Quantitative analysis of the mitochondrial proteome in human ovarian carcinomas

in Endocrine-Related Cancer
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Mitochondria play important roles in growth, signal transduction, division, tumorigenesis and energy metabolism in epithelial ovarian carcinomas (EOCs) without an effective biomarker. To investigate the proteomic profile of EOC mitochondrial proteins, a 6-plex isobaric tag for relative and absolute quantification (iTRAQ) proteomics was used to identify mitochondrial expressed proteins (mtEPs) in EOCs relative to controls, followed by an integrative analysis of the identified mtEPs and the Cancer Genome Atlas (TCGA) data from 419 patients. A total of 5115 quantified proteins were identified from purified mitochondrial samples, and 262 proteins were significantly related to overall survival in EOC patients. Furthermore, 63 proteins were identified as potential biomarkers for the development of an EOC, and our findings were consistent with previous reports on a certain extent. Pathway network analysis identified 70 signaling pathways. Interestingly, the results demonstrated that cancer cells exhibited an increased dependence on mitophagy, such as peroxisome, phagosome, lysosome, valine, leucine and isoleucine degradation and fatty acid degradation pathways, which might play an important role in EOC invasion and metastasis. Five proteins (GLDC, PCK2, IDH2, CPT2 and HMGCS2) located in the mitochondrion and enriched pathways were selected for further analysis in an EOC cell line and tissues, and the results confirmed reliability of iTRAQ proteomics. These findings provide a large-scale mitochondrial proteomic profiling with quantitative information, a certain number of potential protein biomarkers and a novel vision in the mitophagy bio-mechanism of a human ovarian carcinoma.

Downloadable materials

  • Supplementary Table 1. Proteins identified in epithelial ovarian cancer mitochondria relative to controls. (Mitochondrial protein were obtained in 7 EOC tissues and 11 control ovaries with benign gynecologic diseases and identified with iTRAQ-based quantitative proteomics. PSMs = Peptide sequence matches. MW = Molecular weight. Ratio (T/N) = Ratio of tumors to normal controls )
  • Supplementary Table 2: Level 3 RNA-seq V2 data obtained from the TCGA database of ovarian cancer patients (n=419).
  • Supplementary Table 3: Clinical data obtained from the TCGA data of ovarian cancer patients (Overall Survival data were extracted from TCGA database including 419 samples and event=0 means survival status).
  • Supplementary Table 4: 1108 overall survival-related genes were obtained by TCGA database (Overall Survival survival analysis of genes in ovarian cancer was calculated by the Kaplan-Meier method and compared with the log-rank test with R 3.4.2 virsion.)
  • Supplementary Table 5: 262 significant proteins obtained by overlapping analysis between 5115 identified proteins and TCGA database ( PSMs = Peptide sequence matches. MW = Molecular weight. Ratio (T/N) = Ratio of tumors to normal controls)
  • Supplementary Table 6: Protein localization according to biological process (BP) analysis.
  • Supplementary Table 7: The localization of overlapped proteins according to cellular component (CC).
  • Supplementary Table 8: A series of molecular functions (MFs) involved in 262 overlapped proteins.
  • Supplementary Table 9: 992 proteins of 5115 proteins were reported to relate with ovary compared to previous literatures(We searched CooLGeN with key words of ovary or ovarian, to identify studies of related genes on ovary. PSMs = Peptide sequence matches. MW = Molecular weight. Ratio (T/N) = Ratio of tumors to normal controls).
  • Supplementary Table 10: Protein–protein interactions analysised by STRING.
  • Supplementary Table 11: The mRNA-mRNA significant correlation between each other.
  • Supplementary Table 12: Chromosomal location of each significant correlation genes obtained by Ensembl. Ratio (T/N) = Ratio of tumors to normal controls
  • Supplementary Table 13: KEGG pathway analysis mapped the identified proteins to 70 signaling pathways.

