BAF180: Its Roles in DNA Repair and Consequences in Cancer

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BAF180: Its Roles in DNA Repair and Consequences in Cancer Sarah Hopson, and Martin J Thompson ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00541 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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ACS Chemical Biology

Figure 1: Chart of the frequency of different types of mutations of PBRM1 (BAF180) in cancers. Data was collected from COSMIC database.5 261x202mm (150 x 150 DPI)

ACS Paragon Plus Environment


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Figure 2: The domain organization of wild-type BAF180 (A) and various truncated mutant proteins observed in cancers 319x127mm (150 x 150 DPI)


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for table of contents only 277x294mm (96 x 96 DPI)



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BAF180: Its roles in DNA Repair and Consequences in Cancer AUTHORS: Sarah Hopson (1st) and Martin Thompson (2nd)

Sarah Hopson, [email protected], 586-484-4921 Corresponding author: Martin Thompson, [email protected], 906-487-3522


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BAF180: Its Roles in DNA Repair and Consequences in Cancer ABSTRACT (154 WORDS) In 2011, Varela et al. reported that the PBRM1 gene is mutated in approximately 40% of clear cell renal cell carcinoma cases. Since then, the number of studies relating PBRM1 mutations to cancers has substantially increased. BAF180 has now been linked to more than 30 types of cancers, including ccRCC, cholangiocarcinomas, esophageal squamous cell carcinoma, bladder cancer, and breast cancer. The mutations associated with BAF180 are most often truncations, which result in loss of protein expression. This loss has been shown to adversely affect the expression of genes, likely because BAF180 is the chromatin recognition subunit of PBAF. In addition, BAF180 functions in numerous DNA repair mechanisms. Its roles in mediating DNA repair is likely the mechanism by which BAF180 acts a tumor suppressor protein. As research on this protein gains more interest, scientists will begin to piece together the complicated puzzle of the BAF180 protein and why its loss often results in cancer.

Keywords: BAF180, PBRM1, Human Polybromo-1 protein, cancer, cancer mutations, tumor suppressor



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INTRODUCTION The bromodomain-containing protein BAF180 is a large, chromatin-remodelling protein. Encoded by the PBRM1 gene, the BAF180 protein has nine domains: six bromodomains, two BAH-domains, and one HMG-box. Bromodomains (brds) recognize and bind to acetylated lysines. The BAH (bromo-adjacent homology) domains are believed to mediate protein-protein interactions and the HMG-box (high mobility group box) domains bind to DNA. BAF180 has nine isoforms. PBAF (polybromo-associated bromo-adjacent factor) is one of the human homologs of yeast SWI/SNF and the “P” in PBAF stands for polybromo. BAF180 serves as the recognition component of the PBAF transcription-mediating complex. BAF180 is considered to be the fusion of yeast Rsc1, Rsc2, and Rsc4.1, 2 Rsc2 is involved in chromatin remodelling, double strand break repair (via both homologous recombination and nonhomologous end joining), transcription, and chromosomal cohesion.3 The significance of BAF180 in cancer was first noted by Varela et al., who reported it to be mutated in approximately 40% of clear cell renal cell carcinoma (ccRCC) cases.4 Since then, researchers have investigated the significance of BAF180 in numerous cancers. BAF180 has now been linked to more than 30 types of cancers,5-7 including cholangiocarcinomas,8-12 esophageal squamous cell carcinoma,13 and breast cancer (Table 1).14, 15 BAF180 is a known tumor suppressor gene and a driver mutation in ccRCC.16-18 BAF180 has roles in numerous DNA repair mechanisms and its tumor suppressor properties may partially stem from these roles. BAF180 is important for cohesion between centromeres, which is necessary for maintaining genomic stability. Loss of cohesion leads to genomic instability and aneuploidy. BAF180 is also important for silencing transcription near double-stranded DNA breaks (DSB). When transcription is not silenced, DSB repair is


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impaired. Additionally, the BAH domains of BAF180 have been found to be critical for the ubiquitination of PCNA, an early step in the post-replication repair pathway. The tumor suppressor properties of BAF180 may stem from its specific and multiple roles in DNA repair pathways. The PBRM1 gene is located at chromosome 3p21. This location is a mutational hotspot in some cancers, including ccRCC, because 3 tumor suppressor genes are located there.17, 19-23 The 3p21 region is frequently deleted in cancer cells, resulting in loss of heterozygosity. Knudson’s hypothesis proposes that for tumor suppressor genes, “two hits” are required to bring about cancer. In this case, the “first hit” would be loss of heterozygosity (LOH). The “second hit” is the mutation of the remaining copy of the gene; this mutation may result in the production of a protein which has lost its function or is only partially functional, or result in loss of protein expression altogether.17, 24-27 PBRM1, as a tumor suppressor gene, is consistent with this “two hit” approach.18 In addition to chromosomal location, the PBRM1 gene sequence itself has several characteristics that might account for its high mutational frequency. Numerous appearances of base-tracts, particularly A- and T-tracts, occur in the mRNA. Base-tracts have been known to increase the chances of point mutations during replication and poly-A tracks have been found to influence gene transcription both by stopping translation and creating frameshift mutations.28-32 Within the PBRM1 gene, there are numerous instances of two, three, and four base repeats. As of January 2017, COSMIC (Catalogue of somatic mutations in cancer) database has more than 1,100 unique PBRM1 mutations.7 There are three PBRM1 transcripts available on COSMIC and some mutations are recorded under all three transcripts. This leads to redundancy, with the same mutation being reported multiple times. These



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transcripts differ by a few amino acids, thereby changing the numbering of the amino acids. Transcripts ENST00000296302 and ENST00000337303 are identical in numbering until AA1430. Transcript ENST00000356770 numbering is the same as the other two for the first 299 AA. It should be noted that the overall sequence largely remains the same, while the numbering differs. For example, Ser1500 for one transcript might be the same amino acid as Ser1498 for another transcript. So comparing and totalling the number of reported mutations is not straightforward. The individual mutations listed for each transcript must be analyzed and the redundant mutations removed, so they are only recorded once instead of three times. Analysis of the mutations can be done by comparing the mutation location/type and sample IDs for each individual mutation for each transcript. Regardless, mutations of every type (frameshift, synonymous substitution, missense substitution, and nonsense substitution) have been identified, though truncation mutations (introduction of a premature stop codon) are the most common (Figure 1).5-7 Many of the truncation mutations eliminate one or more domains. In extreme cases, the mutated PBRM1 may only code for 2 or 3 domains.4 Figure 2 displays the domain organization of wild-type BAF180 (A) and select cancer-associated mutants (B). Many studies have reported BAF180 mutations in specific cancers, though the implications of these mutations are not always investigated.

