University of Groningen SETD2 and PBRM1 Inactivation in The

University of Groningen SETD2 and PBRM1 inactivation in the development of clear cell renal cell carcinoma Li, Jun IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2016 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Li, J. (2016). SETD2 and PBRM1 inactivation in the development of clear cell renal cell carcinoma. University of Groningen. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 29-09-2021 CHApter 4 PBRM1 LOss IN PRIMARY TUBULAR EPITHELIAL CELLS LEAds TO AberrANT EXpressION OF IMMUNE respONse GENes Jun Li1, Joost Kluiver2, Jan Osinga1, Helga Westers1, Anke van den Berg2, Rolf H. Sijmons1 and Klaas Kok1 1Department of Genetics, and 2Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, PO box 30.001, 9700 RB Groningen, the Netherlands Manuscript in preparation CHAPTER 4 AbstrACT Clear cell Renal Cell Carcinoma (ccRCC) is characterized by loss of the short arm of chromosome 3 in more than 90% of the cases. The Polybromo-1 (PBRM1) gene maps to this region and is the second most frequently mutated gene in ccRCC. PBRM1 is a subunit of the PBAF complex, a subgroup of the SWI/SNF complexes that modify local accessibility of chromatin, and in that way contributes to the regulation of gene expression. With a mutation frequency of 30%, of which about 80% are presumably inactivating, it is clear that inactivation of PBRM1 is a major contributor to the development of ccRCC. However it is unclear how this event contributes to the early steps of ccRCC development. To study the role of PBRM1 in ccRCC initiation, we performed lentiviral-based shRNA knockdown of PBRM1 in kidney primary tubular epithelial cells (PTECs), the presumed normal counterparts of ccRCC. Interestingly, knockdown of PBRM1 did not give the PTECS an clear growth advantage, nor did it extend the proliferative capacity as compared to control PTECs. At the gene expression level, both the gene set enrichment analyses and the Gene Ontology analysis pointed towards a significant effect of PBRM1-KD on the expression on immune responsive genes. Previous studies have already shown aberrant expression of IFN responsive genes in malignant cells with defective SWI/SNF complexes, but mostly without specifying the specific subgroup of these complexes. Based on our data we suggest that functional loss of the wild type PBAF complex could be one of the events triggering the development of ccRCC. 86 PBRM1-loss in PTECs lEads To ExPREssion ChangEs in iFns REsPonsE gEnEs INtrODUCTION Clear cell Renal Cell Carcinoma (ccRCC) is characterized by copy number loss of a large part of the short arm of chromosome 3 (Kok et al., 1997; van den Berg et al., 1997), which occurs in more than 90% of the cases (Hakimi et al., 2013). This frequent allelic loss indicates the location of one or more tumor suppressor genes (TSGs) at this chromosome arm. Any of these genes might be bi-allelicly inactivated due to a mutation in the remaining allele following the model proposed by Knudson (Knudson, 1971). Linkage studies of von Hippel-Lindau cancer syndrome families paved the way for the identification of the Von Hippel–Lindau (VHL) gene, the first identified TSG located at 3p (Latif et al., 1993). In recent years, a series of next generation sequencing studies revealed three additional candidate tumor suppressor genes on 3p, i.e. PBRM1, SETD2 and BAP1 (Duns et al., 2010; Duns et al., 2012; Cancer Genome Atlas Research 2013; Sato et al., 2013). In ccRCC, PBRM1 is the second most frequently mutated gene after VHL. Importantly, more than 80% of the nonsynonymous mutations in PBRM1 are 4 inactivating mutations (COSMIC database). This high mutation frequency indicates that PBRM1 inactivation is a crucial event in the development of ccRCC tumors. PBRM1 encodes the BAF180 protein, a subunit of a specific group of SWI/SNF complexes (Xue et al., 2000; Roberts and Orkin, 2004). In general, SWI/SNF complexes are recruited to chromatin and function to mediate ATP-dependent chromatin remodeling processes. The human SWI/SNF complex consists of multiple subunits including one of two known ATPases (Roberts and Orkin, 2004; Kadoch and Crabtree, 2015). SWI/SNF complexes are divided into two different subtypes known as BAF (BRM-associated factors) and PBAF (polybromo-associated BAF) (Nie et al., 2003). The BCL11, BCL7, CRD9 and ARID1 subunits are specific for BAF complexes, whereas PBRM1 (also known as BAF180), BRD7, and ARID2 (also known as BAF200) are specific for PBAF complexes (Xue et al., 2000; Hohmann and Vakoc, 2014). BAF and PBAF target different genomic segments (Angus-Hill et al., 2001; Lemon et al., 2001). By virtue of its bromodomains, PBRM1 functions as a reader of acetylated Lysines at H3K4 and H3K9 and enables targeting of PBAF to these regions (Kupitz et al., 2008; Thompson, 2009). The specific epigenetic locus recognition mechanism of the BAF complex is still not clear (Kadoch and Crabtree, 2015).The inactivation of one or more subsets of the SWI/SNF complexes can promote the development of cancer (reviewed by Reisman et al., 2009). PBRM1 inactivation will result in loss of the PBAF complex, and this will lead to loss of its tumor suppressive function. Missense mutations in PBRM1 seem to occur more frequently in the 4th bromodomain than in the other functional domains. Since the bromodomains are crucial for the interaction of the PBAF complex with the chromatin, these missense mutations are potentially pathogenic. The consequence of PBRM1 loss in the ccRCC precursor cells, i.e. primary tubular epithelial cells of the kidney (PTECs) (Thoenes et al., 1986), is still unknown. To evaluate this, we generated PTECs stably transduced with viral short hairpin RNA overexpressing 87 CHAPTER 4 constructs. We monitored changes in their phenotype over a period of three to four weeks and determined changes in gene expression profiles at day 6 after transduction. MA TERIAL AND metHOds PTECs isolation and cell culture Kidney primary tubular epithelial cells (PTECs) were isolated from healthy renal cortex segments as previously described (Li et al., 2016). Briefly, the tissue block was cut into small cubes and seed into T25 FCS-pre-coated and Collagen-1-coated T25 flasks (BD Biosciences, San Jose, CA, USA, BD BioCoat 25cm2, Cat#356484). The isolated cells were cultured in DMEM/F-12 GLUTMAX-1 supplemented with 1% ITS (5μg/ml insulin, 5μg/ml transferrin, 5ng/ml selenium ITS), 0.1% EGF (5ng/ml) and 1% P/S (100U/ml penicillin and 100μg/ml streptomycin), at 37°C, 5% CO2. When the cells reached 80%-90% confluence (day 5 to 7), they were split and frozen for use in the experiment as passage 1. At passage 3 the primary PTECs were characterized with the following markers: Cytokeratin 8 (CK8.18), epithelial membrane antigen (EMA), pan cytokeratin (CK AE1.3), C5α receptor (c5αR), and liver-type fatty acid-binding protein 1 (L-FABP). During the experiment the PTECs were maintained in DMEM/F- 12 GLUTMAX-1 containing 10% FBS, 1% ITS, 0.1% EGF and 1% P/S. All the reagents used for cell culturing are from Sigma-Aldrich (St. Louis, MO, USA). CcRCC cell lines RCC1, RCC4, RCC5, and RCC6 are a gift from Dr. C.D. Gerharz (Institute of Pathology, University Hospital, Düsseldorf, Germany), who established these cell lines. CcRCC cell lines RCC-ER, RCC-MF, RCC-JF, RCC-HS, RCC-GW, and RCC-FW were purchased from Cell Line Services, Eppenheim, Germany. The ccRCC cell lines were maintained in RPMI 1640 supplemented with 10% FBS, 1% ITS, and 1% P/S. All the cells were maintained at 37°C in humidified air containing 5% CO2. Construction of shRNA vectors and generation of lentiviral particles Oligonucleotides (Eurogentec, Liège, Belgium) were annealed and subcloned into the pGreenpuro shRNA cloning and expression lentivector (Systems Biosciences, Mountain View, CA, USA). The non-targeting shRNA lentiviral vector was obtained from Systems Biosciences (Mountain View, CA). The insert sequences were confirmed by Sanger sequencing (sh-PB1: 5’-GATCCAGCTAAATTTGCCGAGTTATTCAAGAGATAAC TCGGCAAATTTAGCTTTTTTG-3’; sh-PB2: 5’-GATCCGTTAGGAGTTGTCGGAA TATTCAAGAGATATTCCGACAACTCCTAACTTTTTG-3’). Lentiviral particles were produced by co-transfection of 7x105 HEK293T cells in a 6-well plate by the calcium phosphate (CaPO4)-mediated method, with 2µg pGreenPuro shRNA expression lentivector (sh-PB1, sh-PB2 or non-targeting (NT)) in combination with a plasmid mix containing 1µg pCMV-VSV-G, 1µg pRSV.REV, and 1µg pMDL-gPRRE. Lentiviral particles were harvested 48 hours after transfection and passed through a 0.45µm pore PVDF Millex-HV filter (Millipore, Billerica, MA, USA). 88 PBRM1-loss in PTECs lEads To ExPREssion ChangEs in iFns REsPonsE gEnEs Cell transduction for expression studies and growth competition assay PTECs were transduced with a serial dilution of viral stocks in the presence of 4μg/ ml polybrene (Sigma-Aldrich, St.

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