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FEBS Letters 585 (2011) 3185–3190

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Review Unraveling the enigmatic complexities of BRMS1-mediated suppression ⇑ Douglas R. Hurst a, , Danny R. Welch b,1

a Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA b Department of Cancer Biology, Kansas University Medical Center, Kansas City, KS, USA

article info abstract

Article history: Expression of BRMS1 causes dramatic suppression of metastasis in multiple in vivo model systems. Received 4 July 2011 As we gain further insight into the biochemical mechanisms of BRMS1, we appreciate the impor- Revised 28 July 2011 tance of both molecular and cellular context for functional metastasis suppression. BRMS1 associ- Accepted 29 July 2011 ates with large chromatin remodeling complexes including SIN3:HDAC which are powerful Available online 5 August 2011 epigenetic regulators of expression. Additionally, BRMS1 inhibits the activity of NFjB, a Edited by Wilhelm Just well-known transcription factor that plays significant roles in tumor progression. Moreover, BRMS1 coordinately regulates the expression of metastasis-associated microRNA known as metastamir. How these biochemical mechanisms and biological pathways are linked, either directly or indirectly, Keywords: Metastasis suppression and the influence of molecular and cellular context, are critical considerations for the discovery of BRMS1 novel therapeutic targets for the most deadly aspect of tumor progression-metastasis. Metastamir Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Epigenetic Chromatin remodeling NFjB

1. Introduction MDA-MB-435 (435-BRMS1) cells demonstrated decreased inci- dence and number of metastases to lung and lymph nodes. Ortho- BRMS1 ( Metastasis Suppressor 1) was first de- topic tumor growth rates were similar to parental controls albeit a scribed in 2000 as a gene that suppressed metastasis without pre- lag in growth in the 435-BRMS1 cells for approximately 1 week. venting primary tumor growth [1]. The discovery of BRMS1 was MDA-MB-231 breast cancer cells expressing BRMS1 (231- accomplished using a combination of techniques that began with BRMS1) showed a significant expression-dependent decrease in prior karyotypic observations showing that long and short arms metastatic potential when cells were injected into the lateral tail of 11 are often sites of amplification/deletion and veins of athymic mice. These cells were still capable of forming are associated with the progression of breast cancer [2]. Phillips tumors in the mammary fat pads with similar growth rates as and colleagues hypothesized that transferring an intact copy of the parent controls. These results demonstrated that BRMS1 sup- neomycin-tagged into metastatic breast cancer presses metastasis without blocking tumorigenicity, satisfying cells (MDA-MB-435; neo11/435 with introduced chromosome the functional definition of a metastasis suppressor gene. 11) would alter metastatic potential [3]. Using the technique of Since then, many labs using different model systems showed microcell-mediated chromosomal transfer (MMCT), they found BRMS1 to be a metastasis suppressor for breast carcinoma, mela- significantly fewer metastases in the neo11/435 compared to noma, ovarian carcinoma, and non-small cell lung carcinoma parental cells with no significant changes in orthotopic tumor inci- (reviewed in [4]). Although not formally demonstrated to suppress dence/growth. Following these experiments, Seraj et al. used dif- bladder cancer, Seraj et al. found reduced BRMS1 mRNA in a highly ferential display RT–PCR (DD-RTPCR) to identify transcripts with metastatic variant of a human bladder carcinoma cell line T24T P5-fold higher expression in the neo11/435 [1]. One of these tran- compared to the less metastatic parental line T24 [5]. This pattern scripts was sequenced, cloned, and transfected into MDA-MB-435 of expression was also noted for one other metastasis suppressor, and MDA-MB-231 metastatic breast cancer cells. When injected RhoGDI2. Robertson et al. suggested the presence of a tumor into mammary fat pads of athymic mice, BRMS1-transfected suppressor on the long arm of chromosome 11 in human melano- mas and BRMS1 was subsequently shown to be a bona fide mela- ⇑ Corresponding author. Address: Department of Pathology, 1670 University noma metastasis suppressor gene [6,7]. Quantitative real-time Blvd., VH-G019, Birmingham, AL 35294-0019, USA. Fax: +1 205 975 1126. RT–PCR revealed that BRMS1 mRNA expression is high in melano- E-mail addresses: [email protected] (D.R. Hurst), [email protected] (D.R. cytes, reduced in early stage -derived cell lines, and Welch). barely detectable in advanced or metastatic melanoma cell lines. 1 Address: Department of Cancer Biology, 3901 Rainbow Blvd., Robinson 4030, Introducing BRMS1 into the highly metastatic melanoma cell line Kansas City, KS 66160, USA. Fax: +1 913 588 4701.

