and CD70 antibody were developed from fully human common light chain antibody mice (RenLite®). When compared to EGFR benchmarks, the parental EGFR antibody exhibits similar affinity to both human and cynomolgus monkey EGFR, similar internalization activity in EGFR+ tumor cell lines, and superior blocking activity of EGF to EGFR in vitro. Furthermore, the EGFR parental antibody demonstrates good stability. The parental CD70 antibody exhibits high affinity to both human and monkey CD70, with superior internalization activity to benchmark antibody in CD70+ tumor cell lines, as well as superior blockade of CD70-induced signaling of CD27-Luc when compared to benchmark antibodies. When assembled into a bsAb, BCG020 exhitibs the same high affinity as both parental antibodies, with higher internalization activity than parental antibodies and benchmark antibodies. To evaluate the efficacy of BCG020 in vivo, we established cell line-derived xenograft (CDX) models in immunodeficient (B-NDG) mice. We observed that BCG020 treatment (10 mg/kg) potently inhibited growth of EGFR+CD70+ tumors. Taken together, these data indicate that BCG020 is a first-in-class bispecific antibody with potential to treat solid tumors with high expression of EGFR and CD70. Citation Format: Yongfei Yang, Xiaoxia Sun, Ziyan Li, Yue Yan, Linpeng Xu, Xiaoxue Wu, Luheng Du, Ying Zhu, Chaoshe Guo, W. Frank An. BCG020, a novel bispecific antibody targeting EGFR and CD70, is effective against EGFR and CD70 high expressed tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2724.

Epidermal growth factor receptor inhibition leads to cellular phenotype correction of DSP-mutated keratinocytes.

Desmoplakin (DSP) is a desmosomal component expressed in skin and heart, essential for desmosome stability and intermediate filament connection. Pathogenic variants in the DSP gene encoding DSP, lead to heterogeneous skin, adnexa and heart-related phenotypes, including skin fragility, woolly hair (WH), palmoplantar keratoderma (PPK) and arrhythmogenic/dilated cardiomyopathy (ACM/DCM). The ambiguity of computer-based prediction analysis of pathogenicity and effect of DSP variants, indicates a necessity for functional analysis. Here, we report a heterozygous DSP variant that was not previously described, NM_004415.4:c.3337C>T (NM_004415.4(NP_004406.2):p.(Arg1113*)) in a patient with PPK, WH and ACM. RNA and protein analysis revealed ~50% reduction of DSP mRNA and protein expression. Patient's keratinocytes showed fragile cell-cell connections and perinuclear retracted intermediate filaments. Epidermal growth factor receptor (EGFR) is a transmembrane protein expressed in the basal epidermal layer involved in proliferation and differentiation, processes that are disrupted in the development of PPK, and in the regulation of the desmosome. In skin of the abovementioned patient, evident EGFR upregulation was observed. EGFR inhibition in patient's keratinocytes strongly increased DSP expression at the plasma membrane, improved intermediate filament connection with the membrane edges and reduced the cell-cell fragility. This cell phenotypic recovery was due to a translocation of DSP to the plasma membrane together with an increased number of desmosomes. These results indicate a therapeutic potential of EGFR inhibitors for disorders caused by DSP haploinsufficiency.

MiR-218-5p/EGFR Signaling in Arsenic-Induced Carcinogenesis.

Arsenic is a well-known carcinogen inducing lung, skin, bladder, and liver cancer. Abnormal epidermal growth factor receptor (EGFR) expression is common in lung cancer; it is involved in cancer initiation, development, metastasis, and treatment resistance. However, the underlying mechanism for arsenic-inducing EGFR upregulation remains unclear. RT-PCR and immunoblotting assays were used to detect the levels of miR-218-5p and EGFR expression. The Luciferase assay was used to test the transcriptional activity of EGFR mediated by miR-218-5p. Cell proliferation, colony formation, wound healing, migration assays, tube formation assays, and tumor growth assays were used to study the function of miR-218-5p/EGFR signaling. EGFR and miR-218-5p were dramatically upregulated and downregulated in arsenic-induced transformed (As-T) cells, respectively. MiR-218-5p acted as a tumor suppressor to inhibit cell proliferation, migration, colony formation, tube formation, tumor growth, and angiogenesis. Furthermore, miR-218-5p directly targeted EGFR by binding to its 3'-untranslated region (UTR). Finally, miR-218-5p exerted its antitumor effect by inhibiting its direct target, EGFR. Our study highlights the vital role of the miR-218-5p/EGFR signaling pathway in arsenic-induced carcinogenesis and angiogenesis, which may be helpful for the treatment of lung cancer induced by chronic arsenic exposure in the future.

