Differential expression of MAGEA6 toggles autophagy to promote pancreatic cancer progression
- Department of Molecular and Human Genetics, Baylor College of Medicine, United States
- Cell, Develop & Cancer Biology, Oregon Health & Science University, United States
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, United States
- Michael E DeBakey Department of Surgery, Baylor College of Medicine, United States
- Department of Systems Biology, The University of Texas MD Anderson Cancer Center, United States
Figures
Figure 1
Mutational landscape of MAGEA genes in pan-cancer study.
(A) Histogram of missense mutations and others (nonsense, frameshift, in-frame indels, and splice site mutations) across the entire MAGEA family. (B) Mutation frequency analysis of MAGEA genes. Pink …
Figure 2 with 3 supplements
Cancer-specific mutations of MAGEA genes reduce their protein expression.
(A) Immunoblot analysis of MAGEA3, A4, A6 and A10 variants expressed in HEK293T cells. Variants that are not recognized by the antibody (gray), variants expressed at levels 33% (three standard …
Figure 2—figure supplement 1
Immunoblot analysis of MAGEA12 variants expressed in HEK293T cells.
MAGEA12 and the non-specific band recognized by MAGEA12 antibody are indicated as ← and *, respectively.
Figure 2—figure supplement 2
Immunoblot (top) and densitometry plot (bottom) of the expression deviation analysis.
Standard deviation of all 15 samples = 0.1118.
Figure 2—figure supplement 3
Evolutionary conservation study of MAGEAs.
Wild-type amino acids of recurrent mutations are shown and colored as in Figure 2A.
Figure 3 with 2 supplements
MAGEA variants are degraded through the ubiquitin proteasome pathway.
(A) Correlation study of protein vs. mRNA expression of MAGEA variants. MAGEA variant protein and mRNA expression levels shown in Figure 3B and Figure 3—figure supplement 1, respectively, were …
Figure 3—figure supplement 1
qRT-PCR analysis of the MAGEA variants in Figure 3B.
qRT-PCR primer RT#3 was used, and MAGEA expression is normalized to that of parental HEK293T cells. *P value < 0.05; two-tailed unpaired t-test.
Figure 3—figure supplement 2
Densitometry analysis of protein expression of the MAGEA variants (+MG132/ -MG132) in Figure 3B.
Each dot represents a variant.
Figure 4 with 5 supplements
Cancer-specific mutations and glucose/glutamine depletion stimulate proteasome-dependent MAGEA6 degradation.
(A) Immunoblots (left) and qRT-PCR (right) analysis of HPDE-iKRAS cells expressing GFP (Vec) and MAGEA6 variants with or without MG132. WT: wild-type, GAPDH: glyceraldehyde 3-phosphate …
Figure 4—figure supplement 1
Immunoblot and qRT-PCR analysis of MAGEA6 in PDAC cell models in response to MG132.
Immunoblot (left) and qRT-PCR (right) analysis of MAGEA6 in BxPC-3 (A) and MIA PaCa-2 (B) in the presence or absence of MG132.
Figure 4—figure supplement 2
V5 pull-down assays to examine endogenous polyubiquitination signal on MAGEA6 variants with or without MG132 treatment.
Immunoblots of V5-tag pulled-down samples (left) and total lysate (right) are shown.
Figure 4—figure supplement 3
MAGEA6 protein expression is independent of cell confluency.
(A) Cell proliferation analysis of HPDE-iKRAS cells (mean ± standard deviation of replicates, N = 3). (B) Immunoblot study of the transduced HPDE-iKRAS cells with the estimated cell confluency …
Figure 4—figure supplement 4
MAGEA6 protein expression is regulated by nutrient levels in culture media.
Immunoblot analysis of MAGEA6 in transduced PDAC lines AsPC-1 (left), BxPC-3 (middle), and MIA PaCa-2 (right) in nutrient-depleted DMEM (-) with or without MG132. Fully nutrient-supplemented DMEM …
Figure 4—figure supplement 5
MAGEA6 protein expression is not regulated by AKT and mTORC1 kinases.
HPDE-iKRAS cells expressing wild-type MAGEA6 were treated with AKT and mTORC1 inhibitors (MK2206 and rapamycin, respectively) as indicated. Immunoblots of AKT and S6K phosphorylation status …
Figure 5 with 3 supplements
Overexpression of wild-type MAGEA6, but not mutant MAGEA6, suppresses autophagy in PDAC cell lines.
(A) Immunoblot analysis of autophagy signaling in HPDE-iKRAS cells expressing GFP (Vec) and MAGEA6 variants. (B) Immunofluorescence staining of LC3B puncta in the transduced HPDE-iKRAS cells. …
Figure 5—figure supplement 1
Immunoblot analysis of autophagy signaling of transduced AsPC-1 and MIA PaCa-2.
Figure 5—figure supplement 2
Immunoblot analysis of the accumulation of autophagy substrate SQSTM/p62 in the transduced HPDE-iKRAS cells under BafA1 for the indicated time points.
Figure 5—figure supplement 3
Immunoblot analysis of autophagy activity in transduced HPDE-iKRAS cells under nutrient-depleted conditions as indicated.
Figure 6 with 3 supplements
MAGEA6 mutations and expression variation manipulate autophagy to promote tumor progression at different stages.
(A) Tumor volume plot (50 d after injection) and Kaplan-Meier survival plot (B) of xenograft assay using HPDE-iKRAS cells expressing GFP (Vec) or MAGEA6 (five mice, two injections each, N = 10). P …
Figure 6—figure supplement 1
MAGEA6 expression analysis in PDAC cell line panel.
qRT-PCR of endogenous MAGEA6 expression (A) and immunoblot analysis of endogenous p53 (B) in HPDE and six other PDAC cell lines.
Figure 6—figure supplement 2
Validation of MAGEA6, ATG7 and VPS34 knockdown in BxPC3 cells.
(A) qRT-PCR analysis of MAGEA6 expression of the injected BxPC-3 cells in Figure 6C. P value was calculated by two-tailed unpaired t-test. (B) qRT-PCR analysis of MAGEA6, ATG7 and VPS34 expression …
Figure 6—figure supplement 3
Gene and protein expression analysis of MAGEA6 variants identified from ICGC lung cancer patients.
Immunoblot (A) and qRT-PCR analysis (B) of MAGEA6 variants identified from the ICGC lung cancer database. MAGEA6 variant expression levels are normalized to those of HEK293T cells transfected with …
Figure 7
MAGEA6 drives PDAC via manipulation of autophagy.
In the proposed model, MAGEA6 expression is activated via epigenetic regulation at an early disease stage to suppress autophagy and promote tumor initiation. MAGEA6 mutations and metabolic stress …
Additional files
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Supplementary file 1
Key Resources Table.
- https://cdn.elifesciences.org/articles/48963/elife-48963-supp1-v2.docx
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Supplementary file 2
List of clinical trials of cancer immunotherapy targeting MAGEA antigens.
Clinical trials of MAGEA-targeted immunotherapy reported from clinicaltrials.gov.
- https://cdn.elifesciences.org/articles/48963/elife-48963-supp2-v2.xlsx
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Supplementary file 3
MAGEA variants reported in skin, lung and pancreas cancer patients.
Recurrent mutations of MAGEA3, A4, A6, A10 and A12 genes identified from skin, lung and pancreas cancer patients in ICGC, TCGA and COSMIC databases.
- https://cdn.elifesciences.org/articles/48963/elife-48963-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/48963/elife-48963-transrepform-v2.docx