BAF180 and DNA Repair GENOMIC INSTABILITY Genomic instability is often a hallmark of cancer. The cause of genomic instability in mice was found to be caused by a lack of cohesion between the centromeres of chromosomes. This lack of cohesion can lead to aneuploidy, where a cell contains an abnormal number of chromosomes. When BAF180 was depleted in mouse embryonic stem


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cells, the distance between centromeres increased, suggesting less cohesion between centromeres. This appears to be exclusive to centromeres, as the distances between telomeres and chromosomal arms showed no significant change. To determine the route by which BAF180 affects centromeric cohesion in chromosomes, levels of other cohesion genes were tested in BAF180-deficient cells. It was found that levels were normal, indicating that BAF180 did not affect the transcription of these genes.33 Cancer-associated mutants of BAF180 were unable to restore cohesion to BAF180depleted cells or reduce the cell’s sensitivity to DMSO-induced DNA damage. Yeast cells lacking Rsc2, a yeast homolog of BAF180, are also temperature sensitive and sensitive to DMSO-induced DNA damage. To better understand the mechanism, three different point mutations were introduced in Rsc2: Rsc2-H458P (which corresponds to H1205P in BAF180 isoform 9, located in second BAH domain); Rsc2-T67P (corresponding to T232P in BAF180, brd2); Rsc2-M280I (corresponding to M523I in BAF180, brd4). Mutant Rsc2H458P showed patterns of sensitivity similar to Rsc2 knockout (KO) cells, but mutants Rsc2-T67P and Rsc2-M280I showed reduced sensitivity, indicating that these mutants were able to partially restore normal function. However, none of the created mutants were able to restore cohesion between centromeres.33 Insufficient cohesion between centromeres can have several repercussions including: mis-segregation of chromosomes, formation of micronuclei, and lagging chromosomes. Both acentric chromosomes and lagging chromosomes can cause aneuploidy in a cell,34 wherein the individual cell has a different number of chromosomes than a wildtype cell. Aneuploidy, micronuclei, and lagging chromosomes were identified in BAF180deficient cells.33



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The maintenance of chromosomal cohesion and the stability of the genome, may be two ways in which BAF180 helps prevent tumorigenesis. When BAF180 is depleted from a cell, cohesion between centromeres greatly decreases, and as a result, chromosomal instability increases.33 Levels of other genes involved in maintaining centromeric cohesion and genomic stability were normal. This lack of cohesion and increase in chromosomal instability suggests that BAF180 may have a role in homologous recombination (HR), as other proteins, such as BRCA1, have dual roles in cohesion and HR.35 DNA DOUBLE-STRAND BREAK REPAIR BAF180 also plays a role in double-strand break (DSB) repair by silencing transcription near DNA break sites. When transcription is not silenced, DSB repair is delayed and since most irradiation-induced DSBs are repaired rapidly,36 this delay can lead to chromosomal abberations.37 PBAF was found to silence transcription around DNA breaks. When BAF180 was depleted in reporter cells, transcription of the reporter gene (yellow fluorescent protein) was not silenced, suggesting that BAF180 is needed to silence transcription around DNA breaks. If transcription is not repressed, rapid DSB repair is hindered.36 Additionally, when BAF180-depleted cells were exposed to irradiation, the non-homologous end joining (NHEJ) DNA repair pathway was compromised.38 NHEJ is a key mechanism by which DSBs are repaired. Treatment of siBAF180 cells with the transcription inhibitor DRB (5,6Dichlorobenzimidazole 1-β-D-ribofuranoside), revealed that the role of BAF180 in DSB repair is dependent on active transcription. In siBAF180 cells, DNA damage levels were high at 20 minutes post-IR (irradiation), as indicated by a high number of γH2AX; when BAF180 was re-introduced, levels of γH2AX were similar to control cells 38. γH2AX is used as a biomarker to measure the quantity of DNA double-stranded breaks 39. Wherever


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double-stranded breaks form, H2AX is immediately phosphorylated, becoming γH2AX; therefore, the presence of γH2AX can be used to determine the extent of double-stranded breaks in a 1:1 ratio.39 The phosphorylation of γH2AX is the first step in recruiting DNA repair proteins.39 When siBAF180 cells (which were unable to stop transcription) were treated with DRB (which stops transcription), the DNA damage levels decreased to around that of the control cells, indicating that stopping transcription restored DNA damage repair function. This suggests that when transcription is stopped, BAF180 is not needed for double strand break repair.38 The location and function of proteins is often dependent on the presence of posttranslational modifications (PTMs). A phosphorylation site critical to DSB was identified on BAF180. Ser963 (number corresponding to isoform 8 of BAF180) was found to be phosphorylated by ATM (ataxia telangiectasia mutated).40 ATM is a kinase that is critical for DSB repair.40 This phosphorylation site is located between the sixth brd and the first BAH domain. Two mutant constructs (a phosphomutant S963A and a phosphomimic S963E) were created to determine if phosphorylation was critical to BAF180’s role in DSB repair. When the phosphomutant was added to siBAF180 cells, no significant change was seen, indicating that the phosphomutant did not restore normal DNA repair, suggesting that phosphorylation is important to BAF180’s role in DSB repair. When the phosphomimic was added, levels of damaged DNA were similar to control cells, indicating that proper DNA repair function was restored. This suggests that phosphorylation of BAF180 at S963 is a prerequisite to its role in repair.38 UBIQUITINATION OF PCNA Proliferating Cell Nuclear Antigen (PCNA) regulates replication past sites of DNA damage and its ubiquitination is a necessary step in re-priming past damaged DNA forks



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during post-replication repair (PRR).41 The BAH domains of BAF180 are required to ubiquitinate PCNA.42 The BAF180 orthologue Rsc2 was found to be located near replication forks43 as was BRG1,44 the catalytic subunit of PBAF. Taken together, this suggests that BAF180 (and hence PBAF) may be as well. When BAF180 was depleted, cells were unable to produce the proper levels of ubiquitinated PCNA, which lead to the inability to replicate past DNA lesions.43 After UVirradiation, BAF180-depleted cells had far lower levels of chromatin-bound PCNA (both ubiquitinated and non-ubiquitinated) and decreased levels of Rad18, the E3 ligase that ubiquitinates PCNA, when compared to control cells (where BAF180 was present at normal levels). It has been reported that BAF180 does play a role in regulating the cell cycle.14, 45 Therefore, experiments were conducted to determine if the reduced levels of chromatin-bound PCNA were an indirect consequence of BAF180 impacting the cell cycle. Ubiquitination of PCNA and its binding to chromatin occurs during the S-phase; post-IR, the proportion of cells in S-phase actually increased, indicating that the lower levels of chromatin-bound PCNA and Rad18 are not an indirect consequence of cell cycle changes occurring upon depletion of BAF180.43 This suggests that BAF180 is directly responsible for the observed changes in chromatin-bound PCNA and Rad18, and therefore indirectly responsible for the decreased ubiquitination levels. Interestingly, the levels of BAF180 were higher in S-phase cells than in G1 or G2 phase cells,42 suggesting that the S-phase specific function of BAF180 (i.e. ubiquitination of PCNA) requires higher levels of BAF180 than other phases, which has interesting implications since PBAF (and hence BAF180) is involved in transcription and has not been found to be limited to one phase of the cell cycle. It would be interesting to perform FACS (fluorescence-activated cell sorting) analysis on several components of the PBAF and BAF


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complexes to see how the protein levels change throughout the different phases of the cell cycle. Flag-tagged constructs of the different domains (bromodomains, the BAH domains, and the HMG domain) were created to determine which domains(s) were involved in PCNA ubiqutination. They found that levels of ubiquitinated PCNA in full-length flagtagged BAF180 and flag-BAHs were similar; cells with the flag-brds and flag-HMG constructs were unable to ubiquitinate PCNA, which suggests that the BAH domains are required for PCNA ubiquitination and that the other domains of BAF180 are not necessary. Additionally, BAF180 did not need to be in complex with PBAF in order for the BAH domains to facilitate ubiquitination.42 DNA REPAIR SUMMARY As discussed above, the tumor suppressor properties of BAF180 might stem from its roles in various DNA repair mechanisms. Cancer cells with a mutation in or decreased expression of BAF180 could be compromised with regards to DNA repair in three ways:

(1) Genomic instability: Loss of BAF180 resulted in an increased distance between centromeres. This leads to an increase in genomic instability, which can prevent recombinatorial-based DNA repair mechanisms.33

(2) Inability to ubiquitinate PCNA: Ubiquitination of PCNA is a key step in the PRR pathway. The BAH domains of BAF180 are required for proper ubiquitination levels. Lack of the BAH domains likely leaves much of the DNA unrepaired.42, 43

(3) Silencing of transcription at DNA double-strand breaks: At sites of active transcription near DNA DSBs, BAF180 is needed for the proper repair of these breaks.