 

      Society for Endocrinology

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    Network analysis of identified proteins by iTRAQ. (A) A total of 5115 proteins was classified according to the cell components with PANTHER. (B) KEGG pathway analysis mapped the identified proteins to 70 signaling pathways. (C). The overlapped proteins were obtained between 5115 identified proteins and TCGA overall related survival genes. (D, E and F) The overlapped proteins were classified according to the biological process (BP), cellular component (CC) and molecular function (MF). The less P value and more significant enrichment were shown with the greater node size. The same color indicated the same function group. Among the groups, we chose a representative of the most significant term and lag highlighted. A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0243.

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    The interaction networks of identified proteins. (A) The protein–protein interactions (PPIs) network of 262 overlapped proteins. (B) The mRNA-mRNA pair analysis of 262 overlapped proteins constructed circus chart by R package. Upregulated proteins by iTRAQ in red letters and downregulated proteins in green letters. Red line represents positive correlation and green line represents negative correlation. (C) Cancer cells exhibit an increased dependence on mitophagy, such as peroxisome, phagosome, valine, leucine and isoleucine degradation, fatty acid degradation pathway. D-F. Kaplan-Meier (KM) survival curve of ERBB2, PTBP1 and H2AFX in an epithelial ovarian carcinoma (EOC). A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0243.

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    Peroxisome pathway altered in an ovarian cancer. Green rectangle with red mark means the identified proteins. Green rectangle without red mark means species-specific enzymes. White rectangle means reference pathway. The solid line means molecular interaction. The dot line means indirect effect. The circle means mostly chemical complex. The pathway node in the right panel corresponds to the red marked node in the left diagram. ID number is the Swiss-Prot accession number. Ratio (T/N) = Ratio of tumors to controls. A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0243.

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    Phagosome pathway altered in an ovarian cancer. Green rectangle with red mark means the identified proteins. Green rectangle without red mark means species-specific enzymes. White rectangle means reference pathway. The solid line means molecular interaction. The dot line means indirect effect. The circle means mostly chemical complex. The pathway node in the right panel corresponds to the red marked node in the left diagram. ID number is the Swiss-Prot accession number. Ratio (T/N) = Ratio of tumors to controls. A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0243.

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    Lysosome pathway altered in an ovarian cancer. Green rectangle with red mark means the identified proteins. Green rectangle without red mark means species-specific enzymes. White rectangle means reference pathway. The solid line means molecular interaction. The dot line means indirect effect. The circle means mostly chemical complex. The pathway node in the right panel corresponds to the red marked node in the left diagram. ID number is the Swiss-Prot accession number. Ratio (T/N) = Ratio of tumors to controls. A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0243.

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    Fatty acid degradation pathway altered in an ovarian cancer. Green rectangle with red mark means the identified proteins. Green rectangle without red mark means species-specific enzymes. White rectangle means reference pathway. The solid line means molecular interaction. The dot line means indirect effect. The circle means mostly chemical complex. The pathway node in the upper panel corresponds to the red marked node in the lower diagram. ID number is the Swiss-Prot accession number. Ratio (T/N) = Ratio of tumors to controls. A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0243.

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    Valine, leucine and isoleucine degradation pathway altered in an ovarian cancer. Green rectangle with red mark means the identified proteins. Green rectangle without red mark means species-specific enzymes. White rectangle means reference pathway. The solid line means molecular interaction. The dot line means indirect effect. The circle means mostly chemical complex. The pathway node in the right panel corresponds to the red marked node in the left diagram. ID number is the Swiss-Prot accession number. Ratio (T/N) = Ratio of tumors to controls. A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0243.

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    qRT-PCR and Western blot analyses to validate results of iTRAQ quantitative mitochondrial proteomics. (A) qTR-PCR analysis to quantify the expression levels of GLDC, PCK2, IDH2, CPT2 and HMGCS2 between EOC cells TOV21G and control cells IOSE80. (B) Protein expression levels of GLDC, PCK2, IDH2, CPT2 and HMGCS2 in EOC cells TOV21G and control cells IOSE80. (C) Mitochondrial proteins of EOC and control tissues were analyzed by Western blot using antibodies against GLDC, PCK2, IDH2, CPT2 and HMGCS2. The levels of GLDC, PCK2, IDH2, CPT2 and HMGCS2 were normalized relative to β-actin. Data represent mean ± s.d. A full colour version of this figure is available at https://doi.org/10.1530/ERC-18-0243.

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