Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 14;12(10):945.
doi: 10.1038/s41419-021-04208-3.

Loss of TRIM31 promotes breast cancer progression through regulating K48- and K63-linked ubiquitination of p53

Yafei Guo #  1 , Qin Li #  1 , Gang Zhao #  1 , Jie Zhang  1 , Hang Yuan  1 , Tianyu Feng  1 , Deqiong Ou  1 , Rui Gu  1 , Siqi Li  1 , Kai Li  2   3 , Ping Lin  4   5
Affiliations

Loss of TRIM31 promotes breast cancer progression through regulating K48- and K63-linked ubiquitination of p53

Yafei Guo et al. Cell Death Dis. .

Abstract

Breast cancer is the most common cancer in the world. Relapse and metastasis are important factors endangering the life of breast cancer patients, but the mechanism is still unclear. The stabilization of p53 is essential for preventing carcinogenesis, and ubiquitination is one of the main ways to regulate the stability of p53. Tripartite motif-containing 31 (TRIM31) is a new member of the TRIM family and functions as an E3 ubiquitin ligase. It acts as a cancer promoter or suppressor in the malignant processes of multiple cancers. However, the function of TRIM31 in breast cancer progression remains unknown. In this study, we showed that TRIM31 is downregulated in breast cancer tissues and negatively correlated with breast cancer progression. Both gain- and loss-of-function assays indicated that TRIM31 inhibits the proliferation, colony formation, migration, and invasion of breast cancer cells. Further investigation demonstrated that TRIM31 directly interacts with p53, and inducing the K63-linked ubiquitination of p53 via its RING domain, Meanwhile, TRIM31 suppresses the MDM2-mediated K48-linked ubiquitination of p53 through competitive inhibiting the interaction of MDM2 and p53, leading to the p53 stabilization and activation. Knockdown of p53 reversed the inhibitory effects of TRIM31 on the growth and metastasis of breast cancer cells. Moreover, we found that the RING and coiled-coil (C-C) domains of TRIM31 were essential for its tumor suppressor function. Taken together, our findings reveal a novel mechanism by which TRIM31 suppresses breast cancer development through the stabilization and activation of p53 and define a promising therapeutic strategy for restoring TRIM31 to treat breast cancer.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. TRIM31 was reduced in breast cancer tissues.
a The real-time PCR assay the mRNA expression of TRIM31 in breast cancer tissues and normal tissues based on the data from the TCGA database. b The real-time PCR assay the mRNA expression of TRIM31 in breast cancer tissues and normal breast tissues, and the data were quantified by log10-transformed of 2−Δct values. c Immunohistochemistry (IHC) assays the expression of TRIM31 in 90 human breast cancer tissues and 46 normal tissues. Representative images were shown in the figure. Bar length: 100 μm. d The quantitative analysis of TRIM31 expression in breast cancer and normal breast tissues. e The TRIM31 protein expression in six paired breast cancer tissues was detected by western blot. f Statistical analysis of the TRIM31 expression in breast cancer tissues and their paired adjacent normal tissues. g, h The Kaplan–Meier plot of overall and relapse-free survival by the expression of TRIM31 in the breast cancer patients, the data was carried out from the Kaplan–Meier Plotter. Data are shown as the mean ± SD of at least three independent experiments, and the significant level was identified by *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 2
Fig. 2. TRIM31 suppressed the proliferation, migration and invasion of breast cancer cell lines.
a, b The real-time PCR (a) and western blot (b) assay of TRIM31 interfering in MCF7 and ZR-75-30 cells. c, d CCK8 (c) and colony formation (d) assay were performed to detect the proliferation of MCF7 and ZR-75-30 cells stably interfere with TRIM31 or control. e, f The migration (e) and invasion (f) assay of TRIM31-interference and control of MCF7 and ZR-75-30 cells, bar length: 100 μm. g, h The real-time PCR (g) and western blot (h) assay of TRIM31 overexpression in MCF7 and ZR-75-30 cells. i, j The CCK8 (i) and colony formation (j) assay of MCF7 and ZR-75-30 cells stably overexpress TRIM31 or control. k, l The migration (k) and invasion (l) assay of MCF7 and ZR-75-30 cells stably overexpress TRIM31 or control, Bar length: 100 μm. Data are shown as the mean ± SD of at least three independent experiments, and the significant level was identified by *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 3
Fig. 3. TRIM31 interacted with P53.