Upregulation of the EGFR/MEK1/MAPK1/2 signaling axis as a mechanism of resistance to antiestrogen‑induced BimEL dependent apoptosis in ER+ breast cancer cells.

The epidermal growth factor receptor (EGFR) is commonly upregulated in multiple cancer types, including breast cancer. In the present study, evidence is provided in support of the premise that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitates the escape of breast cancer cells from BimEL‑dependent apoptosis, conferring resistance to therapy. This conclusion is based on the findings that ectopic BimEL cDNA overexpression and confocal imaging studies confirm the pro‑apoptotic role of BimEL in ERα expressing breast cancer cells and that upregulated EGFR/MEK1/MAPK1/2 signaling blocks BimEL pro‑apoptotic action in an antiestrogen‑resistant breast cancer cell model. In addition, the present study identified a pro‑survival role for autophagy in antiestrogen resistance while EGFR inhibitor studies demonstrated that a significant percentage of antiestrogen‑resistant breast cancer cells survive EGFR targeting by pro‑survival autophagy. These pre‑clinical studies establish the possibility that targeting both the MEK1/MAPK1/2 signaling axis and pro‑survival autophagy may be required to eradicate breast cancer cell survival and prevent the development of antiestrogen resistance following hormone treatments. The present study uniquely identified EGFR upregulation as one of the mechanisms breast cancer cells utilize to evade the cytotoxic effects of antiestrogens mediated through BimEL‑dependent apoptosis.

Long Noncoding RNA HLA Complex Group 18 Improves the Cell Proliferation of Myocardial Fibroblasts by Regulating the Hsa-microRNA-133a/Epidermal Growth Factor Receptor Axis.

Hsa-microRNA (has-miR)-133a inactivates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and suppresses the cell proliferation of myocardial fibroblasts by downregulation of the epidermal growth factor receptor (EGFR) expression. Bioinformatics analysis exhibits extended noncoding RNA HLA complex group 18 (lncRNA-HCG18) binds to hsa-miR-133a. The purpose of the current experiment is to explore whether lncRNA-HCG18 adsorbed hsa-miR-133a through sponging, resulting in decreased inhibition of hsa-miR-133a on EGFR and ultimately promoting the proliferation of myocardial fibroblasts. To verify and study the correlation and mechanism between lncRNA-HCG18, hsa-miR-133a, and their target genes. Firstly, after overexpression/silencing of lncRNA-HCG18 in myocardial fibroblasts, the level of hsa-miR-133a expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the EGFR, ERK1/2, and p-ERK1/2 expression levels were assessed by Western blotting to confirm that upregulation of EGFR and p-ERK1/2 protein levels by overexpression of lncRNA-HCG18, siRNA lncRNA-HCG18 (siHCG18) reduced the EGFR and p-ERK1/2 protein levels. Then, the luciferase reporter gene system was used to verify that lncRNA-HCG18 regulated EGFR expression by inhibiting the function of the hsa-miR-133a regulatory target gene. Then, a RAP assay was used to confirm that lncRNA-HCG18 interacted with hsa-miR-133a. Finally, the analysis of CCK-8 results indicated that the cell proliferation of myocardial fibroblasts was significantly reduced by siHCG18 while reversed by overexpression of lncRNA-HCG18. Thus, lncRNA-HCG18 inhibited cell viability of cardiac fibroblasts via the hsa-miR-133a/EGFR axis, which was regarded as a regulator of cell proliferation of cardiac fibroblasts in cardiovascular diseases.

