Overexpression Of Epidermal Growth Factor Receptor Research Articles

LOCL-12 OUTCOMES OF STEREOTACTIC RADIOSURGERY FOR BRAIN METASTASES OF BREAST CANCER IN RELATION TO MOLECULAR SUBTYPES

Abstract PURPOSE To determine intracranial outcomes following stereotactic radiosurgery (SRS) for brain metastases of breast cancer in relation to molecular subtypes of HER2 (human epidermal growth factor receptor) overexpression and estrogen receptor (ER) expression. METHOD From a retrospective institutional database of SRS using VMAT (volumetric modulated arc therapy) carried out between 2012 and 2019, patients with a primary diagnosis of breast cancer were identified. Outcomes following first course of SRS were analyzed using radiotherapy plans and electronic patient records. Systemic anticancer treatments were not registered. Time to intracranial progression (TTP) was calculated using the Kaplan Meier method as the time between SRS and recurrence, whereas dates of death or last follow up without recurrence were censored. The study was approved by the Danish Patient Safety Authority (nr. 31-1521-125). RESULTS The study included 159 patients. 132 patients (83%) underwent a brain MRI examination after SRS and, of these, 101 had recurrence, either local (41%), remote (43%) or both (16%). Tumors were HER2 positive in 50% of cases and 61% were ER positive and 15% were triple negative. The median intracranial TTP was for the whole study group 9.1 months. Median intracranial TTP for patients with the HER2+ / ER+ subtype was 15.4 mos. whereas patients with the triple negative subtype had a significantly shorter median intracranial TTP of 5.1 mos. (p=0.007). Patients with HER2+/ER- and HER2 normal/ER+ had similar median intracranial TTP times of 7.7 mos. and 8.4 mos., respectively (n.s.). CONCLUSION This study found high rates of intracranial recurrence following SRS and poor intracranial TTP that was highly dependent on molecular subtypes, reflecting either biological differences or intracranial effect of systemic treatments or both. The study confirms the universally poor prognosis for breast cancer patients with brain metastases and the high frequency of HER2 overexpression in this patient group.

Investigating the clinical significance of EGFR expression using machine learning in a series of Iraqi patients with triple-negative breast cancer.

Breast cancer is a heterogeneous disease with a distinct profile of the expression of each tumor. Triple-negative breast cancer (TNBC) is a molecular subtype of breast cancer characterized by an aggressive clinical behavior linked to loss or reduced expression of estrogen, progesterone, and Her2/neu receptors. The study's main objective was to investigate the clinical significance of epidermal growth factor receptor (EGFR) overexpression in a series of Iraqi patients with TNBC. The sectional analytic study involved immunohistochemical analysis of EGFR expression in randomly selected 53 formalin fixed paraffin embedded tissue blocks of TNBC cases out of 127 Iraqi patients with TNBC and correlated expression data with clinicopathological parameters including survival time. Machine learning (statistical tests and principal component analysis (PCA)) was used to predict the outcome of the patients using EGFR expression data together with clinicopathological parameters. EGFR was expressed in approximately 28% of TNBC cases. We estimated the risk of mortality and distant metastasis based on EGFR expression and clinicopathologic factors using the principal component analysis (PCA) model. We found a substantial positive correlation between clinical stage and distant metastasis, clinical stage and death, death and distant metastasis, and death and positive EGFR expression. Overall, EGFR expression was linked to a poor prognosis and increased mortality. A higher risk of distant metastasis and death was associated with an advanced clinical stage of the tumor. Furthermore, the existence of distant metastases increased the risk of death. These findings raise the possibility of using EGFR expression data with other clinicopathological parameters to predict the outcome of patients with TNBC.

Epidermal growth factor receptor (EGFR) expression in the serum of patients with triple-negative breast carcinoma: prognostic value of this biomarker.

