Brick1 Is an Essential Regulator of Actin Cytoskeleton Required for Embryonic Development and Cell Transformation

Published OnlineFirst September 22, 2010; DOI: 10.1158/0008-5472.CAN-09-4491 Published OnlineFirst on October 26, 2010 as 10.1158/0008-5472.CAN-09-4491

Tumor and Stem Cell Biology Cancer Research Brick1 Is an Essential Regulator of Actin Cytoskeleton Required for Embryonic Development and Cell Transformation

Beatriz Escobar1, Guillermo de Cárcer1, Gonzalo Fernández-Miranda1, Alberto Cascón4, José J. Bravo-Cordero2, María C. Montoya2, Mercedes Robledo4, Marta Cañamero3, and Marcos Malumbres1

Abstract Brick1 (Brk1) is the less-studied component of the Wave/Scar pathway involved in the branched nucleation of actin fibers. The clinical relevance of Brk1 is emphasized by correlative data showing that Von Hippel-Lindau (VHL) patients that also lose the BRK1 gene are protected against the development of tumors. This contrasts with recent evidence suggesting that the Wave complex may function as an invasion suppressor in epithelial cancers. Here, we show that the downregulation of Brk1 results in abnormal actin stress fiber formation and vinculin distribution and loss of Arp2/3 and Wave proteins at the cellular protrusions. Brk1 is required for cell proliferation and cell transformation by oncogenes. In addition, Brk1 downregulation results in defective direc- tional migration and invasive growth in renal cell carcinoma cells as well as in other tumor cell types. Finally, genetic ablation of Brk1 results in dramatic defects in embryo compaction and development, suggesting an essential role for this protein in actin dynamics. Thus, genetic loss or inhibition of BRK1 is likely to be protective against tumor development due to proliferation and motility defects in affected cells. Cancer Res; 70(22); OF1–11. ©2010 AACR.

Introduction (Scar), and Brick1 (abbreviated as Brk1 and also known as HSPC300, hematopoietic stem progenitor cell 300; ref. 3). Actin nucleation is tightly regulated by the Wiskott- Whereas most members of the Wave/Scar complex have Aldrich syndrome protein (WASP) family Verprolin-homologous been widely studied in recent years, the relevance of the protein (Wave)/Scar (suppressor of cyclic AMP receptor) small protein Brk1 is mostly unknown. Brk1 was originally nucleation-promoting factor (1, 2). The Wave/Scar proteins described as a recessive mutation in the maize orthologue are components of the namesake multiprotein complex, which that results in several morphologic defects in leaf epithelia recruits actin monomers and triggers conformational changes (4). Arabidopsis Brk1 mutants show severe defects in tri- in Arp2/3 complex, bringing its actin-related protein subunits chome morphogenesis, where the participation of actin (Arp2 and Arp3) into closer register, possibly to mimic an actin reorganization is well documented (5–7). RNA interference dimer. The Wave/Scar complex consists of five proteins: Sra1 studies have suggested similar roles for Brk1 in Drosophila (specifically Rac1-associated protein 1; also known as CYFIP1 (8). The mammalian Brk1 orthologue (4) may also be a or Pir121), Nap1 (Nck-associated protein; also known as critical component of the activated Wave/Scar complex, Nap125, Nckap1, Kette or Hem2), Abi (Abl interactor), Wave and depletion of Brk1 in HeLa cells results in defective lamellipodia formation (9, 10). Recent data have suggested an invasion suppressor role for the Wave complex (11). Depletion of Sra1, Nap1, or Wave2 per- Authors' Affiliations: 1Cell Division and Cancer Group, 2Confocal Microscopy and Cytometry Unit, and 3Comparative Pathology Unit, turbs actin dynamics, reduces epithelial adhesion, and leads Spanish Nacional Cancer Research Center (CNIO), and 4Hereditary to disorganization of tissue architecture. This invasion- Endocrine Cancer Group, CNIO and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain suppressive activity contrasts with recent genetic evidence in Von Hippel-Lindau (VHL; OMIM#193300) patients. The hu- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). man BRK1 locus (also known as C3orf10) maps close to the VHL gene (12, 13). VHL patients are predisposed to develop B. Escobar and G. de Cárcer contributed equally to this work. several tumors, including clear cell renal cell carcinomas Present address for M.C. Montoya: Cellomics Unit, Spanish National Cardiovascular Research Center (CNIC), Madrid, Spain. (ccRCC). These patients usually inherit a normal chromosome 3 and a mutant chromosome with a specific mutation or de- Corresponding Author: Marcos Malumbres, Centro Nacional de Inves- tigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, E-28029 letion in 3p25.3, where the VHL gene is located (14). Recent Madrid, Spain. Phone: 34-91-732-8000; Fax: 34-91-732-8033; E-mail: studies in VHL patients have correlated the absence of specific [email protected]. pathologies, such as ccRCC or retinal angioma, with large doi: 10.1158/0008-5472.CAN-09-4491 3p25.3 deletions that also affect BRK1 (12, 13, 15). In these ©2010 American Association for Cancer Research. “protected” patients, the spontaneous second hit (usually a