Abstract LB133: EGFR upregulation in breast cancer cells: A key mechanism of escape from antiestrogen induced death, autophagy, and senescence

Abstract EGFR is upregulated commonly in multiple cancer types, including breast cancer. However, the multiple role(s) of EGFR in breast cancer are not fully elucidated. In this study, we characterized a unique antiestrogen resistant breast cancer subline and provide compelling data that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitated escape from senescence, autophagy, and BimEL-dependent apoptosis. Our studies were conducted with the antiestrogen resistant TR5 cell line established by our laboratory, using a step-wise drug selection with the antiestrogen 4-hydroxytamoxifen (4-OHT) of the estrogen receptor α (ER+) MCF-7 breast cancer cells. A preliminary microarray comparing mRNA expression between 4-OHT-selected TR5 cells and MCF-7 parent cells identified upregulation of the epidermal growth factor receptor (EGFR). Western blot showed a greater than 5-fold upregulation of EGFR expression and downregulation of estrogen receptor alpha (ERα) compared to the levels in MCF-7 and T47-D (antiestrogen sensitive). Selective inhibition of EGFR, phosphorylated at Tyr 1068, was achieved with erlotinib. Inhibition blocked MEK1/MAPK1/2 signaling, which was also upregulated in 4-OHT selected TR5 cells, with detectable increases in the pro-apoptotic BH3 protein BimEL, and a fifty percent reduction in cell viability. However, a high number of cells survived erlotinib therapy due to autophagy induction determined to be cytoprotective. Autophagy induction was identified by analyzing the expression/turnover of autophagy proteins LC3-II and p62, a standard approach to ascertain autophagy levels in cells. Further, erlotinib treatment induced a 2-fold increase in the number of senescent cells in the surviving population. Senescent cells were identified by β-galactosidase staining of erlotinib-treated cells utilizing confocal microscopy. Interestingly, blockade of autophagy with the lysosomotrophic compound chloroquine enhanced the senescence cell population, suggesting that either autophagy blocked senescence induction or lysosome impairment increased senescence. Mechanistically, we have identified phosphorylated AMP-activated protein kinase (pAMPK) as being elevated under conditions of erlotinib treatment, and reduced in 4-OHT-selected TR5 cells relative to MCF-7 parent cells. This correlation supports the hypothesis that pAMPK was downregulated during 4-OHT selection of TR5 cells by elevated EGFR/MEK/MAPK1/2 signaling to allow surviving cells escape senescence induction and/or anti-proliferative autophagy. Overall we propose that EGFR mediated downregulation of pAMPK is a major regulatory pathway in breast cancer cells surviving endocrine treatment. Our current studies are dissecting the role of EGFR-mediated AMPK regulation with somatic cell-based studies and pharmacological intervention. Citation Format: Mackenzie L. Hagan, Michael McGrath, Carol Joseph, Allison Lewis, John R. Barrett, Muthusamy Thangaraju, David Gewirtz, Patricia V. Schoenlein. EGFR upregulation in breast cancer cells: A key mechanism of escape from antiestrogen induced death, autophagy, and senescence [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 LB133.

T-box transcription factor 19 promotes hepatocellular carcinoma metastasis through upregulating EGFR and RAC1.

The effect of targeted therapy for metastatic hepatocellular carcinoma (HCC) is still unsatisfactory. Exploring the underlying mechanism of HCC metastasis is favorable to provide new therapeutic strategies. T-box (TBX) transcription factor family genes, which are crucial regulators in embryo and organ development, are vital for regulating tumor initiation, growth and metastasis. Here we explored the role of TBX19 in HCC metastasis, which is one of the most upregulated TBX family genes in human HCC tissues. TBX19 expression was markedly upregulated in HCC tissues and elevated TBX19 expression predicted poor prognosis. Overexpression of TBX19 enhanced HCC metastasis through upregulating epidermal growth factor receptor (EGFR) and Rac family small GTPase 1 (RAC1) expression. Downregulation of EGFR and RAC1 inhibited TBX19-mediated HCC metastasis, while upregulation of EGFR and RAC1 restored inhibition of HCC metastasis mediated by TBX19 knockdown. Furthermore, epidermal growth factor (EGF)/EGFR signaling upregulated TBX19 expression via the extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-kB axis. Besides, the combined application of EGFR inhibitor Erlotinib and RAC1 inhibitor NSC23766 markedly inhibited TBX19-mediated HCC metastasis. In HCC cohorts, TBX19 expression was positively associated with EGFR and RAC1 expression. Patients with positive coexpression of TBX19/EGFR or TBX19/RAC1 displayed the poorest prognosis. In conclusion, EGF/EGFR signaling upregulated TBX19 expression via ERK/NF-kB pathway and TBX19 fostered HCC metastasis by enhancing EGFR and RAC1 expression, which formed an EGF-TBX19-EGFR positive feedback loop. Targeting this signaling pathway may offer a potential therapeutic strategy to efficiently restrain TBX19-mediated HCC metastasis.

