Inactivation of the PBRM1 Tumor Suppressor Gene Amplifies
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Inactivation of the PBRM1 tumor suppressor gene − − amplifies the HIF-response in VHL / clear cell renal carcinoma Wenhua Gaoa, Wei Lib,c, Tengfei Xiaoa,b,c, Xiaole Shirley Liub,c, and William G. Kaelin Jr.a,d,1 aDepartment of Medical Oncology, Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; bCenter for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA 02215; cDepartment of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard T.H. Chan School of Public Health, Boston, MA 02115; and dHoward Hughes Medical Institute, Chevy Chase, MD 20815 Contributed by William G. Kaelin, Jr., December 1, 2016 (sent for review October 31, 2016; reviewed by Charles W. M. Roberts and Ali Shilatifard) Most clear cell renal carcinomas (ccRCCs) are initiated by somatic monolayer culture and in soft agar (10). These effects were not, inactivation of the VHL tumor suppressor gene. The VHL gene prod- however, proven to be on-target, and were not interrogated in uct, pVHL, is the substrate recognition unit of an ubiquitin ligase vivo. As a step toward understanding the role of BAF180 in that targets the HIF transcription factor for proteasomal degrada- ccRCC, we asked whether BAF180 participates in the canonical tion; inappropriate expression of HIF target genes drives renal car- PBAF complex in ccRCC cell lines and whether loss of BAF180 cinogenesis. Loss of pVHL is not sufficient, however, to cause ccRCC. measurably alters ccRCC behavior in cell culture and in mice. Additional cooperating genetic events, including intragenic muta- tions and copy number alterations, are required. Common examples Results and Discussion of the former are loss-of-function mutations of the PBRM1 and BAP1 We examined the protein levels of BAF180, BAP1, SETD2, and tumor suppressor genes, which occur in a mutually exclusive man- various SWI/SNF components in 16 ccRCC cell lines, together with PBRM1 ner in ccRCC and define biologically distinct subsets of ccRCC. HK-2 immortalized renal epithelial cells, using immunoblot assays. encodes the Polybromo- and BRG1-associated factors-containing All the 16 ccRCCs are pVHL-defective except for SLR20 and SLR21 complex (PBAF) chromatin remodeling complex component BRG1- (21).NPMwasusedasaloadingcontrol. BAF180 was undetectable associated factor 180 (BAF180). Here we identified ccRCC lines in five cell lines (A704, RCC4, SKRC20, SLR24, and SLR25) and whose ability to proliferate in vitro and in vivo is sensitive to barely detectable in another (Caki-2; Fig. 1A and SI Appendix,Fig. wild-type BAF180, but not a tumor-associated BAF180 mutant. Bio- PBRM1 chemical and functional studies linked growth suppression by S1). We also sequenced the cDNAs from these 16 ccRCC BAF180 to its ability to form a canonical PBAF complex containing lines and identified frame-shift, presumably loss-of-function, muta- B BRG1 that dampens the HIF transcriptional signature. tions in the six cell lines with low or undetectable BAF180 (Fig. 1 and C). In addition, we identified a small, in-frame, deletion in the PBRM1 B C kidney cancer | chromatin | PBAF | BAF180 | hypoxia cDNA in SLR26 cells (Fig. 1 and ). The A704 and Caki-2 PBRM1 mutations have been reported previously (10, 22). iallelic inactivation of the VHL tumor suppressor gene is the Four lines had diminished or undetectable levels of the ccRCC Busual initiating or truncal event in clear cell renal cell carci- suppressor protein BAP1 (SLR23, SLR26, UMRC2, and UMRC6), noma (ccRCC), which is the most common form of kidney cancer three had diminished or undetectable levels of the ccRCC (1–6). VHL loss, however, is not sufficient to cause ccRCC (4, 7–9). Other cooperating genetic events in ccRCC include copy Significance number gain of chromosome 5q, copy number loss of chromosome 14q, and intragenic mutations affecting chromatin regulatory genes Mutational inactivation of the VHL tumor suppressor gene is such as PBRM1, BAP1, ARID1A, SETD2, KDM5C,andKDM6A; the signature lesion in the most common form of kidney cancer PI3K pathway genes such as PTEN, PIK3CA, TSC1,andTORC1; and causes inappropriate accumulation of the HIF transcription and redox stress genes such as KEAP1 and NFE2L2 (1, 10–14). factor, which activates genes that normally facilitate adapta- PBRM1 is the gene that, after VHL, is most frequently mu- tion to hypoxia but, in the context of kidney cancer, also pro- tated in ccRCC. Interestingly, PBRM1 and VHL reside at chro- mote tumorigenesis. Additional mutational events are needed, mosomes 3p21 and 3p25, respectively. Accordingly, three genetic in conjunction with VHL loss, to cause kidney cancer. The most hits (intragenic cis mutations affecting PBRM1 and VHL, fol- common of these are inactivating mutations of the PBRM1 tumor lowed by loss of chromosome 3p) can cause biallelic loss of both suppressor gene, which encodes a component [BRG1-associated CELL BIOLOGY PBRM1 and VHL. A similar situation exists for BAP1 