BackgroundEpidermal growth factor receptor (EGFR) overexpression has been considered a poor prognostic factor in breast cancer.MethodologyA prospective study of 206 women with breast cancer analysed by stages (I, II, III and IV) and by immunohistochemical subtype (Luminal A, Luminal B, HER2+ and triple-negative (TN)); 89 healthy controls with normal recent mammography were included. The EGFR measured in the serum (sEGFR) was detected by the Enzyme-Linked Immunosorbent Assay (ELISA) method (R&D Systems kit DY231) collected by blood before any treatment in patients. Kaplan–Meier method and Cox regression were carried out to obtain the prognostic value, considering significance if p < 0.05.ResultsWith a median follow-up of 36.6 months, 47 deaths occurred. Multivariable Cox regression showed difference of overall survival (OS) associated with sEGFR levels (sEGFR ≤ or > 47.8 ng/mL) in patients with TN cancers, but not of Luminal A, Luminal B or HER2+ subtypes; adjusted by stage, the death risk increased by approximately 415% [hazard ratio (HR): 5.149 (1.900–13.955), p = 0.001] for patients with sEGFR > 47.8 ng/mL compared to patients with a lower sEGFR value. There was no significant correlation of sEGFR with staging, histological tumour grade (G1/G2/G3), Ki67 (< or ≥14%) or body mass index.ConclusionsIncreased sEGFR expression in patients with TN tumours is a significant predictor of lower OS and its quantification is inexpensive and straightforward.

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.

Multi-Targeting Approach in Glioblastoma Using Computer-Assisted Drug Discovery Tools to Overcome the Blood-Brain Barrier and Target EGFR/PI3Kp110β Signaling.

Simple SummaryTreatment of glioblastoma is hampered by the activation of compensatory survival mechanisms by malignant cells that lead to drug resistance. Moreover, the blood–brain barrier (BBB) precludes the brain entrance of most drugs. We hypothesized that computer-assisted drug discovery tools would reveal novel multi-targeting drug candidates with BBB-permeant and favorable ADMET properties. We aimed to discover molecules with predicted ability to inhibit the EGFR/PI3Kp110β pathway and to validate their efficacy and safety in biological assays. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Moreover, we tested their anti-tumor efficacy and BBB safety and permeation in cell models. We found two EGFR, two PI3Kp110β, and, mostly, two dual inhibitors with anti-tumor effects. Among them, one EGFR and two PI3Kp110β inhibitors were able to cross the BBB endothelium without compromising it. These studies revealed novel drug candidates for glioblastoma treatment.The epidermal growth factor receptor (EGFR) is upregulated in glioblastoma, becoming an attractive therapeutic target. However, activation of compensatory pathways generates inputs to downstream PI3Kp110β signaling, leading to anti-EGFR therapeutic resistance. Moreover, the blood–brain barrier (BBB) limits drugs’ brain penetration. We aimed to discover EGFR/PI3Kp110β pathway inhibitors for a multi-targeting approach, with favorable ADMET and BBB-permeant properties. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Predictions were validated in in vitro models of the human BBB and BBB-glioma co-cultures. The results disclosed 27 molecules (18 EGFR, 6 PI3Kp110β, and 3 dual inhibitors) for biological validation, performed in two glioblastoma cell lines (U87MG and U87MG overexpressing EGFR). Six molecules (two EGFR, two PI3Kp110β, and two dual inhibitors) decreased cell viability by 40–99%, with the greatest effect observed for the dual inhibitors. The glioma cytotoxicity was confirmed by analysis of targets’ downregulation and increased apoptosis (15–85%). Safety to BBB endothelial cells was confirmed for three of those molecules (one EGFR and two PI3Kp110β inhibitors). These molecules crossed the endothelial monolayer in the BBB in vitro model and in the BBB-glioblastoma co-culture system. These results revealed novel drug candidates for glioblastoma treatment.

Intelligent Nanoparticles With pH-Sensitive Co-Delivery of Temozolomide and siEGFR to Ameliorate Glioma Therapy.

Glioblastoma (GBM) is one of the most lethal forms of human cancer, with very few long-term survivors. In addition to surgery, chemotherapy is still an important strategy. Unfortunately, GBM chemotherapy faces two main challenges: first, in GBM, epidermal growth factor receptor (EGFR) overexpression results in chemoresistance; second, temozolomide (TMZ) lacks target specificity, which can lead to a reduction in the concentration and side effects in GBM. Nowadays, with the development of nanomedicine systems for applications in tumor therapies, increasing anticancer efficacy and reducing side effects with multi-drug delivery are huge advantages. In this study, pH-sensitive and GBM-targeting nanovesicle (Tf-PEG-PAE(SS)) was fabricated. The chemotherapy drug (TMZ) and EGFR inhibitor (EGFR-siRNA) were co-encapsulated in the nanocarrier, and their anticancer outcomes were investigated in detail. In vitro experiments have shown that the nanocarrier transports TMZ and EGFR-siRNA efficiently into U87 cells, causing a vigorous apoptotic response by silencing the proliferative EGFR gene and increasing the drug concentration of TMZ simultaneously. An experimental study in mice bearing orthotropic glioma revealed that the accumulated nanocarriers in the tumor site could inhibit the tumor growth and prolong the mice survival remarkably through the intracranial injection of Tf-PEG-PAE(SS)/TMZ@siEGFR. The drug co-delivery system could extend the blood circulation time and offer a new strategy to treat glioblastoma.

