Published OnlineFirst July 9, 2014; DOI: 10.1158/1541-7786.MCR-14-0005 Molecular Cancer Chromatin, Gene, and RNA Regulation Research SNF5/INI1 Deficiency Redefines Chromatin Remodeling Complex Composition during Tumor Development Darmood Wei1, Dennis Goldfarb2, Shujie Song3,4, Courtney Cannon4,5, Feng Yan4, Donastas Sakellariou- Thompson4, Michael Emanuele4,5, Michael B. Major4,6, Bernard E. Weissman4,7, and Yasumichi Kuwahara4,8 Abstract Malignant rhabdoid tumors (MRT), a pediatric cancer that most frequently appears in the kidney and brain, generally lack SNF5 (SMARCB1/INI1), a subunit of the SWI/SNF chromatin-remodeling complex. Recent studies have established that multiple SWI/SNF complexes exist due to the presence or absence of different complex members. Therefore, the effect of SNF5 loss upon SWI/SNF complex formation was investigated in human MRT cells. MRT cells and primary human tumors exhibited reduced levels of many complex proteins. Furthermore, reexpression of SNF5 increased SWI/SNF complex protein levels without concomitant increases in mRNA. Proteomic analysis, using mass spectrometry, of MRT cells before and after SNF5 reexpression indicated the recruitment of different components into the complex along with the expulsion of others. IP–Western blotting confirmed these results and demonstrated similar changes in other MRT cell lines. Finally, reduced expression of SNF5 in normal human fibroblasts led to altered levels of these same complex members. These data establish that SNF5 loss during MRT development alters the repertoire of available SWI/SNF complexes, generally disrupting those associated with cellular differentiation. These findings support a model where SNF5 inactivation blocks the conversion of growth-promoting SWI/SNF complexes to differentiation-inducing ones. Therefore, restoration of these complexes in tumors cells provides an attractive approach for the treatment of MRTs. Implications: SNF5 loss dramatically alters SWI/SNF complex composition and prevents formation of complexes required for cellular differentiation. Mol Cancer Res; 12(11); 1574–85. Ó2014 AACR. Introduction within a year of diagnosis (1). MRTs are characterized by the Malignant rhabdoid tumors (MRT) are a highly aggressive loss of SNF5/BAF47/INI1/SMARCB1, a core member of pediatric cancer commonly found in the kidneys (RTK) and the SWI/SNF complex, hereafter referred to as SNF5 (2). brain (AT/RT). These tumors have a median onset of 11 Interestingly, SNF5 loss does not affect genetic stability in months and 80% to 90% of children die from the disease MRT, but causes epigenetic instability (3). Thus, this con- sistent and specific molecular change, SNF5 inactivation, provides a unique model for understanding the role of 1Curriculum in Toxicology, University of North Carolina, Chapel Hill, North epigenetics in human tumor development. Numerous stud- Carolina. 2Department of Computer Science, University of North Carolina ies have shown that SNF5 loss affects multiple signaling at Chapel Hill, North Carolina. 3Oncology Center, ZhuJiang Hospital, Southern Medical University, Guangzhou, Guangdong, China. 4Lineberger pathways (4). However, little else is known about the etiology Comprehensive Cancer Center, University of North Carolina, Chapel Hill, of this cancer. North Carolina. 5Department of Pharmacology, University of North Car- The SWI/SNF complex has multiple variants and is olina, Chapel Hill, North Carolina. 6Department of Cell Biology and Phys- fi iology, University of North Carolina, Chapel Hill, North Carolina. 7Depart- classi ed by their mutually exclusive ATPase subunits, BRM ment of Pathology and Laboratory Medicine, University of North Carolina, and BRG1. BRG1-containing SWI/SNF complexes can be Chapel Hill, North Carolina. 8Department of Pediatrics, Kyoto Prefectural — University of Medicine, Kyoto, Japan. subdivided into two mutually exclusive groups BAF180/ PBRM1- and BAF250A/ARID1A-containing complexes. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Although SNF5 appears in all the SWI/SNF complexes, little is known about the effects of SNF5 loss upon SWI/SNF Corresponding Authors: Bernard E. Weissman, Lineberger Comprehen- sive Cancer Center, Room 32-048, University of North Carolina, 450 West activity. One previous study by Doan and colleagues (5) Drive, Chapel Hill, NC 27599-7295. Phone: 919-966-7533; Fax: 919-966- showed that the SWI/SNF complex appeared intact in MRT 3015; E-mail: [email protected]; and Yasumichi Kuwahara, Depart- cell lines despite the loss of SNF5. They also demonstrated ment of Pediatrics, Kyoto Prefectural University of Medicine, 465 Kajii-cyo, Kawaramachi-douri Hirokoji-agaru, Kamigyo-ku, Kyoto-city, Kyoto 