NDUFA12 as a Functional Target of the Anticancer Compound Ertredin in Human Hepatoma Cells As Revealed by Label-Free Chemical Proteomics.

Many attempts have been made to develop new agents that target EGFR mutants or regulate downstream factors in various cancers. Cell-based screening showed that a natural small molecule, Ertredin, inhibited the growth of EGFRvIII mutant cancer cells. Previous studies have shown that Ertredin effectively inhibits anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII mutant cDNA. However, the underlying mechanism remains unclear. In this study, we investigated the target protein of Ertredin by combining drug affinity-responsive target stability (DARTS) assays with liquid chromatography-mass spectrometry using label-free Ertredin as a bait and HepG2 cell lysates as a proteome pool. NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (NDUFA12) was identified as an Ertredin-binding protein that was responsible for its biological activity. The interaction between NDUFA12 and Ertredin was validated by DARTS and cellular thermal shift assays. In addition, the genetic knockdown of the identified target, NDUFA12, was shown to suppress cell proliferation. NDUFA12 was identified as a biologically relevant target protein of Ertredin that is responsible for its antitumor activity, and these results provide insights into the role of NDUFA12 as a downstream factor in EGFRvIII mutants.

Defucosylated Monoclonal Antibody (H2Mab-139-mG2a-f) Exerted Antitumor Activities in Mouse Xenograft Models of Breast Cancers against Human Epidermal Growth Factor Receptor 2.

The clinically approved human epidermal growth factor receptor 2 (HER2)-targeting monoclonal antibodies (mAbs), trastuzumab, and pertuzumab, target domains IV and II, respectively. Trastuzumab is now the standard treatment for HER2-overexpressed breast and gastric cancers, and trastuzumab in combination with pertuzumab showed clinical benefit. However, there still exist patients who do not respond to the therapy. Furthermore, HER2 mutants that cannot be recognized by pertuzumab were found in tumors. Therefore, novel anti-HER2 mAbs and modalities have been desired. In our previous study, we developed a novel anti-HER2 domain I mAb, H2Mab-139 (mouse IgG1, kappa). We herein produced a defucosylated mouse IgG2a type of mAb against HER2 (H2Mab-139-mG2a-f) to enhance antibody-dependent cellular cytotoxicity (ADCC)-mediated antitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in flow cytometry with the dissociation constant (KD) determined to be 3.9 × 10-9 M and 7.7 × 10-9 M against HER2-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/HER2) and HER2-positive BT-474 cells, respectively. Moreover, we showed that H2Mab-139-mG2a-f exerted ADCC and complement-dependent cytotoxicity against CHO/HER2 and BT-474 in vitro and exhibited potent antitumor activities in mouse xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and is useful as an antibody treatment for HER2-positive human cancers.

Pseudomonas Aeruginosa Theft Biofilm Require Host Lipids of Cutaneous Wound.

OBJECTIVE: This work addressing complexities in wound infection, seeks to test the reliance of bacterial pathogen Pseudomonas aeruginosa (PA) on host skin lipids to form biofilm with pathological consequences. BACKGROUND: PA biofilm causes wound chronicity. Both CDC as well as NIH recognizes biofilm infection as a threat leading to wound chronicity. Chronic wounds on lower extremities often lead to surgical limb amputation. METHODS: An established preclinical porcine chronic wound biofilm model, infected with PA or Pseudomonas aeruginosa ceramidase mutant (PA ∆Cer ), was used. RESULTS: We observed that bacteria drew resource from host lipids to induce PA ceramidase expression by three orders of magnitude. PA utilized product of host ceramide catabolism to augment transcription of PA ceramidase. Biofilm formation was more robust in PA compared to PA ∆Cer . Downstream products of such metabolism such as sphingosine and sphingosine-1-phosphate were both directly implicated in the induction of ceramidase and inhibition of peroxisome proliferator-activated receptor (PPAR)δ, respectively. PA biofilm, in a ceram-idastin-sensitive manner, also silenced PPARδ via induction of miR-106b. Low PPARδ limited ABCA12 expression resulting in disruption of skin lipid homeostasis. Barrier function of the wound-site was thus compromised. CONCLUSIONS: This work demonstrates that microbial pathogens must co-opt host skin lipids to unleash biofilm pathogenicity. Anti-biofilm strategies must not necessarily always target the microbe and targeting host lipids at risk of infection could be productive. This work may be viewed as a first step, laying fundamental mechanistic groundwork, toward a paradigm change in biofilm management.