Identification of growth hormone receptor as a relevant target for precision medicine in low-EGFR expressing glioblastoma.

ObjectiveNew therapeutic approaches are needed to improve the prognosis of glioblastoma (GBM) patients.MethodsWith the objective of identifying alternative oncogenic mechanisms to abnormally activated epidermal growth factor receptor (EGFR) signalling, one of the most common oncogenic mechanisms in GBM, we performed a comparative analysis of gene expression profiles in a series of 54 human GBM samples. We then conducted gain of function as well as genetic and pharmocological inhibition assays in GBM patient‐derived cell lines to functionnally validate our finding.ResultsWe identified that growth hormone receptor (GHR) signalling defines a distinct molecular subset of GBMs devoid of EGFR overexpression. GHR overexpression was detected in one third of patients and was associated with low levels of suppressor of cytokine signalling 2 (SOCS2) expression due to SOCS2 promoter hypermethylation. In GBM patient‐derived cell lines, GHR signalling modulates the expression of proteins involved in cellular movement, promotes cell migration, invasion and proliferation in vitro and promotes tumourigenesis, tumour growth, and tumour invasion in vivo. GHR genetic and pharmacological inhibition reduced cell proliferation and migration in vitro.ConclusionThis study pioneers a new field of investigation to improve the prognosis of GBM patients.

Physiologically Based Pharmacokinetic (PBPK) Modeling to Predict PET Image Quality of Three Generations EGFR TKI in Advanced-Stage NSCLC Patients.

Introduction: Epidermal growth factor receptor (EGFR) mutated NSCLC is best treated using an EGFR tyrosine kinase inhibitor (TKI). The presence and accessibility of EGFR overexpression and mutation in NSCLC can be determined using radiolabeled EGFR TKI PET/CT. However, recent research has shown a significant difference between image qualities (i.e., tumor-to-lung contrast) in three generation EGFR TKIs: 11C-erlotinib, 18F-afatinib and 11C-osimertinib. In this research we aim to develop a physiological pharmacokinetic (PBPK)-model to predict tumor-to-lung contrast and as a secondary outcome the uptake of healthy tissue of the three tracers. Methods: Relevant physicochemical and drug specific properties (e.g., pKa, lipophilicity, target binding) for each TKI were collected and applied in established base PBPK models. Key hallmarks of NSCLC include: immune tumor deprivation, unaltered tumor perfusion and an acidic tumor environment. Model accuracy was demonstrated by calculating the prediction error (PE) between predicted tissue-to-blood ratios (TBR) and measured PET-image-derived TBR. Sensitivity analysis was performed by excluding each key component and comparing the PE with the final mechanistical PBPK model predictions. Results: The developed PBPK models were able to predict tumor-to-lung contrast for all EGFR-TKIs within threefold of observed PET image ratios (PE tumor-to-lung ratio of −90%, +44% and −6.3% for erlotinib, afatinib and osimertinib, respectively). Furthermore, the models depicted agreeable whole-body distribution, showing high tissue distribution for osimertinib and afatinib and low tissue distribution at high blood concentrations for erlotinib (mean PE, of −10.5%, range −158%–+190%, for all tissues). Conclusion: The developed PBPK models adequately predicted the image quality of afatinib and osimertinib and erlotinib. Some deviations in predicted whole-body TBR lead to new hypotheses, such as increased affinity for mutated EGFR and active influx transport (erlotinib into excreting tissues) or active efflux (afatinib from brain), which is currently unaccounted for. In the future, PBPK models may be used to predict the image quality of new tracers.

Exploring Degradation of Mutant and Wild-Type Epidermal Growth Factor Receptors Induced by Proteolysis-Targeting Chimeras.