Neurofibromin Deficiency Causes Epidermal Growth Factor Receptor Upregulation through the Activation of Ras/ERK/SP1 Signaling Pathway in Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheet Tumor.

Neurofibromatosis type 1 (NF1) is an autosomal dominant human genetic disorder. The progression of benign plexiform neurofibromas to malignant peripheral nerve sheet tumors (MPNSTs) is a major cause of mortality in patients with NF1. Although elevated epidermal growth factor receptor (EGFR) expression plays a crucial role in the pathogenesis of MPNST, the cause of EGFR overexpression remains unclear. Here, we assessed EGFR expression levels in MPNST tissues of NF1 patients and NF1 patient-derived MPNST cells. We found that the expression of EGFR was upregulated in MPNST tissues and MPNST cells, while the expression of neurofibromin was significantly decreased. Manipulation of NF1 expression by NF1 siRNA treatment or NF1-GAP-related domain overexpression demonstrated that EGFR expression levels were closely and inversely correlated with neurofibromin levels. Notably, knockdown of the NF1 gene by siRNA treatment augmented the nuclear localization of phosphorylated SP1 (pSP1) and enhanced pSP1 binding to the EGFR gene promoter region. Our results suggest that neurofibromin deficiency in NF1-associated MPNSTs enhances the Ras/ERK/SP1 signaling pathway, which in turn may lead to the upregulation of EGFR expression. This study provides insight into the progression of benign tumors and novel therapeutic approaches for treatment of NF1-associated MPNSTs.

METTL3 induces PLX4032 resistance in melanoma by promoting m6A-dependent EGFR translation.

Acquired resistance often limits therapeutic efficacy of the BFAF (V600E) kinase inhibitor PLX4032 in patients with advanced melanoma. Epitranscriptomic modification of mRNAs by N6-methyladenosine (m6A) modification contributes to melanoma pathogenesis; however, its role in acquired PLX4032 resistance remains unexplored. Here, we showed that m6A methyltransferase METTL3 expression is upregulated in A375R cells, a PLX4032-resistant subline of A375 melanoma cells, compared with the parental cells. Moreover, METTL3 increased the m6A modification of epidermal growth factor receptor (EGFR) mRNA in A375R cells, which promoted its translation efficiency. In turn, increased EGFR expression facilitated rebound activation of the RAF/MEK/ERK pathway in A375R cells, inducing PLX4032 resistance. In contrast, knockout of METTL3 in A375R cells reduced EGFR expression and restored PLX4032 sensitivity. PLX4032 treatment following METTL3 knockout induced apoptosis and reduced colony formation in A375R cells and reduced A375R cell-derived tumor growth in BALB/c nude mice. These findings indicate that METTL3 promotes rebound activation of the RAF/MEK/ERK pathway through EGFR upregulation and highlight a critical role for METTL3-induced m6A modification in acquired PLX4032 resistance in melanoma, implicating METTL3 as a potential candidate for targeted chemotherapy.