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.07.045 3186 D.R. Hurst, D.R. Welch / FEBS Letters 585 (2011) 3185–3190

C8161 significantly inhibited metastases in both spontaneous and metastasis was significantly suppressed while orthotopic tumor experimental assays without preventing orthotopic tumor growth. growth was indistinguishable from parental or vector-only trans- Similar findings were noted for models of ovarian carcinoma [8] fectants [19,20]. Interaction with HDAC1 was demonstrated by and NSCLC [9]. co-immunoprecipitation suggesting an association with similar The BRMS1 gene maps to 11q13.1–13.2 [1]. This is within a re- chromatin remodeling complexes. Altogether, these results suggest gion that is often lost in late stage breast cancers and is near sites that the mouse ortholog has similar functionalities as human that are among the most common amplifications and deletions BRMS1. Interestingly, a genetic mapping study showed that meta- associated with breast cancer progression [2]. Within the 50 up- static ability of mammary tumors using inbred mouse strains was stream region there are several putative regulatory elements linked to allelic variations of Brms1 [21,22]. Recent, as yet unpub- including GATA-1, CREB, GATA-2, and CdxA. There is no TATA lished data from our laboratories shows that transgenic expression box suggesting that transcription of BRMS1 proceeds in a TATA- of Brms1 in F1 generation MMTV-PyMT crosses yields significantly independent mechanism [1]. Two putative CpG islands were iden- reduced lung metastases while not affecting primary tumor devel- tified in the promoter region of BRMS1 (3477 to 2214 and 531 opment (L.M. Cook, D.R. Hurst, and D.R. Welch, in preparation). to +608) and using methylation specific PCR it was determined the Although it has been clearly demonstrated that BRMS1 sup- distal CpG island (3477 to 2214) was methylated in tumori- presses metastasis, determining specific functions and delineating genic and metastatic cell lines leading to the suppression of BRMS1 mechanisms of suppression have proven to be difficult. BRMS1 expression [10]. does not completely block any single step in the metastatic cas- In addition to the wild-type transcript, the mRNA for several cade. Rather, it inhibits several hallmarks of metastasis leading to splice variants was identified. Wild-type BRMS1 has 10 exons span- 80–90% inhibition in mouse models of metastasis. Proteomic and ning 741 nucleotides. BRMS1.v2 contains an alternative splice site gene array studies have identified multiple changes in cod- in exon 10, BRMS1.v3 has an alternative splice site in exon 5, lacks ing and non-coding when BRMS1 is expressed [23–26]. exons 6–9, and alternatively splices exon 10, and BRMS1.v4 lacks These expression changes may be responsible for driving the mul- exon 9 [11]. These splice variants are differentially expressed in tiple phenotypic changes observed including invasion and migra- metastatic compared to non-metastatic breast cancer cell lines. tion, restoration of gap junctional intercellular communication However, the functional relevance of each variant is presently un- (GJIC), and anoikis. Two mechanisms have been identified that known since translation to protein has not yet been verified. could lead to changes that may be linked includ- The BRMS1 protein is 246 amino acids and is predicted to be ing interaction with chromatin remodeling complexes and inhibi- 28.5 kDa [1]; however, it migrates closer to 35 kDa on SDS–PAGE tion of NFjB activity. These are discussed in greater detail below. [11]. This is presumably due to the highly charged N-terminal glu- tamate rich region within the first 50 amino acids. There are two coiled-coil regions between AA51-81 and AA147-180 and two pre- 2. Regulation of gene expression...but not a transcription factor dicted nuclear localization sequences between AA198-205 and AA239-245. The first NLS is required for localization to the nucleus The coordinate expression of genetic programs is necessary to [12,13]. It is not yet clear if the second NLS has any functional rel- enable a cancer cell to complete all the required steps of the evance to localization and/or trafficking although we have preli- metastatic cascade [27–29]. Molecules that regulate gene tran- minary data suggesting roles in nuclear retention (D.R. Hurst and scription could dramatically impact the metastatic process. There D.R. Welch, unpublished). Several putative phosphorylation sites have been multiple reports showing that BRMS1 alters the exist however there have been no reports of phosphorylation. expression level of metastasis associated genes. The questions Other members of the SIN3 complex including BRMS1 family that have not yet been fully answered are how? and which are members have been shown to be phosphorylated by cell cycle reg- the most relevant complexes? There is no evidence for BRMS1 ulatory kinases [14,15]. However, whether BRMS1 phosphoryla- functioning as a transcription factor. However, there is concrete tion occurs and whether the modifications by cell cycle evidence that it associates with transcriptional repressive chro- machinery are responsible for, or a consequence of, different matin remodeling complexes. BRMS1 presumably regulates the growth in orthotopic versus ectopic tissues remains to be deter- transcription of genes by interaction with large SIN3:HDAC chro- mined. There are no predicted glycosylation sites. The protein is matin remodeling complexes through the direct interaction with stabilized by HSP90 and turnover is proteasome dependent [16]. AT rich interacting domain 4A (ARID4A) and SUDS3 leading to BRMS1 is part of a family that includes suppressor of defective the suppression of basal transcription [30,31]. These findings silencing 3 (SUDS3 or mSds3) and BRMS1-like (BRMS1L or p40) have been confirmed by protein–protein interaction studies of [17,18]. A conserved region known as the Sds3-like domain is pres- other known to be a part of these complexes in addition ent in each of these proteins. This region has not yet been function- to BRMS1 [18,32–36]. ally defined although it is likely important for protein–protein Chromatin remodeling complexes are diverse and incompletely interactions. BRMS1 is 23% identical and 49% similar in amino acid characterized. Much more is known regarding lower species and, sequence to SUDS3 and 57% identical and 79% similar to BRMS1L. although there are many similarities with the complexes in mam- Expression of SUDS3 in metastatic breast cancer cell lines does mals, they are much more complex and exist in many forms com- not significantly suppress metastasis as would be predicted, sug- pared to yeast or lower organisms. For example, in Saccharomyces gesting that, although similar, these proteins have distinct func- cerevisiae, two distinct SIN3 complexes have been isolated known tions and do not functionally compensate for one another [17]. as Rpd3L and Rpd3S (note: a yeast ortholog of BRMS1 has not been High at both the DNA and amino acid levels identified) [37]. However, we and others have demonstrated that of BRMS1 is found in multiple species. In particular, the murine SIN3 complexes in mammalian cells exist in many sizes and are ortholog (Brms1) is 85% homologous at the DNA level and the ami- not limited to only two. The mammalian core components have no acid sequence is 95% identical [19]. In related subpopulations of been detected in multiple size exclusion FPLC fractions from cell tumor cells derived from a single mammary carcinoma, but vary- lysates in sizes ranging from several hundred kDa to those exceed- ing in metastatic potential, Brms1 expression was inversely ing 2 MDa. Because these components are found in a range of sizes correlated with increased aggressiveness. Importantly, Brms1 demonstrates the existence of multiple complexes. Understanding functions as a metastasis suppressor in murine mammary the composition of these differently sized complexes will be carcinomas. Upon transfection of Brms1 into 66c14 or 4T1, important to determine how they function. D.R. Hurst, D.R. Welch / FEBS Letters 585 (2011) 3185–3190 3187

