BRCA1/BARD1-Dependent Ubiquitination of NF2 Regulates Hippo-YAP1 Signaling

BRCA1/BARD1-dependent ubiquitination of NF2 regulates Hippo-YAP1 signaling

Sachin Vermaa,1, Narayana Yeddulaa,1, Yasushi Sodaa,2, Quan Zhua,3, Gerald Paoa, James Morescoa, Jolene K. Diedricha, Audrey Hongb,c, Steve Plouffeb,c, Toshiro Moroishib,c, Kun-Liang Guanb,c,4, and Inder M. Vermaa,4

aLaboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA 92037; bDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; and cMoores Cancer Center, University of California, San Diego, La Jolla, CA 92093

Contributed by Inder M. Verma, February 28, 2019 (sent for review December 31, 2018; reviewed by Jixin Dong and David M. Livingston) Coordination of growth and genomic stability is critical for normal by serum stimulation and changes in cell density (24). BRCA1 ac- cell physiology. Although the E3 ubiquitin ligase BRCA1 is a key player tivity is further regulated via changes in its levels, phosphorylation in maintenance of genomic stability, its role in growth signaling state, and subnuclear distribution, depending on different stages of remains elusive. Here, we show that BRCA1 facilitates stabilization of cell cycle and signaling (25–27). Furthermore, BRCA1/BARD1 YAP1 protein and turning “off” the Hippo pathway through ubiquiti- activity is also regulated by proteosomal degradation (28). There- nation of NF2. In BRCA1-deficient cells Hippo pathway is “turned On.” fore, BRCA1/BARD1 activity is tightly controlled to coordinate Phosphorylation of YAP1 is crucial for this signaling process because a growth-factor-stimulated downstream signaling and genomic stabil- YAP1 mutant harboring alanine substitutions (Mt-YAP5SA) in LATS1 ity. Although BRCA1 is known to play key roles in maintenance of kinase recognition sites not only resists degradation but also rescues genomic stability, whether it cooperates with signaling pathways YAP1 transcriptional activity in BRCA1-deficient cells. Furthermore, to regulate physiological processes such as cell growth is not fully an ectopic expression of the active Mt-YAP5SA, but not inactive explored. Mt-YAP6SA, promotes EGF-independent proliferation and tumori- The Hippo pathway transducer, YAP1 protein, is a mitogen- −/− genesis in BRCA1 mammary epithelial cells. These findings es- responsive transcriptional coactivator. YAP1 is stabilized in the tablish an important role of BRCA1 in regulating stability of YAP1 presence of serum which turns “Off” the Hippo pathway (29). protein that correlates positively with cell proliferation. Upon serum stimulation, nuclear YAP1 in association with TEAD CELL BIOLOGY proteins promotes expression of genes involved in cell proliferation. Hippo | BRCA1 | NF2 | ubiquitination | cancer In absence of serum, Hippo signaling kinases LATS1/2 are activated which, in turn, phosphorylate YAP1 to cause its cytoplasmic nherited mutations in breast cancer early onset gene BRCA1 localization and degradation (30), thereby enabling homeostatic Ipredispose individuals to highly aggressive form of breast and growth-restrictive regulation. Although YAP1/TAZ activation ovarian cancers (1, 2). Studies in a knockout mouse model have shown that mouse BRCA1 plays a key role in cell proliferation Significance during embryonic development (3). Mouse BRCA1 function can be compensated by replacing it with human BRCA1 gene despite Normal cells harbor protective mechanisms to sense DNA poor sequence homology (4, 5). BRCA1 is involved in multiple damage, halt cell growth, and repair chromatin lesions to cellular functions, e.g., transcription, heterochromatin structure maintain genomic stability. How mitogen signaling in pro- formation, replication fork stability, homologous recombination – liferating cells is affected upon BRCA1 loss as part of these repair, centrosome regulation, and mitotic spindle formation (6 11). protective checkpoints is an intriguing question. This study These diverse roles are specified by interaction of BRCA1 with its reveals a unique finding of linking BRCA1 to Hippo signaling