a The diagram showed the process for the quantitative proteomics and CO-IP-MS. b The Venn diagram showed the number of significant upregulated (black) and downregulated (blue) proteins identified in response to TRIM31 knockdown, and the protein expression fold change is MCF7-1/MCF7-NC > 1.5 or <0.67, and unique peptide ≧ 2 is defined as a significantly different protein. TRIM31 interaction candidates were identified in the Co-IP process (red). The below showed potential substrates of the TRIM31. c, d The HEK293 cells were co-transfected with Flag-TRIM31 and HA-P53 plasmids for 48 h, and then the cells were treated with MG132 (20 μM) for 4 h. The lysates of cells were immunoprecipitated with Flag-tag antibody and then western blot assay with HA-tag antibody (c); immunoprecipitated with HA-tag antibody and immunoblotted with Flag-tag antibody (d). e The MCF7 cell lysates were immunoprecipitated with Ig G and anti-p53 antibody and the expression of TRIM31 and p53 were detected by western blot. f GST-pull down was performed to verify the direct interaction of TRIM31 and P53 and the components were detected by the western blot assay with anti-GST and anti-His antibody. g The schematic diagram showed structural domains of TRIM31 and p53 protein. h HEK293 cells were transfected with HA-P53 and Falg-TRIM31 or several TRIM31 deletion mutants. The whole-cell lysates were immunoprecipitated with anti-Flag beads and immunoblotted with anti-Flag and anti-HA antibodies. i HEK293 cells were co-transfected with Falg-TRIM31 and HA-P53 or several P53 deletion mutants. The whole-cell lysates were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-Flag and anti-HA antibodies.
Fig. 4
Fig. 4. TRIM31 stabilized and activated p53 by inducing the K63 ubiquitination of p53 and inhibiting its K48 ubiquitination by disrupting the MDM2-P53 complex.
a The MCF7 and ZR-75-30 cells stably interfere with TRIM31 or control, the cell lysates were immunoblotted with anti-TRIM31, anti-p53, and anti-β-actin antibodies. b The qPCR was used to detect the mRNA expression of TRIM31 and P53 in MCF7 and ZR-75-30 cells. c The MCF7 and ZR-75-30 cells stably interfere with TRIM31 or control were treated with cyclohexamide (CHX) for 0, 1, 2 and 4 h, the expression of TRIM31 and P53 were detected by western blot. The line chart was further quantitatively analyzed TRIM31 expression. d The MCF7 cells were co-transfected with HA-TRIM31 and Falg-ub plasmids. The cell lysates were immunoprecipitated by anti-P53 antibody, and then western blot assay with anti-Falg, anti-P53, and anti-HA antibody. e The vitro ubiquitination assay was performed and then the western blot assay with the anti-p53 antibody. f The MCF7 cells were co-transfected with HA-TRIM31, Flag-Ub-K48, and Flag-Ub-K63 plasmids, then the cells lysates were pulled down with P53 antibody and analyzed by western blot. g The MCF7 cells were transfected with HA-TRIM31, Flag-Ub-k48R, and Flag-Ub-K63R plasmids. The cell lysates were immunoprecipitated with anti-P53 antibody and then western blot assay with Flag-tag antibody. h, i The MCF7 cells was transfected with HA-TRIM31 or HA-TRIM31-36A together with Flag-Ub-K63 (h) and Flag-Ub-K48 (i) plasmids. The cell lysates were immunoprecipitated with anti-P53 antibody and then western blot assay with anti-Flag antibody. j The MCF7 cells were co-transfected with HA-P53 and Flag-TRIM31 plasmids, the lysates of cells were immunoprecipitated with anti-HA beads and then western blot assay with anti-Flag, anti-HA, and anti-MDM2 antibody. k Pull-down assay was performed to detect the interaction of TRIM31 and p53 and the proteins were immunoblotted with anti-His, GST antibody. l, m The MCF7 and ZR-75-30 cells stably interfere with TRIM31 or control, the real-time PCR assay of the mRNA expression of TRIM31, P53, P21, and BAX in MCF7 and ZR-75-30 cells (l). Western blot assay of the expression of TRIM31, P53, P21, and BAX protein in MCF7 and ZR-75-30 cells (m). n, o The MCF7 and ZR-75-30 cells stably overexpress TRIM31 or control, the real-time PCR assay of the mRNA expression of TRIM31, P53, P21 and BAX in MCF7 and ZR-75-30 cells (n). Western blot assay of the expression of TRIM31, P53, P21 and BAX protein in MCF7 and ZR-75-30 cells (o). Data are shown as the mean ± SD of at least three independent experiments, and the significant level was identified by *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 5
Fig. 5. TRIM31 exerts the tumor-suppressive function via p53.