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large deletion at least in ccRCC) results in the inactivation of V-9131), Brk1 (a gift from Theresia Stradal, Helmholtz Zen- not only the remaining VHL gene but also the remaining BRK1 trum fur Infektionsforschung, Munster, Germany; and Alexis gene. “Unprotected” VHL patients develop renal tumors in Gautreau, Laboratoire d’Enzymologie et de Biochimie Struc- which at least one allele of BRK1 is conserved. turales, Gif sur Yvette, France), anti-Wave1 (BD 612276), In this article, we analyze the cellular effects on eliminating anti-Wave2 (Santa Cruz sc-10394), anti-Arp2 (Santa Cruz Brk1 expression using RNA interference or genetic alleles and sc-15389), anti-AbiI (a kind gift of Giorgio Scita, IFOM, Fonda- propose that Brk1 is required for cell proliferation, migration, zione Istituto FIRC di Oncologia Molecolare, Milan, Italy), and and metastasis of tumor cells. These data support the notion that anti-Actin (Sigma A4700). Filamentous actin was visualized VHL patients with large 3p25.3 deletions affecting Brk1 may using Alexa488 conjugated phalloidin (Invitrogen). have better prognosis due to the inability of affected cells to properly support tumor development in the absence of this protein. Generation and characterization of Brk1 mutant mice Mouse ES cells carrying a β-galactosidase gene in Brk1 in- Materials and Methods tron 1 (clone DC0002; Sanger Institute) were used to generate Brk1-deficient mice. The identification of the insertion site Cell culture and short hairpin RNA expression was done by PCR amplification using several oligonucleotides All cell lines were obtained from the American Type Cul- in the intron 1 of Brk1 and in the neoR gene (Supplementary ture Collection (ATCC) and were characterized at this cell Fig. S1). Fertilized embryos were collected by flushing the bank by morphologic and cytogenetic studies. The VHL gene uteri of pregnant females from crosses between Brk1(+/−) was scored for mutations in renal cells as reported previously mice, with HEPES-buffered Medium 2 (M2; Sigma) at E1.5- (13) and by direct sequencing to confirm known mutations. E2.5. Embryos were individually cultured in vitro in potassium All cells were used immediately for this work after receipt simplex optimized medium (KSOM; Chemicon International from ATCC without further authentication. Proliferation Inc.). To allow embryo “hatching,” blastocysts were then and transformation assays were done as reported previously transferred to gelatinized 96-well plates and cultured without (16, 17). Silencing experiments were done by using short hair- leukemia inhibitory factor in DMEM supplemented with 15% pin RNAs (shRNA; ref. 13) or small interfering RNAs (siRNAs; fetal bovine serum. All animal procedures were done accord- Dharmacon) against BRK1 transcripts. Cell movement was ing to a protocol approved by the corresponding Animal Care scored using videomicroscopy (Leica DM1600) and Meta- Committee (CBBA/4.2; no. 139/07). morph sofware (Molecular Devices). Wound-healing assays were quantified by evaluating the wound area recovered after Statistical analysis 16 hours using ImageJ software. Velocity and migratory direc- All statistical analyses were done using GraphPad Prism tionality were determined by tracking the positions of cell 4.0 software, and means ± SEM are indicated in the nuclei (MetaMorph). Directionality results from the division corresponding figures unless otherwise indicated. of the direct distance from start point to end point between the total track distance. For migration and invasion experiments, cells were seeded into Transwell chambers covered with Results an 8-μm pore membrane filter (BD Biosciences). Twenty- four hours later, cells were fixed with 4% paraformaldehyde Brk1 downregulation induces cell morphology and and stained with 4′,6-diamidino-2-phenylindole (DAPI). Cell proliferation defects in ccRCC cells migration and/or invasion was analyzed by detection of DAPI Human BRK1 is deleted along VHL in some VHL patients at the top (green nuclei) and bottom (orange) compartments of and it is thought to protect these patients from ccRCC. How- the chamber using a Leica SP5-MP confocal microscope and ever, the Brk1 expression levels have not been tested in these the IMARIS software. tumor cells. We therefore quantified BRK1 mRNA levels in 15 ccRCC primary tumors. As depicted in Fig. 1A, BRK1 is ex- In vivo metastasis assays pressed in all these tumors and, in fact, its expression levels We made tail vein injection of 5 × 105 luciferase-expressing are increased by 2- to 4-fold in most tumors and by more B16F10 cells in 10-week-old female CB17/Icr severe com- than 10-fold in two of these ccRCC. The only tumor with nor- bined immunodeficient (SCID) mice (Charles River Laborato- mal levels of expression corresponded to a mixed sarcoma- ries). Whole-body optical imaging was done using D-luciferin toid renal tumor with a reduced (<20%) component of clear (Promega) and the IVIS imaging system (Xenogen). cells. About half of these primary tumors carry diverse mutations (including point mutations, small insertions, and dele- Reverse transcription-PCR and tions) in the VHL gene without any specific correlation with protein immunodetection BRK1 expression levels (Fig. 1A). BRK1 transcripts (468 bp) were amplified using the follow- BRK1 is also expressed in human ccRCC cell lines such as ing oligonucleotides: Brk1_F: 5′-CGTTCGATATGTCTTGT‐ 786O and HTB46 (13), as well as CAKI2, SN12C, and U031 (data CGT-3′ and Brk1_R, 5′-ACTTTAAGGAAAGTTTGAGA-3′. not shown). We therefore analyzed the consequences of limit- For immunodetection, specific antibodies against the follow- ing the expression of Brk1 in two different ccRCC cell lines using proteins were used: Ki67 (Master Diagnostica), active ing previously validated shRNAs (13). These shRNAs result in a caspase-3 (RyD Systems), α-tubulin (YL1/2), Vinculin (Sigma significant downregulation (>60% in U031 and >80% in SN12C