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BAF180 silences transcription at sites of DSBs, allowing for the rapid repair of damaged DNA.38

The Impact of BAF180 in Cancer BAF180 has been found to be mutated in more than 30 types of cancers, most notably ccRCC.4 Loss of BAF180 has been linked to cell proliferation and deregulation of gene expression, including apoptotic and cell cycle regulator genes. CELL PROLIFERATION Cancer cell lines lacking BAF180 show various effects on cell proliferation. The effect of loss of BAF180 on cell proliferation differs between cell lines and sometimes within the same cell line (Tables 2 and 3).4, 14, 24, 25, 45-47 For example, Varela et al. and Xiao et al. demonstrated that knockdown of BAF180 results in increased proliferation in cell line 786-O (kidney adenocarcinoma, expresses wild-type BAF180), while Murakami et al. reported that BAF180 knockdown reduced cell growth in this cell line (meaning BAF180 had a positive correlation with cell growth) and Gao et al. found no effect on proliferation.4, 24, 46, 47

Some researchers have attempted to identify other genes that might influence the impact of BAF180 with regards to cell proliferation. One group noted that in order for BAF180 re-expression to have a tumor-suppressive effect, cells needed to express HIF1α, though another group who also examined hypoxia factors did not notice any correlation between the effect of BAF180 on cell proliferation and HIF.24, 47 This is interesting, as VHL, the most frequently mutated gene in ccRCC, is known to regulate the expression of hypoxia-related genes. One group found that the presence of the BRG1, the ATPase subunit


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of the PBAF complex, was necessary for re-expression or knockdown of BAF180 to have an effect on cell proliferation.24 The effects of cancer-associated mutations on growth rate have been studied as well. A tumor-associated BAF180 mutation (missing the HMG-box and C-terminus) was not able to suppress proliferation in A704 cells (kidney adenocarcinoma cells, lacking BAF180), but was able to suppress growth in Caki2 cells (clear cell renal cell carcinoma cells, lacking BAF180). Researchers also showed that a mutant lacking the first two bromodomains was able to reduce proliferation (in A704 cells), though a mutant lacking all six bromodomains was unable to.24 In contrast, a recent study demonstrated that the introduction of single point mutations into the binding pocket of the individual bromodomains of BAF180 abrogated its tumor-suppressive effects in Caki2 cells. When wild type BAF180 was introduced into Caki2 cells, the growth rate decreased significantly. However, introduction of the mutant brd 1, 2, 4, and 5 BAF180 constructs had no effect on cellular growth, meaning the cells continued to grow at a rate similar to control cells that lacked BAF180.26 Overall, the lack of agreement on the effect of BAF180 knockdown on cell proliferation may be due to the different knockdown techniques and the cell lines used. It should be noted that Varela et al., Gao et al., Xiao et al., Chowdhury et al., and Murakami et al. performed their studies in renal cells, while Xia et al. and Burrows et al. used mammary cells; Burrows et al. also used fibroblasts, and Huang et al. used bladder cells. The reasons behind the different proliferation responses of cell lines to BAF180 is still unknown, though Gao et al. presents several plausible explanations, such as the presence of additional mutations which cause the cells to be unaffected by BAF180. In addition, Gao et al. suggests that the results of Varela et al. may not be accurate due to off-target effects. To



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clarify the proliferative effects of BAF180, experiments in each of these cells lines using the same knockdown or re-expression technique are necessary in order to understand the importance of BAF180 in cancer cell proliferation. CLEAR CELL RENAL CELL CARCINOMA Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma (~92% of cases).16, 48 Of these, approximately 40% of cases carry a mutation in the PBRM1 gene, though mutations are rarely found in other types of renal carcinomas 49. This makes PBRM1 the second most commonly mutated gene in ccRCC, after VHL (mutated in 75-80% of cases).17, 23 Interestingly, in ~90% of ccRCC cases, the 3p arm of one copy of chromosome 3 is deleted, which encompasses four tumor suppressor genes: VHL, PBRM1, SETD2, and BAP1.16-19, 21-23, 50 This deletion results in loss of heterozygosity, the first “hit” in Knudson’s two-hit hypothesis for inactivation of tumor suppressor proteins. MUTATIONS OF BAF180 IN CCRCC Of the 40% of PBRM1 mutations, approximately 82% of them are truncating mutations either by frameshifts or nonsense mutations.51 In one study, 53% of ccRCC tumor samples tested had no detectable expression of BAF180. Of these, 90% had a mutation in the gene. In the 47% of samples that were positive for BAF180, 90% had wildtype PBRM1/BAF180.27 Another study reported that, in 112 immunostained ccRCC samples, 34 (30.4%) had no detectable expression and 78 (69.6%) showed positive expression of BAF180.52 It should be noted that the researchers in both studies concluded that the negative IHC results meant that there was no BAF180 present, though a mutant BAF180 protein lacking the antibody’s immunogen could be expressed.


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Interestingly, mutations in BAF180 are mutually exclusive with mutations of BAP1 (BRCA1-associated protein 1), a nuclear deubiquitinase protein.18-20, 27, 53, 54 Genes whose mutations are mutually exclusive usually have redundant function. However, the functions of BAF180 and BAP1 are not redundant: BAF180 is a chromatin-remodeller and BAP1 is a nuclear deubiquitinase. The significance of the mutual exclusivity is not fully understood, though it is generally accepted that patients with a mutation in BAP1 have a worse prognosis, with regard to overall survival.18-20, 27, 53, 54 Silencing of the PBRM1 gene at its promoter via hypermethylation has also been proposed as a possible mechanism for PBRM1 inactivation.51 Promoter hypermethylation of tumor-suppressor genes is commonly observed in cancer.55 However, Ibragimova and colleagues found that hypermethylation of PBRM1 is a rare event in ccRCC, as is the hypermethylation of BAP1, SETD2, and some other chromatin modifying genes. This result is not unprecedented, as some tumor-suppressor proteins exhibit promoter hypermethylation, while other do not. They propose that deletion of the 3p21 region (which affects four tumor-suppressor genes) is more favorable to oncogenesis than the inactivation of one gene by hypermethylation.55 This study favours an explanation that aberrant BAF180 function is due to mutation of the gene and not suppression of gene expression. GENE EXPRESSIONSIGNATURE IN BAF180- CELLS As expected, the gene expression profile of BAF180-deficient cells is substantially different from that of BAF180-proficient cells. Most of the affected genes were found to be down-regulated upon loss of BAF180. Three groups investigated the gene expression patterns of ccRCC cells and noted that genes involved in cell adhesion, cell proliferation, chromosomal instability, and hypoxia response were often affected.18, 25, 56 Levels of miRNA were also down-regulated.56