Heparin-binding epidermal growth factor inhibits apoptosis in cisplatin-resistant pancreatic cancer cells via upregulation of EGFR and ERCC 1 expressions

Purpose: To investigate the influence of heparin-binding epidermal growth factor (HB-EGF) on apoptosis in cisplatin-resistant pancreatic cancer cells, as well as its mechanism of action.
 Methods: Pancreatic cancer cisplatin-resistant cells (BXPC-3/CDDP) were transfected with HB-EGF small interfering RNA (siRNA). The cells were randomly assigned to four groups, namely, BXPC-3 group (group A), BXPC-3/CDDP group (group B), transfected group A (group Asi) and transfected group B (group Bsi). Cell proliferation was determined using MTT assay, and the levels of expression of HBEGF, epidermal growth factor receptor (EGFR) and excision repair cross-complementation group 1 (ERCC 1) were determined using Western blotting. The extent of apoptosis was determined by flow cytometry.
 Results: Cell proliferation was increased in group B, relative to group A, but was significantly decreased after transfection with HB-EGF siRNA (p < 0.05). The half-maximal inhibitory concentration (IC50) of group Bsi was reduced, relative to group Asi (p < 0.05). The expression of HB-EGF was significantly upregulated in group B, relative to group A (p < 0.05). In contrast, HB-EGF siRNA transfection of the cells significantly down-regulated HB-EGF expression (p < 0.05). Early apoptosis was significantly higher in group A than in groups B and Bsi. Higher levels of apoptosis were seen in group Bsi, relative to group B after inhibition of HB-EGF expression (p < 0.05).
 Conclusion: These results indicate that HB-EGF is resistant to cisplatin, and it inhibits apoptosis in pancreatic cancer cells via the upregulation of EGFR and ERCC 1 expressions.

Lapatinib ditosylate rescues memory impairment in D-galactose/ovariectomized rats: Potential repositioning of an anti-cancer drug for the treatment of Alzheimer's disease

Epidermal growth factor receptor (EGFR) signaling plays a substantial role in learning and memory. The upregulation of EGFR has been embroiled in the pathophysiology of Alzheimer's disease (AD). Nevertheless, most of EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have been extensively studied for non-CNS diseases such as cancer and rheumatoid arthritis. TKIs targeting-based research in neurodegenerative disorders sounds to be lagging behind those of other diseases. Hence, this study aims to explore the molecular signaling pathways and the efficacy of treatment with lapatinib ditosylate (LAP), as one of EGFR-TKIs that has not yet been investigated in AD, on cognitive decline induced by ovariectomy (OVX) with chronic administration of D-galactose (D-gal) in female Wistar albino rats. OVX rats were injected with 150 mg/kg/day D-gal ip for 8 weeks to induce AD. Administration of 100 mg/kg/day LAP p.o. for 3 weeks starting after the 8th week of D-gal administration improved memory and debilitated histopathological alterations. LAP decreased the expression of GFAP, p-tau, and Aβ 1-42. Besides, it reduced EGFR, HER-2, TNF-α, NOX-1, GluR-II, p38 MAPK, and p-mTOR. LAP increased nitrite, and neuronal pro-survival transduction proteins; p-PI3K, p-AKT, and p-GSK-3β levels. Taken together, these findings suggest the role of LAP in ameliorating D-gal-induced AD in OVX rats via activating the pro-survival pathway; PI3K-Akt-GSK-3β, while inhibiting p-mTOR, NOX-1, and p38 MAPK pathways. Moreover, this research offered a significant opportunity to advance awareness of the repositioning of TKI anti-cancer drugs for the treatment of AD.

Corrigendum] Tumor‑penetrating peptide fused EGFR single‑domain antibody enhances radiation responses following EGFR inhibition in gastric cancer.

Following the publication of the above article, the authors noticed that data shown in certain of the panels in Figs.4 and5 were selected incorrectly and presented wrongly in these figures. Essentially, in Fig.4, the data shown for the Tunel, anti‑EGFR‑iRGD and Tunel, anti‑EGFR‑iGRD+IR data panels (i.e., the panels in the third row, columns 2 and 4), were chosen incorrectly, and in Fig.5, the data panel for the Lung, IR experiment (fourth row, third column) was selected incorrectly. The revised versions of Figs.4 and 5, featuring all the correct data panels, are shown on the next page. Furthermore, the results were re‑analyzed based on the correct data. The errors made in the compilation of these Figures did not affect the overall conclusions reported in the paper. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in Oncology Reports 40: 1583-1591, 2018; DOI: 10.3892/or.2018.6532].