Several epidermal growth factor receptor (EGFR) proteolysis-targeting chimeras (PROTACs), including MS39 and MS154 developed by us, have been reported to effectively degrade the mutant but not the wild-type (WT) EGFR. However, the mechanism underlying the selectivity in degrading the mutant over the WT EGFR has not been elucidated. Here, we report comprehensive structure-activity relationship studies that led to the discovery of two novel EGFR degraders, 31 (MS9449) and 72 (MS9427), and mechanistic studies of these EGFR degraders. Compounds 31 and 72 selectively degraded the mutant but not the WT EGFR through both ubiquitination/proteasome and autophagy/lysosome pathways. Interestingly, we found that the mutant but not the WT EGFR can effectively form EGFR-PROTAC-E3 ligase ternary complexes. Furthermore, we found that PI3K inhibition sensitized WT EGFR to PROTAC-induced degradation and combination treatment with a PI3K inhibitor enhanced antiproliferation activities of EGFR degraders in cancer cells harboring WT EGFR, providing a potential therapeutic strategy for patients with WT EGFR overexpression.

STRIvE-01: Phase I study of EGFR806 CAR T-cell immunotherapy for recurrent/refractory solid tumors in children and young adults.

2541 Background: The epidermal growth factor receptor (EGFR) is a cell surface tyrosine kinase receptor associated with cell proliferation and differentiation. EGFR expression and activating mutations are associated with aggressive neoplastic disease, chemotherapy resistance, and increased metastatic potential. Published data and EGFR immunohistochemistry (IHC) performed on tissue microarrays indicate that 15-40% of pediatric solid tumors (ST) express EGFR. The unique EGFR monoclonal antibody (mAb) 806 selectively binds to an epitope that is conformationally hidden when EGFR is tethered but revealed when tethering is perturbed as occurs with EGFR overexpression, truncation, or through extra-cellular domain missense mutations. Methods: Children and young adults (CYA) with EGFR-expressing recurrent/refractory (R/R) ST were enrolled on a Phase 1 trial to examine the safety and feasibility of administering autologous chimeric antigen receptor (CAR) T cells derived from autologous T cells genetically modified to express a second generation EGFR806-specific scFV-IgG4hinge-CD28tm/cyto-4-1-BB-zeta and EGFRt tracking/suicide contract. All subjects received lymphodepleting chemotherapy with fludarabine and cyclophosphamide prior to the administration of cryopreserved CAR T cells a the prescribed dose level. The biologically effective dose (BED) or maximum tolerated dose was determined based upon observed toxicity through day 28 from initial CAR-T infusion and using a 3+3 statistical design. Results: Eleven subjects (n=10 evaluable, age range 9-25, median 18) were enrolled and received either dose level (DL) 1 (0.5 x 106 CAR-T/kg, n=4) or DL2 (1 x 106 CAR-T cells/kg, n=7). CAR T were manufactured successfully in all subjects. Most common toxicities were fatigue, tumor-related pain and cytokine release syndrome (n=2, maximum CTCAE grade 1). Dose limiting toxicity of CTCAE grade 4 transaminase level and hyperbilirubinemia occurred at DL2 (n=1). Maximum circulating CAR-T expansion was 29.66 cells/uL (range 0.05-29.66 cells/uL) with median persistence of 28 days (range 0-90). Two subjects on DL1 and one subject on DL2 demonstrated mixed response on day 28 and tolerated additional CAR T infusion without dose limiting toxicity. Conclusions: EGFR806 directed CAR-T cells have an acceptable toxicity profile in CYA with R/RST and demonstrate anti-tumor activity in some patients. Additional analyses are ongoing to identify biomarkers of response and toxicity. Clinical trial information: NCT03618381.

