EGFR-positive Cancer Research Articles

A Combination of Two Receptor Tyrosine Kinase Inhibitors, Canertinib and PHA665752 Compromises Ovarian Cancer Cell Growth in 3D Cell Models

IntroductionAdvanced ovarian cancer is often a fatal disease as chemotherapeutic drugs have limited effectiveness. Better targeted therapy is needed to improve the survival and quality of life for these women. Receptor tyrosine kinases including EGFR, Her-2 and c-Met are associated with a poor prognosis in ovarian cancer. Therefore, the co-activation of these receptors may be crucial for growth promoting activity. In this study, we explored the effect of combining two small molecule inhibitors that target the EGFR/Her-2 and c-Met receptor tyrosine kinases in two ovarian cancer cell lines. The aim of this study was to investigate the combined inhibition activity of a dual EGFR/Her-2 inhibitor (canertinib) and a c-Met inhibitor (PHA665752) in ovarian cancer cell lines in 3D cell aggregates.MethodsOVCAR-5 and SKOV-3 ovarian cancer cell lines were cultured on a non-adherent surface to produce 3D cell clusters and aggregates. Cells were exposed to canertinib and PHA665752, both individually and in combination, for 48 h. The effect on growth, metabolism and the expression/phosphorylation of selective signaling proteins associated with EGFR, Her-2 and c-Met were investigated.ResultsThe single drug treatments significantly decreased cell growth and altered the expression of signaling proteins in OVCAR-5 and SKOV-3 cell lines. The combination treatment showed greater reduction of cell numbers for both cell lines. Total expression and phosphorylation of signaling proteins were further reduced in the combination drug treatments, compared to the single inhibitor treatments.ConclusionOur findings suggest that the concurrent targeting of more than one receptor tyrosine kinase may be useful in developing more effective targeted drug regimens for patients, who have EGFR, Her-2 and c-Met positive ovarian cancer cells.

Cyanidin-3-o-glucoside directly binds to ERα36 and inhibits EGFR-positive triple-negative breast cancer.

Anthocyanins have been shown to inhibit the growth and metastatic potential of breast cancer (BC) cells. However, the effects of individual anthocyanins on triple-negative breast cancer (TNBC) have not yet been studied. In this study, we found that cyanidin-3-o-glucoside (Cy-3-glu) preferentially promotes the apoptosis of TNBC cells, which co-express the estrogen receptor alpha 36 (ERα36) and the epidermal growth factor receptor (EGFR). We demonstrated that Cy-3-glu directly binds to the ligand-binding domain (LBD) of ERα36, inhibits EGFR/AKT signaling, and promotes EGFR degradation. We also confirmed the therapeutic efficacy of Cy-3-glu on TNBC in the xenograft mouse model. Our data indicates that Cy-3-glu could be a novel preventive/therapeutic agent against the TNBC co-expressed ERα36/EGFR.

Dimerization of EGFR and HER2 induces breast cancer cell motility through STAT1-dependent ACTA2 induction.

The dimerization of EGFR and HER2 is associated with poor prognosis such as induction of tumor growth and cell invasion compared to when EGFR remains as a homodimer. However, the mechanism for events after dimerization in breast cancer models is not clear. We found that expressions of alpha-smooth muscle actin (ACTA2) and signal transducer and activator of transcription 1 (STAT1) significantly increased with transient or stable overexpression of HER2 in EGFR-positive breast cancer cells. ACTA2 and STAT1 expression was also increased in HER2-positive breast cancer patients. In contrast, ACTA2 expression was decreased by HER2 siRNA. Next, we investigated the co-relation between STAT1 and ACTA2 expression. Basal ACTA2 expression was significantly decreased by treatment with the STAT1 inhibitor fludarabine or the JAK2 inhibitor AG490. In contrast, ACTA2 expression was increased by STAT1 overexpression. Levels of ACTA2, STAT1, and HER2 were increased and relapse free survival was decreased in high-risk breast cancer patients. We also investigated the effect of ACTA2 on cell motility, which was suppressed by ACTA2 shRNA overexpression in MDA-MB231 HER2 and 4T1 mammary carcinoma cells. The number of lung metastatic nodules was significantly decreased in ACTA2 knockdown mice. Taken together, these results demonstrated that induction of ACTA2 by EGFR and HER2 dimerization was regulated through a JAK2/STAT1 signaling pathway, and aberrant ACTA2 expression accelerated the invasiveness and metastasis of breast cancer cells.