Many components of the SIN3 complexes play tumor suppres- nearly every cell compartment. They have many diverse functions sive roles in cancer. Typical studies have been done by ectopically since they are not limited to deacetylation of chromatin modifying expressing one gene and characterizing the nature of tumor inhibi- histones in the nucleus. It is important to understand how HDACs tion. Often, clinical studies of expression of a protein in cancer pro- and other enzymes are functioning in cancer progression so that gression do not correlate because it is important to define the inhibitors can be used appropriately. Matrix metalloproteinase nature of the complex as a whole and not an individual protein. (MMP) inhibitors were rapidly advanced into clinical trials because How the total composition of SIN3 complexes is playing a role in they very effectively inhibited invasion [43]. Unfortunately, clinical cancer is currently poorly-defined. However, it has been shown trials with MMP inhibitors failed. The failure was in part due to the that altering the composition of SIN3 complexes by expressing fact that the biology of MMPs was not well understood and their BRMS1 or BRMS1 mutants results in 90% suppression of metasta- activities were much more diverse than originally thought. A sim- sis [30]. The function of these SIN3 complexes is dependent upon ilar situation could occur with the HDAC inhibitors if we fail to the specific modification of the protein composition and further understand the biology and fail to identify proper targets specifi- emphasizes the need to characterize these complexes as a whole cally for metastasis. rather than by individual protein expression. Recently Smith et al. showed that select HDAC inhibitors cause Gene expression regulated through chromatin remodeling com- changes to the composition of SIN3 complexes independent of plexes occurs by multiple mechanisms [38]. In simple terms, HDAC inhibition that could lead to significant changes in complex although it is much more complicated, histone acetyltransferase specificity [34]. This may lead to differential efficacy of HDAC (HAT) containing complexes add acetyl moieties to specific histone inhibitors in the treatment of cancer caused by these secondary ef- lysine side chains resulting in an open conformation of DNA fects. Since many of these inhibitors are being tested in clinical tri- (euchromatin) that is more accessible to transcription factors. His- als, it is important to understand how SIN3 complexes are tone deacetylase (HDAC) containing complexes remove acetyl moi- functioning and how the composition could be affecting enzyme eties from the lysine side chains resulting in a more compact activity. It is not completely clear how (or even if) BRMS1 alters conformation of DNA (heterochromatin) causing a repression of HDAC activity although some reports suggest it functions by transcription. SIN3 (switch-independent 3) is a scaffold that is recruiting HDAC1 to enhance HDAC activity at specific locations well-known for recruitment of HDACs capable of transcriptional [44,45]. silencing [39,40]. While the majority of current studies with chro- The recruitment of HDAC1, and presumably SIN3 complexes, matin remodeling complexes are focused on HAT or HDAC activity, by BRMS1 leads to inhibition of NFjB activity. BRMS1 has been these complexes recruit many other protein and DNA modifying shown to recruit HDAC1 to NFjB consensus binding regions using enzymes in addition to deacetylases including glycosyl transfer- ChIP assays [44,45]. It has been suggested that HDAC1 leads to H3 ases and methyl transferases. It is now realized that many of these deacetylation and reduced binding of p65 at the NFjB site of enzymatic modifications are linked and influence the function of specific promoters. These data imply that BRMS1 is recruited, pos- protein and DNA interacting molecules. The functional specificity sibly via SIN3 complexes, to promoter binding regions where it (i.e., which genes they regulate and, as a result, the phenotypes inhibits HDAC1 activity leading to deacetylation of p65 and re- that are altered) of these complexes is dictated by their protein duced transactivation by NFjB. Acetylation is an important regula- composition and resulting enzyme activity. tory mechanism in BRMS1-dependent gene regulation of NFjB HDAC inhibitors are currently in clinical trials since they pro- transactivation. The p65 subunit of NFjB is acetylated at lysine mote apoptosis, inhibit invasion and angiogenesis, and cause cell residues by several co-activators including p300, CBP and p/CAF. cycle arrest [41,42]. However, the mechanisms for how HDAC K310 is a prominent residue playing a key role in p65 inhibitors are functioning are not clear. There are currently 11 zinc transcriptional regulation. Interestingly, BRMS1 abolishes TNF- containing HDACs and 7 NAD+-dependent HDACs that are found in dependent acetylation of p65 on K310 and results in a substantial