a The MCF7 cells stably interfere with TRIM31 were transfected with Flag-p53 plasmid, and the expression of TRIM31 and p53 were detected by western blot. b The cell survival of MCF7 at 72 h was detected by CCK8 assay. c The colony formation assay was performed to detect the cell proliferation of MCF7 cells. d The transwell migration assay was performed to detect the migration capability of MCF7 cells. e The ZR-75-30 cells stably overexpress TRIM31 were transfected with p53 special shRNA, and the TRIM31 and p53 expression were detected by western blot. f The CCK8 analyzed the cell survival of ZR-75-30 at 72 h. g The colony formation was performed to detect the cell proliferation of ZR-75-30 cells. h The transwell migration assay was performed to detect the migration capability of ZR-75-30, Bar length: 100 μm. Data are shown as the mean ± SD of at least three independent experiments, and the significant level was identified by *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 6
Fig. 6. The coiled-coil domain and the RING domain of TRIM31 were critical for the suppressor role of TRIM31.
The MCF7 and ZR-75-30 cells were transfected with Flag-TRIM31 plasmid or TRIM31 truncation mutant (TRIM31-ΔRING or TRIM31-ΔC–C) plasmids or Flag-TRIM31-C36A plasmid, and the expression of TRIM31 and its truncation mutants were detected by western blot (b–d). The proliferation (b), colony formation (c), and migration (d) of breast cancer cells were further detected and analyzed, Bar length: 100 μm. Data are shown as the mean ± SD of at least three independent experiments, and the significant level was identified by *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 7
Fig. 7. Overexpression of TRIM31 suppresses the tumorigenicity in nude mice models and correlation analysis of TRIM31 and p53 in breast cancer tissues.
a–d About 5 × 106 of ZR-75-30 cells stably overexpressing TRIM31 and control were injected into both backs of mice. The images showed the excised tumors from TRIM31-overexpression ZR-75-30 cells and control cells (a). The growth curve (b), tumor volume (c), and tumor weight (d) of the TRIM31-overexpression group compared with the control group were statistically analyzed at 30 days after injection. e About 5 × 106 of the ZR-75-30 cells stably overexpress TRIM31 or control were injected into nude mice by tail-vein injection. Representative pictures of HE staining of lung tissue (left), the arrows indicated the metastatic nodules on the surface of lung tissues. Bar length: 100 μm. The number of nodules on lung tissues was quantified at 45 days after tail–vein injection (right). f–i About 3 × 106 of ZR-75-30 cells stably express HA-TRIM31, sh-p53, and control (Mock+sh-NC, TRIM31 + sh-NC, Mock + sh-p53, TRIM31 + sh-p53) were injected into mammary fat pads of 6-week old female nude mice. The images showed the tumors from these four groups (f). The growth curve (g), tumor volume (h), and tumor weight (i) of these four groups were statistically analyzed at 40 days after injection. j Representative pictures of HE staining of lung tissues (left), the arrows indicated the metastatic nodules on the surface of lung tissues. Bar length: 100 μm. The number of nodules on the lungs was quantified at 40 days after injection (right). k The expression of TRIM31 and p53 in eight paired breast cancer patient tissues were analyzed by western blot (up). Pearson correlation analysis was used to measure the correlation of TRIM31 and p53 (down). l Representative images of IHC staining of TRIM31 and p53 in normal and breast cancer tissues (up). Bar length: 100 μm. Correlations analysis of the expression of TRIM31 and p53 protein (down). m The model of the antitumor function of TRIM31 in breast cancer progression. In normal cells, the expression of TRIM31 was upregulated. On the one hand, TRIM31 can directly bind to p53 and induce the K63 ubiquitination of p53 to stabilize and activate the p53 signal, on the other hand, TRIM31 can suppress the K48-linked ubiquitination and proteasome degradation of p53 by disrupting the binding of MDM2 and p53. In breast cancer cells, the expression of TRIM31 was downregulated, the p53 was mainly controlled by MDM2 that mediate the K48 polyubiquitination of p53 for degradation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Steeg PS. Targeting metastasis. Nat Rev Cancer. 2016;16:201–18. doi: 10.1038/nrc.2016.25. - DOI - PMC - PubMed
    1. Wellings E, Vassiliades L, Abdalla R. Breast cancer screening for high-risk patients of different ages and risk—which modality is most effective? Cureus. 2016;8:e945. - PMC - PubMed
    1. Kruse JP, Gu W. Modes of p53 regulation. Cell. 2009;137:609–22. doi: 10.1016/j.cell.2009.04.050. - DOI - PMC - PubMed
    1. Lane DP. Cancer. p53, guardian of the genome. Nature. 1992;358:15–6. doi: 10.1038/358015a0. - DOI - PubMed
    1. Vousden KH, Prives C. Blinded by the light: the growing complexity of p53. Cell. 2009;137:413–31. doi: 10.1016/j.cell.2009.04.037. - DOI - PubMed

Publication types

MeSH terms

Grants and funding