heterodimeric partner BARD1 that greatly enhances the E3 ubiq- pathway to explain this conundrum. Our work shows that uitin ligase activity of the complex (12). How loss of BRCA1 activity serum-responsive expression of BRCA1 is required for YAP1 in heterozygous carriers leads to tumorigenesis remains only par- – stability. Ubiquitination of NF2 by BRCA1/BARD1 in proliferating tially understood (13 15). cells inhibits NF2/LATS association and Hippo signaling. These Ubiquitination is a highly regulated and reversible event in- findings suggest Hippo signaling activation as a protective bar- duced by various stimuli that not only regulate protein stability rier in BRCA1-deficient cells, which upon inactivation, promotes but also functional interaction, localization, and signaling dy- cell proliferation and tumorigenesis. namics (16). These changes in protein activity by ubiquitination are governed by number of ubiquitin molecules attached and Author contributions: S.V., K.-L.G., and I.M.V. designed research; S.V., N.Y., Y.S., J.M., nature of linkage involved. The enzymatic activity of BRCA1/ J.K.D., and A.H. performed research; Q.Z., G.P., J.M., J.K.D., S.P., and T.M. contributed BARD1 ubiquitin ligase complex is unique because it can generate new reagents/analytic tools; S.V., N.Y., Y.S., K.-L.G., and I.M.V. analyzed data; and S.V., different kinds (mono and poly) of atypical ubiquitin linkages (K6, Q.Z., A.H., K.-L.G., and I.M.V. wrote the paper. K29, K48, and K63) depending on the substrate and interacting Reviewers: J.D., University of Nebraska Medical Center; and D.M.L., Dana Farber Cancer Institute. E2 subunit (17–19). Several cellular proteins have been identified Conflict of interest statement: K.-L.G. is a cofounder and has equity interest in Vivace as substrates of this ubiquitin ligase activity, e.g., NPM1, RPB1, Therapeutics, Inc. The terms of this arrangement have been reviewed and approved by CtiP,RPB8,PR-A,TFIIE,HistonesH2A,H2B,H3,H4,Ƴ-Tubulin, the University of California, San Diego in accordance with its conflict of interest policies. ER-α, Aurora A/B, and BRCA1 itself (20–23). These proteins are Published under the PNAS license. known to be involved in regulating key signaling steps in dividing 1Present address: Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037. cells, e.g., in regulating gene expression, proliferation, chromosome 2Present address: Project Division of ALA Advanced Medical Research, Institute of Medical maintenance, duplication, repair, and segregation. However, unlike Science, University of Tokyo, 108-8639 Tokyo, Japan. substrates of K48 ubiquitination, which are directed for proteasomal 3Present address: Ludwig Institute for Cancer Research, La Jolla, CA 92093. degradation, many substrates with other atypical ubiquitin linkages 4To whom correspondence may be addressed. Email: [email protected] or imohanv1@ are not degraded but involved in signaling transduction. Thus, gmail.com. multiple pathways are integrated by BRCA1/BARD1 activity to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ensure genomic stability in normal cells. Not only is BRCA1 1073/pnas.1822155116/-/DCSupplemental. highly expressed in proliferating cells, it is transcriptionally regulated Published online March 27, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1822155116 PNAS | April 9, 2019 | vol. 116 | no. 15 | 7363–7370 Downloaded by guest on September 23, 2021 has been reported in various cancers (31), mutations involving BARD1 activity in cells by expressing shRNA against BRCA1 and core Hippo signaling components are not frequent (32), suggesting BARD1. As shown (Fig. 1F; lane 3 compared with lane 2), possible involvement of upstream regulators which are yet to be ubiquitination of NF2 was greatly enhanced upon stimulation identified. In a recent analysis performed using 9,125 tumors of cell with serum. On the other hand, NF2 ubiquitination was profiled by The Cancer Genome Atlas (TCGA) from 33 cancer significantly reduced in BRCA1/BARD1-deficient cells (Fig. 1F;lane types, multiple