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Figure 1. Downregulation of Brk1 in clear cell renal carcinoma (ccRCC) cells results in proliferative and motility defects. A, BRK1 mRNA was scored by real-time reverse transcription-PCR in 15 different primary ccRCC samples and a normal kidney (N). Numbers in brackets indicate the Furhman grade. The relative levels of BRK1 mRNA are represented as a fold-increase versus the control tissue. VHL exons were sequenced in these primary tumors, and the mutations found are indicated according to the corresponding Ensembl sequence (ENSG00000134086). wt, wild-type sequence; N.T., not tested. B, reduction in BRK1 mRNA levels after expression of shBrk1 vectors as detected by real-time PCR. These data were normalized versus actin considering the endogenous expression levels in U031 cells as 1. Brk1 knockdown is also verified by immunoblot in U031 and SN12C cells stably expressing the shRNA constructs. C, stable shBrk1 SN12C clones display rounded morphology with a clear impairment in the formation of the actin stress fibers (phalloidin in red) observed in control cultures (arrows). α-Tubulin (α-tub), green; DNA (DAPI), blue. D, antiproliferative effect of shBrk1 vectors versus a scramble sequence (shCtrl) in U031 cells. DNA content was measured by propidium iodide. Representative micrographs are also shown illustrating the cell clamps formed in U031 shBrk1 cells.

