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Synthetic Genetic Array Screen Identifies PP2A As a Therapeutic Target in Mad2-Overexpressing Tumors

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Synthetic Genetic Array Screen Identifies PP2A As a Therapeutic Target in Mad2-Overexpressing Tumors Synthetic genetic array screen identifies PP2A as a therapeutic target in Mad2-overexpressing tumors Yang Biana, Risa Kitagawaa, Parmil K. Bansalb, Yo Fujiia, Alexander Stepanovb, and Katsumi Kitagawaa,c,1 aCenter for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Children’s Hospital, Columbus, OH 43205; bDepartment of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and cDepartment of Pediatrics, College of Medicine, The Ohio State University, Columbus, OH 43205 Edited by Douglas Koshland, University of California, Berkeley, CA, and approved December 13, 2013 (received for review August 21, 2013) The spindle checkpoint is essential to ensure proper chromosome subunit A, and it serves as a scaffolding molecule (11) to co- segregation and thereby maintain genomic stability. Mitotic arrest ordinate the assembly of the catalytic subunit and a variable deficiency 2 (Mad2), a critical component of the spindle checkpoint, is regulatory B subunit (12). overexpressed in many cancer cells. Thus, we hypothesized that On the basis of the hypothesis that a candidate gene’s inhibition Mad2 overexpression could specifically make cancer cells susceptible can induces lethality specifically in Mad2-overexpressing tumors, to death by inducing a synthetic dosage lethality defect. Because the we investigated the role of PPP2R1A in Mad2-overexpressing tu- spindle checkpoint pathway is highly conserved between yeast and mor cells. Inhibition of PP2A killed Mad2-overexpressing cells by humans, we performed a synthetic genetic array analysis in yeast, increasing Mad2 phosphorylation while suppressing Mad2 protein which revealed that Mad2 overexpression induced lethality in 13 levels. We propose that the inhibition of PP2A to target Mad2- gene deletions. Among the human homologs of candidate genes, overexpressing tumors can be a unique strategy for developing knockdown of PPP2R1A, a gene encoding a constant regulatory sub- potential anticancer therapies. unit of protein phosphatase 2, significantly inhibited the growth of Mad2-overexpressing tumor cells. PPP2R1A inhibition induced Mad2 Results phosphorylation and suppressed Mad2 protein levels. Depletion of Synthetic Genetic Array Screen Identifies Target Genes Whose PPP2R1A inhibited colony formation of Mad2-overexpressing HeLa Deletion Causes Synthetic Dosage Lethality in Mad2-Overexpressing cells but not of unphosphorylated Mad2 mutant-overexpressing cells, Yeast Cells. As Mad2 is overexpressed in many cancer cells (Table suggesting that the lethality induced by PP2A depletion in Mad2- S1) (8), this phenotype can be used to specifically kill Mad2- overexpressing cells is dependent on Mad2 phosphorylation. Also, overexpressing tumor cells. We hypothesized that a specific gene the PP2A inhibitor cantharidin induced Mad2 phosphorylation and whose inhibition causes synthetic dosage lethality (SDL) with inhibited the growth of Mad2-overexpressing cancer cells. Aurora B Mad2 overexpression can be a target of cancer therapy (13, 14). knockdown inhibited Mad2 phosphorylation in mitosis, resulting in Because the spindle checkpoint and cell cycle regulation are the blocking of PPP2R1A inhibition–induced cell death. Taken to- highly conserved between yeast and humans (15), we used syn- gether, our results strongly suggest that PP2A is a good therapeutic thetic genetic array (SGA) technology (16, 17) to screen the target in Mad2-overexpressing tumors. haploid deletion MATa library (4,541 strains) for candidate genes whose deletion kills Mad2-overexpressing yeast cells. Deletion cancer therapy target | anticancer drug | aneuploidy | yeast genetics mutant strains carrying pGAL-MAD2 were spotted onto dextrose or galactose plates. Because galactose induces the pGAL1 pro- he spindle checkpoint is a surveillance mechanism that ensures moter to overexpress MAD2, SDL interactions were determined Tfaithful chromosome segregation during mitosis by monitoring by comparing the dextrose and galactose plates (Dataset S1). the attachment of chromosomes to the spindle microtubules. Several key components of the spindle checkpoint have been Significance identified, such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, and Aurora B kinase. The mitotic arrest deficiency 2 (MAD2) gene was The spindle checkpoint is required for proper chromosome the first gene of the spindle checkpoint pathway to be characterized segregation. Mitotic arrest deficiency 2 (Mad2), a component of (1, 2). Mad2 is a key component of the spindle checkpoint, playing the spindle checkpoint, is overexpressed in many cancer cells. an important role in preventing the loss or