Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection.
BACKGROUND
Nitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time.


RESULTS
With this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle.


CONCLUSIONS
The dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs.
Bivi
Nicoletta
N
Department of Biomedical Sciences and Technologies, University of Udine, Piazzale Kolbe, 33100, Udine, Italy. nicoletta.bivi@uniud.it
Romanello
Milena
M
Harrison
Richard
R
Clarke
Ian
I
Hoyle
David C
DC
Moro
Luigi
L
Ortolani
Fulvia
F
Bonetti
Antonella
A
Quadrifoglio
Franco
F
Tell
Gianluca
G
Delneri
Daniela
D
D019386:Alendronate / Q000494:pharmacology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology / Q000648:ultrastructure; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004164:Diphosphonates / Q000494:pharmacology*; D012968:Etidronic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D017353:Gene Deletion; D006801:Humans; D000077557:Ibandronic Acid; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D034622:RNA Interference; D000068296:Risedronic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; C485678:DBF4 protein, human; C088096:Dbf4 protein, S cerevisiae; D004164:Diphosphonates; D011106:Polyisoprenyl Phosphates; D029701:Saccharomyces cerevisiae Proteins; D000068296:Risedronic Acid; D012968:Etidronic Acid; C002963:geranylgeranyl pyrophosphate; D000077557:Ibandronic Acid; D019386:Alendronate