Abnormal oxidative metabolism in a quiet genomic background underlies clear cell papillary renal cell carcinoma
- Memorial Sloan Kettering Cancer Center, United States
- University of Michigan, United States
- Human Genome Sequencing Center, Baylor College of Medicine, United States
- Baylor College of Medicine, United States
- University of Washington, United States
- Ludwig-Maximilians University, Germany
- Metabolon Inc, United States
- Cedars-Sinai Medical Center, United States
- cBio Center, Dana-Farber Cancer Institute, United States
- Harvard Medical School, United States
- Siteman Cancer Center, Washington University, United States
- Rogel Cancer Center, University of Michigan, United States
Figures
Figure 1 with 1 supplement
CCPAP is a metabolic outlier compared to conventional ccRCC.
(A) t-distributed stochastic neighbor embedding (t-SNE) metabolomic data from 140 nominal ccRCC identified two outlier tumors (dataset RC12). Pathological review confirmed that these tumors were clear cell papillary renal cell carcinoma (CCPAP). (B) t-SNE of metabolomic data of a validation cohort (RC13) of 9 CCPAP and 10 ccRCC tumors confirmed that CCPAP tumors cluster distinctly from ccRCC tumors.
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Figure 1—source data 1
Metabolomics results.
- https://doi.org/10.7554/eLife.38986.004
Figure 1—figure supplement 1
Principal components analysis of RC13 data.
https://doi.org/10.7554/eLife.38986.003
Figure 2 with 2 supplements
Metabolic landscape of CCPAP.
(A) Heatmap of differentially abundant metabolites among CCPAP (n = 9), ccRCC (n = 10) and matching normal (n = 10). For visual clarity, only metabolites with Mann-Whitney q < 0.1 and absolute log2 fold-change >1 are displayed; a full table of results is available in Figure 2—source data 1. Yellow arrows indicate high-sorbitol ccRCC tumors. (B) Pathway map depicting metabolite changes (tumors relative to normal kidney tissue) in central carbon metabolism. Metabolites in ccRCC (left half of each box) significantly different from CCPAP (right half of each box) are indicated. (C) Relative abundances of key metabolites in CCPAP, ccRCC or normal tissue in polyol pathway. Yellow dots in ccRCC (Log Sorbitol plot) indicate high-sorbitol ccRCC tumors. (D) Relative abundances of reduced and oxidized glutathione (GSH and GSSG) and NADH/NAD ratio in CCPAP, ccRCC or normal tissue. NS, q > 0.1;*,q < 0.1; **, q < 0.01, Mann-Whitney U test., BH-corrected for multiple hypothesis testing.
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Figure 2—source data 1
Differential abundance of metabolites.
- https://doi.org/10.7554/eLife.38986.008
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Figure 2—source data 2
Metabolite information related to metabolomics data in RC13.
- https://doi.org/10.7554/eLife.38986.009
Figure 2—figure supplement 1
Additional metabolomic analysis of CCPAP tumors.
(A) Correlation between differential abundance calculations when using either low stage ccRCC (horizontal axis) or all ccRCC (vertical axis) as the reference set. (B) Quantification of sorbitol in 6 CCPAP tumors by two independent measurements from Metabolon (RC13, horizontal axis) and University of Michigan (RC15, vertical axis). In RC15 dataset, four technical replicates were run for each sample, with a line fitted through the mean of the four technical replicates. (C) Correlation of all metabolites measured in both Michigan and RC13 datasets.
Figure 2—figure supplement 2
Comparison of metabolite ratios across different tissue types for metabolites involved in NAD reduction reactions.
https://doi.org/10.7554/eLife.38986.007
Figure 3 with 1 supplement
CCPAP tumors exhibit suppressed mitochondrial respiration.
(A) Plots of log transformed mtDNA copy number of 322 TCGA nominal ccRCC samples. Two misclassified samples (BP-4760 and BP-4795) were pathologically re-confirmed as CCPAP. (B) Quantification of transcription from the mitochondrial genomes of tumor or normal samples from different subtypes of kidney cancers. ***, p<0.001, t-test. (C) Gene set enrichment analysis of transcriptome of CCPAP compared with adjacent normal tissue showing that CCPAP tumors exhibit a downregulation of genes related to mitochondria, and oxidative phosphorylation. (D) The relative abundance of aspartate and fumarate in CCPAP, ccRCC or normal tissue. NS, q > 0.1; **, q < 0.01, Mann-Whitney U test following multiple hypothesis correction.
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Figure 3—source data 1
RNA (including miRNA) Sequencing Results.
- https://doi.org/10.7554/eLife.38986.012
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Figure 3—source data 2
GSEA results.
- https://doi.org/10.7554/eLife.38986.013
Figure 3—figure supplement 1
H and E image of 5 misclassified TCGA-KIRC samples, which has been re-evaluated to be CCPAP.
https://doi.org/10.7554/eLife.38986.011
Figure 4 with 1 supplement
CCPAP tumors show few somatic alterations to the nuclear genome, but recurrent depletion of the mitochondrial genome.