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking

A fascinating conundrum in cell signaling is how stimulation of the same receptor tyrosine kinase with distinct ligands generates specific outcomes. To decipher the functional selectivity of EGF and TGF-α, which induce epidermal growth factor receptor (EGFR) degradation and recycling, respectively, we devised an integrated multilayered proteomics approach (IMPA). We analyzed dynamic changes in the receptor interactome, ubiquitinome, phosphoproteome, and late proteome in response to both ligands in human cells by quantitative MS and identified 67 proteins regulated at multiple levels. We identified RAB7 phosphorylation and RCP recruitment to EGFR as switches for EGF and TGF-α outputs, controlling receptor trafficking, signaling duration, proliferation, and migration. By manipulating RCP levels or phosphorylation of RAB7 in EGFR-positive cancer cells, we were able to switch a TGF-α-mediated response to an EGF-like response or vice versa as EGFR trafficking was rerouted. We propose IMPA as an approach to uncover fine-tuned regulatory mechanisms in cell signaling.

EGF-coated gold nanoparticles provide an efficient nano-scale delivery system for the molecular radiotherapy of EGFR-positive cancer

Purpose Radiolabeled antibodies and peptides hold promise for molecular radiotherapy but are often limited by a low payload resulting in inadequate delivery of radioactivity to tumour tissue and, therefore, modest therapeutic effect. We developed a facile synthetic method of radiolabeling indium-111 (111In) to epidermal growth factor (EGF)-gold nanoparticles (111In-EGF-Au NP) with a high payload.Materials and methods EGF-Au NP were prepared via an interaction between gold and the disulphide bonds of EGF and radiolabeled using 111InCl3. Targeting efficiency was investigated by quantitating internalized radioactivity and by confocal imaging following exposure of MDA-MB-468 (1.3 × 106 EGFR/cell) and MCF-7 (104 EGFR/cell) cells to Cy3-EGF-Au NP. Cytotoxicity was evaluated in clonogenic assays.Results The proportion of total administered radioactivity that was internalized by MDA-MB-468 and MCF-7 cells was 15% and 1.3%, respectively (mixing ratio of EGF:Au of 160). This differential uptake in the two cell lines was confirmed using confocal microscopy. 111In-EGF-Au NP were significantly more radiotoxic to MDA-MB-468 than MCF-7 cells with a surviving fraction of 17.1 ± 4.4% versus 89.8 ± 1.4% (p < 0.001) after exposure for 4 h.Conclusions An 111In-labeled EGF-Au nanosystem was developed. It enabled targeted delivery of a high 111In payload specifically to EGFR-positive cancer cells leading to radiotoxicity that can be exploited for molecularly targeted radiotherapy.

An Anti-EGFR IgA That Displays Improved Pharmacokinetics and Myeloid Effector Cell Engagement In Vivo.

Antibodies of IgA isotype effectively engage myeloid effector cells for cancer immunotherapy. Here, we describe preclinical studies with an Fc engineered IgA2m(1) antibody containing the variable regions of the EGFR antibody cetuximab. Compared with wild-type IgA2m(1), the engineered molecule lacked two N-glycosylation sites (N166 and N337), two free cysteines (C311 and C472), and contained a stabilized heavy and light chain linkage (P221R mutation). This novel molecule displayed improved production rates and biochemical properties compared with wild-type IgA. In vitro, Fab- and Fc-mediated effector functions, such as inhibition of ligand binding, receptor modulation, and engagement of myeloid effector cells for antibody-dependent cell-mediated cytotoxicity, were similar between wild-type and engineered IgA2. The engineered antibody displayed lower levels of terminal galactosylation leading to reduced asialoglycoprotein-receptor binding and to improved pharmacokinetic properties. In a long-term in vivo model against EGFR-positive cancer cells, improved serum half-life translated into higher efficacy of the engineered molecule, which required myeloid cells expressing human FcαRI for its full efficacy. However, Fab-mediated effector functions contributed to the in vivo efficacy because the novel IgA antibody demonstrated therapeutic activity also in non-FcαRI transgenic mice. Together, these results demonstrate that engineering of an IgA antibody can significantly improve its pharmacokinetics and its therapeutic efficacy to inhibit tumor growth in vivo.