and factor 180 (BAF180)] of a multiprotein complex [Polybromo- and SETD2, which are also located on chromosome 3p21. PBRM1 BRG1-associated factors-containing complex (PBAF)] that regu- and BAP1 mutations are largely mutually exclusive in ccRCC and lates the positions of nucleosomes throughout the genome. We define biologically distinct ccRCC subtypes (12). describe here kidney cancer cell-based models for monitoring The PBRM1 gene product, BRG1-associated factor 180 (BAF180), BAF180 function and show that loss of BAF180 accentuates the is part of the multisubunit Polybromo- and BRG1-associated factors- transcriptional response to HIF. containing complex (PBAF) switch/sucrose nonfermentable (SWI/ SNF) chromatin remodeling complex (15–18). Mutations affecting Author contributions: W.G. and W.G.K. designed research; W.G. and T.X. performed re- search; T.X. performed MNase-Seq; W.G., W.L., X.S.L., and W.G.K. analyzed data; and W.G. SWI/SNF components have been linked to multiple forms of cancers and W.G.K. wrote the paper. – (15 18). For reasons that are not clear, however, there is a strong bias Reviewers: C.W.M.R., St. Jude Children’s Research Hospital; and A.S., Stowers Institute for to mutate specific SWI/SNF components in specific types of cancer. Medical Research. PBRM1 In this regard, inactivating mutations are most common in Conflict of interest statement: W.G.K. receives consulting income and equity from Peloton ccRCC, followed by cholangiocarcinoma (19, 20), but are otherwise Therapeutics, which is developing HIF2 inhibitors for treatment of kidney cancer. relatively uncommon in cancer. 1To whom correspondence should be addressed. Email: [email protected]. PBRM1 siRNA-mediated knockdown of wild-type was repor- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ted to increase the proliferation of multiple ccRCC cell lines in 1073/pnas.1619726114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1619726114 PNAS | January 31, 2017 | vol. 114 | no. 5 | 1027–1032 Downloaded by guest on September 29, 2021 could be immunoprecipitated with either an anti-FLAG antibody A or an anti-HA antibody from these cells (786-O-BKI), but not A498 HK-2 A704 Caki-2 RCC4 SKRC20SLR24SLR25769-P786-O SLR20SLR21SLR23SLR26UMRC2UMRC6UOK101 from parental 786-O cells, although recovery of the tagged BAF180 BAF180 was reproducibly higher with the anti-FLAG antibody than with the anti-HA antibody (Fig. 2B). In parallel, we infected BAP1 A704 cells, which lack detectable endogenous BAF180 (Fig. 1A), SETD2 with a lentivirus encoding FLAG-HA-tagged wild-type BAF180, a BAF250a tumor-associated BAF180 mutant with a frame-shift mutation (Q1298*) (10), or the empty vector (EV) (Fig. 2C). BAF200 We next performed preparative anti-FLAG immunoprecipi- BAF155 tations with these cell lines under stringent wash conditions. BAF47 The bound proteins were then eluted with a FLAG-peptide, BRG1 immunoprecipitated with an anti-HA antibody, eluted with an HA peptide, and either resolved by SDS/PAGE and detected BRM by silver staining (Fig. 2D)oridentifiedbymassspectrometry NPM (Fig. 2F and SI Appendix,TableS1). As expected, we observed silver-stained protein bands with predicted molecular weights B consistent with epitope-tagged BAF180 in immunoprecipitates Cell Protein Annotation cDNA Annotation prepared from the 786-O-BKI and A704-BAF180 cells and a A704 p.E572fs*16 c.1713-1714insTT slightly faster migrating band in the immunoprecipitates from Caki-2 p.E860fs*4 c. 2582-2585delTATA A704-BAF180 (Q1298*) cells (Fig. 2D). No such bands were RCC4 p.H897fs*3 c.2685-2686insA observed with the control samples prepared from parental 786-O SKRC20 p.F1012fs*1 c.3034delT cells and A704-EV cells. SLR24 p.H897fs*3 c.2685-2686insA Mass spectrometry analysis confirmed the recovery of BAF180 SLR25 p.Q477fs*17 c.1431_1443delGCAAGTTATGCAG itself, as well as other members of the canonical PBAF complex, SLR26 p.M1209_E1214delIMFYKKE c.3625_3642delATGTTCTACAAAAAAGAA including BAF170, BAF155, BAF200, BAF57, BAF60a, BAF45a, BAF60b, BAF53a, and BAF47 and the SWI/SNF-associated C proteins BCL7A and BCL7C. Surprisingly, we also recovered multiple peptides that are unique to the BRM DNA-dependent ATPase or unique to the BRG1 DNA-dependent ATPase, even A704 SLR25 SKRC20 RCC4/SLR24 SLR26 Caki-2 though only BRG1 is believed to participate in PBAF com- 1 1634 plexes (15–18). BAF180WT We next confirmed that exogenous wild-type BAF180, but not BAF180Δ2BD mutant (Q1298*) BAF180, coimmunoprecipitated with PBAF E BAF180Δ6BD components in A704 cells (Fig. 2 ). Consistent with this finding, the exogenous wild-type BAF180 in A704 cells, similar to the BAF180Q1298* endogenous wild-type BAF180 in 786-O cells and UMRC2 cells, was detected in a high-molecular-weight complex containing Bromodomain (BD): binding to acetylated histone other PBAF components after glycerol gradient centrifugation. In contrast, BAF180 (Q1298*) was not detected in this higher- Bromo-Adjacent Homology (BAH): putative protein-protein interaction order complex (Fig. 2 G–J).