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cells) of Brk1 expression (Fig. 1B). Brk1 knockdown provokes a that Brk1 is required for proper proliferation of a wide spec- dramatic reduction in actin stress fibers and reduced prolifer- trum of normal and tumor cells. ation (Fig. 1C and D). These phenotypes are frequently accom- To test the consequences of Brk1 depletion in cell motility, panied by dramatic morphologic changes in shBrk1 cultures, we first did a wound healing assay in vitro in different cell which frequently display rounded clusters of cells rarely types. Wound closure is significantly impaired in U2OS and observed in control cultures (Fig. 1D and Supplementary ccRCC shBrk1 cells, which only manage to cover <30% to 40% Videos 1 and 2). of the wound area whereas control cells spread over 50% to We next tested the specificity of the Brk1 downregulation 65% of the wound (Fig. 3C). This is not likely a consequence phenotype in cell morphology by rescue experiments using of defective proliferation because the doubling time of these RNAi-resistant forms of Brk1. Due to the difficulties in trans- cultures is ∼35 hours and the test is done only in 16 hours. fecting ccRCC cells, we first reproduced the cell morphology Additionally, we have done the same assay in ccRCC cells defects generated after Brk1 knockdown in U2OS cells. that harbor VHL mutations (786O cell line) and we have ob- Transfection of U2OS cells with siRNAs against the coding served similar defects in migration and wound recovery region (siBrk1) or the 3′-untranslated region (siBrk1 3UTR) (Supplementary Fig. S3A), suggesting that the effect mediat- of BRK1 transcripts results in lack of actin stress fibers and ed by Brk1 downregulation is independent of the VHL status. rounded cells, similar to what was observed in ccRCC cells Interestingly, time-lapse imaging of wound healing revealed (Fig. 2A). Whereas control cells are scattered through the differences in the motility of the cells located at the wound culture plates and exhibit a polarized morphology, Brk1 edges. To quantify these differences, U2OS cells were seeded downregulation results in rounded cells that frequently form at low confluence and allowed to migrate randomly during epithelial-like cellular groups with few or no prolongations 16 hours (Fig. 3D). shBrk1 cells display a significantly reduced and altered actin cytoskeleton (Fig. 2 and Supplementary speed (21.04 ± 0.7 μm/h; n = 21) when compared with control Videos 3 and 4). Coexpression of a human Brk1-GFP fusion cells (32.44 ± 2.1 μm/h; n = 25). In addition, whereas control protein (sensitive to siBrk1 but resistant to siBrk1-3UTR) re- cells are able to “travel” linear distances longer than 100 μm, sults in the recovery of actin stress fibers in siBrk1-3UTR cells shBrk1 cells only perform small random movements with a but not in siBrk1 cells, indicating that the defects observed reduction in directionality (D/T ratio). Downregulation of after RNA interference are specifically due to Brk1 knock- Brk1 in ccRCC cells also results in similar defects in motility down. In Brk1-deficient cells, not only Brk1 but also other (see Supplementary Videos 1 and 2). Wave complex proteins are downregulated, suggesting that Brk1 is required for the stability of other Wave complex pro- Brk1 downregulation results in impaired migration, teins as previously reported (10, 18). Downregulation of Brk1 invasion, and metastatic potential also results in defective concentration of Arp2/3 at actin-rich Downregulation of Brk1 also results in similar defects in protrusions (Supplementary Fig. S1), indicating that although chemotactic responses on Transwell filters. U2OS or ccRCC Arp2/3 total levels are maintained (Fig. 2B), they are not shBrk1 cells were seeded in the upper compartment of properly localized in the absence of Brk1. Transwell chambers and serum-supplemented medium was shBrk1 cells also display a dramatic reorganization of focal used as a chemoattractant in the lower compartment. After adhesions as revealed by staining of vinculin, one of the 48 hours, almost half of the control U2OS cells were able to major components of these structures (Fig. 2C). Vinculin- cross the porous membrane, whereas only 15% of shBrk1 dependent adhesion sites become larger at the cell periphery cells were able to migrate (Fig. 4A). Similar defects were ob- with an average area of 18.8 ± 0.78 μm2 per individual focal served in ccRCC cells which displayed a significant reduction adhesion (n = 1224 individual focal adhesions in more than 50 of 20% to 50% in the ratio of cells that migrated through the cells), compared with the control cells that have an average of membrane. Downregulation of Brk1 also affects the perfor- 12.09 ± 0.47 μm2 (n = 558). mance of these tumor cells in invasion assays using collagen IV–coated Transwell chambers. As depicted in Fig. 4B, U2OS Brk1 is required for cell transformation and motility and ccRCC shBrk1 cells suffered a significant reduction in Similarly to ccRCC cells, downregulation of Brk1 in U2OS their invasion capacity. These faults are not due to defects cells results in a significant inhibition of cell proliferation in the secretion of matrix metalloproteinases and the degra- (Fig. 3A and Supplementary Fig. S2). Because VHL patients dation of the extracellular membrane because all these cul- with germline loss of Brk1 seem to be protected against tu- tures displayed similar behavior in collagen degradation mor formation, we decided to analyze the susceptibility of assays (Supplementary Fig. S3). Hence, Brk1 seems to be re- primary cells to malignant transformation by oncogenes after quired for cell migration and invasion of tumor cells, likely as knocking down Brk1. We used primary Cdk4-R24C murine a consequence of its critical role in actin dynamics and a sub- embryonic fibroblasts that have increased susceptibility to sequent alteration in cell motility. malignant transformation (17). As shown in Fig. 3B, Brk1 is Finally, we did in vivo metastasis assays in mice. We gener- required for full malignant transformation of primary cells by ated several stable shBrk1 clones using the highly metastatic oncogenes such as Ras or a combination of Ras plus E1A, B16F10 mouse melanoma cell line, carrying a constitutive ex- suggesting a protective effect of Brk1 loss against malignant pression of the luciferase reporter gene. First, we tested these transformation of primary cells. Taken together, the results clones in vitro using wound-healing assays to confirm that obtained in ccRCC cells, U2OS, and primary cultures suggest Brk1 repression affects B16F10 cells in a similar manner to

