55P - Inhibition of the ubiquitin-conjugating enzyme E2B restores the BCNU sensitivity of cancer cells by regulating MGMT ubiquitination

Date 11 September 2017
Event ESMO 2017 Congress
Session Poster display session
Topics Basic Science
Presenter Shih Han Hsu
Citation Annals of Oncology (2017) 28 (suppl_5): v1-v21. 10.1093/annonc/mdx361
Authors S.H. Hsu1, S. Chen2, L. Chen2, J. Chang1
  • 1Institute Of Basic Medical Sciences, College of Medicine, National Cheng Kung University, 701 - Tainan City/TW
  • 2National Institute Of Cancer Research, National Health Research Institutes, 704 - Tainan/TW

Abstract

Background

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes the mutagenic O6-alkyl groups from guanines. 1, 3-Bis (2-chloroethyl)-1-nitrosourea (BCNU), a DNA damage reagent, is known to induce cell death of tumors and the ubiquitin dependent proteolysis of MGMT. The present study aims to enhance BCNU cytotoxicity toward cancer cells by modulating MGMT dynamics.

Methods

Human nasopharygeal carcinoma cells, including HONE-1 and TW01, and human colon carcinoma HT-29 cells were used for the BCNU treatments, siRNA knockdown, immunoprecipitation and western blot experiments. The BCNU cytotoxicity was determined using methylene blue assay. Proteins involved in MGMT ubiquitination were confirmed with immunofloresence staining and in vitro protein ubiquitination assays.

Results

We previously identified ubiquitin-conjugating enzyme E2B (UBE2B), a DNA repair enzyme with ubiquitin-conjugating abilities, as a critical regulator of the cell cycle in oral cancer cells. A novel role of UBE2B was further revealed in regulating MGMT dynamics in nasopharygeal carcinoma cells and colon carcinoma cells. Increased colocalization of UBE2B with MGMT was found in BCNU treated cancer cells. Depletion of MGMT or UBE2B in cancer cells resulted in decreased IC50 for BCNU. Lower MGMT expression levels were observed in UBE2B deficient cells. Overexpression of MGMT rescued the UBE2B-depleted cells from the cytotoxic concentrations of BCNU, suggesting that MGMT is a downstream target of UBE2B. The E3 ubiquitin ligase RAD18, that is known as a partner of UBE2B in facilitating PCNA ubiquitination, was analyzed to investigate the mechanism of the UBE2B regulation on MGMT. Interaction of RAD18 and MGMT was observed in cancer cells, and was enhanced under the BCNU treatments. Our results also showed that UBE2B and RAD18 contribute to MGMT ubiquitination under in vitro conditions.

Conclusions

Our study indicated that the UBE2B-RAD18 regulation on MGMT plays an important role in BCNU-induced cancer cell death. Thus, UBE2B inhibition may be considered as a potential strategy for cancer treatment. (This work was supported by Taiwan Ministry of Science and Technology under the grants no. NSC 103-2320-B006-036-MY3).

Clinical trial identification

Legal entity responsible for the study

National Health Research Institutes, Tainan, Taiwan

Funding

Taiwan Ministry of Science and Technology

Disclosure

All authors have declared no conflicts of interest.