Analysis of defective protein ubiquitylation associated to adriamycin resistant cells

Abstract

DNA damage activated by Adriamycin (ADR) promotes ubiquitin-proteasome system-mediated proteolysis by stimulating both the activity of ubiquitylating enzymes and the proteasome. In ADR-resistant breast cancer MCF7 (MCF7) cells, protein ubiquitylation is significantly reduced compared to the parental MCF7 cells. Here, we used tandem ubiquitin-binding entities (TUBEs) to analyze the ubiquitylation pattern observed in MCF7 or MCF7 cells. While in MCF7, the level of total ubiquitylation increased up to six-fold in response to ADR, in MCF7 cells only a two-fold response was found. To further explore these differences, we looked for cellular factors presenting ubiquitylation defects in MCF7 cells. Among them, we found the tumor suppressor p53 and its ubiquitin ligase, Mdm2. We also observed a drastic decrease of proteins known to integrate the TUBE-associated ubiquitin proteome after ADR treatment of MCF7 cells, like histone H2AX, HMGB1 or β-tubulin. Only the proteasome inhibitor MG132, but not the autophagy inhibitor chloroquine partially recovers the levels of total protein ubiquitylation in MCF7 cells. p53 ubiquitylation is markedly increased in MCF7 cells after proteasome inhibition or a short treatment with the isopeptidase inhibitor PR619, suggesting an active role of these enzymes in the regulation of this tumor suppressor. Notably, MG132 alone increases apoptosis of MCF7 and multidrug resistant ovarian cancer A2780DR1 and A2780DR2 cells. Altogether, our results highlight the use of ubiquitylation defects to predict resistance to ADR and underline the potential of proteasome inhibitors to treat these chemoresistant cells.

Copy number variation is highly correlated with differential gene expression: a pan-cancer study

“…In contrast, DDGs were significantly related with ubiquitin mediated proteolysis, wnt signaling pathway (Fig. 5a), whose dysfunction tend to lead to tumorigenesis [29], metastasis [30], resistance [31], etc. Interestingly, both AUGs and DDGs are remarkably enriched in the ubiquitin-proteasome system, which has been increasingly reported to be highly related to cancer recently [32, 33].…”

“…In contrast, DDGs were significantly related with ubiquitin mediated proteolysis and wnt signaling pathway (Fig. 5b), whose dysfunction tend to lead to tumorigenesis [29], metastasis [49], resistance [31], etc. With respect to wnt signaling, lost function of DDGs such as inhibitory SMAD4 and APC would definitely enhance the function of wnt signaling leading to tumorigenesis [50–57], while attenuated function of ubiquitin mediated proteolysis facilitate proliferation [58].…”

Using Ubiquitin Binders to Decipher the Ubiquitin Code

“…TUBE2 which is based in the UBA domain of the UV excision repair protein HR23A has been used to demonstrate the role of Ub in multiple types of stress, including chemotherapy [74,55].…”

“…Pan-specific TUBEs have been used to show that Rac1 is ubiquitylated by the HECT E3 Hace1 [74,75], and to discriminate the ubiquitylation pattern of cells that are sensitive or resistant to doxorubicin chemotherapy, underlining the potential use of TUBEs in the search for biomarkers [55,74,75].…”

E3 Ubiquitin Ligase in Anticancer Drugdsla Resistance: Recent Advances and Future Potential

“…CUL2 knockdown enhanced cell sensitivity to doxorubicin treatment by regulating MAF1-mediated actin stress fiber integrity and apoptosis ( Wang et al, 2019 ). MDM2, an E3 targeting p53 for degradation, can influence PC and BC cell sensitivity to doxorubicin ( Lang et al, 2017 ; Cheteh et al, 2020 ). FKBP12 attenuated the cell toxicity of doxorubicin by binding to and degrading MDM2, disrupting the MDM2/MDM4 interaction, and inducing MDM2 self-ubiquitination ( Liu et al, 2017 ).…”