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In addition to investigating the differential expressed genes, methylation of CpG sites was investigated. Of the 1405 differently methylated sites, 1229 were hypermethylated, which would result in gene silencing. However, not all of these genes may be directly regulated by PBRM1.56 Re-expression of BAF180 into Caki2 cells led to the up-regulation of genes involved in cell adhesion and the actin cytoskeleton.25 This is in accordance with phenomenological data demonstrating that knockdown of BAF180 results in increased cell migration.4 Two other studies reported that BAF180-deficient cells had down-regulation of the same genes, as compared to control samples.18, 25, 56 Re-expression of BAF180 also resulted in the up-regulation of apoptotic processes, carbohydrate metabolic processes, and hypoxia response genes. Down-regulation of genes involved in the cell cycle was observed in the same cells.25 It is not uncommon for ccRCC tumors to have a hypoxia signature, as VHL (the most frequently mutated gene in ccRCC) is involved in the recruitment of several hypoxiarelated genes.57 BAF180-deficient cells have also been noted as having a hypoxia signature.4, 25 When BAF180 was re-expressed in cancer cells, the expression of HIF-target genes (hypoxia inducible factor) decreased, though no change was observed in HIF1α and HIF2α levels.24 In contrast, a second study found that re-expression of BAF180 resulted in increased expression of HIF-target genes.25 A third study found that knockdown of BAF180 in hepatitis B cells and ccRCC cells resulted in down-regulation of HIF-target genes,47 correlating with data from the second study.25 In Caki2 cells, point mutations in the binding sites of bromodomains 1, 2, 4, 5, and 6 were unable to bring about the upregulation of apoptotic genes that is usually observed when wild type BAF180 is


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introduced, though the mutation in brd3 had no effect on gene expression as compared to wild type BAF180.26 CLINICAL SIGNFICANCE OF BAF180 MUTATIONS IN CCRCC Multiple groups have attempted to correlate the presence of BAF180 mutations to clinical relevance. However, studies provide conflicting data with regards to correlation between BAF180 mutations and tumor size, differentiation, and patient prognosis. Some researchers have found that patients with BAF180 mutations presented with advanced tumor stage,16, 17, 20, 54, 58 while others found no statistically significant correlation 23, 51, 59. Similarly, some studies saw that patients with BAF180 mutations have a poor prognosis,16, 52, 54, 58

while others report that there is no correlation.20, 23, 59 Despite disagreement

regarding the prognosis impact of BAF180 mutations, it is generally accepted that patients with mutations in BAP1 have a worse prognosis than patients with a mutation in BAF180.17, 18, 20, 23, 51, 59 It has been proposed that BAF180 be used as a biomarker and prognostic indicator, but more investigations need to be conducted before that can happen.18, 22, 24, 51, 54, 60, 61 Other Cancers Mutations in PBRM1 have been identified in more than 30 types of cancers. Though mutations in ccRCC are well studied, due to the prominence of PBRM1 mutations in that cancer, there has been some investigation into PBRM1 mutations in other cancers. EPITHELIOID SARCOMA Epithelioid sarcoma is a rare sarcoma that occurs in the soft tissue of the extremities (referred to as “conventional type”), most often in young adults.62-64 It has a high metastatic rate. A second type of epithelioid sarcoma, referred to as “proximal-type”, occurs in the pelvic area.62-64 Loss of PBRM1 expression was found in 82.6% of epithelioid sarcoma



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cases. However, the sample size in this study was relatively small (23 cases), so this percentage may be artificially high. There was no difference in PBRM1 expression between the two locations of epithelioid sarcoma (conventional and proximal) nor between the three types of tumors (spindle, epithelioid, rhaboid); loss of BAF180 expression was observed in all of them. No correlation between PBRM1 expression and cellular morphology was found, nor between PBRM1 mutations and prognosis. However, there was a correlation between PBRM1 mutations and mutations in INI1 (also called SMARCB1, a core subunit of SWI/SNF complexes), which frequently occurred together.62 SMARCB1 is found in both the BAF and PBAF complexes. The mutation of SMARCB1 affects both BAF and PBAF complexes, while mutations in PBRM1 affect PBAF complexes. The co-occurrence of mutations in both BAF and PBAF might contribute to the high metastatic rate of this cancer. BLADDER CARCINOMA In bladder cancer, PBRM1 acts as a tumor suppressor by repressing gene expression of cyclin B1, a cell cycle checkpoint protein.45 Huang et al. found that mRNA and protein levels of PBRM1 were significantly lower in bladder cancer cells than in normal cells. Additionally, lower levels of PBRM1 were associated with poor patient survival rates in bladder cancer.45, 52 Cancer cell proliferation, migration, and tumor sizes all increased when expression levels of PBRM1 were reduced. Overall, PBRM1 is mutated in 2-10% of bladder cancers and is being investigated as a potential biomarker.5-7 BREAST CARCINOMA Several studies have analyzed potential mutations and expression levels in breast cancer. Pereira and colleagues found that, in ER- (estrogen receptor negative) tissues, 25% of the samples had PBRM1 mutations and most of these resulted in loss of heterozygosity


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(LOH).65 In-depth examination of PBRM1 mutations was performed by Xia et al. who showed (via PCR) that duplications in various exons were common and 25 of the 52 tumor samples had LOH for BAF180. Further analysis revealed that BAF180 plays a role in the regulation of p21 expression and induces G1 arrest upon reintroduction into BAF180deficient breast cancer cell lines.14 Real time PCR and western blotting have demonstrated that breast cancer tumor samples and cell lines have reduced expression of the BAF180 protein, as compared to paired normal tissues.15, 66 Additionally, low BAF180 expression had a strong correlation with higher tumor stage and worse survival prognosis.66 MALIGNANT MESOTHELIOMA PBRM1 has been found to be deleted in several cases of malignant mesothelioma (MM). Loss of chromosome 3p21 is common in epithelial MM, resulting in the deletion of nine genes. This region includes PBRM1, as it is located at 3p21.1. Interestingly, this deletion also encompasses BAP1, which is sometimes deleted in ccRCC. Mutations in these chromatin modifiers are mutually exclusive in ccRCC, though that does not appear to be the case in MM.67 PBRM1 was found to be deleted in both types of MM (pleural and peritoneal), though the loss was more common in pleural MM.68, 69 Two point mutations in PBRM1 were found and determined to be hemizygous mutations, because there was deletion of the other copy of the gene.70 Monoallelic gene loss was observed in 12/33 (36%) cases and biallelic deletion in 3/33 (9%) cases.70 They also confirmed that depletion of BAF180 caused increased proliferation in a malignant mesothelioma cell line.70 cBioPortal reports deletion of PBRM1 gene in a frequency of 5% in patients with pleural MM.5, 6 CANCERS OF THE BILIARY TRACT



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Cancers of the biliary tract include intrahepatic and extrahepatic cholangiocarcinomas and gallbladder carcinoma. As is the case for different types of renal carcinomas, the mutational frequency of PBRM1 differs between biliary tract locations. PBRM1 seems to be most frequently mutated in intrahepatic cholangiocarcinoma (ICC), ranging from 11-26% of samples.8-12 The most frequently mutated genes in ICC are KRAS5 and TP53, while PBRM1, ARID1A, BAP1, and SMARCB1 seem to be tied for third most mutated gene in ICC.11 PBRM1 has been suggested to be a driver mutation for ICC,9, 10 though recent evidence indicates otherwise.8 Jiao et al. and Churi et al. found that patients had a worse prognosis when PBRM1 was mutated; Luchini et al. did not identify any correlation between PBRM1 mutations and patient survival. In extrahepatic cholangiocarcinomas, the frequency of mutation was 3.5-5%.10, 11 Additionally, BAF180 is under-expressed in 53% of gallbladder carcinomas.71 CONCLUSION PBRM1 was first acknowledged as a major player in cancer after Varela et al. reported a mutational frequency of 40% in ccRCC. Since then, mutations in the PBRM1 gene have been linked to more than 30 types of cancers, including cholangiocarcinomas,8-12 esophageal squamous cell carcinoma,13 and breast cancer.14, 15 Interest in this gene continues to grow as further evidence of its importance is expounded. The significance of BAF180 mutations in cancer likely stems from its roles in DNA repair and as interest in this protein expands, researchers will likely be able to better understand the intricate mechanisms and develop targeted treatments.

