Similar deficiencies, different outcomes: succinate dehydrogenase loss in adrenal medulla vs. fibroblast cell culture models of paraganglioma

Abstract

Heterozygosity for loss-of-function alleles of the genes encoding the four subunits of succinate dehydrogenase (SDHA, SDHB, SDHC, SDHD), as well as the SDHAF2 assembly factor predispose affected individuals to pheochromocytoma and paraganglioma (PPGL), two rare neuroendocrine tumors that arise from neural crest-derived paraganglia. Tumorigenesis results from loss of the remaining functional SDHx gene copy, leading to a cell with no functional SDH and a defective tricarboxylic acid (TCA) cycle. It is believed that the subsequent accumulation of succinate competitively inhibits multiple dioxygenase enzymes that normally suppress hypoxic signaling and demethylate histones and DNA, ultimately leading to increased expression of genes involved in angiogenesis and cell proliferation. Why SDH loss is selectively tumorigenic in neuroendocrine cells remains poorly understood. In the absence of SDH-loss tumor-derived cell models, the cellular burden of SDH loss and succinate accumulation have been investigated through conditional knockouts of SDH subunits in pre-existing murine or human cell lines with varying degrees of clinical relevance. Here we characterize two available murine SDH-loss cell lines, immortalized adrenally-derived premature chromaffin cells vs. immortalized fibroblasts, at a level of detail beyond that currently reported in the literature and with the intention of laying the foundation for future investigations into adaptive pathways and vulnerabilities in SDH-loss cells. We report different mechanistic and phenotypic manifestations of SDH subunit loss in the presented cellular contexts. These findings highlight similarities and differences in the cellular response to SDH loss between the two cell models. We show that adrenally-derived cells display more severe morphological cellular and mitochondrial alterations, yet are unique in preserving residual Complex I function, perhaps allowing them to better tolerate SDH loss, thus making them a closer model to SDH-loss PPGL relative to fibroblasts.(281 words).

Keywords: Complex I; Hypoxia; Paraganglioma; Pheochromocytoma; Succinate dehydrogenase; Tricarboxylic acid cycle.

Fig. 1

SDH-deficiency induces phenotypic changes in imCCs and iMEFs. (A) Maximum velocity of SDH catalysis (SDH_max) calculated according to Eq. 3 (n = 20 cells per line). (B) Quantitation of cell doubling time based on Eq. 2 for imCCs and iMEFs (n = 6 wells per line). (C) Average cell diameter for control and SDH-loss imCCs and iMEFs. Data represent average calculated diameter for 6 fields per line. (D) Percentage of detached cells following trypsinization for 5 min compared to a counting control treated with excess trypsin and mechanical force for total detachment (100%, indicated by red line). P-values are calculated using student’s t-test and the number of asterisks indicates degree of significance. (E) Examples of Flowjo-generated cell cycle analysis histograms showing percentage of the indicated cells in each of the indicated cell cycle phases (G1, S or G2) based on DNA content (measured by fluorescence intensity) using a Watson analysis model (n = 50,000 cells per sample). (F) average percentage of cells in each of the indicated cell cycle phases (G1, S or G2) for each control or SDH-loss line shown as mean ± SEM (n = 3 samples per line processed in a single run). RMSD values for the depicted histograms are Sdhb^+/+imCCs: 4.94; Sdhb^−/−imCCs: 4.59; Sdhc^+/−iMEFs: 4.31 and Sdhc^−/−iMEFs: 5.77. These values are representative of the replicate samples. Totals do not all reach 100% because of the presence of cells with DNA content not assigned to any of the indicated phases. P-values are calculated using Bonferroni correction for multiple t-tests and the number of asterisks indicates degree of significance

Fig. 2

Metabolite profiling and effects on DNA methylation. (A) Schematic overview of TCA cycle highlighting the loss of SDH function (red “X”). (B-C) Relative metabolite levels (log scale) in SDH-loss cells compared to their respective controls (n = 3 samples of million cells per line, data shown as mean ± SEM) normalized to protein content (B) or per cell (C). Cell normalization data were accounted for mathematically by calculating the number of cells in each sample using a standard curve for protein content per 1 M cells. Lac: lactate; Asp: aspartate; Mal: malate; Fum: fumarate; Succ: succinate; Glu: glucose; 2-hg, 2-hydroxyglutarate; AKG, 2-oxoglutarate; c-Acon: cis-aconitate; Isoc: isocitrate; Cit: citrate. (D) Western blot of H3K9me3, detected at 17 kDa, where hypermethylation serves as a marker of succinate inhibition of JMJD demethylases, a subfamily of OG-dependent dioxygenases. Anti-histone H3 is used as loading control, also detected at 17 kDa. (E) Western Blot analysis for HIF1/2α levels in various indicated oxygen concentrations. Protein extracts from the indicated cell lines were probed with anti-HIF1α or HIF2α, both detected at 120 kDa (two upper panels). α-tubulin was used as a loading control and detected at 50 kDa (lower panel)

Fig. 3

Altered mitochondrial and cellular morphology following SDH loss. (A) Representative electron micrographs of mitochondria for the indicated cell lines at the indicated magnifications. Individual examples of mitochondria are indicated by red arrows in the far-right panels. (B) Staining of the same cell lines to show unusual circumferential F-actin staining (red arrows) around enlarged mitochondria in SDH-loss cells. (C) Mitochondrial volume density measurement by 3D microscopy for the four indicated lines (n = 20 cells per line). (D) Average chromosome counts per cell for each control and SDH-loss line (n = 20 cells per line). Red line symbolizes the normal chromosomal count of a diploid mouse cell. Error bars in panels C-D indicate mean ± SD. P-values are calculated using student’s t-test for each experimental and control pair and the number of asterisks indicate degree of significance. (E) Micrographs of single-cell FISH analysis with a mouse pan centromere probe (green) to mark the centromeres on DAPI stained chromosomes

Fig. 4

imCCs, but not iMEFs maintain substantial metabolic fitness following loss of SDH. (A-B) Seahorse mitochondrial stress test profiles for the indicated cell lines in 20% O₂ (A) or 5% O₂ (B). Rote/AA: rotenone and antimycin A. (C) Complex-specific Seahorse-based assay utilizing Complex I, II and III inhibitors or substrates to assess activities of Complexes I and II in the indicated cell lines. (D-E) Bar graphs representing data for Complex I (D) or Complex II (E) activity from panel C. Seahorse tests were performed in replicates of three (n = 3), error bars represent mean ± SEM. P-values are calculated using student’s t-test and the degree of significance is indicated with number of asterisks

Fig. 5

Distinct transcriptomic changes upon SDH loss in imCCs (A) or iMEFs (B). Data are displayed as volcano plots where genes shown in back are differentially expressed [p-value < 0.05, Log₂(FC) > 1.5]. (C) Scatter plot showing relatively poor correlation between Log₂(FC) values for RNA transcripts changed upon SDH loss in imCCs (x-axis) and iMEFs (y-axis). (D) For comparison, relatively strong correlation is observed for expressed transcripts between WT imCCs (x-axis) and SDH-loss imCCs (y-axis). E and F. Volcano plots show differential expression of RNAs encoding components of Complex I upon SDH loss in imCCs (E) or in iMEFs (F). Labelled genes are differentially expressed [p-value < 0.05, Log₂(FC) > 1.5]