title: ANKHD1 represses p21 (WAF1/CIP1) promoter and promotes multiple myeloma cell growth.
abstract: ANKHD1 (Ankyrin repeat and KH domain-containing protein 1) is highly expressed and plays an important role in the proliferation and cell cycle progression of multiple myeloma (MM) cells. ANKHD1 downregulation modulates cell cycle gene expression and upregulates p21 irrespective of the TP53 mutational status of MM cell lines. The present study was aimed to investigate the role of ANKHD1 in MM in vitro clonogenicity and in vivo tumourigenicity, as well as the role of ANKHD1 in p21 transcriptional regulation. ANKHD1 silencing in MM cells resulted in significantly low no. of colonies formed and in slow migration as compared to control cells (p < 0.05). Furthermore, in xenograft MM mice models, tumour growth was visibly suppressed in mice injected with ANKHD1 silenced cells compared to the control group. There was a significant decrease in tumour volume (p = 0.006) as well as in weight (p = 0.02) in the group injected with silenced cells compared to those of the control group. Co-immunoprecipitation and chromatin immunoprecipitation (ChIP) assays confirmed the interaction between p21 and ANKHD1. Moreover, overexpression of ANKHD1 downregulated the activity of a p21 promoter in luciferase assays. Decrease in luciferase activity suggests a direct role of ANKHD1 in p21 transcriptional regulation. In addition confocal analysis after U266 cells were treated with Leptomycin B (LMB) for 24 h showed accumulation of ANKHD1 inside the nucleus as compared to untreated cells where ANKHD1 was found to be predominantly in cytoplasm. This suggests ANKHD1 might be shuttling between cytoplasm and nucleus. In conclusion, ANKHD1 promotes MM growth by repressing p21 a potent cell cycle regulator.
authors.affiliation: Hematology and Hemotherapy Center-University of Campinas/Hemocentro-Unicamp, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, Brazil. Electronic address: anamikadhyani2009@gmail.com.
authors.forename: Anamika
authors.initials: A
authors.lastname: Dhyani
authors.affiliation: Hematology and Hemotherapy Center-University of Campinas/Hemocentro-Unicamp, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, Brazil.
authors.forename: João A
authors.initials: JA
authors.lastname: Machado-Neto
authors.affiliation: Department of Biological Sciences, Federal University of Sao Paulo, Diadema, São Paulo, Brazil.
authors.forename: Patricia
authors.initials: P
authors.lastname: Favaro
authors.affiliation: Hematology and Hemotherapy Center-University of Campinas/Hemocentro-Unicamp, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, Brazil.
authors.forename: Sara T Olalla
authors.initials: ST
authors.lastname: Saad
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D018613:Microscopy, Confocal; D009101:Multiple Myeloma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D034622:RNA Interference; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D047368:Tumor Burden / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: C504171:ANKHD1 protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D005231:Fatty Acids, Unsaturated; D016601:RNA-Binding Proteins; C038753:leptomycin B
keywords: ANKHD1; Clonogenicity; Multiple myeloma; Transcriptional regulation; Tumourigenicity; p21 (WAF1/CIP1)