title: Krüppel-like factors KLF2 and 6 and Ki-67 are direct targets of zoledronic acid in MCF-7 cells.
abstract: Bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases due to breast and prostate cancer. Recent clinical studies indicated a benefit in survival and tumor relapse with the supportive treatment of breast cancer using zoledronic acid (ZA), thus stimulating the debate about its putative anti-tumor activity in vivo. MCF-7 breast cancer cells were treated for 3 h (pulse treatment) and 72 h (permanent treatment) with ZA, and apoptosis rates and cell viability, defined as ATP content, were determined after 72 h. Permanent and pulse stimulation with ZA inhibited the viability of MCF-7 cells, which could partly be rescued by atorvastatin (Ator) pre-treatment but not by geranylgeranyl pyrophosphate (GGPP) co-treatment. Microarray analysis of ZA treated MCF-7 cells identified genes of the mevalonate pathway as significantly upregulated, which was verified by qPCR. Additionally the putative tumor suppressors krüppel-like factor 2 and 6 (KLF2 and KLF6) were markedly upregulated, while the classical proliferation marker Ki-67 was clearly downregulated. The expression of all three genes was confirmed by qPCR on mRNA level and by immunocytochemistry or Western blot staining. Expression of target genes were also analyzed in other breast (MDA-MB-231, BT-20, ZR75-1, T47D) and prostate (LNCaP, PC3) cancer cell lines by qPCR. ZA responsiveness of KLF2, KLF6 and Ki-67 could be verified in PC3 and T47D cells, KLF6 responsiveness in LNCaP and KLF2 responsiveness in MDA-MB-231 and BT-20 cells. Here we demonstrate in the apoptosis insensitive MCF-7 cell line a remarkable impact of ZA exposure on cell viability and on the regulation of putative tumor suppressors of the KLF family. The molecular mechanism involved might be the accumulation of isopentenyl pyrophosphate (IPP) and ApppI, since we could partly rescue the ZA effect by Ator pre-treatment and GGPP co-treatment. These data should stimulate further research into both the role of the mevalonate pathway and the accumulation of pyrophosphate compounds like ApppI in tumorigenesis and differentiation and their potential apart from the inhibition of mitochondrial ADP/ATP translocase and apoptosis, since such effects might well be responsible for the adjuvant ZA treatment benefit of patients suffering from breast cancer.
authors.affiliation: Orthopedic Center for Musculoskeletal Research, University of Würzburg, Brettreichstrasse 11, 97074 Würzburg, Germany. r-ebert.klh@uni-wuerzburg.de
authors.forename: Regina
authors.initials: R
authors.lastname: Ebert
authors.forename: Sabine
authors.initials: S
authors.lastname: Zeck
authors.forename: Jutta
authors.initials: J
authors.lastname: Meissner-Weigl
authors.forename: Barbara
authors.initials: B
authors.lastname: Klotz
authors.forename: Tilman D
authors.initials: TD
authors.lastname: Rachner
authors.forename: Peggy
authors.initials: P
authors.lastname: Benad
authors.forename: Ludger
authors.initials: L
authors.lastname: Klein-Hitpass
authors.forename: Maximilian
authors.initials: M
authors.lastname: Rudert
authors.forename: Lorenz C
authors.initials: LC
authors.lastname: Hofbauer
authors.forename: Franz
authors.initials: F
authors.lastname: Jakob
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001940:Breast / Q000187:drug effects / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004164:Diphosphonates / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D019394:Ki-67 Antigen / Q000378:metabolism*; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D000077211:Zoledronic Acid
chemical_list: D004164:Diphosphonates; D007093:Imidazoles; C096161:KLF2 protein, human; C105465:KLF6 protein, human; D019394:Ki-67 Antigen; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D011518:Proto-Oncogene Proteins; D000077211:Zoledronic Acid