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Figure 2. Brk1 regulates the actin cytoskeleton and cell adhesion. A, cellular defects in U2OS cells expressing siRNAs against the coding region (siBrk1) or the 3′-UTR (siBrl1-3UTR) of human Brk1. These reagents provoke changes in morphology and loss of actin stress fibers (arrows). A Brk1-GFP fusion transcript (devoid of the 3′-UTR) rescues the defects observed in siBrk1-3UTR but not siBrk1 siRNAs, which also target the transcript of the fusion protein, resulting in decreased levels of the GFP signal. B, downregulation of Brk1 in U2OS cells stably expressing Brk1-specific shRNAs as verified by reverse transcription-PCR and Western blot (WB). C, shBrk1 U2OS cultures form cellular clusters with rounded morphology and cortical distribution of actin and vinculin (red; arrowheads). Details of vinculin (white insets) staining in control and shBrk1 cells suggest enlarged focal adhesions in the absence of Brk1. The area of each focal adhesion located at the cell periphery was quantified in more than 50 cells per assay. Focal adhesion sites (n = 1224) range up to an area of 250 μm2 in siBrk1 cells, whereas control cells (n = 558) have a maximum area of 100 μm2 (P < 0.0001; unpaired t test). Bars represent 10 μm.

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Figure 3. Brk1 is required for cell proliferation, transformation, and motility. A, representative growth curve and DNA content analysis of two different U2OS clones expressing shRNAs against Brk1 (shBrk1). B, focus formation assay in control and shBrk1 mouse primary fibroblasts transfected with 2 μg of Ras- or E1A-expressing vectors or empty vectors. C, representative pictures and quantification of wound healing in U2OS cells at t =0andt =16h after the wound. The histograms represent the percentage of area recovered in U2OS or ccRCC (SN12C or U031) cells 16 h after the wound. D, quantification of random motility in U2OS cultures. The velocity of these cells (μm/h) and directionality, scored as distance from the origin (D)/total distance (T) ratio, are quantified showing significant defects upon Brk1 downregulation.