A novel nanobody as therapeutics target for EGFR-positive colorectal cancer therapy: exploring the effects of the nanobody on SW480 cells using proteomics approach

BackgroundThe epidermal growth factor receptor (EGFR) overexpression is found in metastatic colorectal cancer (mCRC). Targeted molecular therapies such as monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKI) are becoming more precise, targeting specifically for cancer therapeutics. However, there are adverse effects of currently available anti-EGFR drugs, including drug-resistant and side effects. Nanobodies can overcome these limitations. Our previous study has found that cell-penetrable nanobodies targeted at EGFR-tyrosine kinase were significantly reduced EGFR-positive lung cancer cells viability and proliferation. The aim of the present study was to determine the effect of cell-penetrable nanobody (R9VH36) on cell viability and proteomic profile in EGFR-positive human colorectal cancer cell lines.MethodsThe human colorectal carcinoma cell line (SW480) was treated with R9VH36, compared with gefitinib. Cell viability was monitored using the MTT cell viability assay. The proteomic profiling was analyzed by LC–MS/MS .ResultsThe half-maximal inhibitory concentration (IC50) values determined for R9VH36 and gefitinib against SW480 were 527 ± 0.03 nM and 13.31 ± 0.02 μM, respectively. Moreover, both the gefitinib-treated group and nanobody-treated group had completely different proteome profiles. A total 6626 differentially expressed proteins were identified. PCA analysis revealed different proteome profiling in R9VH36 experiment. There were 8 proteins in R9VH36 that significantly exhibited opposite expression directions when compared to gefitinib. These proteins are involved in DNA-damage checkpoint processes.ConclusionThe proteomics explored those 6,626 proteins had different expressions between R9VH36 and gefitinib. There were 8 proteins in R9VH36 exhibited opposite expression direction when comparing to gefitinib. Our findings suggest that R9VH36 has the potential to be an alternative remedy for treating EGFR-positive colon cancer.

Critical roles for EGFR and EGFR-HER2 clusters in EGF binding of SW620 human carcinoma cells.

Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR–HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR–EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

Clinical Outcomes of Patients with Peritoneal Metastasis-Only Colorectal Cancer Treated with First-Line Bevacizumab and FOLFIRI Through Irinotecan Dose Escalation According to UGT1A1 Polymorphism: Compared to Liver Metastasis-Only, and Lung Metastasis-Only.

BackgroundThe prognosis of metastatic colorectal cancer (mCRC) depends on the metastatic site and systemic therapy regimen. Peritoneal metastases are associated with a relatively unfavorable prognosis among patients with mCRC. In this article, we present the treatment outcomes of patients with peritoneal carcinomatosis (PC)-only, liver metastasis (LiM)-only, and lung metastasis (LuM)-only CRC.MethodsOverall, 206 mCRC patients with single-site metastasis and who had received treatment from January 2014 to December 2018 were recruited. Among 206 patients with mCRC, 15 had PC-only mCRC, 145 had LiM-only mCRC, and 46 had LuM-only mCRC. They attended regular follow-ups until November 2020, and the median follow-up period was 24.7 months (5.1–41.3 months). Patients’ characteristics, including clinical data, gene mutation profiles, and clinical outcomes, were evaluated. All patients with PC-only CRC were treated with first-line bevacizumab and FOLFIRI, and the irinotecan dose escalation depended on UGT1A1 polymorphism.ResultsOf the 206 patients, no statistical difference was observed between the PC-only, LiM-only, and LuM-only groups in terms of age, primary tumor location, RAS mutation status, BRAF mutation status, and epidermal growth factor receptor overexpression (all P > 0.05). Patients with PC-only CRC had a median progression-free survival (mPFS) of 18.0 months and a median overall survival (mOS) of 24.6 months. Patients with LiM-only or LuM-only CRC had mPFS of 18.2 and 26.6 months and mOS of 25.0 and 44.5 months, respectively. No significant differences regarding PFS and OS (both P > 0.05) between the three groups of patients with mCRC were observed.ConclusionOur study revealed that in patients with PC-only mCRC treatment of first-line bevacizumab and FOLFIRI through irinotecan dose escalation according to UGT1A1 polymorphism could confer such patients with comparable outcomes to that of patients with LiM-only and LuM-only mCRC.

Analysis of the epidermal growth factor receptor/phosphoinositide-dependent protein kinase-1 axis in tumor of the external auditory canal in response to epidermal growth factor stimulation.