EGFR-rich extracellular vesicles derived from highly metastatic nasopharyngeal carcinoma cells accelerate tumour metastasis through PI3K/AKT pathway-suppressed ROS.

Nasopharyngeal carcinoma (NPC) is the most common cancer with high metastatic potential that occurs in the epithelial cells of the nasopharynx. Distant metastases are the primary cause for treatment failure and mortality of NPC patients. However, the underlying mechanism responsible for the initiation of tumour cell dissemination and tumour metastasis in NPC is not well understood. Here, we demonstrated that epidermal growth factor receptor (EGFR) was highly expressed in tumour tissues of NPC patients with distant metastases and was associated with a decrease in reactive oxygen species (ROS). We also revealed that extracellular vesicles (EVs) transfer occurred from highly to poorly metastatic NPC cells, mediating cell–cell communication and enhancing the metastatic potential of poorly metastatic NPC cells. Further experiments indicated that EVs derived from highly metastatic NPC cells induced the up‐regulation of EGFR and down‐regulation of ROS in low metastatic NPC cells. Mechanistically, EGFR‐rich EVs‐mediated EGFR overexpression down‐regulated intracellular ROS levels through the PI3K/AKT pathway, thus promoting the metastatic potential of poorly metastatic NPC cells. Strikingly, treatment with EVs secreted from highly metastatic NPC cells was significantly associated with rapid NPC progression and shorter survival in xenografted mice. These findings not only improve our understanding of EVs‐mediated NPC metastatic mechanism but also have important implications for the detection and treatment of NPC patients accompanied by aberrant EGFR‐rich EVs transmission.

Prognostic impact of cytoplasmatic EGFR upregulation in patients with oral squamous cell carcinoma: A pilot study.

In various tumors, epidermal growth factor-receptor (EGFR) serves a role in tumorigenesis and has an impact on survival. Usually the EGF-receptor is located on the surface of the cell membrane and is involved in various signaling pathways. The dimerization of EGFR with other ErbB family proteins, such as HER2, is important for the tumor progression. Nevertheless, a second EGFR-associated signaling pathway appears to be important for tumor cells, which is cytoplasmic/nuclear EGFR. The present study examined the influence of membranous or cytoplasmic localized EGFR on the prognosis of patients with oral squamous cell carcinoma (OSCC). Slides from 45 OSCC tumor samples were stained against EGFR using immunohistochemistry and analysed by the Remmele score system. The association with histopathological parameters and survival data was analyzed. Cytoplasmatic EGFR localization was identified as an independent predictive biomarker for overall survival in the examined OSCC cohort according to multivariate Cox regression analysis. Positive cytoplasmatic EGFR staining was correlated with a higher risk of early death (RR=3.0; P=0.035), while membranous EGFR localization did not affect patient survival. To the best of our knowledge, the present study is the first study to demonstrate that cytoplasmatic-localized EGFR is an independent prognostic biomarker for the overall survival of patients with OSCC.

Abstract 5197: A systems approach to evaluating the effects of two EGFR-targeted therapies on cellular models of human skin and lung cancer using an integrated imaging and analysis platform and the rapid quantification of multiple key biomarkers