(A) Mutational burden of CCPAP tumors from both MSKCC and TCGA as well as ccRCC from TCGA. Highlighted in the boxes are COSMIC cancer gene census genes which are mutated in either MSKCC (red) or TCGA (blue) CCPAP tumors. Notably, there is not a single gene which is non-synonymously mutated more than once in CCPAP. (B) Copy number profile of CCPAP tumor sample 284. (C) The fraction of copy-number altered genome from CCPAP tumors profiled by both MSKCC and TCGA, as well as ccRCC tumors profiled by TCGA. (D) The relative mtDNA copy number in CCPAP and adjacent-normal tissues that were sequenced by either whole-exome-sequencing or whole-genome-sequencing. (E) Somatic variants identified by ultra-deep targeted duplex sequencing of mtDNA of CCPAP tumors.
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Figure 4—source data 1
Mutation annotation file.
- https://doi.org/10.7554/eLife.38986.016
Figure 4—figure supplement 1
Mitochondrial gene expression analysis of CCPAP tumors.
(A) Differential expression analysis of nuclear-DNA-encoded mitochondrial genes. (B) Relative abundance of transcription of 13 mitochondrial genomes-encoded genes of CCPAP, CCRCC and matching normal tissues.
Figure 5 with 3 supplements
Immunohistochemical and histological characterization of CCPAP tumors.
(A) Representative IHC staining of mtDNA-encoded MT-CO1 and nuclear-DNA-encoded/mitochondrially-localized TOM20 of CCPAP, ccRCC tumors and adjacent normal tissues. (B) H-scores of immunohistochemical (IHC) staining for the mtDNA-encoded MT-CO1 protein across different subtypes of kidney cancers (pRCC, papillary RCC; chRCC, chromophobe RCC) **, p<0.01, ***, p<0.001, ****, p<0.0001, t-test. (CCPAP, n = 11; ccRCC, n = 7; pRCC, n = 2; chRCC, n = 4; oncocytoma, n = 4) (C) Representative IHC staining of 8-oxo-dG of CCPAP and ccRCC tumors. (D) H-score of IHC staining for 8-oxo-dG across different subtypes of kidney cancers. *, p<0.05, t-test. (CCPAP, n = 14; ccRCC, n = 3; pRCC, n = 3; oncocytoma, n = 2) (E) Representative images showing the CCPAP and ccRCC tumor regions stained with Oil-Red-O, Periodic acid–Schiff (PAS), and PAS diastase (n = 3 for each staining).
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Figure 5—source data 1
Score for MT-CO1 IHC and 8-oxo-dG IHC.
- https://doi.org/10.7554/eLife.38986.021
Figure 5—figure supplement 1
MicroRNA and methylation analysis of CCPAP tumors.
(A) Plot of Adjusted p-value and relative abundance of miRNA (comparing CCPAP and CCRCC). (B) Principal components analysis of 450 k methylation data from the KIRC TCGA project. Green dots indicate ccRCC tumors, blue dots indicate adjacent normal tissue, and red dots indicate the 4 CCPAP samples. CCPAP tumors primarily cluster with normal tissue.
Figure 5—figure supplement 2
Mutation signature analysis of CCPAP tumor samples.
https://doi.org/10.7554/eLife.38986.019
Figure 5—figure supplement 3
Heatmaps showing intersample correlations (red, positive; blue, negative) between profiles of 5 CCPAP tumors profiled by TCGA as well as average ccRCC (KIRC), chRCC (KICH) and pRCC(KIRP) (rows) and profiles of human and mouse kidney nephron sites (column).
Glom, kidney glomerulus; S1/S3, kidney proximal tubule; mTAL, kidney medullary thick ascending limb of Henle’s loop; cTAL, kidney cortical thick ascending limb of Henle’s loop; DCT, kidney distal convoluted tubule.
Author response image 1
Sorbitol Labeling from Glucose or Fructose in Mock Condition (A), Antimycin A Condition (B), and Rho-Zero Condition (C).
Left panel indicates total ion count. Right panel indicates labeling proportions, corrected for abundance of natural isotopes. Leftmost data of each panel is glucose labeling, rightmost data of each panel is fructose labeling.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rabbit Anti- TOM20 Antibody | Santa Cruz Biotechnology | Cat# sc-11415, RRID:AB_2207533 | IHC (1:100) |
| Antibody | Mouse Anti- MTCO1 Antibody | Abcam | Cat# ab14705, RRID:AB_2084810 | IHC (1:2000) |
| Antibody | Mouse Anti-8- Oxo-dG Antibody | Genox Corpooration | Cat# MOG-020P, RRID:AB_1106819 | IHC (1:400) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.38986.022
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Abnormal oxidative metabolism in a quiet genomic background underlies clear cell papillary renal cell carcinoma
eLife 8:e38986.
https://doi.org/10.7554/eLife.38986