ObjectivesThe epidermal growth factor receptor (EGFR) is related to the invasion and metastasis of external auditory canal (EAC) squamous cell carcinoma (SCC). The phosphoinositide‐dependent protein kinase‐1 (PDPK1) accelerates tumor cell growth through anti‐apoptotic signaling under the influence of downstream EGFR‐mediated signaling pathways. In this study, we investigated the EGFR/PDPK1 axis in the EAC under EGF stimulation.MethodsWe confirmed EGFR and PDPK1 expression in human EACSCC specimens immunohistochemically. We next transfected the EGF expression vector in the mouse EAC and then conducted a PDPK1 inhibitory experiment. Immunohistochemical analysis was performed in the mouse EAC, using anti‐EGF, anti‐EGFR, anti‐PDPK1, and anti‐Ki67 antibodies. Immunohistochemical analysis of cleaved caspase‐3 and terminal deoxy(d)‐UTP nick end labeling (TUNEL) detection assays were also performed for the assessment of apoptosis in the inhibitory experiment.ResultsImmunohistochemical analysis revealed overexpression and colocalization of EGFR and PDPK1 in human EACSCC specimens. The growth of a protuberant tumor was observed in the mouse EAC in which EGF expression vector was transfected, and EGF, EGFR, PDPK1, and Ki67 labeling indexes (LIs) were significantly increased. PDPK1 inhibition then induced normal epithelial appearance in the EAC. Moreover, EGF, EGFR, PDPK1, and Ki67 LIs were decreased, and cleaved caspase‐3 and TUNEL LIs were increased in the EAC.ConclusionWe demonstrated the possibility that PDPK1 plays an important role in EACSCC.Level of Evidence: NA.

Antitumor Efficacy of EGFR-Targeted Recombinant Immunotoxin in Human Head and Neck Squamous Cell Carcinoma.

Over 90% of head and neck squamous cell carcinoma (HNSCC) overexpresses the epidermal growth factor receptor (EGFR). However, the EGFR-targeted monotherapy response rate only achieves 10-30% in HNSCC. Recombinant immunotoxin (RIT) often consists of an antibody targeting a tumor antigen and a toxin (e.g., diphtheria toxin [DT]) that kills cancer cells. We produced a humanized RIT, designated as hDT806, targeting overexpressed EGFR and investigated its effects in HNSCC. Distinct from the EGFR-targeted tyrosine kinase inhibitor erlotinib or antibody cetuximab, hDT806 effectively suppressed cell proliferation in the four HNSCC lines tested (JHU-011, -013, -022, and -029). In JHU-029 mouse xenograft models, hDT806 substantially reduced tumor growth. hDT806 decreased EGFR protein levels and disrupted the EGFR signaling downstream effectors, including MAPK/ERK1/2 and AKT, while increased proapoptotic proteins, such as p53, caspase-9, caspase-3, and the cleaved PAPR. The hDT806-induced apoptosis of HNSCC cells was corroborated by flow cytometric analysis. Furthermore, hDT806 resulted in a drastic inhibition in RNA polymerase II carboxy-terminal domain phosphorylation critical for transcription and a significant increase in the γH2A.X level, a DNA damage marker. Thus, the direct disruption of EGFR signaling, transcription inhibition, DNA damage, as well as apoptosis induced by hDT806 may contribute to its antitumor efficacy in HNSCC.

Cetuximab Combined With Sonodynamic Therapy Achieves Dual-Modal Image Monitoring for the Treatment of EGFR-Sensitive Non-Small-Cell Lung Cancer.

BackgroundBlocking signaling by epidermal growth factor receptor (EGFR), can effectively inhibit the proliferation and differentiation of non-small-cell lung cancer (NSCLC). Additionally, an increasing number of NSCLC patients have treatment limitations caused by EGFR overexpression or mutations. Therefore, we constructed a nanotherapy platform consisting of cetuximab (CTX) to target EGFR-sensitive NSCLC with an iron tetroxide core loading the sound-sensitive agent IR780 for dual-mode imaging diagnosis by combining targeting and sonodynamic therapy (SDT) to reshape the tumor microenvironment (TME), enhance the SDT antitumor effects and improve the therapeutic effects of EGFR sensitivity.MethodsIR780@INPs were prepared by reverse rotary evaporation, CTX was adsorbed/coupled to obtain IR780@INPs-CTX, and the morphology and structure were characterized. Intracellular ROS levels and cell apoptosis first verified its killing effects against tumor cells. Then, a nude mouse lung cancer subcutaneous xenograft model was established with HCC827 cells. A real-time fluorescence IVIS imaging system determined the targeting and live distribution of IR780@INPs-CTX in the transplanted tumors and the imaging effects of the T2 sequence of the INPs by magnetic resonance imaging (MRI) 0 h, 2 h, 4 h and 6 h after administration to confirm drug efficacy.Results In vitro, US+IR780@INPs-CTX produced a large amount of ROS after SDT to induce cell apoptosis, and significant cell death after live/dead staining was observed. In vivo fluorescence imaging showed the IR780@INPs-CTX was mainly concentrated in the tumor with a small amount in the liver. MRI displayed rapid enrichment of the IR780@INPs into tumor tissue 0h after injection and the T2 signal intensity gradually decreases with time without obvious drug enrichment in the surrounding tissues. In vivo, at the end of treatment, the US+IR780@INPs-CTX group showed disappearance or a continued decrease in tumor volume, indicating strong SDT killing effects.ConclusionThe combination of CTX and SDT is expected to become a novel treatment for EGFR-sensitive NSCLC.