Fig. 1. Pathways regulated by BRMS1. The composition of the SIN3 complex dictates which genes are regulated. Multiple metastasis associated genes are regulated by BRMS1 including metastamir (green). The dotted lines indicate either indirect mechanisms or multiple steps not shown. References are provided in the text. 3188 D.R. Hurst, D.R. Welch / FEBS Letters 585 (2011) 3185–3190 decrease in the transactivation potential of p65 [45]. In that same by loss of estrogen or progesterone receptor (ER, PR) or expression study, BRMS1 promoted HDAC1 binding to the RelA/p65 subunit of of HER2 [54]. Laser capture microdissection is necessary to ensure NFjB where HDAC1 promoted the deacetylation of K310 on p65 at purity of the material from a heterogeneous mass and was used to NFjB binding sites at the promoter regions of cIAP2 and Bfl-1/A1 show that BRMS1 localization shifting from nuclear to cytoplasmic leading to the loss of NFjB-dependent transcriptional activation. is associated with highly proliferative ER-negative breast cancers An interesting regulatory feedback loop with microRNA-146a [55]. More recently, decreased BRMS1 expression was identified (miR-146a) has been described for NFjB by Baltimore’s group in metastatic that is thought to contribute to angiogen- [46]. Upon stimulation by LPS, NFjB upregulates miR-146a which esis and melanoma progression [56]. in turn targets the translation of the upstream signaling molecules Consistent with the protein data, BRMS1 mRNA correlated with IRAK1 and TRAF6 [47]. BRMS1 also upregulates miR-146a and PR and loss of HER2 and loss of BRMS1 mRNA correlated with poor expression of miR-146a or miR-146b in MDA-MB-231 cells is suf- prognosis [57]. However, not all studies identified a correlation of ficient to suppress metastasis [48]. It is not clear how BRMS1 decreased BRMS1 expression with cancer progression. In fact, clin- upregulates miR-146a since it negatively regulates NFjB although ical studies with BRMS1 have been relatively inconsistent with re- it could be dependent on the particular environmental stimulus. gard to patient survival and likelihood of metastasis. The Other metastasis-associated microRNAs (metastamir) have been inconsistencies are thought to occur mainly because most clinical found to be coordinately regulated by BRMS1 [23]. Molecular path- studies were performed with measurements of mRNA; and it has ways, including metastamir, regulated by BRMS1 (and presumably been observed that BRMS1 protein and mRNA do not necessarily SIN3 complexes) are beginning to be elucidated that significantly correlate [11]. As techniques are more refined and the target pop- impact hallmarks of metastasis [48–51] (Fig. 1). Understanding ulations are more defined, studies with BRMS1 are starting to be the biochemical mechanisms of BRMS1:SIN3:NFjB and how these stratified. Moreover, considering the biochemical mechanisms of molecules are affected by the microenvironment will prove impor- BRMS1 function, it is unlikely that BRMS1 expression alone will tant to map key pathological pathways. be useful as a prognostic indicator of metastatic potential in cancer patients. As is the case with the majority of cancer biomarkers, 3. BRMS1 itself is epigenetically regulated expression of sets of genes rather than individual genes is more useful to predict disease progression. This further supports the As described above it has become increasingly clear that epige- need to understand the nature of the protein complexes with netic modifications play significant roles in the regulation of genes which BRMS1 is interacting and functioning so that clinically rele- that drive cancer progression. In fact, the metastasis suppressor vant biomarkers may be identified. Future studies may show iden- gene nm23 is silenced through promoter methylation and