Current Targeted Therapy for Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of cancer deaths worldwide. Surgery or surgery plus radiotherapy and/or chemotherapy for patients with metastatic CRC (mCRC) were accepted as the main therapeutic strategies until the early 2000s, when targeted drugs, like cetuximab and bevacizumab, were developed. The use of targeted drugs in clinical practice has significantly increased patients' overall survival. To date, the emergence of several types of targeted drugs has opened new possibilities and revealed new prospects for mCRC treatment. Therapeutic strategies are continually being updated to select the most suitable targeted drugs based on the results of clinical trials that are currently underway. This review discusses the up-to date molecular evidence of targeted therapy for mCRC and summarizes the Food and Drug Administration-approved targeted drugs including the results of clinical trials. We also explain their mechanisms of action and how these affect the choice of a suitable targeted therapy.

Short peptides derived from hGAPDH exhibit anti-cancer activity.

Peptides have become an attractive drug discovery modality alongside small molecule compounds and high molecular weight biomolecules because they bind strongly to their target molecules. Previously, we found that secreted extracellular human GAPDH exhibits inhibitory activity against cancer cell growth. We sought to identify the minimal peptide sequence required for GAPDH activity in an effort to develop a small GAPDH-derived peptide with anti-cancer activity. Moreover, derivatives of the identified peptide, in which some amino acid residues were substituted with unnatural amino acids, were found to show stronger anti-cancer activity than non-substituted peptides.

Human pancreatic cancer cells under nutrient deprivation are vulnerable to redox system inhibition.

Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in GSH; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment.

Inhibition of mitochondria ATP synthase suppresses prostate cancer growth through reduced insulin-like growth factor-1 secretion by prostate stromal cells.

Modulation of prostate stromal cells (PrSCs) within tumor tissues is gaining attention for the treatment of solid tumors. Using our original in vitro coculture system, we previously reported that leucinostatin (LCS)-A, a peptide mycotoxin, inhibited prostate cancer DU-145 cell growth through reduction of insulin-like growth factor 1 (IGF-I) expression in PrSCs. To further obtain additional bioactive compounds from LCS-A, we designed and synthesized a series of LCS-A derivatives as compounds that target PrSCs. Among the synthesized LCS-A derivatives, LCS-7 reduced IGF-I expression in PrSCs with lower toxicity to PrSCs and mice than LCS-A. As LCS-A has been suggested to interact with mitochondrial adenosine triphosphate (ATP) synthase, a docking study was performed to elucidate the mechanism of reduced IGF-I expression in the PrSCs. As expected, LCS-A and LCS-7 directly interacted with mitochondrial ATP synthase, and like LCS-A and LCS-7, other mitochondrial ATP synthase inhibitors also reduced the expression of IGF-I by PrSCs. Furthermore, LCS-A and LCS-7 significantly decreased the growth of mouse xenograft tumors. Based on these data, we propose that the mitochondrial ATP synthases-IGF-I axis of PrSCs plays a critical role on cancer cell growth and inhibition could be a potential anticancer target for prostate cancer.

Tumor-derived exosomes in the regulation of macrophage polarization.