ccRCC or U2OS cells and verify Brk1 dowregulation by reverse Taken together, these observations suggest that Brk1 is re- transcription-PCR. Additionally, we could observe that quired for proper proliferation and migration of tumor cells, shBrk1 B16F10 cells display as well aberrant actin stress fiber and suppression of Brk1 expression impairs the formation of distribution (Supplementary Fig. S4). Subsequently, these aggressive metastatic nodules in vivo. cells were injected in the tail vein of CB17/Icr SCID mice, and the animals were examined weekly for luciferase signal. Brk1 is a ubiquitous protein essential for After 9 or 16 days, control animals showed a remarkable lu- mouse development ciferase bioluminescent signal at the thorax, whereas mice Because large genomic deletions in VHL patients may af- boosted with shBrk1 cells barely showed a residual luciferase fect the BRK1 gene, we next tested the cellular defects signal (Fig. 4C). In addition, the Kaplan-Meier survival curve caused by complete genetic ablation of Brk1 using a of these mice indicates that shBrk1 cells are less aggressive in Brk1-null allele in the mouse. Brk1(+/−) mice were generat- inducing the lethal pulmonary metastasis (Fig. 4D). Macro- ed by microinjecting Brk1(+/−) ES cells into developing scopic (Fig. 4C) and histologic (Supplementary Fig. S4) anal- blastocysts. Brk1(+/−) mice are viable and fertile and do ysis of lungs from treated animals reveal a dramatic reduction not develop evident abnormalities during the first 18 in the number of tumor masses upon Brk1 downregulation. months of life. Because the Brk1(−) mutant allele expresses

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the β-geo gene under the endogenous Brk1 transcriptional during midgestation (Supplementary Fig. S6). At this stage, regulatory sequences (Supplementary Fig. S5), we analyzed β-galactosidase is clearly detected in neuroepithelium, the pattern of expression of mouse Brk1 transcripts using spinal cord, root ganglia, ependidymus, vascular endothelia, β-galactosidase as a reporter. Brk1 is ubiquitously ex- fibroblasts, and heart. Similar results have been observed pressed in the developing embryo as detected by whole- using in situ hybridization with specific antisense oligonu- mount β-galactosidase activity at embryonic day (E)6.5 or cleotides (Supplementary Fig. S6). Brk1 is also expressed

Figure 4. Brk1 is required for tumor cell spreading. A, migration (Transwell) and (B) invasion (collagen-coated Transwell) assays showing significant deficiencies in U2OS, SN12C, and U031 cells after downregulation of Brk1 (shBrk1). Cells that crossed the Transwell membrane are in orange, whereas the green signal indicates cells that remain at the upper compartment. C, bioluminescent imaging of living SCID mice i.v. injected with B16F10 cells expressing a luciferase gene and a control vector or shBrk1 vectors. Quantification of the luciferase signal into living animals 9 and 16 d postinjection (Turkey's multiple comparison test P < 0.001). Macroscopic view of the lungs taken from mice number 1, 4, and 5 at 2 wk postinjection. D, Kaplan-Meier survival curve. Median survival is 18 d in control mice and 24 d in shBrk1 mice (survival log-rank test analysis P < 0.005).

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Figure 5. Early embryonic lethality in the absence of Brk1. A, representative images of E4.5 and E6.5 embryos in culture. Most Brk1(+/+) and Brk1(+/−) embryos hatch out of the zona pellucida producing trophoblasts that attach to the plate (open arrow), liberating the inner cell mass (asterisk). B, immunofluorescence of wild-type and Brk1-deficient embryos after 4 d in culture. Brk1(+/+) embryos display a localization of actin filaments (green) at the cellular cortex. Brk1(−/−) embryos display aberrant structures within cells that are not compacted and actin is not properly concentrated. C, an unusual E8.5 Brk1(−/−) embryo (out of 27 E8.5 embryos analyzed) was identified by PCR analysis showing abnormal size and structure. D, E7.5 Brk1(−/−) in decidua embryos (dashed yellow lines) are composed of a small and disordered mass of cells with abundant apoptotic figures (filled arrowheads). H&E, hematoxylin and eosin staining. Bars indicate 200 μm (top and left) or 50 μm (bottom right). Fluorescence analysis (right) indicates that actin (as detected with a red-tagged phalloidin) is polarized in wild-type embryos (white arrowheads) but generates a diffuse signal in Brk1-deficient embryos (bottom left and right inset). The few remaining mutant cells display positive signal (yellow arrowheads) for active caspase-3 (green; bottom right). Asterisks indicate trophoblast cells highly loaded with actin. DNA (DAPI), blue signal. Bars indicate 20 μm (upper and bottom left), 10 μm (top inset) or 50 μm (bottom right inset).

in most adult tissues with a particular overrepresentation in man and mouse, after meta-analysis of available microarray brain or kidney as observed by Northern blot analysis or or expressed sequence tag data (Supplementary Fig. S8). β-galactosidase activity (Supplementary Fig. S7). A ubiquitous Complete genetic ablation of Brk1 induces embryonic distribution of Brk1 transcripts is also detected, both in hu- lethality as no Brk1(−/−) mice can be detected at birth.