Abstract The physiological function of the epidermal growth factor receptor (EGFR) is to regulate epithelial tissue development and homeostasis, but its overexpression and activation are strongly linked to oncogenesis and tumor progression in a variety of human cancers. Many EGFR-targeted therapies have been developed which alone have proven to be effective, but resistance often develops, indicating a need for more studies probing the efficacy of combinatorial drug treatments. These studies will further require the discovery and characterization of biomarkers that can predict therapeutic efficacy and resistance. The purpose of this study was to examine the effects of individual and combined treatment with two classical EGFR-targeted therapies with different modes of action on cellular models of human skin cancer and non-small cell lung carcinoma (NSCLC). We characterized the effects of a two-day treatment with varying concentrations of EGF on cellular health and proliferation and on the expression of several key biomarker proteins in A431 and A549 cell lines. We then examined the individual and combined effects of treatment with the tyrosine kinase inhibitor (TKI) gefitinib and the humanized monoclonal antibody cetuximab. Cellular growth, proliferation and health were assessed with ATPlite luminescence assays and well-imaging and cytometry measures using a high-throughput multimodal imager and plate reader. Biomarkers measured included EGFR, immune checkpoint proteins (PD-L1 and CD276/B7-H3), chemokines (IL-8/CXCL8 and GRO-α/CXCL1), a protease inhibitor (TIMP-2), and growth factor (VEGF). All these biomarker proteins were rapidly assessed using AlphaLISA no-wash, mix-and-read assays on 5 μL samples collected from the same wells of the culture plate. We observed that EGF treatment resulted in decreased EGFR expression and upregulation of PD-L1 protein in a dose-dependent manner in both culture models. CD276, VEGF and TIMP-2 expression levels, however, were upregulated with EGF treatment only in the A431 cells. Complex drug treatment effects on biomarker expression are presented. In addition, treatment effects on EGFR phosphorylation and downstream signaling pathways (including MEK/ERK and AKT) were probed using AlphaLISA SureFire Ultra phosphorylation assays and demonstrate the different pathways effected by either drug or a combination of both. These data illustrate the effects of EGFR stimulation on the expression of an array of biomarkers and signaling pathways and in the cellular tolerance for individual and combined treatment with two EGFR-targeted therapies with different mechanisms of action between two different cellular models of human cancer. Citation Format: Jeanine M. Hinterneder, Lauren Berstler, Adam Carlson, Jen Carlstrom. A systems approach to evaluating the effects of two EGFR-targeted therapies on cellular models of human skin and lung cancer using an integrated imaging and analysis platform and the rapid quantification of multiple key biomarkers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5197.

Pediatric Soft Tissue Tumors With BCOR ITD Express EGFR but Not OLIG2.

Somatic internal tandem duplication of 3' of BCOR (BCOR ITD) has been found in clear cell sarcomas of the kidney (CCSK), soft tissue undifferentiated round cell sarcomas/primitive myxoid mesenchymal tumors of infancy (URCS/PMMTI), and a subgroup of central nervous system high-grade neuroepithelial tumors (CNS-HGNET). BCOR ITD+ tumors share morphologic features. Expression of OLIG2 and epidermal growth factor receptor (EGFR) has been reported in CNS-HGNET with BCOR ITD. Here, we characterize OLIG2 and EGFR expression in URCS/PMMTI with BCOR ITD. Paraffin blocks of 9 polymerase chain reaction-confirmed soft tissue BCOR ITD+ tumors (URCS/PMMTI) were immunophenotyped for OLIG2 and EGFR expression and scored semiquantitatively by percentage of positive cells and intensity of staining as negative, 1+, 2+, and 3+. Fluorescence in situ hybridization (FISH) for EGFR amplification was performed (amplification EGFR/CEP7 ratio ≥2.0). All 9 tumors showed membrane/cytoplasmic expression of EGFR, strong and diffuse (3+) in 8 cases; weak (+2) in 1. FISH detected no EGFR amplification. OLIG2 was negative in all. EGFR is overexpressed in pediatric URCS/PMMTI with BCOR ITD and may be related to transcriptional upregulation of EGFR by BCOR ITD. OLIG2 negative staining differentiates URCS/PMMTI from CNS-HGNET. This finding may further support the possibility that these tumors have a different stem cell of origin.

Abstract 4935: T-DM1-resistant cells gain high invasive activity via EGFR and integrin cooperated pathways