BACKGROUND: This review focuses on exosomes derived from various cancer cells. The review discusses the possibility of differentiating macrophages in alternatively activated anti-inflammatory pro-tumorigenic M2 macrophage phenotypes and classically activated pro-inflammatory, anti-tumorigenic M1 macrophage phenotypes in the tumor microenvironment (TME). The review is divided into two main parts, as follows: (1) role of exosomes in alternatively activating M2-like macrophages-breast cancer-derived exosomes, hepatocellular carcinoma (HCC) cell-derived exosomes, lung cancer-derived exosomes, prostate cancer-derived exosomes, Oral squamous cell carcinoma (OSCC)-derived exosomes, epithelial ovarian cancer (EOC)-derived exosomes, Glioblastoma (GBM) cell-derived exosomes, and colorectal cancer-derived exosomes, (2) role of exosomes in classically activating M1-like macrophages, oral squamous cell carcinoma-derived exosomes, breast cancer-derived exosomes, Pancreatic-cancer derived modified exosomes, and colorectal cancer-derived exosomes, and (3) exosomes and antibody-dependent cellular cytotoxicity (ADCC). This review addresses the following subjects: (1) crosstalk between cancer-derived exosomes and recipient macrophages, (2) the role of cancer-derived exosome payload(s) in modulating macrophage fate of differentiation, and (3) intracellular signaling mechanisms in macrophages regarding the exosome's payload(s) upon its uptake and regulation of the TME. EVIDENCE: Under the electron microscope, nanoscale exosomes appear as specialized membranous vesicles that emerge from the endocytic cellular compartments. Exosomes harbor proteins, growth factors, cytokines, lipids, miRNA, mRNA, and DNAs. Exosomes are released by many cell types, including reticulocytes, dendritic cells, B-lymphocytes, platelets, mast cells, and tumor cells. It is becoming clear that exosomes can impinge upon signal transduction pathways, serve as a mediator of signaling crosstalk, thereby regulating cell-to-cell wireless communications. CONCLUSION: Based on the vesicular cargo, the molecular constituents, the exosomes have the potential to change the fate of macrophage phenotypes, either M1, classically activated macrophages, or M2, alternatively activated macrophages. In this review, we discuss and describe the ability of tumor-derived exosomes in the mechanism of macrophage activation and polarization.

Anti-Metastatic Activity of an Anti-EGFR Monoclonal Antibody against Metastatic Colorectal Cancer with KRAS p.G13D Mutation.

The now clinically-used anti-epidermal growth factor receptor (EGFR) monoclonal antibodies have demonstrated significant efficacy only in patients with metastatic colorectal cancer (mCRC), with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS). However, no effective treatments for patients with mCRC with KRAS mutated tumors have been approved yet. Therefore, a new strategy for targeting mCRC with KRAS mutated tumors is desired. In the present study, we examined the anti-tumor activities of a novel anti-EGFR monoclonal antibody, EMab-17 (mouse IgG2a, kappa), in colorectal cancer (CRC) cells with the KRAS p.G13D mutation. This antibody recognized endogenous EGRF in CRC cells with or without KRAS mutations, and showed a high sensitivity for CRC cells in flow cytometry, indicating that EMab-17 possesses a high binding affinity to the endogenous EGFR. In vitro experiments showed that EMab-17 exhibited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities against CRC cells. In vivo analysis revealed that EMab-17 inhibited the metastases of HCT-15 and HCT-116 cells in the livers of nude mouse metastatic models, unlike the anti-EGFR monoclonal antibody EMab-51 of subtype mouse IgG1. In conclusion, EMab-17 may be useful in an antibody-based therapy against mCRC with the KRAS p.G13D mutation.

Interferon-Induced Transmembrane Protein 1 (IFITM1) Promotes Distant Metastasis of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a severe malignancy associated with early and widespread metastasis. To study SCLC metastasis, we previously developed an orthotopic transplantation model using the human SCLC cell line DMS273. In the model, metastatic foci were found in distant tissues such as bone and the adrenal gland, similarly as observed in patients with SCLC. In this study, we evaluated the differentially expressed genes between orthotopic and metastatic tumors in the model. We isolated tumor cells from orthotopic and metastatic sites, and the tumor cell RNA was analyzed using DNA microarray analysis. We found that 19 genes in metastatic tumors were upregulated by more than 4-fold compared with their expression in orthotopic tumors. One of these genes encodes a transmembrane protein, interferon (IFN)-induced transmembrane protein 1 (IFITM1), and immunohistochemical analysis confirmed the higher expression of the protein in metastatic sites than in orthotopic sites. IFITM1 was also detected in some SCLC cell lines and lung tumors from patients with SCLC. The overexpression of IFITM1 in DMS273 cells increased their metastatic formation in the orthotopic model and in an experimental metastasis model. Conversely, the silencing of IFITM1 suppressed metastatic formation by DMS273 cells. We also found that IFITM1 overexpression promoted the metastatic formation of NCI-H69 human SCLC cells. These results demonstrate that IFITM1 promotes distant metastasis in xenograft models of human SCLC.

Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy.