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References [1] Thompson, M. (2009) Polybromo-1: the chromatin targeting subunit of the PBAF complex, Biochimie 91, 309-319. [2] Xue, Y., Canman, J. C., Lee, C. S., Nie, Z., Yang, D., Moreno, G. T., Young, M. K., Salmon, E. D., and Wang, W. (2000) The human SWI/SNF-B chromatinremodeling complex is related to yeast Rsc and localizes at kinetochores of mitotic chromosomes, Proc. Natl. Acad. Sci. U. S. A. 97, 13015-13020. [3] Cairns, B.R., Schlichter, A., Erdjument-Bromage, H., Tempst, P., Kornberg, R.D. and Winston, F. (1999) Two functionally distinct forms of the RSC nucleosomeremodeling complex, containing essential AT hook, BAH, and bromodomains. Mol. Cell 4, 715-723. [4] Varela, I., Tarpey, P., Raine, K., Huang, D., Ong, C. K., Stephens, P., Davies, H., Jones, D., Lin, M.-L., Teague, J., Bignell, G., Butler, A., Cho, J., Dalgliesh, G. L., Galappaththige, D., Greenman, C., Hardy, C., Jia, M., Latimer, C., Lau, K. W., Marshall, J., McLaren, S., Menzies, A., Mudie, L., Stebbings, L., Largaespada, D. A., Wessels, L. F. A., Richard, S., Kahnoski, R. J., Anema, J., A.Tuveson, D., Perez-Mancera, P. A., Mustonen, V., Fischer, A., Adams, D. J., Rust, A., Chan-on, W., Subimerb, C., Dykema, K., Furge, K., Campbell, P. J., Teh, B. T., Stratton, M. R., and Futreal, P. A. (2011) Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma, Nature (London, U. K.) 469, 539-542. [5] Cerami, E., Gao, J., Dogrusoz, U., Gross, B. E., Sumer, S. O., Aksoy, B. A., Jacobsen, A., Byrne, C. J., Heuer, M. L., Larsson, E., Antipin, Y., Reva, B., Goldberg, A. P., Sander, C., and Schultz, N. (2012) The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data, Cancer Discov. 2, 401-404. [6] Gao, J., Aksoy, B. A., Dogrusoz, U., Dresdner, G., Gross, B., Sumer, S. O., Sun, Y., Jacobsen, A., Sinha, R., Larsson, E., Cerami, E., Sander, C., and Schultz, N. (2013) Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal, Sci. Signaling 6, pl1. [7] Forbes, S. A., Beare, D., Gunasekaran, P., Leung, K., Bindal, N., Boutselakis, H., Ding, M., Bamford, S., Cole, C., Ward, S., Kok, C. Y., Jia, M., De, T., Teague, J. W., Stratton, M. R., McDermott, U., and Campbell, P. J. (2015) COSMIC: exploring the world's knowledge of somatic mutations in human cancer, Nucleic Acids Res. 43, D805-D811. [8] Luchini, C., Robertson, S. A., Hong, S. M., Felsenstein, M., Anders, R. A., Pea, A., Nottegar, A., Veronese, N., He, J., Weiss, M. J., Capelli, P., Scarpa, A., Argani, P., Kapur, P., and Wood, L. D. (2017) PBRM1 loss is a late event during the development of cholangiocarcinoma, Histopathology 71, 375-382. [9] Jiao, Y., Pawlik, T. M., Anders, R. A., Selaru, F. M., Streppel, M. M., Lucas, D. J., Niknafs, N., Guthrie, V. B., Maitra, A., Argani, P., Offerhaus, G. J., Roa, J. C., Roberts, L. R., Gores, G. J., Popescu, I., Alexandrescu, S. T., Dima, S., Fassan, M., Simbolo, M., Mafficini, A., Capelli, P., Lawlor, R. T., Ruzzenente, A., Guglielmi, A., Tortora, G., de Braud, F., Scarpa, A., Jarnagin, W., Klimstra, D., Karchin, R., Velculescu, V. E., Hruban, R. H., Vogelstein, B., Kinzler, K. W., Papadopoulos, N., and Wood, L. D. (2013) Exome sequencing identifies frequent inactivating



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas, Nat. Genet. 45, 1470-1473. [10] Churi, C. R., Shroff, R., Wang, Y., Rashid, A., Kang, H. C., Weatherly, J., Zuo, M., Zinner, R., Hong, D., Meric-Bernstam, F., Janku, F., Crane, C. H., Mishra, L., Vauthey, J.-N., Wolff, R. A., Mills, G., and Javle, M. (2014) Mutation Profiling in Cholangiocarcinoma: Prognostic and Therapeutic Implications, PLoS ONE 9, e115383. [11] Simbolo, M., Fassan, M., Ruzzenente, A., Mafficini, A., Wood, L. D., Corbo, V., Melisi, D., Malleo, G., Vicentini, C., Malpeli, G., Antonello, D., Sperandio, N., Capelli, P., Tomezzoli, A., Iacono, C., Lawlor, R. T., Bassi, C., Hruban, R. H., Guglielmi, A., Tortora, G., de Braud, F., and Scarpa, A. (2014) Multigene mutational profiling of cholangiocarcinomas identifies actionable molecular subgroups, Oncotarget 5, 2839-2852. [12] Misumi, K., Hayashi, A., Shibahara, J., Arita, J., Sakamoto, Y., Hasegawa, K., Kokudo, N., and Fukayama, M. (2017) Intrahepatic cholangiocarcinoma frequently shows loss of BAP1 and PBRM1 expression, and demonstrates specific clinicopathological and genetic characteristics with BAP1 loss, Histopathology 70, 766-774. [13] Nakazato, H., Takeshima, H., Kishino, T., Kubo, E., Hattori, N., Nakajima, T., Yamashita, S., Igaki, H., Tachimori, Y., Kuniyoshi, Y., and Ushijima, T. (2016) Early-Stage Induction of SWI/SNF Mutations during Esophageal Squamous Cell Carcinogenesis, PLoS One 11, e0147372. [14] Xia, W., Nagase, S., Montia, A. G., Kalachikov, S. M., Keniry, M., Su, T., Memeo, L., Hibshoosh, H., and Parsons, R. (2008) BAF180 Is a Critical Regulator of p21 Induction and a Tumor Suppressor Mutated in Breast Cancer, Cancer Res. 68, 1667-1674. [15] Decristofaro, M. F., Betz, B. L., Rorie, C. J., Reisman, D. N., Wang, W., and Weissman, B. E. (2001) Characterization of SWI/SNF protein expression in human breast cancer cell lines and other malignancies, J. Cell. Physiol. 186, 136-145. [16] Pawlowski, R., Muhl, S. M., Sulser, T., Krek, W., Moch, H., and Schraml, P. (2013) Loss of PBRM1 expression is associated with renal cell carcinoma progression, Int. J. Cancer 132, E11-17. [17] Pena-Llopis, S., Christie, A., Xie, X. J., and Brugarolas, J. (2013) Cooperation and antagonism among cancer genes: the renal cancer paradigm, Cancer Res. 73, 41734179. [18] Kapur, P., Pena-Llopis, S., Christie, A., Zhrebker, L., Pavia-Jimenez, A., Rathmell, W. K., Xie, X. J., and Brugarolas, J. (2013) Effects on survival of BAP1 and PBRM1 mutations in sporadic clear-cell renal-cell carcinoma: a retrospective analysis with independent validation, Lancet Oncol. 14, 159-167. [19] Brugarolas, J. (2013) PBRM1 and BAP1 as novel targets for renal cell carcinoma, Cancer J 19, 324-332. [20] Hakimi, A. A., Chen, Y. B., Wren, J., Gonen, M., Abdel-Wahab, O., Heguy, A., Liu, H., Takeda, S., Tickoo, S. K., Reuter, V. E., Voss, M. H., Motzer, R. J., Coleman, J. A., Cheng, E. H., Russo, P., and Hsieh, J. J. (2013) Clinical and pathologic impact of select chromatin-modulating tumor suppressors in clear cell renal cell carcinoma, Eur. Urol. 63, 848-854.