Development of self-assembling peptide nanovesicle with bilayers for enhanced EGFR-targeted drug and gene delivery

Development of rational vectors for efficient drug and gene delivery is crucial for cancer treatment. In this study, epidermal growth factor receptor (EGFR)-binding peptide amphiphile (PA) were used as the primary bilayer skeleton material to construct ultra-stable self-assembling peptide nanovesicle (SPV). The resulted EGFR-targeted SPV (ESPV) could efficiently encapsulate therapeutic cargos (drugs or small interfering RNAs [siRNAs]) or labelled fluorescent cargo (quantum dots [QDs]) and exhibited excellent affinity for EGFR-positive cancer cells. Moreover, ESPV could deliver more drug or plasmid DNA to tumour sites and promote gene expression (a three-fold ratio of ESPVs vs cationic liposomes). Notably, the individual delivery or co-delivery of doxorubicin (DOX) and the acetylcholinesterase (AChE) gene via the ESPVs resulted in excellent drug/gene delivery both in vitro and in vivo and exerted a significant growth-suppressing effect on a liver cancer xenograft. This nanoscale, targeted cargo-packaging technology may provide a new strategy for the design of highly targeted cancer therapy vectors.

EGFR-Targeted Polymeric Mixed Micelles Carrying Gemcitabine for Treating Pancreatic Cancer.

The objective of this study was to design GE11 peptide (YHWYGYTPQNVI) linked micelles of poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-gemcitabine-graft-dodecanol (PEG-b-PCC-g-GEM-g-DC) for enhanced stability and target specificity of gemcitabine (GEM) to EGFR-positive pancreatic cancer cells. GE11-PEG-PCD/mPEG-b-PCC-g-GEM-g-DC mixed micelles showed EGFR-dependent enhanced cellular uptake, and cytotoxicity as compared to scrambled peptide HW12-PEG-PCD/mPEG-b-PCC-g-GEM-g-DC mixed micelles and unmodified mPEG-b-PCC-g-GEM-g-DC micelles. Importantly, GE11-linked mixed micelles preferentially accumulated in orthotopic pancreatic tumor and tumor vasculature at 24 h post systemic administration. GE11-linked mixed micelles inhibited orthotopic pancreatic tumor growth compared to HW12-linked mixed micelles, unmodified mPEG-b-PCC-g-GEM-g-DC micelles, and free GEM formulations. Tumor growth inhibition was mediated by apoptosis of tumor cells and endothelial cells as determined by immunohistochemical staining. In summary, GE11-linked mixed micelles is a promising approach to treat EGFR overexpressing cancers.

Modification of cytokine-induced killer cells with chimeric antigen receptors (CARs) enhances antitumor immunity to epidermal growth factor receptor (EGFR)-positive malignancies.

Epidermal growth factor receptor (EGFR, ErbB1, Her-1) is a cell surface molecule overexpressing in a variety of human malignancies and, thus, is an excellent target for immunotherapy. Immunotherapy targeting EGFR-overexpressing malignancies using genetically modified immune effector cells is a novel and promising approach. In the present study, we have developed an adoptive cellular immunotherapy strategy based on the chimeric antigen receptor (CAR)-modified cytokine-induced killer (CAR-CIK) cells specific for the tumor cells expressing EGFR. To generate CAR-CIK cells, a lentiviral vector coding the EGFR-specific CAR was constructed and transduced into the CIK cells. The CAR-CIK cells showed significantly enhanced cytotoxicity and increased production of cytokines IFN-γ and IL-2 when co-cultured with EGFR-positive cancer cells. In tumor xenografts, adoptive immunotherapy of CAR-CIK cells could inhibit tumor growth and prolong the survival of EGFR-overexpressing human tumor xenografts. Moreover, tumor growth inhibition and prolonged survival in mice with EGFR(+) human cancer were associated with the increased persistence of CAR-CIK cells in vivo. Our study indicates that modification with EGFR-specific CAR strongly enhances the antitumor activity of the CIK cells against EGFR-positive malignancies.

Near Infrared Photoimmunotherapy Targeting EGFR Positive Triple Negative Breast Cancer: Optimizing the Conjugate-Light Regimen.