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Because Brk1 is highly expressed in mouse at the blastocyst largely overlooked in mammals. Brk1 is the highest con- stage (Supplementary Fig. S8), we then analyzed fertilized served member of the Wave/Scar pathway (10) with clear embryos in vitro. E1.5 embryos were isolated and cultured orthologues in plants and animals but not in yeast. Brk1 is during 6 days in vitro. By E4.5, some embryos display an ab- not an essential component of the Wave/Scar complex be- normal phenotype with undeveloped blastocysts showing ab- cause Sra1, Nap1, Wave, and Abi can still form a complex normal morphologies (Fig. 5A). One day later, both Brk1(+/+) in the absence of Brk1 (22). However, partial downregulation and Brk1(+/−) blastocysts hatched, producing a compacted of Brk1 has a dramatic effect on the total levels of the com- inner cell mass surrounded by trophoblasts, whereas the ab- ponents of the Wave complex (Fig. 2 and refs. 10, 23) and in normal Brk1(−/−) blastocysts remain as a degenerated blas- the localization of Wave and Arp proteins to cellular protru- tocyst. Interestingly, all Brk1(−/−) embryos show an aberrant sions. Brk1 is also known to be present in a large pool of free actin network (Fig. 5B). Whereas in wild-type compacted protein that associates to the Wave complex during the as- blastomeres, actin is precisely localized at the cell cortex, sembly of these macromolecular structures (10, 22). Brk1(−/−) blastocysts display an irregular actin staining The in vivo requirements for Wave/Scar complex proteins indicating the absence of proper actin fibers at the apical have revealed a rich variety of phenotypes associated to actin and basolateral boundary. Lack of the proper actin structure defects. Wave2-null embryos die by E10.5-E12.5 due to defects is likely to prevent the proper compaction of the embryos in cell migration and cardiovascular development (23, 24). and the establishment of intercellular junctions required at Genetic ablation of other members of the complex, such as the eight-cell stage as previously proposed in E-cadherin– Nap1 or Abi, suggests similar roles for these proteins in mid- deficient mice (19–21). Despite these severe defects in vitro, gestation, cytoskeleton dynamics, organogenesis, and neural afewBrk1(−/−) embryos get implanted, although they are biology (25–27). Lack of Brk1 results in abnormal distribution dramatically underdeveloped (Fig. 5C). Whereas wild-type of actin at the cell cortex of blastomeres. At this stage, a mor- cells are well organized into the different embryonic layers phologic reorganization called compaction results in outer with polarized actin, Brk1(−/−) cells are disorganized and polarized blastomeres assembling typical epithelial intercel- display a diffuse distribution of actin. These mutant embryos lular junctions rich in actin filaments at the apical pole are reduced to a mass of abnormal cells with evident signs of underlying and adjacent to the contact zone between blasto- apoptosis depicted by cleavage caspase-3 staining (Fig. 6D). All meres (28). Cytochalasin D, a potent inhibitor of actin together, these results show the essential roles of Brk1 in the polymerization, disrupts these structures and inhibits inter- reorganization of the actin cytoskeleton and cell survival, at cellular flattening (29). Lack of Brk1 is likely to impair the es- least during embryonic development. tablishment of specialized cellular junctions required for a fully polarized epithelium. The developmental arrest caused Discussion by Brk1 deficiency occurs earlier than in the absence of any other member of the Wave complex, a difference that may be Despite the extensive information on the different Wave/ explained by the presence of diverse paralogues for the later Scar complex components, the small protein Brk1 has been proteins (e.g., Wave1-3, Abi1-3, Sra1-2). In addition, Brk1 may

Figure 6. A model for the genetic relationships between VHL and BRK1 in renal cell carcinoma. Both genes map very close to human Chr. 3. Individuals may inherit (A) a small deletion or a VHL mutation not affecting BRK1 or (B) a large deletion affecting both VHL and BRK1. In case A, the second hit (usually a large deletion in renal cells) results in complete deficiency in VHL in the presence of a wild-type copy of BRK1. These patients develop renal cell carcinoma. On the other hand, in case B, the second hit results in the concomitant ablation of VHL and BRK1 in affected cells. These cells are defective in proliferation and motility and cannot generate a tumor.

