Hypoxia-induced proteasomal degradation of DBC1 by SIAH2 in breast cancer progression
- State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Center for Cell Responses, Tianjin Key Laboratory of Protein Science, College of Life Sciences, Haihe Laboratory of Cell Ecosystem, Nankai University, China
- School of Statistics and Data Science, LPMC and KLMDASR, Nankai University, China
Figures
Figure 1 with 1 supplement
Hypoxia induces DBC1 degradation.
(A) MDA-MB-231 cells were cultured under normoxia or hypoxia for 24 hr and then the total RNA was extracted from cells using a total RNA extraction kit. RNA-sequence analysis of gene expression and …
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Figure 1—source data 1
Hypoxia induces DBC1 degradation.
- https://cdn.elifesciences.org/articles/81247/elife-81247-fig1-data1-v2.zip
Figure 1—figure supplement 1
The stability of DBC1 was regulated under hypoxic conditions.
(A) MDA-MB-231 cells were exposed to normoxia or hypoxia for 24 hr, with or without HIF1 Inhibitor (10 μM) for 12 hr, and then DBC1 and indicated proteins were detected by Western blotting. (B, C) …
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Figure 1—figure supplement 1—source data 1
Hypoxia induces DBC1 degradation.
- https://cdn.elifesciences.org/articles/81247/elife-81247-fig1-figsupp1-data1-v2.zip
Figure 2 with 1 supplement
SIAH2 interacts with DBC1 and regulates its stability.
(A) HeLa cells were transfected with Myc-DBC1 and several hypoxia-relative E3 ligases and cultured under normoxic conditions for 24 hr. The protein level of DBC1 was detected by Western blotting. (B)…
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Figure 2—source data 1
SIAH2 interacts with DBC1.
- https://cdn.elifesciences.org/articles/81247/elife-81247-fig2-data1-v2.zip
Figure 2—figure supplement 1
The stability of DBC1 was regulated by SIAH2.
(A) HeLa cells were transfected with several hypoxia-relative E3 ligases and cultured for 24 hr. The proteins levels of E3 ligases were detected by Western blotting. (B, C) MCF10A and MCF7 cells …
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Figure 2—figure supplement 1—source data 1
SIAH2 interacts with DBC1.
- https://cdn.elifesciences.org/articles/81247/elife-81247-fig2-figsupp1-data1-v2.zip
Figure 3 with 1 supplement
SIAH2 promotes DBC1 polyubiquitination and degradation under hypoxia.
(A) HEK293T cells were transfected with Myc-DBC1, HA-Ub, and Flag-SIAH2 or Flag-SIAH2RM for 24 hr and then treated with MG132 (10 μM) for 6 hr. Ubiquitylation assays were performed and the …
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Figure 3—source data 1
SIAH2 promotes DBC1 polyubiquitination and degradation.
- https://cdn.elifesciences.org/articles/81247/elife-81247-fig3-data1-v2.zip
Figure 3—figure supplement 1
Hypoxia-induced DBC1 degradation is dependent on E3 Ligase SIAH2.
(A, B) HEK293T cells were transfected with HA-tagged wildtype ubiquitin or the different lysine-to-arginine mutants together with Myc-DBC1 and Flag-SIAH2 or the empty Flag-vector for 24 hr and then …
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Figure 3—figure supplement 1—source data 1
SIAH2 regulate DBC1 polyubiquitination and its degradation.
- https://cdn.elifesciences.org/articles/81247/elife-81247-fig3-figsupp1-data1-v2.zip
Figure 4 with 1 supplement
OTUD5 interacts with DBC1 and regulates its stability.
(A) MDA-MB-231 cells were cultured under normoxia or hypoxia for indicated time, then one group was recovered to normoxia for 6 hr, and then treated with MG132 (10 μM) for 6 hr. Cells were …
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Figure 4—source data 1
OTUD5 interacts with DBC1.
- https://cdn.elifesciences.org/articles/81247/elife-81247-fig4-data1-v2.zip
Figure 4—figure supplement 1
The stability of DBC1 was regulated by OTUD5.
(A) HEK293T cells were co-transfected with Myc-DBC1 and a series of Flag-Dubs, and immunoprecipitation was performed with an anti-Flag bead. Co-immunoprecipitated DBC1 was detected by Western …
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Figure 4—figure supplement 1—source data 1
OTUD5 regualte the deubiquitination of DBC1.
- https://cdn.elifesciences.org/articles/81247/elife-81247-fig4-figsupp1-data1-v2.zip
Figure 5 with 1 supplement
Hypoxia regulates tumor progression via the SIAH2-DBC1 axis.
(A) SIAH2 siRNA was transfected into indicated cell lines. The cells were cultured under normoxia or hypoxia for 24 hr and then cell lysates were analyzed by immunoblotting using the indicated …
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Figure 5—source data 1
Source data of Hypoxia regulates tumor progression via the SIAH2-DBC1 axis.
- https://cdn.elifesciences.org/articles/81247/elife-81247-fig5-data1-v2.zip
Figure 5—figure supplement 1
Hypoxia regulates DBC1 stability to affect tumor progression.
(A) Apoptosis assay analysis of wildtype, SIAH2 knockout, and SIAH2/CCAR2 double-knockout MDA-MB-231 cells under normoxia or hypoxia. (B) Colony formation analysis of wildtype, SIAH2 knockout, and SI…
Figure 6 with 1 supplement
Correlation of SIAH2 and DBC1 expression with tumor progression in BRCA.
(A) SIAH2 expression level in different human cancers from TCGA data in TIMER. *p<0.05, **p<0.01, ***p<0.001. (B) Volcano plot analysis of SIAH2 DBC1 and OTUD5 expression levels in breast cancer. (C)…
Figure 6—figure supplement 1
The expression levels of SIAH2 and DBC1 were negative correlation in BRCA.
(A) Expression levels of SIAH2 in breast cancer tissues were classified into three grades (negative, +, ++, or +++) according to the percentage of immunopositive cells and immunostaining intensity. …
Author response image 1
HEK293T cells were transfected with Myc-DBC1 (K287R) mutant and Flag-OTUD5 for 24 h.Cells were collected for immunoprecipitation with anti-Flag antibody.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/81247/elife-81247-mdarchecklist1-v2.docx
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Supplementary file 1
Identification of DBC1 as a SIAH2-associated protein.
List of representative proteins identified by SIAH2 Co-IP/MS and the number of peptides for each protein-identified peptide are indicated.
- https://cdn.elifesciences.org/articles/81247/elife-81247-supp1-v2.docx
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Supplementary file 2
Patient characteristics based on DBC1 expression.
The relationship between the clinicopathological characteristics of breast cancer patients and the expression level of DBC1.
- https://cdn.elifesciences.org/articles/81247/elife-81247-supp2-v2.docx