AimTriple-negative breast cancer (TNBC) is considered one of the most aggressive subtypes of breast cancer. Near infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that employs an antibody-photosensitizer conjugate (APC) followed by exposure of NIR light for activating selective cytotoxicity on targeted cancer cells and may have application to TNBC. In order to minimize the dose of APC while maximizing the therapeutic effects, dosing of the APC and NIR light need to be optimized. In this study, we investigate in vitro and in vivo efficacy of cetuximab (cet)-IR700 NIR-PIT on two breast cancer models MDAMB231 (TNBC, EGFR moderate) and MDAMB468 (TNBC, EGFR high) cell lines, and demonstrate a method to optimize the dosing APC and NIR light.MethodAfter validating in vitro cell-specific cytotoxicity, NIR-PIT therapeutic effects were investigated in mouse models using cell lines derived from TNBC tumors. Tumor-bearing mice were separated into 4 groups for the following treatments: (1) no treatment (control); (2) 300 μg of cet-IR700 i.v., (APC i.v. only); (3) NIR light exposure only, NIR light was administered at 50 J/cm2 on day 1 and 100 J/cm2 on day 2 (NIR light only); (4) 300 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm2 on day 1 after injection and 100 J/cm2 of light on day 2 after injection (one shot NIR-PIT). To compare different treatment regimens with a fixed dose of APC, we added the following treatments (5) 100 μg of cet-IR700 i.v., NIR light administered at 50 J/cm2 on day 1 and 50 μg of cet-IR700 i.v. immediately after NIR-PIT, then NIR light was administered at 100 J/cm2 on day 2, which were performed two times every week (“two split” NIR-PIT) and (6) 100 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm2 on day 1 and 100 J/cm2 on day 2, which were performed three times per week (“three split” NIR-PIT).ResultBoth specific binding and NIR-PIT effects were greater with MDAMB468 than MDAMB231 cells in vitro. Tumor accumulation of cet-IR700 in MDAMB468 tumors was significantly higher (p < 0.05) than in MDAMB231 tumors in vivo. Tumor growth and survival of MDAMB231 tumor bearing mice was significantly lower in the NIR-PIT treatment group (p < 0.05). In MDAMB468 bearing mice, tumor growth and survival was significantly improved in the NIR-PIT treatment groups in all treatment regimens (one shot NIR-PIT; p < 0.05, “two split” NIR-PIT; p < 0.01, “three split” NIR-PIT; p < 0.001) compared with control groups.ConclusionNIR-PIT for TNBC was effective regardless of expression of EGFR, however, greater cell killing was shown with higher EGFR expression tumor in vitro. In all treatment regimens, NIR-PIT suppressed tumor growth, resulting in significantly prolonged survival that further improved by splitting the APC dose and using repeated light exposures.

A comparison of non-biologically active truncated EGF (EGFt) and full-length hEGF for delivery of Auger electron-emitting 111In to EGFR-positive breast cancer cells and tumor xenografts in athymic mice