alterations in Hippo pathway members were identi- 5 compared with 3). We further tested if BRCA1 overexpression fied per tumor samples (33), suggesting that cooccurring copy can also augment NF2 ubiquitination. Overexpression of BRCA1 number alteration events correlate with oncogenic activation of greatly enhanced NF2 ubiquitination (Fig. 1G; lane 3 compared YAP1. Among core pathway components (scaffold protein: NF2; with 2). Also, to test involvement of BARD1 in this process we kinases: MST1, MST2, LATS1, LATS2; accessory molecules: coexpressed shRNA against BARD1 in the same cells, which re- SAV1, MOB1, MOB2; transcriptional coactivators: YAP1, TAZ), versed enhanced NF2 ubiquitination (Fig. 1G; lane 4 compared with NF2 is critical for Hippo signaling as its inactivation impairs 3). Similarly, ubiquitination of endogenous NF2 was confirmed to be LATS1/2 activation and subsequent phosphorylation of YAP1 sensitive to shRNA-mediated inhibition of BRCA1/BARD1 (Fig. (34). The current model in the field suggests that NF2 activates 1H). We also inhibited BRCA1 expression in HEK293T-Cas9 cells Hippo signaling pathway via binding LATS1/2 to facilitate LATS1/ (stably transduced with Cas9 gene) by transient transfection of single 2 activation by MST1/2 (35). Also, NF2 activity is regulated by guide RNA (sgRNA) targeting BRCA1 and observed inhibition of intramolecular interactions between N-terminal Ferm domain and NF2 ubiquitination (SI Appendix,Fig.S1C). Mutations in BRCA1 C-terminal coil domains, directing interconversion between open N-terminus ring finger and C-terminus BRCT domains have been to closed conformation (36). What molecular mechanism restricts identified in human patients (43). We therefore tested the patho- NF2 activity during mitogen signaling and assembly of these sig- genic mutant defective in BARD1 interaction and ubiquitin ligase naling complexes however remains unclear. Recently reported in- activity (BRCA1 C61G) or BRCT domain mutants (BRCA1 teractions of Hippo pathway components with various ubiquitin M1755R) for their ability to promote NF2 ubiquitination. Both of ligases and deubiquitinase enzymes (37–41) suggest existence of the pathogenic mutants BRCA1 C61G or BRCA1 M1755R failed ubiquitin signaling switches during Hippo signaling regulation. to enhance NF2 ubiquitination (Fig. 1I,lanes5and6).Onthe In this article, we show an important role of BRCA1 expres- contrary, expression of ring-finger domain mutant, BRCA1-I31M sion in Hippo signaling pathways. During mitogen signaling, that does not alter ubiquitin ligase activity, was found to be equally NF2 is inhibited by BRCA1/BARD1-mediated ubiquitination, potent as wild-type protein in promoting NF2 ubiquitination (Fig. leading to YAP1 stabilization. Our findings suggest a previously 1I,lanes3and4).Basedontheseobservations, we speculated that uncharacterized protective mechanism in normal cells for fine- BRCA1-dependent ubiquitination of NF2 in proliferating cells tuning growth-factor signaling and genomic stability, which when may inhibit Hippo signaling. bypassed in BRCA1-deficient mammary epithelial cells, promote To dissect molecular consequences of BRCA1-mediated in- cell growth and tumorigenesis. hibition of Hippo signaling, we examined effects of BRCA1 expression or knockdown on YAP1 transcriptional activity. Measured Results by YAP1-responsive UAS-luciferase/TEAD-Gal4 reporter (30), we BRCA1/BARD1 Promote NF2 Ubiquitination and Inhibits Hippo observed a profound increase in YAP1 transcriptional output upon Signaling. We first tested if ubiquitin signaling could be involved BRCA1 coexpression (Fig. 1J). On the other hand, we observed in Hippo signaling regulation in proliferating cells. We observed strong inhibition of reporter activity in BRCA1 knockdown cells. that among all of the core Hippo components, only NF2 and to a These data suggest that BRCA1 directly regulates Hippo signaling lesser extent LATS1 were heavily ubiquitinated in serum-stimulated through NF2 ubiquitination and promotes YAP1 activation. cells (Fig. 1A). LATS1/2 