Coxsackievirus and adenovirus receptor (CAR) is a single-pass transmembrane protein that is associated with adenoviral infection. CAR is involved in the formation of epithelial tight junctions and promotes tumor growth in some cancers. Previously, we developed mouse monoclonal antibodies against human CAR and found that one, mu6G10A, significantly inhibited tumor growth in xenografts of human cancer cells. Herein, we generated and characterized a mouse-human chimeric anti-CAR antibody (ch6G10A) from mu6G10A. ch6G10A had binding activity, inducing antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, and in vivo anti-tumor activity against CAR-expressing prostate cancer DU-145 cells. In addition, cancer tissue array analysis confirmed that CAR is highly expressed in neuroendocrine lung cancers including small cell lung cancer, and treatment with ch6G10A effectively inhibited in vivo subcutaneous tumor growth of NCI-H69 small cell lung cancer cells in nude mice. Moreover, treatment with mu6G10A effectively inhibited both in vivo orthotopic tumor growth and distant metastatic formation in mouse xenograft models of a highly metastatic subline of human small cell lung cancer DMS273 cells. These results suggest that targeting therapy to CAR with a therapeutic antibody might be effective against several cancer types including small cell lung cancer.

Leucinostatin Y: A Peptaibiotic Produced by the Entomoparasitic Fungus Purpureocillium lilacinum 40-H-28.

Leucinostatin Y, a new peptaibiotic, was isolated from the culture broth of the entomoparasitic fungus Purpureocillium lilacinum 40-H-28. The planar structure was elucidated by detailed analysis of its NMR and MS/MS data. The absolute configurations of the amino acids were partially determined by an advanced Marfey's method. The biological activities of leucinostatin Y were assessed using human pancreatic cancer cells, revealing the importance of the C-terminus of leucinostatins for preferential cytotoxicity to cancer cells under glucose-deprived conditions and inhibition of mitochondrial function.

Potential Anticancer Activity of Auranofin.

Gold compounds have a long history of use in medicine. Auranofin was developed more than 30 years ago as an oral therapy for rheumatoid arthritis. Now, however, auranofin is rarely used in clinical practice despite its efficacy for treating rheumatoid arthritis because more novel antirheumatic medications are available. Although its use in clinical practice has decreased, studies on auranofin have continued and it shows promise for the treatment of several different diseases, including cancer and bacterial and parasitic infections. Several potential novel applications of auranofin for treating human disease have been proposed. Auranofin inhibits the activity of thioredoxin reductase (TrxR), an enzyme of the thioredoxin (Trx) system that is important for maintaining the intracellular redox state. Particularly in cancers, TrxR inhibition leads to an increase in cellular oxidative stress and induces apoptosis. TrxR overexpression is associated with aggressive tumor progression and poor survival in patients with breast, ovarian, and lung cancers. The Trx system may represent an attractive target for the development of new cancer treatments. Therefore, the TrxR inhibitor auranofin may be a potent anticancer agent. This review summarizes the current understanding of auranofin for cancer therapy.

Monotherapy with a novel intervenolin derivative, AS-1934, is an effective treatment for Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori (H. pylori) infection causes various gastrointestinal diseases including gastric cancer. Hence, eradication of this infection could prevent these diseases. The most popular first-line treatment protocol to eradicate H. pylori is termed "triple therapy" and consists of a proton pump inhibitor (PPI), clarithromycin, and amoxicillin or metronidazole. However, the antibiotics used to treat H. pylori infection are hindered by the antibiotics-resistant bacteria and by their antimicrobial activity against intestinal bacteria, leading to side effects. Therefore, an alternative treatment with fewer adverse side effects is urgently required to improve the overall eradication rate of H. pylori. OBJECTIVE: The aim of this study was to assess the effectiveness and mechanism of action of an antitumor agent, intervenolin, and its derivatives as an agent for the treatment of H. pylori infection. RESULTS: We demonstrate that intervenolin, and its derivatives showed selective anti-H. pylori activity, including antibiotic-resistant strains, without any effect on intestinal bacteria. We showed that dihydroorotate dehydrogenase, a key enzyme for de novo pyrimidine biosynthesis, is a target and treatment with intervenolin or its derivatives decreased the protein and mRNA levels of H. pylori urease, which protects H. pylori against acidic conditions in the stomach. Using a mouse model of H. pylori infection, oral monotherapy with the intervenolin derivative AS-1934 had a stronger anti-H. pylori effect than the triple therapy commonly used worldwide to eradicate H. pylori. CONCLUSION: AS-1934 has potential advantages over current treatment options for H. pylori infection.