IntroductionEGFt is a truncated form of human epidermal growth factor (hEGF) that is non-biologically active but retains binding and internalization into EGFR-positive cells. Our aim was to compare EGFt and hEGF for delivery of 111In to human breast cancer (BC) cells and tumors and evaluate its cytotoxicity against EGFR-positive BC cells, mediated by the Auger electron emissions of 111In. MethodsThe binding, internalization and nuclear localization of EGFt and hEGF in MDA-MB-468 human BC cells were first assessed by confocal fluorescence microscopy. Subcellular fractionation was then used to quantify the cellular and nuclear uptake of 111In-EGFt and 111In-hEGF in MDA-MB-468 cells. The effect of exposure in vitro to 111In-EGFt or 111In-hEGF on the clonogenic survival of MDA-MB-468 (106 EGFR/cell) or MCF-7 cells (104 EGFR/cell) was determined. The pharmacokinetics and tumor and normal tissue biodistribution of 111In-EGFt was compared to 111In-hEGF in CD-1 athymic mice with s.c. MDA-MB-468 and MCF-7 tumors. Nuclear importation in MDA-MB-468 tumors was determined ex vivo by subcellular fractionation. ResultsFluorescently-labeled EGFt and hEGF were bound, internalized and localized in the nucleus of MDA-MB-468 cells. Binding of 111In-EGFt to MDA-MB-468 cells was 8-fold lower than 111In-hEGF, but nuclear importation as a proportion of cell-bound 111In was 3.6-fold greater than 111In-hEGF. Nuclear uptake of 111In-EGFt was lower than 111In-hEGF when differences in cell binding were taken into account. The cytotoxicity of 111In-EGFt (1.0MBq/mL; 10 nmols/L) against MDA-MB-468 cells was 9-fold lower than 111In-hEGF but only 2-fold lower at a higher concentration (1.85MBq/mL; 40 nmols/L). 111In-EGFt and 111In-hEGF exhibited greater cytotoxicity against MDA-MB-468 cells than MCF-7 cells. 111In-EGFt was eliminated more slowly from the blood of tumor-bearing mice and exhibited lower liver uptake but higher kidney accumulation. Uptake of 111In-EGFt in MDA-MB-468 tumors was 2.2-fold lower than 111In-hEGF, and was blocked by anti-EGFR monoclonal antibody, nimotuzumab. Nuclear uptake into MDA-MB-468 tumor cells was higher for 111In-EGFt than 111In-hEGF, but when the lower tumor uptake of 111In-EGFt was considered, there were no overall differences. ConclusionWe conclude that the absence of biological activity of EGFt makes it attractive for delivery of Auger electron-emitting 111In to EGFR-overexpressing BC, but its lower cellular and tumor uptake would limit its effectiveness compared to 111In-hEGF. Advances in Knowledge and Implications for Patient Care111In-EGFt may reduce the adverse effects previously observed in patients administered 111In-hEGF since it is not biologically active, but its lower uptake by BC cells and tumors would limit its effectiveness for treatment of breast cancer.

Targeted therapy of solid tumors by monoclonal antibody specific to epidermal growth factor receptor.

Tumor growth and progression depends largely on the activity of cell membrane receptors like epidermal growth factor receptor (EGFR) that plays a pivotal role in the progression and invasion of different solid tumors. As one of the most promising approaches for targeting and therapy, monoclonal antibodies (mAbs) have widely been used in the treatment of various malignancies. However, the clinical effects of mAbs appear to be dependent upon its specificity and potency. In the current investigation, a series of mAbs were produced against human EGFR using hybridoma technology. Balb/c mice were immunized against EGFR-positive A431 cancer cells and the most immune mouse was selected for fusion and generation of anti-EGFR mAbs. Isotyping of the generated mAbs was performed by ELISA method. Of various monoclones produced, IgG1 subclass (mAb BF4) displayed specific binding to the EGFR-expressing A431 cells. Flow cytometry and immunofluorescence staining revalidated its specific reactivity with EGFR and MTT assay revealed significant growth inhibition of A431 cells treated with mAb BF4 mainly through induction of apoptosis. Based on these findings, we propose the produced anti-EGFR mAb BF4 to be exploited for diagnostic and possibly treatment of various malignancies with overexpression of EGFR.

Antibody-mediated delivery of anti-KRAS-siRNA in vivo overcomes therapy resistance in colon cancer.

KRAS mutations are frequent driver mutations in multiple cancers. KRAS mutations also induce anti-EGFR antibody resistance in adenocarcinoma such as colon cancer. The aim of this study was to overcome anti-EGFR antibody resistance by coupling the antibody to KRAS-specific siRNA. The anti-EGFR antibody was chemically coupled to siRNA. The resulting complex was tested for antibody binding efficiency, serum stability and ability to deliver siRNA to EGFR-expressing cells. Western blotting, viability, apoptosis, and colony formation assays were performed for efficacy evaluation in vitro. Furthermore, therapeutic activity of the antibody-KRAS-siRNA complexes was examined in in vivo xenograft mouse tumor models. Antibody-siRNA complexes were targeted and internalized via the EGFR receptor. Upon internalization, target gene expression was strongly and specifically repressed, followed by a reduced proliferation and viability, and induced apoptosis of the cells in vitro. Clonogenic growth of mutant KRAS-bearing cells was suppressed by KRAS-siRNA-anti-EGFR antibody complexes. In xenograft mouse models, anti-EGFR antibody-KRAS-siRNA complexes significantly slowed tumor growth in anti-EGFR-resistant cells. The coupling of siRNA against KRAS to anti-EGFR antibodies provides a novel therapy approach for KRAS-mutated EGFR-positive cancer cells in vitro and in vivo. These findings provide an innovative approach for cancer-specific siRNA application and for enhanced therapeutic potential of monoclonal antibody therapy and personalized treatment of cancer entities.