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[21] The Cancer Genome Atlas Research, N., ,. (2013) Comprehensive molecular characterization of clear cell renal cell carcinoma, Nature (London, U. K.) 499, 4349. [22] Czarnecka, A. M., Kornakiewicz, A., Kukwa, W., and Szczylik, C. (2014) Frontiers in clinical and molecular diagnostics and staging of metastatic clear cell renal cell carcinoma, Future Oncol. 10, 1095-1111. [23] Gossage, L., Murtaza, M., Slatter, A. F., Lichtenstein, C. P., Warren, A., Haynes, B., Marass, F., Roberts, I., Shanahan, S. J., Claas, A., Dunham, A., May, A. P., Rosenfeld, N., Forshew, T., and Eisen, T. (2014) Clinical and pathological impact of VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in clear cell renal cell carcinoma, Genes, Chromosomes Cancer 53, 38-51. [24] Gao, W., Li, W., Xiao, T., Liu, X. S., and Kaelin, W. G. (2017) Inactivation of the PBRM1 tumor suppressor gene amplifies the HIF-response in VHL−/− clear cell renal carcinoma, Proc. Natl. Acad. Sci. U. S. A. 114, 1027-1032. [25] Chowdhury, B., Porter, E. G., Stewart, J. C., Ferreira, C. R., Schipma, M. J., and Dykhuizen, E. C. (2016) PBRM1 Regulates the Expression of Genes Involved in Metabolism and Cell Adhesion in Renal Clear Cell Carcinoma, PLOS ONE 11, e0153718. [26] Porter, E. G., and Dykhuizen, E. C. (2017) Individual Bromodomains of Polybromo-1 Contribute to Chromatin Association and Tumor Suppression in Clear Cell Renal Carcinoma, J. Biol. Chem. 292, 2601-2610. [27] Peña-Llopis, S., Vega-Rubín-de-Celis, S., Liao, A., Leng, N., Pavía-Jiménez, A., Wang, S., Yamasaki, T., Zhrebker, L., Sivanand, S., Spence, P., Kinch, L., Hambuch, T., Jain, S., Lotan, Y., Margulis, V., Sagalowsky, A. I., Summerour, P. B., Kabbani, W., Wong, S. W. W., Grishin, N., Laurent, M., Xie, X.-J., Haudenschild, C. D., Ross, M. T., Bentley, D. R., Kapur, P., and Brugarolas, J. (2012) BAP1 loss defines a new class of renal cell carcinoma, Nat. Genet. 44, 751759. [28] Schaaper, R. M., Danforth, B. N., and Glickman, B. W. (1986) Mechanisms of spontaneous mutagenesis: An analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene, J. Mol. Biol. 189, 273-284. [29] Fan, H., and Chu, J.-Y. (2007) A Brief Review of Short Tandem Repeat Mutation, Genomics, Proteomics Bioinf. 5, 7-14. [30] Gragg, H., Harfe, B. D., and Jinks-Robertson, S. (2002) Base Composition of Mononucleotide Runs Affects DNA Polymerase Slippage and Removal of Frameshift Intermediates by Mismatch Repair in Saccharomyces cerevisiae, Mol. Cell. Biol. 22, 8756-8762. [31] Goula, A.-V., and Merienne, K. (2013) Abnormal Base Excision Repair at Trinucleotide Repeats Associated with Diseases: A Tissue-Selective Mechanism, Genes 4, 375-387. [32] Lang, G. I., Parsons, L., and Gammie, A. E. (2013) Mutation rates, spectra, and genome-wide distribution of spontaneous mutations in mismatch repair deficient yeast, G3: Genes, Genomes, Genet. 3, 1453-1465. [33] Brownlee, Peter M., Chambers, Anna L., Cloney, R., Bianchi, A., and Downs, Jessica A. (2014) BAF180 Promotes Cohesion and Prevents Genome Instability and Aneuploidy, Cell Rep. 6, 973-981.



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[34] Griffiths, A., Miller, J., and Suzuki, D. (2000) Aneuploidy, In Introduction to Genetic Analysis 7th ed., W. H. Freeman, New York. [35] Brownlee, P. M., Meisenberg, C., and Downs, J. A. (2015) The SWI/SNF chromatin remodelling complex: Its role in maintaining genome stability and preventing tumourigenesis, DNA Repair 32, 127-133. [36] Kakarougkas, A., Downs, J. A., and Jeggo, P. A. (2015) The PBAF chromatin remodeling complex represses transcription and promotes rapid repair at DNA double-strand breaks, Mol. Cell. Oncol. 2, e970072. [37] Downs, J. A. (2007) Chromatin structure and DNA double-strand break responses in cancer progression and therapy, Oncogene 26, 7765-7772. [38] Kakarougkas, A., Ismail, A., Chambers, Anna L., Riballo, E., Herbert, Alex D., Künzel, J., Löbrich, M., Jeggo, Penny A., and Downs, Jessica A. (2014) Requirement for PBAF in Transcriptional Repression and Repair at DNA Breaks in Actively Transcribed Regions of Chromatin, Mol. Cell 55, 723-732. [39] Kuo, L. J., and Yang, L.-X. (2008) γ-H2AX - A Novel Biomarker for DNA Doublestrand Breaks, In Vivo 22, 305-309. [40] Matsuoka, S., Ballif, B. A., Smogorzewska, A., McDonald, E. R., Hurov, K. E., Luo, J., Bakalarski, C. E., Zhao, Z., Solimini, N., Lerenthal, Y., Shiloh, Y., Gygi, S. P., and Elledge, S. J. (2007) ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage, Science 316, 1160-1166. [41] Kannouche, P. L., Wing, J., and Lehmann, A. R. (2004) Interaction of Human DNA Polymerase η with Monoubiquitinated PCNA: A Possible Mechanism for the Polymerase Switch in Response to DNA Damage, Mol. Cell 14, 491-500. [42] Niimi, A., Hopkins, S. R., Downs, J. A., and Masutani, C. (2015) The BAH domain of BAF180 is required for PCNA ubiquitination, Mutat. Res. 779, 16-23. [43] Niimi, A., Chambers, A. L., Downs, J. A., and Lehmann, A. R. (2012) A role for chromatin remodellers in replication of damaged DNA, Nucleic Acids Res. 40, 7393-7403. [44] Cohen, S. M., Chastain, P. D., Rosson, G. B., Groh, B. S., Weissman, B. E., Kaufman, D. G., and Bultman, S. J. (2010) BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression, Nucleic Acids Res. 38, 6906-6919. [45] Huang, L., Peng, Y., Zhong, G., Xie, W., Dong, W., Wang, B., Chen, X., Gu, P., He, W., Wu, S., Lin, T., and Huang, J. (2015) PBRM1 suppresses bladder cancer by cyclin B1 induced cell cycle arrest, Oncotarget 6, 16366-16378. [46] Xiao, X., Tang, C., Xiao, S., Fu, C., and Yu, P. (2013) Enhancement of proliferation and invasion by MicroRNA-590-5p via targeting PBRM1 in clear cell renal carcinoma cells, Oncol Res 20, 537-544. [47] Murakami, A., Wang, L., Kalhorn, S., Schraml, P., Rathmell, W. K., Tan, A. C., Nemenoff, R., Stenmark, K., Jiang, B. H., Reyland, M. E., Heasley, L., and Hu, C. J. (2017) Context-dependent role for chromatin remodeling component PBRM1/BAF180 in clear cell renal cell carcinoma, Oncogenesis 6, e287. [48] American Cancer Society: Cancer Facts and Figures 2010. Atlanta, GA: American Cancer Society, 2010. [49] Ho, T. H., Kapur, P., Joseph, R. W., Serie, D. J., Eckel-Passow, J. E., Parasramka, M., Cheville, J. C., Wu, K. J., Frenkel, E., Rakheja, D., Stefanius, K., Brugarolas, J., and Parker, A. S. (2015) Loss of PBRM1 and BAP1 Expression Is Less Common in


