Research ArticleEVOLUTIONARY BIOLOGY

MAGE cancer-testis antigens protect the mammalian germline under environmental stress

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Science Advances  29 May 2019:
Vol. 5, no. 5, eaav4832
DOI: 10.1126/sciadv.aav4832
  • Fig. 1 MAGEs are mammalian-specific genes that are restricted to expression in defined cell types of the testis.

    (A) Identification of testis-specific transcripts. (B) Many MAGE genes evolved in eutherian mammals. (C and D) Unsupervised hierarchical clustering of human (C) and mouse (D) MAGE genes based on reverse transcription quantitative polymerase chain reaction (RT-QPCR) expression data. Color key indicates relative log2 expression (0 to 12). (E) Unsupervised hierarchical clustering of MAGE genes during the first wave of spermatogenesis in the mouse testis (P5 to P56) as measured by RT-QPCR. Color key indicates relative expression (0 to 1). (F) Summary of differential expression of MAGE genes during spermatogenesis.

  • Fig. 2 Mage-a genes promote the maintenance of SSCs.

    (A and B) In situ hybridization (A) and immunohistochemistry (B) show that Mage-a genes are expressed in early stages of spermatogenesis. Staining was performed using human and mouse anti-MAGE-A antibodies that recognize multiple MAGE-A proteins, including mouse Mage-a1/Mage-a2/Mage-a3/Mage-a5/Mage-a6/Mage-a8. (C) Primary spermatogonial stem cell cultures were treated with dimethyl sulfoxide (DMSO) or RA for 24 or 48 hours before expression of Mage-a genes were detected by RT-QPCR (n = 3). Data are means ± SEM. (D to F) Mage-a genes are required to maintain ID4-EGFPbright stem cells in primary SSC cultures. Knockdown efficiency after a 24-hour transfection is shown (D). Log2 fold change of ID4-EGFPbright (E) and ID4-EGFPdim (F) cells after knockdown of indicated genes (n = 3 biological replicates on a single ID4-EGFP SSC cell line). Data are means ± SD. (G and H) Mage-a genes are required for robust stem cell repopulation of testis (n = 3 technical replicates). Note that siMAGE-A depletes multiple Mage-a genes. Data shown are mean ± SEM. P values determined by Student’s t test, *P < 0.05, **P < 0.01, ***P <0.001. n.s., not significant.

  • Fig. 3 Male germline of Mage-a KO mice is hypersensitive to genotoxic stress.

    (A) Schematic of allelic series deleting 2, 6, or all 8 mouse Mage-a genes through CRISPR-Cas9 technology. (B to D) Confirmation of Mage-a KO by genotyping (B), immunoblotting of testis lysates (C), and immunostaining of testis (D). WT, wild type. (E to G) Mage-a genes are not required for fertility under basal conditions; testis weights (n ≥ 6), pregnancy rate (n = 6), and litter size (n ≥ 16) are shown. (H to K) Mage-a genes are required to recover (8 weeks) from busulfan (20 mg/kg) genotoxic stress in mice. Testis weights (n = 4) (H), histology (I), and quantification (n > 200 tubules) (J) are shown. Fertility of mice from weeks 8 to 10 after busulfan treatment was determined (n = 4) (K). Data are means ± SEM. P values determined by Student’s t test, **P < 0.01 and ***P <0.001.

  • Fig. 4 Mage-a genes are required for robust spermatogenesis upon long-term starvation of mice.

    (A and B) Long-term starvation of mice (n = 5 to 6) resulted in a 20% body weight decrease. (C) Serum glucose was measured after starvation of ad libitum feeding of wild-type or Mage-a Δ6 mice (n = 3 to 9). (D to G) Starvation of Mage-a Δ6 mice decreases testis size (n = 7 to 13) (D), increases seminiferous tubule damage (>200 tubules analyzed) (E and F), and decreases sperm count (n = 7 to 12) (G). Data are means ± SEM. P values were determined by Student’s t test, *P < 0.05.

  • Fig. 5 MAGE-A genes promote resistance to a glycolysis inhibitor in human cancer and mouse spermatogonia stem cells.

    (A) Expression of human MAGE-A6 in MIA PaCa-2 cancer cells in complete media or treated with 2 mM 2-DG for 4 days. (B and C) Human MAGE-A6 expression promotes sustained growth of MIA PaCa-2 cancer cells treated with 2 mM 2-DG glycolysis inhibitor (B), but not without (C). (D) Mage-a Δ8 SSCs are more sensitive to 2-DG than littermate control wild-type SSCs. Cells were treated with indicated doses of 2-DG for 16 hours in the absence of feeder cells before the percentage of live cells was determined by annexin-V/4′,6-diamidino-2-phenylindole staining and flow cytometry analysis. (E and F) Metabolomic analysis of MAGE-A6 expressing MIA PaCa-2 cells grown in standard media or 2 mM 2-DG for 2 to 10 days. Nontargeted metabolomics was performed by the National Institutes of Health (NIH) West Coast Metabolomics Core (n = 6). Principal components analysis (E) and relative quantities of the indicated fatty acids (F) are shown. (G and H) The carnitine palmitoyltransferase inhibitor etomoxir (Eto) reverses MAGE-A6–induced growth in the presence of 2-DG after 7 days (G) but has no effect in cells grown in complete medium without 2-DG (H). (I) Pan-cancer gene expression analysis reveals that testis-specific MAGE genes are frequently expressed in tumors (n = 5532). Data are means ± SEM from n = 3 independent experiments. P values determined by Student’s t test, *P < 0.05, **P < 0.01, ***P <0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaav4832/DC1

    Table S1. Expression (TPM) of testis-specific genes using the GTex dataset.

    Table S2. Ortholog analysis of testis-specific genes using OrthoDB v9.1.

    Table S3. Primers and siRNAs for MAGE genes.

    Fig. S1. Human MAGE gene expression analysis.

    Fig. S2. Mouse anatomical MAGE gene expression map in an adult organism.

    Fig. S3. Tissue expression map of mouse MAGE genes during embryonic development and in embryonic stem cells.

    Fig. S4. MAGE gene expression in mouse testis.

    Fig. S5. Validation of SCC cultures.

    Fig. S6. Phenotyping of Mage-a KO mice.

    Fig. S7. p53 protein levels are increased in Mage-a KO mouse testis.

  • Supplementary Materials

    The PDF file includes:

    • Legend for table S1
    • Table S2. Ortholog analysis of testis-specific genes using OrthoDB v9.1.
    • Table S3. Primers and siRNAs for MAGE genes.
    • Fig. S1. Human MAGE gene expression analysis.
    • Fig. S2. Mouse anatomical MAGE gene expression map in an adult organism.
    • Fig. S3. Tissue expression map of mouse MAGE genes during embryonic development and in embryonic stem cells.
    • Fig. S4. MAGE gene expression in mouse testis.
    • Fig. S5. Validation of SCC cultures.
    • Fig. S6. Phenotyping of Mage-a KO mice.
    • Fig. S7. p53 protein levels are increased in Mage-a KO mouse testis.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Expression (TPM) of testis-specific genes using the GTex dataset.

    Files in this Data Supplement:

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