A novel dual-labeled small peptide as a multimodal imaging agent for targeting wild-type EGFR in tumors.

The epidermal growth factor receptor (EGFR) is over-expressed in various human cancer. The over-expression of EGFR in tumors is an excellent target for the development of cancer imaging agents. In the present study, we developed Tc-99m SYPIPDT-GHEG-ECG-K-tetramethylrhodamine (SYPIPDT-ECG-TAMRA) as a molecular imaging agent targeting wild-type EFGR (wtEGFR)-positive tumor cells, and verified its feasibility as molecular imaging agent. SYPIPDT-ECG-TAMRA was synthesized using Fmoc solid-phase peptide synthesis. The radiolabeling of SYPIPDT-ECG-TAMRA with Tc-99m was accomplished using ligand exchange via tartrate. Cellular uptake and binding affinity studies were performed. In vivo gamma camera imaging, ex vivo imaging and biodistribution studies were performed using NCI-H460 and SW620 tumor-bearing murine models. After radiolabeling procedures with Tc-99m, Tc-99m SYPIPDT-ECG-TAMRA complexes were prepared at high yield (> 95%). The binding affinity value (Kd) of Tc-99m SYPIPDT-ECG-TAMRA for NCI-H460 cells was estimated to be 76.5 ± 15.8 nM. In gamma camera imaging, the tumor to normal muscle uptake ratios of Tc-99m SYPIPDT-ECG-TAMRA increased with time (2.7 ± 0.6, 4.0 ± 0.9, and 6.2 ± 1.0 at 1, 2, and 3 h, respectively). The percentage injected dose per gram of wet tissue for the NCI-H460 tumor was 1.91 ± 0.11 and 1.70 ± 0.22 at 1 and 3 h, respectively. We developed Tc-99m SYPIPDT-ECG-TAMRA, which is dual-labeled with both radioisotope and fluorescence. In vivo and in vitro studies demonstrated specific uptake of Tc-99m SYPIPDT-ECG-TAMRA into wtEGFR-positive NCI-H460 cells and tumors. Thus, the results of the present study suggest that Tc-99m SYPIPDT-ECG-TAMRA is a potential dual-modality imaging agent targeting wtEGFR.

Fluorescence Imaging of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer.

Simple SummaryLung cancer is the leading cause of cancer-related deaths, with a low (<21%) 5-year survival rate. Lung cancer is often driven by the misfunction of molecules on the surface of cells of the epithelium, which orchestrate mechanisms by which these cells grow and proliferate. Beyond common non-specific treatments, such as chemotherapy or radiotherapy, among molecular-specific treatments, a number of small-molecule drugs that block cancer-driven molecular activity have been developed. These drugs initially have significant success in a subset of patients, but these patients systematically develop resistance within approximately one year of therapy. Substantial efforts towards understanding the mechanisms of resistance have focused on the genomics of cancer progression, the response of cells to the drugs, and the cellular changes that allow resistance to develop. Fluorescence microscopy of many flavours has significantly contributed to the last two areas, and is the subject of this review. Non-small cell lung cancer (NSCLC) is a complex disease often driven by activating mutations or amplification of the epidermal growth factor receptor (EGFR) gene, which expresses a transmembrane receptor tyrosine kinase. Targeted anti-EGFR treatments include small-molecule tyrosine kinase inhibitors (TKIs), among which gefitinib and erlotinib are the best studied, and their function more often imaged. TKIs block EGFR activation, inducing apoptosis in cancer cells addicted to EGFR signals. It is not understood why TKIs do not work in tumours driven by EGFR overexpression but do so in tumours bearing classical activating EGFR mutations, although the latter develop resistance in about one year. Fluorescence imaging played a crucial part in research efforts to understand pro-survival mechanisms, including the dysregulation of autophagy and endocytosis, by which cells overcome the intendedly lethal TKI-induced EGFR signalling block. At their core, pro-survival mechanisms are facilitated by TKI-induced changes in the function and conformation of EGFR and its interactors. This review brings together some of the main advances from fluorescence imaging in investigating TKI function and places them in the broader context of the TKI resistance field, highlighting some paradoxes and suggesting some areas where super-resolution and other emerging methods could make a further contribution.