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Non–Clear Cell Renal Cell Carcinoma Than in Clear Cell Renal Cell Carcinoma, Urol. Oncol. 33, 23.e29-23.e14. [50] Duns, G., Hofstra, R. M., Sietzema, J. G., Hollema, H., van Duivenbode, I., Kuik, A., Giezen, C., Jan, O., Bergsma, J. J., Bijnen, H., van der Vlies, P., van den Berg, E., and Kok, K. (2012) Targeted exome sequencing in clear cell renal cell carcinoma tumors suggests aberrant chromatin regulation as a crucial step in ccRCC development, Hum. Mutat. 33, 1059-1062. [51] Piva, F., Santoni, M., Matrana, M. R., Satti, S., Giulietti, M., Occhipinti, G., Massari, F., Cheng, L., Lopez-Beltran, A., Scarpelli, M., Principato, G., Cascinu, S., and Montironi, R. (2015) BAP1, PBRM1 and SETD2 in clear-cell renal cell carcinoma: molecular diagnostics and possible targets for personalized therapies, Expert Rev. Mol. Diagn. 15, 1201-1210. [52] da Costa, W. H., Rezende, M., Carneiro, F. C., Rocha, R. M., da Cunha, I. W., Carraro, D. M., Guimaraes, G. C., and de Cassio Zequi, S. (2014) Polybromo-1 (PBRM1), a SWI/SNF complex subunit is a prognostic marker in clear cell renal cell carcinoma, BJU Int. 113, E157-163. [53] Joseph, R. W., Kapur, P., Serie, D. J., Parasramka, M., Ho, T. H., Cheville, J. C., Frenkel, E., Parker, A. S., and Brugarolas, J. (2016) Clear Cell Renal Cell Carcinoma Subtypes Identified by BAP1 and PBRM1 Expression, J. Urol. (N. Y., NY, U. S.) 195, 180-187. [54] Kluzek, K., Bluyssen, H. A., and Wesoly, J. (2015) The epigenetic landscape of clearcell renal cell carcinoma, Journal of Kidney Cancer and VHL 2, 90-104. [55] Ibragimova, I., Maradeo, M. E., Dulaimi, E., and Cairns, P. (2013) Aberrant promoter hypermethylation of PBRM1, BAP1, SETD2, KDM6A and other chromatinmodifying genes is absent or rare in clear cell RCC, Epigenetics 8, 486-493. [56] Wang, Y., Guo, X., Bray, M. J., Ding, Z., and Zhao, Z. (2016) An integrative genomics approach for identifying novel functional consequences of PBRM1 truncated mutations in clear cell renal cell carcinoma (ccRCC), BMC Genomics 17 Suppl 7, 515. [57] UniProtKB - Q64259 (VHL_RAT) [58] Nam, S. J., Lee, C., Park, J. H., and Moon, K. C. (2015) Decreased PBRM1 expression predicts unfavorable prognosis in patients with clear cell renal cell carcinoma, Urol. Oncol. 33, 340.e9-340.e16. [59] Joseph, R. W., Kapur, P., Serie, D. J., Parasramka, M., Ho, T. H., Cheville, J. C., Frenkel, E., Parker, A. S., and Brugarolas, J. (2015) Clear Cell Renal Cell Carcinoma Subtypes Identified by BAP1 and PBRM1 Expression, J. Urol. (N. Y., NY, U. S.) 195, 180-187. [60] Fay, A. P., de Velasco, G., Ho, T. H., Van Allen, E. M., Murray, B., Albiges, L., Signoretti, S., Hakimi, A. A., Stanton, M. L., Bellmunt, J., McDermott, D. F., Atkins, M. B., Garraway, L. A., Kwiatkowski, D. J., and Choueiri, T. K. (2016) Whole-Exome Sequencing in Two Extreme Phenotypes of Response to VEGFTargeted Therapies in Patients With Metastatic Clear Cell Renal Cell Carcinoma, J. Natl. Compr. Cancer Network 14, 820-824. [61] Gulati, S., Martinez, P., Joshi, T., Birkbak, N. J., Santos, C. R., Rowan, A. J., Pickering, L., Gore, M., Larkin, J., Szallasi, Z., Bates, P. A., Swanton, C., and Gerlinger, M. (2014) Systematic evaluation of the prognostic impact and



