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. 2015 Aug 25;112(34):10732-7.
doi: 10.1073/pnas.1512863112. Epub 2015 Aug 10.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

Sebastian Stahl  1 , Ana Rita da Silva Mateus Seidl  2 , Axel Ducret  3 , Sabine Kux van Geijtenbeek  3 , Sven Michel  3 , Tomas Racek  4 , Fabian Birzele  4 , Alexander K Haas  1 , Ruediger Rueger  4 , Michael Gerg  5 , Gerhard Niederfellner  2 , Ira Pastan  6 , Ulrich Brinkmann  7
Affiliations

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor

Sebastian Stahl et al. Proc Natl Acad Sci U S A. .

Abstract

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

Keywords: ADP-ribosylation of eEF2; DPH gene knockout; Pseudomonas exotoxin; diphtheria toxin; translation.

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Conflict of interest statement

Conflict of interest statement: S.S., A.R.d.S.M.S., A.D., S.K.G., S.M., T.R., F.B., A.K.H., R.R., M.G., G.N., and U.B. are employed by Roche, and I.P. by the National Cancer Institute. All parties are interested in and hold patents (I.P.’s patent has been assigned to the NIH and he has a Cooperative Research and Development Agreement with Roche Pharmaceuticals) for the development of Pseudomonas exotoxin-derived entities in targeted cancer therapy.

Figures

Fig. S1.
Fig. S1.
Generation of MCF7 derivatives. (A) ZFN transfected cells are either exposed to PE followed by isolation and characterization of surviving cells (Left branch), or individually cloned and subjected to PCR-based genetic analyses without toxin exposure (Right branch and example for HRM analyses below). (B) ZFN target sequences and allele sequences of mutated MCF7 clones.
Fig. 1.
Fig. 1.
Diphthamide modification and ADP ribosylation of eEF2. (A) Detection of unmodified eEF2 in parent and mutated MCF7 by a rabbit mAb that specifically detects eEF2 without diphthamide (SI Text S2). (B) Semiquantitative assessment of eEF2 modifications by MS (46) (SI Text S3). (C) ADP ribosylation of eEF2 was assessed in extracts exposed to PE Bio-NAD, followed by Western blot detection of Bio-ADPR-eEF2 (32).
Fig. S2.
Fig. S2.
Western blot identification of antibodies that specifically detect eEF2 without diphthamide.
Fig. S3.
Fig. S3.
MS-based determination of eEf2 H715 modifications.
Fig. 2.
Fig. 2.
Influence of DPH inactivation on sensitivity to ADP ribosylating toxins and TNF. Dose–responses of cells with inactivation of DPH1, DPH2, DPH4, and DPH5 in comparison with parent MCF7 (bold line), exposed to PE (A), DT (B) or TNF (C). Complete knockouts (Upper) and partial knockous (Lower) are shown. Parent MCF7 (bold line) and partial knockouts show the same sensitivity to PE, DT, and TNF. Cells with complete inactivation of DPH1, DPH2, DPH4, and DPH5 are resistant to PE and DT and hypersensitive to TNF.
Fig. S4.
Fig. S4.
(A) Growth, (B) susceptibility toward saporin and CHX, and (C) chromosome composition of MCF7 derivatives. (C) Images were captured with a Zeiss Plan-Neofluar microscope at 100× magnification.
Fig. 3.
Fig. 3.
Inactivation of DPH5 induces NF-κB and death receptor pathways. RNAseq data were obtained for untreated MCF7, TNF treated MCF7, and DPH5 inactivated MCF7 (SI Text S5). Genes that are significantly changed in their expression levels (MCF7 vs. TNF-MCF7, MCF7 vs. DPH5ko) were subjected to ingenuity upstream pathway analyses. Interestingly, differentially regulated pathways and regulators in both conditions overlap largely and share a common core of TNF- and IFNG-regulated genes, leading to a preactivation of death receptor signaling regulators as consequence of DPH5 deficiency and loss of diphthamide (orange: consequence of TNF treatment as well as DPH5ko; green: TNF treatment only; blue: DPH5ko only). Arrows indicate direct activating/inhibiting interactions between two nodes (based on literature findings) and dashed arrows indicate indirect interactions. Different protein types are represented by different symbols.
Fig. S5.
Fig. S5.
(A) Analyses of TNFR expression by FACS with TNFR specific antibodies. (B) Transcriptome (mRNAseq) comparisons of MCF7.
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