Small ubiquitin-related modifier pathway is a major determinant of doxorubicin cytotoxicity in Saccharomyces cerevisiae.
Development of drug resistance is a major challenge in cancer chemotherapy using doxorubicin. By screening the collection of Saccharomyces cerevisiae deletion strains to identify doxorubicin-resistant mutants, we have discovered that the small ubiquitin-related modifier (SUMO) pathway is a major determinant of doxorubicin cytotoxicity in yeast. Mutants lacking UBA2 (SUMO activating enzyme; E1), UBC9 (conjugating enzyme; E2), and ULP1 and ULP2 (desumoylation peptidases) are all doxorubicin resistant, as are mutants lacking MLP1, UIP3, and NUP60, which all interact with ULP1. Most informatively, mutants lacking the SUMO E3 ligase Siz1 are strongly doxorubicin resistant, whereas mutants of other SUMO ligases are either weakly resistant (siz2) or hypersensitive (mms21) to doxorubicin. These results suggest that doxorubicin cytotoxicity is regulated by Siz1-dependent sumoylation of specific proteins. Eliminating SUMO attachment to proliferating cell nuclear antigen or topoisomerase II does not affect doxorubicin cytotoxicity, whereas reducing SUMO attachment to the bud neck-associated septin proteins has a modest effect. Consistent with these results, doxorubicin resistance in the siz1Delta strain does not seem to involve an effect on DNA repair. Instead, siz1Delta cells accumulate lower intracellular levels of doxorubicin than wild-type (WT) cells, suggesting that they are defective in doxorubicin retention. Although siz1Delta cells are cross-resistant to daunorubicin, they are hypersensitive to cisplatin and show near WT sensitivity to other drugs, suggesting that the siz1Delta mutation does not cause a general multidrug resistance phenotype. Cumulatively, these results reveal that SUMO modification of proteins mediates the doxorubicin cytotoxicity in yeast, at least partially, by modification of septins and of proteins that control the intracellular drug concentration.
Huang
Ruea-Yea
RY
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, and Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. raya.huang@roswellpark.org
Kowalski
David
D
Minderman
Hans
H
Gandhi
Nishant
N
Johnson
Erica S
ES
D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D004317:Doxorubicin / Q000493:pharmacokinetics / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D017353:Gene Deletion; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D004317:Doxorubicin; D044767:Ubiquitin-Protein Ligases; C436837:Siz1 protein, S cerevisiae