Evaluation of Biomarkers for HER3-targeted Therapies in Cancer

Evaluation of Biomarkers for HER3-targeted Therapies in Cancer

Enhanced cytotoxic activity of cetuximab in EGFR-positive lung cancer by conjugating with gold nanoparticles.

Cetuximab (C225) is a unique agent, targeting epidermal growth factor receptor (EGFR)-positive cancer. However, the therapeutic effect of C225 in EGFR high-expressing non-small cell lung cancer (NSCLC) remains poor. Here, we report that conjugation of C225 with gold nanoparticles (AuNPs) enhances the cytotoxicity of C225 in NSCLC both in vitro and in vivo. The NSCLC cell lines A549 (EGFRhigh) and H1299 (EGFRlow) were employed to investigate different responses to C225, IgG-AuNPs and C225-AuNPs. The antitumor properties of C225-AuNPs were explored in vivo by establishing a tumor xenograft model in nude mice. Overall, the therapeutic effect of C225-AuNPs was more pronounced in EGFRhigh A549 cells compared with EGFRlow H1299 cells. The cytotoxic effect of C225-AuNPs in A549 cells increased in a dose-dependent manner. C225-AuNPs significantly suppressed A549 cell proliferation and migration capacity and accelerated apoptosis compared with C225, and this effect was probably due to enhanced EGFR endocytosis and the subsequent suppression of downstream signaling pathway. Finally in the tumor xenograft of nude mice, treatment with C225-AuNPs also led to a significant reduction in tumor weight and volume with low toxicity. Our findings suggest that C225-AuNPs conjugate has promising potential for targeted therapy of EGFR positive NSCLC patients.

Calcitriol and its analogues enhance the antiproliferative activity of gefitinib in breast cancer cells

Coexpression of EGFR and HER2 has been associated with poor disease outcome, high rates of metastasis and resistance to conventional treatments in breast cancer. Gefitinib, a tyrosine kinase inhibitor, reduces both cell proliferation and tumor growth of breast cancer cells expressing EGFR and/or HER2. On the other hand, calcitriol and some of its synthetic analogs are important antineoplastic agents in different breast cancer subtypes. Herein, we evaluated the effects of the combined treatment of gefitinib with calcitriol or its analogs on cell proliferation in breast cancer cells.The presence of EGFR, HER2 and vitamin D receptor were evaluated by Western blot in two established breast cancer cell lines: SUM-229PE, SKBR3 and a primary breast cancer-derived cell line. The antiproliferative effects of gefitinib alone or in combination with calcitriol and its analogs, calcipotriol and EB1089, were assessed by growth assay using a DNA content-based method. Inhibitory concentrations on cell proliferation were calculated by non-linear regression analysis using sigmoidal fitting of dose-response curves. Pharmacological effects of the drug combinations were calculated by the Chou–Talalay method. Phosphorylation of ERK1/2 MAPK was evaluated by Western blot. Gene expression of EGFR, HER2 and BIM was assessed by real time PCR. BIM protein levels were analyzed in cells by flow cytometry. The effects of the drugs alone or combinated on cell cycle phases were determined using propidium iodide. Apoptosis was evaluated by detection of subG1 peak and determination of active caspase 3 by flow cytometry.Gefitinib, calcitriol, calcipotriol and EB1089 inhibited cell proliferation in a dose dependent manner. The combinations of gefitinib with calcitriol or its analogs were more effective to inhibit cell growth than each compound alone in all breast cancer cells studied. The gene expression of EGFR and HER2 was downregulated and not affected, respectively, by the combined treatment. Furthermore, phosphorylation of ERK 1/2 was inhibited a greater extent in co-treated cells than in the cells treated with alone compounds. The combination of gefitinib with calcitriol or their synthetic analogs induced apoptosis in SUM-229PE cells, this was shown by the significant upregulation of BIM protein levels, higher percentages of cells in subG1 peak and increase of caspase 3-positive cells.The combination of gefitinib with calcitriol or their synthetic analogs resulted in a greater antiproliferative effect than with either of the agents alone in EGFR and HER2 positive breast cancer cells. The mechanistic explanation for these results includes downregulation of MAPK signaling pathway, decrease of cells in G2/M phase and induction of apoptosis mediated by upregulation of BIM and activation of caspase 3.This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’.