gain of chromosomes This phenotype can be used to specifically kill Mad2-over- during cell division (3). Sotillo et al. reported that mice genetically expressing tumor cells. Because the spindle checkpoint path- engineered to overexpress Mad2 developed chromosome instability way is highly conserved between yeast and humans, we (CIN) and aneuploidy (4). High levels of Mad2 also resulted in the performed a screen to identify mutants in which Mad2 formation of aggressive tumors in multiple organs (5). Recent overexpression kills yeast cells. The screen revealed that studies show that overexpression of Mad2 is caused by loss of the Mad2 overexpression induced lethality in 13 gene deletions. tumor suppressor Rb or p53 (6, 7). Mad2 is overexpressed in many Among the human homologs of the 13 candidate genes, cancers (Table S1), such as malignant lymphoma, liver cancer, lung a gene encoding protein phosphatase 2 (PP2A) significantly cancer, soft tissue sarcoma, hepatocellular carcinoma, gastric can- inhibited the growth of Mad2-overexpressing tumor cells. cer, colorectal carcinoma, and human osteosarcoma (8). These results indicate that PP2A can be a specific therapeutic Protein phosphatase 2A (PP2A), an important and ubiquitously target in Mad2-overexpressing tumors. expressed serine/threonine phosphatase, dephosphorylates many key cellular molecules such as Akt, p53, and c-Myc. It plays an Author contributions: Y.B., Y.F., and K.K. designed research; Y.B., R.K., P.K.B., Y.F., and A.S. performed research; Y.B., R.K., P.K.B., Y.F., A.S., and K.K. analyzed data; and Y.B., important role in cellular processes such as cell proliferation, signal R.K., and K.K. wrote the paper. transduction, and apoptosis (9). PP2A holoenzymes negatively and The authors declare no conflict of interest. positively regulate cell cycle progression by dephosphorylating This article is a PNAS Direct Submission. pocket proteins and multiple cyclin-dependent kinase substrates 1To whom correspondence should be addressed. E-mail: Katsumi.Kitagawa@ (10). PP2A consists of a common heteromeric core enzyme, which is nationwidechildrens.org. composed of a catalytic subunit and a constant regulatory sub- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. unit. PPP2R1A encodes an α isoform of the constant regulatory 1073/pnas.1315588111/-/DCSupplemental. 1628–1633 | PNAS | January 28, 2014 | vol. 111 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1315588111 Downloaded by guest on September 28, 2021 Eighteen candidates showed lethality on galactose plates, and HeLa-Mad2O/E HeLa-GFP A B HeLa HeLa their specificity was confirmed by comparing them with candidate Mad2O/E GFP strains carrying the pGAL1 vector only (Fig. 1A). Of the 18 de- Mad2 O/E HeLa- letion mutant strains, a growth defect was induced by galactose in 5 HeLa strains (black) and by Mad2 overexpression in 13 strains (red). IB: Mad2 IB: Hsc70 siRNA Depletion of PPP2R1A Impairs Growth of Mad2-Overexpressing Cells. transfection The 13 genes that were identified by the SGA screen have putative human homologs (Fig. 1B). Assuming that the SDL between Culture for 3 days Mad2 overexpression and gene deletion might be conserved be- tween yeast and humans, we studied whether siRNA-mediated FACS knockdown of candidate genes shows lethality with Mad2 over- analysis Cell count expression in human cells. Mad2-overexpressing HeLa cell lines GFP were established (Fig. 2A), and GFP-expressing HeLa cells and C Mad2-overexpressing HeLa cells were mixed in a 1:1 ratio. Mixed 120 cells were transfected with siRNA targeting the candidate genes, 100 and the ratio of Mad2-overexpressing cells to HeLa-GFP cells was measured by flow cytometry after 72 h. The percentage of Mad2- 80 overexpressing cells was calculated over GFP-negative cells and normalized against that of control luciferase siRNA-treated cells 60 B C (Fig. 2 and ). Depletion of PPP2R1A significantly reduced the negative cells) (%) P 40 growth of Mad2-overexpressing cells (Fig. 2C). Relative Mad2 O/E cells (GF 20 Depletion of PPP2R1A Inhibits Colony Formation of Mad2-Overexpressing Cells. Potential cell growth inhibition of PPP2R1A on clonogenic 0 survival in Mad2-overexpressing HeLa cells was studied by the siRNA ALIX SNF7 RAB7WDR5VBP1 PTPN23 CENP-FTsg101RNF20MED31VPS25 Luciferase PPP2R1A SUPT4H1 DEX GAL Fig. 2. Synthetic lethality with Mad2 overexpression in HeLa cells. (A) Cell A lysates from HeLa or Mad2-overexpressing HeLa (HeLa-Mad2 O/E) cells were Deletion gene pGAL-MAD2 pGAL only pGAL-MAD2 immunoblotted with anti-Mad2 antibody or Hsc70 antibody (a loading WT control). (B) A schematic figure of the assay. GFP-expressing HeLa cells and SNF7 YPT7 Mad2-overexpressing HeLa cells were mixed in a 1:1 ratio. Mixed cells were SWD3 transfected with the candidate gene’s siRNA. After 72 h, the ratio of Mad2- STP22 RSC1 overexpressing cells to HeLa-GFP
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