602- that BRG1-dependent genes did not show altered expression 8856, Japan. Phone: 81-75-251-5571; Fax: 81-75-252-1399; E-mail: in MRT cell lines (5). However, they did not address the [email protected] stoichiometry of the complex components in MRT cell lines doi: 10.1158/1541-7786.MCR-14-0005 or the interaction of the complex with chromatin. Other Ó2014 American Association for Cancer Research. studies have indicated that loss of a SWI/SNF complex 1574 Mol Cancer Res; 12(11) November 2014 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst July 9, 2014; DOI: 10.1158/1541-7786.MCR-14-0005 SNF5/INI1 Regulates SWI/SNF Complex Composition in MRTs member could affect the stability of other complex members Protein extracts and Western blotting in mammalian cells and in flies (6–12). Furthermore, a Isolation of nuclear protein and Western blotting were growing number of studies have identified multiple SWI/ carried out as described previously (16, 23). Western blot SNF complexes defined by the presence or absence of analyses for protein expression were carried out using the different components (4, 13–15). Importantly, these differ- antibodies listed in Supplementary Table S1. Densitometry ent complexes can promote either growth or differentiation was carried out using the Bio-Rad Image Lab 4.1 software. depending upon their composition (4, 13–15). To address whether SNF5 loss affects SWI/SNF complex RNA extraction and quantitative real-time reverse composition during MRT development, we examined the transcription PCR analysis effects of SNF5 expression in MRT cell lines. Our studies RNA was extracted using the RNeasy Mini Kit (Qiagen), demonstrate that reexpression of SNF5 in MRT cell lines and 1 mg was used for cDNA synthesis primed with random altered total protein levels of many components, whereas primers (Invitrogen). cDNA was analyzed using TaqMan knockdown of SNF5 in normal human fibroblasts showed (Applied Biosystems) quantitative real-time reverse tran- the opposite effect without altering mRNA expression. After scription PCR (qPCR) analysis, with b-actin as the reference SNF5 reexpression, many components were recruited into gene in each reaction. Reactions were performed on an ABI SWI/SNF complexes that are generally associated with 7900 HT sequence detection system (Applied Biosystems), ÀDDCt induction of differentiation. Therefore, SNF5 loss may and relative quantification was determined using the 2 promote MRT development by preventing a switch from method (Chai and colleagues, ref. 16). The TaqMan gene SWI/SNF complexes that promote proliferation to those expression assay primer/probe sets used in these studies are associated with differentiation. These studies emphasize the listed in Supplementary Table S2. need to resolve the scope and composition of SWI/SNF complexes among different tissues and may account for the Immunoprecipitation low incidence of these deadly childhood cancers. Cells were infected with adenovirus expressing either HA- tagged SNF5 (Ad-SNF5-HA) or empty vector (Ad-CMV). Materials and Methods Proteins were isolated from the infected cells using IP buffer Cell lines [50 mmol/L Tris, 400 mmol/L NaCl, 2 mmol/L EDTA, Our laboratory previously described the A204.1, D98OR, 10% Glycerol, 1% NP-40, 0.5% sodium deoxycholate, 0.1 and G401.6 cell lines (16–18). The MCF-7, A673, Jurkat, mmol/L PMSF, 4 mmol/L sodium fluoride, 40 nmol/L DAOY, and RD cell lines were obtained from the American sodium orthovanadate, and 1Â complete Mini protease Type Tissue Collection (ATCC). The TTC642 and inhibitor cocktail (Roche Diagnostics)]. The isolated protein TTC549 cell lines were kindly provided from Dr. Timothy was quantified and adjusted to 1 mg/mL. One milligram of Triche, Children's Hospital of Los Angeles. Dr. Peter protein from each sample was incubated with BRG-1 (A303- Houghton, Nationwide Childrens Hospital, kindly provid- 877A; Bethyl), or normal rabbit IgG (sc-2027; Santa Cruz ed the BT-12 AT/RT cell line. 293FT cells were kindly Biotechnology) rotating overnight at 4 C with 30 mLofa provided by Dr. Inder Verma, Salk Institute. All cell lines 50% slurry of protein A/G Sepharose beads. The beads were were cultured in RPMI-1640 plus 10% fetal bovine serum then washed three times with IP wash buffer (1Â PBS, 10% (Gibco; or Benchmark FBS, Gemini Biosciences) and were glycerol, and 1% Triton) and then suspended in 1Â used within 30 passages of their initial arrival to minimize the NuPAGE LDS Loading Buffer supplemented with 125 chances of cross-contamination. mmol/L DTT and boiled for 5 minutes. The supernatants were then run on 4% to 12% Bis-Tris polyacrylamide gel, Adenovirus infection transferred to polyvinylidene