were previously shown to be ubiquitinated by Itch and CRLA4(DCAF1) ubiquitin ligase complex that direct BRCA1 Binds NF2 Through FERM and C-Terminal Domains. We further LATS1/2 for degradation (38, 39). The nature of ubiquitination analyzed direct role of BRCA1 protein in suppressing Hippo and ligase(s) specific for NF2 as well as its possible implication in signaling through protein interactions. We tested interaction of Hippo signaling has not been previously explored. Enrichment of BRCA1 with core Hippo pathway components by pull-down as- ubiquitinated NF2 in denaturing conditions followed by mass spec- says. HA-tagged NF2, but not any other core component (MOB, trometry analyses revealed that NF2 ubiquitination is K63-linked SAV, MST1, LATS, YAP1, and TAZ), was able to interact with and involves multiple lysines (K159, K269, K274, K364, K387, GST-BRCA1 fusion protein (Fig. 2A). The intracellular interac- K396, K439, and K449) residues located not only in unstructured tion of BRCA1 with NF2 was further confirmed by immunopre- coiled coil domain but also highly ordered N-terminus FERM cipitation of endogenous BRCA1, which also recovered NF2 domain (Fig. 1B and SI Appendix,Fig.S1A and B). Since FERM protein in eluted fractions (Fig. 2B). Also, HA-NF2 was able to domain is known to mediate interaction with LATS1 kinase (35), specifically bind to GST-BRCA1 fusion protein but not GST we examined binding of equal amounts of free and ubiquitinated negative control in vitro (Fig. 2C), confirming direct interaction be- NF2 with LATS1 from cell extract. Compared with free NF2 that tween the two proteins. We tested pathogenic mutants in BRCT- is efficiently bound to LATS1 (Fig. 1C; lane 1), ubiquitination domain (P1744R and M1775R) or ring-finger domain (I31M, of NF2 inhibited LATS1 binding (Fig. 1C; lane 2). Enrichment C39Y, T37R, C61G, V11A, and ring-domain deleted dRing), for of ubiquitinated proteins after cell fractionation revealed that their ability to interact with NF2. As shown, both wild-type and ubiquitinated NF2 resided mainly in the nucleus (Fig. 1D). Using all ring-finger mutants were equally potent in NF2 binding (Fig. mutants of ubiquitin with single lysine available for linkage (Ub- 2D). However, interaction with NF2 was abolished in BRCT- K6, Ub-K11, Ub-K27, Ub-K29, Ub-K33, Ub-K48, and Ub-K63), domain mutants. We also compared patient-derived BRCA1- we found that NF2 ubiquitination preferentially incorporates mutant HCC1937 cell line (harboring mutation in BRCT domain Ub-K6, Ub-K27, Ub-K29, and Ub-K63 but not Ub-K48, Ub-K11, and loss of WT allele) with HCC1937-WTBRCA1 cells (recon- and Ub-K33 (Fig. 1E) ubiquitin mutants, suggesting its possible stituted with WTBRCA1), for their ability to bind NF2. Consistent involvement in nonproteosomal regulation. These results are in with our observations, endogenous NF2 binds BRCA1 in WT- agreement with the long half-life (exceeding 24 h) time of NF2 in BRCA1 reconstituted HCC1937 cells, but not Mt-BRCA1– cells (42). Expression of E3 ligase BRCA1 in cultured cell is expressing HCC1937 cells (SI Appendix,Fig.S2A). We next serum responsive and known to catalyze formation of K6, K29, mapped the protein domain involved in BRCA1/NF2 interaction. K48, and K63 linked ubiquitin chains (17–19,24).Totestinvolvement The N-terminal half of NF2, which possesses FERM domain, is re- of BRCA1/BARD1 with NF2 ubiquitination, we inhibited BRCA1/ quired for BRCA1 binding (Fig. 2E). Reciprocally, the C-terminal