Isolating epidermal growth factor receptor overexpressing carcinoma cells from human whole blood by bio-ferrography.

The epidermal growth factor receptor (EGFR) is overexpressed in carcinoma. In some cases, including in colorectal cancer, it is used as a therapeutic target. Bio-Ferrography is a nondestructive method for isolating magnetized cells and tissues from a fluid onto a glass slide based on their interaction with an external, strong, and focused magnetic field. Here, we implement Bio-Ferrography to separate EGFR-positive cancer cells from EGFR-negative noncancer cells, mixed at a ratio of 1 to 1 × 10(6) , from either phosphate-buffered saline or human whole blood. Incubation of the cells with an anti-EGFR antibody and magnetic microbeads coupled to a secondary antibody was used to magnetize the target cells prior to the ferrographic analysis. A procedure was developed for "a proof of concept" isolation. Recovery values as high as 78% for 1 mL phosphate-buffered saline, and 53% for 1 mL human whole blood, with a limit-of-detection of 30 and 100 target cells, respectively, were achieved. These capture efficiency values are considered significant and, therefore, warrant further study on isolation of real circulating tumor cells from blood samples of patients, aiming at early diagnosis of EGFR-overexpressing tumor types.

Abstract 4661: The regulatory mechanism of fibronectin in EGFR and/or HER2 positive breast cancer

Abstract Fibronectin (FN) is a glycoprotein of the extracellular matrix and plays a major role in cell adhesion, migration and oncogenic transformation. Aberrant FN expression is associated with poor prognosis in a variety of cancers including breast cancer. However, the regulatory mechanism between FN and HER2 is not fully elucidated in breast cancer. Our clinical data showed that both disease free survival (DFS) and overall survival (OS) was lower in EGFR (+) and HER2 (+) breast cancer patients than in EGFR (+) breast cancer patients. To verify the causing of poor prognosis, we analyzed secreted proteins in EGFR and/or HER2 overexpressed MDA-MB231 cells. Our results showed that the level of FN expression was significantly increased in HER2 overexpressed MDA-MB231 cells. On the other hand, the induction of FN by HER2 overexpression was decreased by trastuzumab (HER2 monoclonal antibody) and AG825 (HER2 inhibitor), respectively. In addition, we observed that the expression of FN was dose-dependently increased by EGFR ligands in SKBR3 cells which are expressed endogenous EGFR and HER2. EGF ligands-induced FN expression also decreased through the suppression of HER2 in SKBR3 cell. However, these effects did not detect in trastuzumab-resistant breast cancer cell line, JIMT-1. Taken together, we demonstrated that the induction of FN by HER2 correlates poor clinical outcome in breast cancer patients. Citation Format: Myeongjin Jeon. The regulatory mechanism of fibronectin in EGFR and/or HER2 positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4661. doi:10.1158/1538-7445.AM2014-4661

Development of EGFR-Targeted Nanoemulsion for Imaging and Novel Platinum Therapy of Ovarian Cancer

Platinum-based chemotherapy is the treatment of choice for malignant epithelial ovarian cancers, but generalized toxicity and platinum resistance limits its use. Theranostic nanoemulsion with a novel platinum prodrug, myrisplatin, and the pro-apoptotic agent, C6-ceramide, were designed to overcome these limitations. The nanoemulsions, including ones with an EGFR binding peptide and gadolinium, were made using generally regarded as safe grade excipients and a high shear microfluidization process. Efficacy was evaluated in ovarian cancer cells, SKOV3, A2780 and A2780CP. The nanoemulsion with particle size <150nm were stable in plasma and parenteral fluids for 24h. Ovarian cancer cells in vitro efficiently took up the non-targeted and EGFR-targeted nanoemulsions; improved cytotoxicity was observed for the these nanoemulsions with the latter showing a 50-fold drop in the IC50 in SKOV3 cells as compared to cisplatin alone. The addition of gadolinium did not affect cell viability in vitro, but showed relaxation times comparable to Magnevist(®). The myrisplatin/C6-ceramide nanoemulsion synergistically enhanced in vitro cytotoxicity. An EGFR binding peptide addition further increased in vitro cytotoxicity in EGFR positive cancer cells. The diagnostic version showed MR imaging similar to the clinically relevant Magnevist® and may be suitable as a theranostic for ovarian cancer.