re- tification of specific ‘complexome’ markers associated with cancer expression following treatment with 5-aza-20-deoxycytidine was progression and metastasis. found to cause a decrease in motility of the breast cancer cells [52]. Breast cancer cell lines with differing tumorigenic and meta- 5. Concluding thoughts static potential have been utilized to demonstrate decreased levels of BRMS1 with increasing aggressiveness [10]. The promoter re- It has become increasingly recognized that metastasis is a rele- gion of BRMS1 contains two putative CpG islands leading to the vant therapeutic target for cancer patients. Many genes involved in hypothesis that BRMS1 expression is downregulated by promoter metastasis are regulated epigenetically and are therefore influ- hypermethylation in advanced stages of cancer. Methylation spe- enced by their surrounding environments. Understanding how cific PCR (MSP) was used to show that metastatic cells are hyper- metastasis regulatory genes are regulated by the microenviron- methylated in the distal CpG island (3477 to 2214) of the ment in which tumor cells find themselves will be a significant BRMS1 promoter and treatment with 5-aza-20-deoxycytidine re- advancement to the understanding of the mechanisms of metasta- versed the methylation pattern leading to an increase in BRMS1 sis. By definition, metastasis suppressors are involved in differen- expression. However, one must be cautious since the expression le- tial responses to exogenous signals since cells grow in orthotopic vel of BRMS1 in the cell lines tested has not been noted to be di- but not ectopic locations. BRMS1 exemplifies how expressing and rectly proportional to the CpG methylation pattern. At this time non-expressing cells respond differentially to the same environ- it remains to be determined if there are feedback or feedforward mental cues [49]. Further dissection of the mechanisms by which regulatory loops involving the expression level of BRMS1 and the the metastasis suppressors control cellular responses to signals interconnectivity of HDAC and methyltransferase activity. (growth factors, matrix molecules, cell adhesion proteins, etc.) might allow design of new therapeutic strategies targeting metas- 4. Clinical relevance of BRMS1 expression tasis by controlling those interactions. BRMS1 and SIN3 chromatin remodeling complexes regulate Promoter hypermethylation of BRMS1 in aggressive tumor cells specific coding and non-coding genes, depending on the composi- might suggest that decreased expression of BRMS1 could serve as a tion of the complex. These complexes are likely regulated by sig- clinically relevant biomarker. Indeed, a significant number of nals from the environment surrounding the cell. We hypothesize lymph node metastases in multiple matched patient tissues were that complex composition is likely regulated by signals from the hypermethylated in the distal CpG island (3477 to 2214) [10]. environments surrounding tumor cells. As metastatic cells travel Hypermethylation occurred in 60% of the lymph node metastases to distant sites, they continuously receive different signals from and 45% of the primary tumors, suggesting that an increase in the changing microenvironments. Depending upon the signal(s) methylation coincides with cancer development and progression. received, different complexes would be assembled. Then, depend- A recent study examining circulating tumor cells isolated from ing on the complexes and the genes with which they interact, the peripheral blood of operable breast cancer patients identified pro-survival, pro-apoptotic, or senescence ‘‘operons’’ would be ini- hypermethylation of BRMS1 promoter sequences in patients with tiated. BRMS1 may, in fact, alter chromatin remodeling complex metastases compared to healthy control population [53]. Other assembly thereby leading to different cellular responses to the studies found that loss of BRMS1 protein has been correlated with changing microenvironment. Experiments are currently underway reduced disease-free survival when patient samples were stratified to test these hypotheses and corollaries. D.R. Hurst, D.R. Welch / FEBS Letters 585 (2011) 3185–3190 3189

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