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Fig. 1. BRCA1/BARD1 promotes NF2 ubiquitination to inhibit Hippo signaling. (A) HEK293T cells were cotransfected with His-Ub with HA-MOB, HA-SAV, HA- MST1, HA-NF2, HA-LATS1, HA-YAP1, or HA-TAZ. After 36 h cells were treated with MG132 for 8 h followed by lysis in denaturation buffer, and then total ubiquitinated proteins were pulled down with the use of Ni-NTA beads, and ubiquitination was checked by immunoblotting with anti-HA antibody. (B)A schematic structure of NF2, indicating the positions of the ubiquitination sites and peptide sequences identified by mass spectrometry analysis. (C) HEK293T cells were cotransfected with HA-NF2 alone or in combination with His-Ub followed by purification of free and ubiquitinated NF2 using HA-binding beads. The HA-bead–bound proteins were then incubated with cell lysate for LATS1 binding as described in Materials and Methods.(D) NF2 ubiquitination was checked by Ni-NTA pull-downs from HEK293T cells transfected with His–Ub and HA-NF2, after an initial cell fractionation step using MF, CF, and NF. MF, membrane fraction; CF, cytoplasmic fraction; NF, nuclear fraction. Successful cell fractionation was confirmed by immunoblotting for specific markers of MF (E-cadherine), CF (GAPDH), and NF (BRG1). (E) Indicated mutants of HA-ubiquitin were cotransfected into HEK293T cells with Flag-NF2. HA pull-downs from transfected cells were then analyzed by immunoblotting using NF2 antibody. (F) HEK293T cells transfected with ubiquitin and HA-NF2 constructs were first starved for 12 h, then stimulated with serum for 24 h and tested for NF2 ubiquitination as described above. (G) HEK293T cells were cotransfected with His-Ub, HA-NF2, BRCA1 and shBRCA1, and shBARD1 as indicated and NF2 ubiquitination was checked. (H and I) HEK293T cells transfected with His-Ub and indicated plasmids, to measure NF2 ubiquitination. (J) HEK293T cells were cotransfected with UAS-luciferase/TEAD-GAL4, renilla luciferase (normalization control), shBRCA1, and BRCA1 as indicated, followed by measurement of luciferase activity which is normalized to control renilla luciferase reading. Results are expressed as means ± SEMs. Only values of P < 0.05 were considered significant (****P < 0.0001), by ANOVA test.

fragment of BRCA1, but not its N-terminal ring-finger domain or ined the cellular localization of YAP1, which is regulated upon central intrinsically disordered region, is required for NF2 binding phosphorylation through Hippo signaling. As expected, control cells (Fig. 2F). These results suggested that BRCA1 could regulate with Hippo signaling suppressed, show strong nuclear localization of Hippo signaling through direct NF2 interaction. YAP1 protein (Fig. 3E). On the other hand, BRCA1 knockdown promoted cytoplasmic localization of YAP1 protein. In contrast, the Serum-Responsive Expression of BRCA1 Contributes to YAP1 Stabilization YAP1 protein level remained unaffected in NF2-null HEK293A Through Regulation of Hippo Signaling. Because NF2-mediated Hippo cells (Fig. 3F; lane 4 compared with lane 2), suggesting that NF2 signaling is involved in regulation of YAP1 stability, we further downstream Hippo signaling is required for suppression of YAP1 probed a possible relationship between BRCA1 expression and levels in BRCA1-deficient cells. Suppression of YAP1 protein levels YAP1 protein. As shown, stimulation of various cell lines with serum by Hippo signaling requires phosphorylation of YAP1 at multiple led to significant increase in BRCA1 expression (Fig. 3 A and B;lane serine residues by LATS1/2 kinases. We therefore compared cel- 2 compared with lane 1). As expected, protein levels of YAP1, pAKT, lular levels of WT-YAP1 with Mt-YAP5SA protein that has mu- and pEGFR, which are growth signaling markers, were also elevated tations in all five LATS phosphorylation sites. As expected, expression in these cells. In contrast, BRCA1 knockdown by shRNA suppressed of shBRCA1 strongly suppressed total levels of WT-YAP1 (Fig. 3G; YAP1 levels in multiple cell types (Fig. 3 C and D; lane 2 compared lane 2 compared with lane 1). Also, the pYAP1 levels in WT-YAP1– with lane 1). As anticipated, BRCA1 knockdown activated DNA expressing cell remained comparable to control cells, suggesting damage signaling as judged by levels of ɣH2AX. All of the other accumulation of phosphorylated WT-YAP1 in BRCA1 knockdown serum-responsive markers tested using specific antibodies, e.g., cells. However, BRCA1 knockdown had little or no effect on pAKT, pEGFR, and pERK1/2, remained unchanged. We exam- Mt-YAP5SA levels (Fig. 3G; lane 5 compared with lane 4).