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have additional Wave-independent roles in which the large ic ablation of Wave2 also results in significant deficiency in pool of free Brk1 may be involved (22). The latter possibility proliferation (23), and original data from other Wave com- is further supported by the fact that Nap1-deficient embryos plex members suggest a requirement for Wave2/3 and Abi in develop morphogenetic defects and lethality by E9 once the tumor cell migration and invasion (31–33). Thus, different epiblast and mesoderm layers are already organized despite sets of Wave complexes may have distinct roles in favoring the complete absence of Wave complexes (26). proliferation or invasion. Alternatively, it is also possible that How does Brk1 function affect tumor susceptibility in VHL Brk1 may have additional unknown, Wave-independent patients? Approximately 20% to 30% of VHL patients have functions. However, the essential roles of Brk1 in actin orga- small or large (0.5 to 250 kb) germline deletions affecting nization and cell function reported here, along with the re- VHL and, in some cases, flanking genes (15). For the tumor cent identification of the strong requirements for other actin to develop, a spontaneous second hit occurs during the pa- regulators in tumor development (34), suggests the thera- tient's lifetime that inactivates the wild-type VHL allele. In a peutic value of inhibiting Brk1 or actin dynamics in ccRCC significant number of tumors, this second hit consists of large or other tumors. deletions due to Alu-mediated recombination because the BRK1 and VHL genes lie in a region of high Alu density (15). Disclosure of Potential Conflicts of Interest Thus, the final retention of BRK1 is mostly determined by the inherited allele. Those individuals that inherit small deletions No potential conflicts of interest were disclosed. or point mutations affecting VHL but not BRK1 (Fig. 6A) are susceptible to develop tumors upon the second hit (usually a Acknowledgments large deletion). However, individuals that inherit large dele- We are indebted to Amancio Carnero for supplying the B16F10 cells with tions affecting BRK1 and VHL (Fig. 6B) will be protected be- luciferase expression, to Theresia Stradal and Alexis Gautreau for the anti-Brk1 cause the second hit will produce Brk1-deficient cells, which antibody, and to Giorgio Scita for the anti-AbiI antibody. are defective in proliferation, polarization, and motility and are likely to be inefficient in tumor formation. In fact, recent Grant Support analysis has shown a perfect correlation between the loss of BRK1 and protection from ccRCC in a recent survey of 127 The Cell Division and Cancer Group is funded by grants from the Fondo Investigaciones Sanitarias (FIS05/1186), Ministerio de Ciencia e Innovación individuals with germline VHL deletions (30). (SAF2009-07973) and Consolider-Ingenio (CSD2007-00017), Comunidad de The fact that other components of the Wave complex Madrid (S-BIO-0283-2006), Fundación Médica Mutua Madrileña Automovilís- tica, Fundación Ramón Areces and the Association for International Cancer (e.g., Sra1) may act as invasion suppressors (11) suggests Research (AICR). that the Wave complex may have different activities depend- The costs of publication of this article were defrayed in part by the payment ing on the cellular scenario or the protein composition. For of page charges. This article must therefore be hereby marked advertisement in instance, whereas downregulation of Sra1 does not alter cell accordance with 18 U.S.C. Section 1734 solely to indicate this fact. proliferation, downregulation of the close paralogue Sra2 re- Received 12/15/2009; revised 07/14/2010; accepted 07/28/2010; published sults in a dramatic reduction in cell proliferation (11). Genet- OnlineFirst 09/22/2010.

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www.aacrjournals.org Cancer Res; 70(22) November 15, 2010 OF11

Brick1 Is an Essential Regulator of Actin Cytoskeleton Required for Embryonic Development and Cell Transformation

Beatriz Escobar, Guillermo de Cárcer, Gonzalo Fernández-Miranda, et al.

Cancer Res Published OnlineFirst September 22, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-4491

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