Dihydroorotate Dehydrogenase as a Target for the Development of Novel Helicobacter pylori-Specific Antimicrobials.

Helicobacter pylori (H. pylori) infection is the world's most common bacterial infection, affecting approximately 50% of the global population. H. pylori is the strongest known risk factor for stomach diseases, including cancer. Hence, treatment for H. pylori infection can help reduce the risk of these diseases. However, the emergence of drug-resistant strains of H. pylori and the occurrence of adverse effects resulting from current therapies have complicated the successful eradication of H. pylori infection. Although various antibiotics that target several bacterial enzymes have been discovered, dihydroorotate dehydrogenase (DHODH) may hold potential for the development of novel anti-H. pylori agents with reduced toxicity and side effects. Here we review the existing literature that has focused on strategies for developing novel therapeutic agents that target the DHODH of H. pylori.

Impact of MET inhibition on small-cell lung cancer cells showing aberrant activation of the hepatocyte growth factor/MET pathway.

Small-cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers, and is characterized as extremely aggressive, often displaying rapid tumor growth and multiple organ metastases. In addition, the clinical outcome of SCLC patients is poor due to early relapse and acquired resistance to standard chemotherapy treatments. Hence, novel therapeutic strategies for the treatment of SCLC are urgently required. Accordingly, several molecular targeted therapies were evaluated in SCLC; however, they failed to improve the clinical outcome. The receptor tyrosine kinase MET is a receptor for hepatocyte growth factor (HGF), and aberrant activation of HGF/MET signaling is known as one of the crucial mechanisms enabling cancer progression and invasion. Here, we found that the HGF/MET signaling was aberrantly activated in chemoresistant or chemorelapsed SCLC cell lines (SBC-5, DMS273, and DMS273-G3H) by the secretion of HGF and/or MET copy number gain. A cell-based in vitro assay revealed that HGF/MET inhibition, induced either by MET inhibitors (crizotinib and golvatinib), or by siRNA-mediated knockdown of HGF or MET, constrained growth of chemoresistant SCLC cells through the inhibition of ERK and AKT signals. Furthermore, treatment with either crizotinib or golvatinib suppressed the systemic metastasis of SBC-5 cell tumors in natural killer cell-depleted SCID mice, predominantly through cell cycle arrest. These findings reveal the therapeutic potential of targeting the HGF/MET pathway for inhibition, to constrain tumor progression of SCLC cells showing aberrant activation of HGF/MET signaling. We suggest that it would be clinically valuable to further investigate HGF/MET-mediated signaling in SCLC cells.

Mitochondrial uncoupler exerts a synthetic lethal effect against ß-catenin mutant tumor cells.

The wingless/int-1 (Wnt) signal transduction pathway plays a central role in cell proliferation, survival, differentiation and apoptosis. When ß-catenin: a component of the Wnt pathway, is mutated into an active form, cell growth signaling is hyperactive and drives oncogenesis. As ß-catenin is mutated in a wide variety of tumors, including up to 10% of all sporadic colon carcinomas and 20% of hepatocellular carcinomas, it has been considered a promising target for therapeutic interventions. Therefore, we screened an in-house natural product library for compounds that exhibited synthetic lethality towards ß-catenin mutations and isolated nonactin, an antibiotic mitochondrial uncoupler, as a hit compound. Nonactin, as well as other mitochondrial uncouplers, induced apoptosis selectively in ß-catenin mutated tumor cells. Significant tumor regression was observed in the ß-catenin mutant HCT 116 xenograft model, but not in the ß-catenin wild type A375 xenograft model, in response to daily administration of nonactin in vivo. Furthermore, we found that expression of an active mutant form of ß-catenin induced a decrease in the glycolysis rate. Taken together, our results demonstrate that tumor cells with mutated ß-catenin depend on mitochondrial oxidative phosphorylation for survival. Therefore, they undergo apoptosis in response to mitochondrial dysfunction following the addition of mitochondrial uncouplers, such as nonactin. These results suggest that targeting mitochondria is a potential chemotherapeutic strategy for tumor cells that harbor ß-catenin mutations.