MiR-875-5p suppresses cervical cancer cell proliferation and metastasis via negative regulation of EGFR

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To explore miRNA-875-5p and epidermal growth factor receptor (EGFR) activities in tissues or cells from cervical cancer, and their underlying molecular mechanisms.&#x0D; Methods: Tissues were obtained from cervical cancer patients and their miR-875-5p expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Caski or HeLa cells were transfected with miR-875-5p mimics or miR-875-5p inhibitor to assess the effect of miR-875- 5p expression on cell viability, cell cycle, migration, and invasion using Cell Counting Kit-8 (CCK-8), flow cytometry, wound healing, and Transwell assays. Potential target genes of miR-875-5p were predicted and verified using a dual luciferase reporter assay. In addition, EGFR expression was evaluated by western blot.&#x0D; Results: MicroRNA-875-5p was expressed at low levels in cervical cancer tissues and was related to FIGO stage, lymph node metastasis, pathological grade, vascular involvement, and deep stromal invasion in patients with cervical cancer. MicroRNA-875-5p overexpression inhibited cell viability, migration, and invasion, and caused G0/G1 phase block of Caski and HeLa cells. Moreover, EGFR was the target gene of miR-875-5p and was negatively regulated by miR-875-5p. Reductions in cell viability, migration, invasion, and the number of G0/G1-phase cells were inhibited by EGFR overexpression.&#x0D; Conclusion: MiR-875-5p suppresses cervical cancer cell growth and metastasis by negatively regulating EGFR. Therefore, miR-875-5p can potentially be exploited for the management of cervical cancer.&#x0D; &#x0D; &#x0D; &#x0D;

Breast cancer recurrence: factors impacting occurrence and survival

BackgroundBreast cancer mortality has decreased due to improved screening and treatment options. Nevertheless, 25–30% of patients develop disease recurrence and die from the disease dissemination. Patients who develop metastatic disease represent a heterogeneous group and management plans are dependent on molecular subtype, disease burden and metastatic site.AimTo determine predictive clinicopathological factors of disease recurrence and their impact on survival in the molecular era.MethodsConsecutive patients who breast cancer developed recurrence at our tertiary referral centre between 2000 and 2015 were included. Clinicopathological and treatment data were assessed using descriptive statistics. Oncological outcome was assessed using Cox regression and Kaplan Meier analyses.ResultsTwo hundred sixty-five consecutive patients who developed breast cancer recurrence were included; median age at metastasis was 59.3 years (range 27–87 years), and median time to recurrence (TTR) was 47.7 ± 38.5 months (range 3.0–194.3 months). Survival was 24.2% (64/265) 53.2% were luminal A (LABC) (141/265), 18.5% were luminal B (LBBC) (49/265), 18.5% were triple negative (TNBC) (49/265), and 9.8% were human epidermal growth factor receptor-2 overexpressing (HER2 +) (26/265). TTR for patients with LABC was 56.0 ± 41.3 months, LBBC was 48.4 ± 41.1 months, TNBC was 26.9 ± 28.5 months and HER2 + was 34.3 ± 21.8 months. Increased grade (P < 0.001), Nottingham Prognostic Indices (P < 0.001), TNBC (P < 0.001), HER2 + subtype (P < 0.001) and receiving targeted therapy (P = 0.006) predicted shorted TTR. Estrogen receptor positivity (P < 0.001), progesterone receptor positivity (P = 0.010), invasive lobular carcinoma (P = 0.009) and receiving endocrine therapy (P = 0.001) predicted longer TTR.ConclusionReadily available clinicopathological factors predict risk of metastatic dissemination. Developing a tailored program to identify patients at risk of recurrence is crucial in controlling metastatic dissemination of breast cancer.