Analysis of defective protein ubiquitylation associated to adriamycin resistant cells

Cell Cycle. 2017;16(24):2337-2344. doi: 10.1080/15384101.2017.1387694. Epub 2017 Nov 20.


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 (MCF7ADR) 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 MCF7ADR cells. While in MCF7, the level of total ubiquitylation increased up to six-fold in response to ADR, in MCF7ADR cells only a two-fold response was found. To further explore these differences, we looked for cellular factors presenting ubiquitylation defects in MCF7ADR 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 MCF7ADR cells. p53 ubiquitylation is markedly increased in MCF7ADR 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 MCF7ADR 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.

Keywords: Adriamycin; TUBEs; Ubiquitylation; breast cancer; chemoresistance; degradation; ovarian cancer; p53; proteasome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Cell Line, Tumor
  • Doxorubicin / pharmacology*
  • Drug Resistance, Neoplasm / drug effects*
  • Female
  • Humans
  • Leupeptins / pharmacology
  • MCF-7 Cells
  • Proteasome Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-mdm2 / metabolism
  • Tumor Suppressor Protein p53 / metabolism
  • Ubiquitination / drug effects


  • Leupeptins
  • Proteasome Inhibitors
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • Doxorubicin
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde

Grants and funding

This work was supported by the Spanish Ministry of Economy and Competiveness (MINECO, CTQ2011- 27874 grant), the Government of the Basque Country, (Etortek grants 2013, 2014), Diputación Foral de Gipuzkoa (VL, FA) Saiotek program (FA, MSR) and grant 2016/22/E/ NZ5/00381 from the Polish National Science Centre (RJ).