Catalytic Asymmetric Total Synthesis and Stereochemical Revision of Leucinostatin†A: A Modulator of Tumor-Stroma Interaction.

Total synthesis of leucinostatin A, a modulator of tumor-stroma interactions, using asymmetric catalyses, a nitroaldol reaction, thioamide-aldol reaction, Strecker-type reaction, and alcoholysis of 3-methylglutaric anhydride, is described. We demonstrated the applicability of the established catalytic asymmetric processes to the synthesis of molecules with a complex structure. Careful analysis of the NMR data, HPLC profiles, and biological activity revealed that the correct structure of leucinostatin A is the epimeric form of the reported structure; the secondary alcohol within the AHMOD residue has an R configuration.

Report on the use of non-clinical studies in the regulatory evaluation of oncology drugs.

Non-clinical studies are necessary at each stage of the development of oncology drugs. Many experimental cancer models have been developed to investigate carcinogenesis, cancer progression, metastasis, and other aspects in cancer biology and these models turned out to be useful in the efficacy evaluation and the safety prediction of oncology drugs. While the diversity and the degree of engagement in genetic changes in the initiation of cancer cell growth and progression are widely accepted, it has become increasingly clear that the roles of host cells, tissue microenvironment, and the immune system also play important roles in cancer. Therefore, the methods used to develop oncology drugs should continuously be revised based on the advances in our understanding of cancer. In this review, we extensively summarize the effective use of those models, their advantages and disadvantages, ranges to be evaluated and limitations of the models currently used for the development and for the evaluation of oncology drugs.

New anti-cancer chemicals Ertredin and its derivatives, regulate oxidative phosphorylation and glycolysis and suppress sphere formation in vitro and tumor growth in EGFRvIII-transformed cells.

BACKGROUND: EGFRvIII is a mutant form of the epidermal growth factor receptor gene (EGFR) that lacks exons 2-7. The resulting protein does not bind to ligands and is constitutively activated. The expression of EGFRvIII is likely confined to various types of cancer, particularly glioblastomas. Although an anti-EGFRvIII vaccine is of great interest, low-molecular-weight substances are needed to obtain better therapeutic efficacy. Thus, the purpose of this study is to identify low molecular weight substances that can suppress EGFRvIII-dependent transformation. METHODS: We constructed a new throughput screening system and searched for substances that decreased cell survival of NIH3T3/EGFRvIII spheres under 3-dimensional (3D)-culture conditions, but retained normal NIH3T3 cell growth under 2D-culture conditions. In vivo activity was examined using a mouse transplantation model, and derivatives were chemically synthesized. Functional characterization of the candidate molecules was investigated using an EGFR kinase assay, immunoprecipitation, western blotting, microarray analysis, quantitative polymerase chain reaction analysis, and measurement of lactate and ATP synthesis. RESULTS: In the course of screening 30,000 substances, a reagent, "Ertredin" was found to inhibit anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII cDNA. Ertredin also inhibited sphere formation in cells expressing wild-type EGFR in the presence of EGF. However, it did not affect anchorage-dependent 2D growth of parental NIH3T3 cells. The 3D-growth-inhibitory activity of some derivatives, including those with new structures, was similar to Ertredin. Furthermore, we demonstrated that Ertredin suppressed tumor growth in an allograft transplantation mouse model injected with EGFRvIII- or wild-type EGFR-expressing cells; a clear toxicity to host animals was not observed. Functional characterization of Ertredin in cells expressing EGFRvIII indicated that it stimulated EGFRvIII ubiquitination, suppressed both oxidative phosphorylation and glycolysis under 3D conditions, and promoted cell apoptosis. CONCLUSION: We developed a high throughput screening method based on anchorage-independent sphere formation induced by EGFRvIII-dependent transformation. In the course of screening, we identified Ertredin, which inhibited anchorage-independent 3D growth and tumor formation in nude mice. Functional analysis suggests that Ertredin suppresses both mitochondrial oxidative phosphorylation and cytosolic glycolysis in addition to promoting EGFRvIII degradation, and stimulates apoptosis in sphere-forming, EGFRvIII-overexpressing cells.