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

Abstract

BackgroundEGFRvIII 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.MethodsWe 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.ResultsIn 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2521-9) contains supplementary material, which is available to authorized users.

FBXO22 Possesses Both Protumorigenic and Antimetastatic Roles in Breast Cancer Progression

“…Colonies were washed 3 times in PBS, fixed in methanol, stained with 0.1% crystal violet, and counted using an inverted microscope. To examine anchorage-independent 3D growth, MDA-MB-231 cells stably expressing pCDH and HA-FBXO22 (500 cells in 2 mL media) were seeded on 24-well ultra-low attachment surface plates (Corning) as described previously (29). After 2 weeks of culture, cells were observed and counted under a phase-contrast microscopy.…”

“…S1D). Anchorage-independent 3D growth assays (29) showed that overexpression of FBXO22 in MDA-MB-231 cells increased the size but not number of formed spheroidal clumps ( Supplementary Fig. S1E and S1F).…”

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

“…Previous studies also indicated that Ertredin inhibits ATP synthesis to inhibit cell proliferation. 5,6 Indeed, NDUFA12 knockdown reduced the NDUFA12 expression levels, resulting in the suppression of cell proliferation (Figure 5F). Taken together, these results suggest that NDUFA12 is a biologically relevant target of Ertredin and is responsible for its antiproliferative activity in cancer cells.…”

“…Ertredin exhibited potent anticancer activity against EGFRvIII-derived cancer cells. 5,6 However, it is unclear how Ertredin exerts an inhibitory effect on these cells. To address this question and identify the target of Ertredin, we first examined the effect of Ertredin on the proliferation of various cultured cells.…”

“…In the previous study, we showed that Ertredin inhibits the function of mitochondrial complex 1. 5 We performed mitochondrial oxygen consumption analysis to evaluate the effect of Ertredin on mitochondrial function. Indeed, Ertredin can effectively inhibit the function of complex 1, while the inhibitory effect on complex 2 is small (Figure S2).…”