Abstract Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate developed to treat trastuzumab-resistant cancer. Despite initial favorable outcomes, most patients eventually cease to respond by developing acquired resistance to T-DM1. Currently, there is no targeted therapy to treat T-DM1-resistant cancer. To explore novel therapeutic targets to overcome T-DM1 resistance, we generated T-DM1-resistant cells using trastuzumab-resistant JIMT1 cells. We found that the loss of human epidermal growth factor receptor 2 (HER2) confers T-DM1 resistance, which in turn activates a compensatory mechanism that increases epidermal growth factor receptor (EGFR) expression. Upregulation of EGFR increases the protein levels of α5β1 integrin, resulting in enhanced motility and invasion of T-DM1-resistant cells. However, silencing β1 integrin expression by siRNA or β1 integrin blockage induced by an inhibitory antibody, MAB 13, significantly increases invasion of T-DM1-resistant cells. The discovery of functional cooperation between EGFR and integrin in regulating cell growth and invasion provides an opportunity to develop novel therapeutic strategy by dual-targeting EGFR and specific integrin to overcome T-DM1 resistance. We now try to identify downstream molecules involved in the increased invasion using next generation sequencing (NGS) technology in β1 integrin-deficient T-DM1 resistant cells. Citation Format: Yukinori Endo, Zachary Olson, Wen Jin Wu. T-DM1-resistant cells gain high invasive activity via EGFR and integrin cooperated pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4935.

FCHSD2 controls oncogenic ERK1/2 signaling outcome by regulating endocytic trafficking

The evolution of transformed cancer cells into metastatic tumors is, in part, driven by altered intracellular signaling downstream of receptor tyrosine kinases (RTKs). The surface levels and activity of RTKs are governed mainly through clathrin-mediated endocytosis (CME), endosomal recycling, or degradation. In turn, oncogenic signaling downstream of RTKs can reciprocally regulate endocytic trafficking by creating feedback loops in cells to enhance tumor progression. We previously showed that FCH/F-BAR and Double SH3 Domain-Containing Protein (FCHSD2) has a cancer-cell specific function in regulating CME in non-small-cell lung cancer (NSCLC) cells. Here, we report that FCHSD2 loss impacts recycling of the RTKs, epidermal growth factor receptor (EGFR) and proto-oncogene c-Met (MET), and shunts their trafficking into late endosomes and lysosomal degradation. Notably, FCHSD2 depletion results in the nuclear translocation of active extracellular signal-regulated kinase 1 and 2 (ERK1/2), leading to enhanced transcription and up-regulation of EGFR and MET. The small GTPase, Ras-related protein Rab-7A (Rab7), is essential for the FCHSD2 depletion-induced effects. Correspondingly, FCHSD2 loss correlates to higher tumor grades of NSCLC. Clinically, NSCLC patients expressing high FCHSD2 exhibit elevated survival, whereas patients with high Rab7 expression display decreased survival rates. Our study provides new insight into the molecular nexus for crosstalk between oncogenic signaling and RTK trafficking that controls cancer progression.

Noggin is associated with a poor prognosis of gastric cancer by promoting the proliferation of gastric cancer cells via the upregulation of EGFR.

Noggin is an antagonist of bone morphogenetic proteins(BMP), being indispensable for certain developmental events. The present study aimed to examine the role of Noggin in the development and prognosis of gastric cancer(GC) and to elucidate the underlying mechanisms. The expression of Noggin in GC was evaluated by RT‑qPCR, immunohistochemistry and by the analyses of publicly available databases. The effects of Noggin on proliferation, cell cycle, adhesion, invasion, colony formation and tumour spheroid were examined following both the knockdown and overexpression of Noggin in GC cell lines. The involvement of epidermal growth factor receptor(EGFR) signalling was examined by western blot analysis and by using small molecule inhibitors. As a result, a higher expression of Noggin in GC was found to be associated with a poorer overall survival. Noggin overexpression promoted the proliferation and colony formation of GC cells by promoting cell cycle progression. The knockdown of Noggin in HGC27 cells exerted an opposite effect on proliferation, colony formation and cell cycle progression. Noggin expression positively correlated with EGFR expression in both GC cell line models and The Cancer Genome Atlas human GC cohort. Targeting EGFR and its downstream pathways diminished cell proliferation which was promoted by Noggin. Furthermore, Noggin overexpression resulted in an enhanced nuclear translocation of β‑catenin, leading to an upregulation of EGFR. Thus, the findings of the present study demonstrate that Noggin promotes the proliferation of GC cells by upregulating EGFR and enhancing a vicious circle formed by β‑catenin, EGFR, ERK and Akt.