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intratumour heterogeneity of clear cell renal cell carcinoma biomarkers, Eur. Urol. 66, 936-948. [62] Li, L., Fan, X. S., Xia, Q. Y., Rao, Q., Liu, B., Yu, B., Shi, Q. L., Lu, Z. F., and Zhou, X. J. (2014) Concurrent loss of INI1, PBRM1, and BRM expression in epithelioid sarcoma: implications for the cocontributions of multiple SWI/SNF complex members to pathogenesis, Hum. Pathol. 45, 2247-2254. [63] Chbani, L., Guillou, L., Terrier, P., Decouvelaere, A. V., Gregoire, F., TerrierLacombe, M. J., Ranchere, D., Robin, Y. M., Collin, F., Freneaux, P., and Coindre, J. M. (2009) Epithelioid sarcoma: a clinicopathologic and immunohistochemical analysis of 106 cases from the French sarcoma group, Am. J. Clin. Pathol. 131, 222227. [64] Hosseinzadeh, P., and Cheung, F. (2009) Epithelioid Sarcoma, p Epithelioid sracoma. [65] Pereira, B., Chin, S.-F., Rueda, O. M., Vollan, H.-K. M., Provenzano, E., Bardwell, H. A., Pugh, M., Jones, L., Russell, R., Sammut, S.-J., Tsui, D. W. Y., Liu, B., Dawson, S.-J., Abraham, J., Northen, H., Peden, J. F., Mukherjee, A., Turashvili, G., Green, A. R., McKinney, S., Oloumi, A., Shah, S., Rosenfeld, N., Murphy, L., Bentley, D. R., Ellis, I. O., Purushotham, A., Pinder, S. E., Børresen-Dale, A.-L., Earl, H. M., Pharoah, P. D., Ross, M. T., Aparicio, S., and Caldas, C. (2016) The somatic mutation profiles of 2,433 breast cancers refine their genomic and transcriptomic landscapes, Nat. Commun. 7, 11479. [66] Mo, D., Li, C., Liang, J., Shi, Q., Su, N., Luo, S., Zeng, T., and Li, X. (2015) Low PBRM1 identifies tumor progression and poor prognosis in breast cancer, Int. J. Clin. Exp. Pathol. 8, 9307-9313. [67] Yoshikawa, Y., Sato, A., Tsujimura, T., Otsuki, T., Fukuoka, K., Hasegawa, S., Nakano, T., and Hashimoto-Tamaoki, T. (2015) Biallelic germline and somatic mutations in malignant mesothelioma: Multiple mutations in transcription regulators including mSWI/SNF genes, Int. J. Cancer 136, 560-571. [68] Borczuk, A. C., Pei, J., Taub, R. N., Levy, B., Nahum, O., Chen, J., Chen, K., and Testa, J. R. (2016) Genome-wide analysis of abdominal and pleural malignant mesothelioma with DNA arrays reveals both common and distinct regions of copy number alteration, Cancer Biol. Ther. 17, 328-335. [69] Yoshikawa, Y., Sato, A., Tsujimura, T., Morinaga, T., Fukuoka, K., Yamada, S., Murakami, A., Kondo, N., Matsumoto, S., Okumura, Y., Tanaka, F., Hasegawa, S., Hashimoto-Tamaoki, T., and Nakano, T. (2011) Frequent deletion of 3p21.1 region carrying semaphorin 3G and aberrant expression of the genes participating in semaphorin signaling in the epithelioid type of malignant mesothelioma cells, Int. J. Oncol. 39, 1365-1374. [70] Yoshikawa, Y., Emi, M., Hashimoto-Tamaoki, T., Ohmuraya, M., Sato, A., Tsujimura, T., Hasegawa, S., Nakano, T., Nasu, M., Pastorino, S., Szymiczek, A., Bononi, A., Tanji, M., Pagano, I., Gaudino, G., Napolitano, A., Goparaju, C., Pass, H. I., Yang, H., and Carbone, M. (2016) High-density array-CGH with targeted NGS unmask multiple noncontiguous minute deletions on chromosome 3p21 in mesothelioma, Proc. Natl. Acad. Sci. U. S. A. 113, 13432-13437. [71] Sicklick, J. K., Fanta, P. T., Shimabukuro, K., and Kurzrock, R. (2016) Genomics of gallbladder cancer: the case for biomarker-driven clinical trial design, Cancer Metastasis Rev. 35, 263-275.


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Table 1: The frequency of PBRM1 alterations in different types of cancers. PBRM1, the gene for BAF180, has been found to be mutated in more than 30 types of cancers. Data was collected from cBioPortal February 2017.5, 6

Cancer

Frequency of alterations in PBRM1

Clear cell renal cell carcinoma (ccRCC) Neuroendocrine Prostate Carcinoma Cutaneous Squamous Cell Carcinoma Breast Cancer Bladder Cancer Uterine Carcinomas Stomach Adenocarcinoma Diffuse Large B-Cell Lymphoma Esophageal Carcinoma Pancreatic Carcinomas Prostate Adenocarcinoma Lung Carcinoma Melanomas Mesothelioma Papillary RCC Colorectal Adenocarcinoma Head and Neck Carcinoma Thymic Carcinomas Extrahepatic Cholangiocarcinomas Intrahepatic Cholangiocarcinomas Hepatocellular Carcinoma Thyroid Carcinomas non ccRCC Gallbladder Carcinoma Cervical Squamous Cell Carcinoma Sarcoma Adenoid Cystic Carcinoma Ovarian Carcinoma Neuroblastoma Chromophobe Renal Cell Carcinoma Gliomas Adrenocortical Carcinoma Chronic Lymphocytic Leukemia Pediatric Ewing Sarcoma Multiple Myeloma


39% 24% 10% 2-14% 2-10% 4-9% 3-8% 3-8% 2-8% 1-7% 1-7% 2-6% 1-6% 5% 4% 3-4% 2-4% 2-4% 3.5-5% 11-26% 1-4% 3% 3% 3% 3% 1-3% 2% 2% 2% 2% 1-2% 1% 1% 1% 1%


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Table 2: Effects of BAF180 Knockout on Cell Lines. Reported results on the effect of BAF180 knockdown on cell proliferation differ. The origin of the cell lines are as follows: bladder carcinoma: UM-UC-3; renal cell carcinoma: ACHN, 786-O, SN12C, TK10, UMRC2, KC-10, 769-P, RCC10, A498, A704; Clear cell renal cell carcinoma: Caki1. Knockdown BAF180, increased proliferation Author

Cell Line ACHN

Cell line +/- for BAF180 +

Varelab

KD method siRNA

Varelab

786-O

+

siRNA

Varelab

SN12C

+

siRNA

Varelab

TK10

+

siRNA

Xiaod

786-O

+

Xiaod

ACHN

+

miRNA targeting PBRM1 miRNA targeting PBRM1

Knockdown BAF180, decrease proliferation Gaoc

UMRC2

+

shRNA

Murakamie

786-O

+

shRNA

Murakamie

KC-12

+

shRNA

Murakamie

769-P

+

shRNA

Murakamie

RCC10

+

shRNA

Murakamie

A498

+

shRNA

Knockdown BAF180, no effect on proliferation Gaoc

786-O

+

Gaoc

SN12C

+

shRNA

Chowdhuryf Caki1

+

Chowdhuryf A498

+

lentiviral

Varelab

-

siRNA

A704

Table 3: Differing results of the effect of BAF180 re-expression on cellular proliferation. The origin of the cell lines are as follows: bladder carcinoma: UM-UC-3, 5637; renal cell a: Huang, et al. (2015).42 b: Varela, et al. (2011).2 c: Gao, et al. (2017).22 d: Xiao, et al. (2013).43 e: Murakami, et al. (2017).44 f: Chowdhury, et al. (2016).23


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carcinoma: ACHN, 786-O, A704, SKRC20, SKR24; Clear cell renal cell carcinoma: Caki2, RCC4; breast cancer: HCC1143; endometrial adenocarcinoma: EJ. Re-introduction of BAF180, decrease proliferation Author Cell Line Cell line +/- KD Method for BAF180 Gaoa UMRC2 + shRNA Murakamib Murakamib Murakamib Murakamib Murakamib

786-O KC-12 769-P RCC10 A498

a: Gao, et al. (2017).22 b: Murakami, et al. (2017).44

+ + + + +

shRNA shRNA shRNA shRNA shRNA



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Figure 1: Chart of the frequency of different types of mutations of PBRM1 (BAF180) in cancers. Data was collected from COSMIC database.7 Figure 2: The domain organization of wild-type BAF180 (A) and various truncated mutants observed in cancers. The sequences presented here are the predicted coding sequences of mutations in the gene.


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BAF180: Its Roles in DNA Repair and Consequences in Cancer

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