Verma et al. PNAS | April 9, 2019 | vol. 116 | no. 15 | 7365 Downloaded by guest on September 23, 2021 Fig. 2. BRCA1 binds NF2 through FERM and BRCT domains. (A) HEK293T cells expressing HA-tag Hippo proteins and GST or GST-BRCA1 fusion proteins were subjected to immunoprecipitation as described in Materials and Methods.(B) Endogenous NF2 was detected in anti-BRCA1 beads, but not control, immu- noprecipitates. (C) HA-NF2 directly binds GST-BRCA1, as revealed by immunoprecipitation upon in vitro binding experiment, performed as described in Materials and Methods.(D) HEK293T cells expressing various mutants of BRCA1 and HA-NF2 were subjected to immunoprecipitation using HA-binding beads. The resulting eluent was used to measure HA-NF2–bound BRCA1 by immunoblotting with anti-BRCA1 antibody. (E) HEK293T cells expressing either full- length NF2, N-terminus fragment (1–332) or C-terminus fragment (308–590), and GST-BRCA1 were subjected to immunoprecipitation using GST-binding beads. The resulting eluent was used to measure GST-BRCA1–bound NF2 by immunoblotting with anti-NF2 antibody. (F) HEK293T cells expressing the indicated BRCA1 Myc-tag fragments (1–303, 303–772, 772–1314, and 1314–1863) were tested for interaction with HA-NF2.

Correspondingly, shRNA-mediated inhibition of LATS1/2 kinases However, cells expressing Mt-YAP5SA exhibited significantly higher rescued WT-YAP1 protein degradation in BRCA1 knockdown reporter activity compared with WT-YAP1–expressing cells. These re- cells (Fig. 3H; lane 6 compared with lane 3). YAP1 degradation sults show that BRCA1 deficiency activates Hippo signaling and leads to was also rescued by treatment of cells with proteasome inhibitor subsequent proteosomal degradation of WT-YAP1 protein (Fig. 3J). MG132 or by coexpression of dominant negative mutant of ubiquitin (SI Appendix,Fig.S2B and C). Furthermore, we tested rescue of Mt-YAP5SA Expression in BRCA1-Deficient Mammary Epithelial Cells YAP1 transactivation activity by Mt-YAP5SA using UAS-Luciferase/ Confers EGF-Independent Proliferation and Tumorigenesis. Having TEAD-Gal4/YAP1 reporter system. Expression of WT-YAP1 and established that BRCA1 deficiency activates Hippo signaling, we Mt-YAP5SA protein promoted luciferase output from UAS-driven next examined whether Hippo signaling contributes to growth in- luciferase/Gal-4-TEAD reporter. As expected, BRCA1 knockdown hibition observed in BRCA1-deficient cells. As expected, knockdown suppressed reporter output in cells expressing WT-YAP1 (Fig. 3I). of BRCA1 expression suppressed cell growth in HEK293A cells

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Fig. 3. BRCA1 expression regulates YAP1 activity. (A and B) Cells were starved of serum for 12–16 h followed by stimulation with serum in complete medium for 24 h. (C and D) Cells were transduced with lentiviral vectors expressing shRNA against BRCA1 for 72 h, and immunoblot analysis was done. (E) Immu- nofluorescence analysis to check localization of YAP1 protein in BRCA1 knockdown U2OS cells 36-h postinfection. (F) HEK293A cells or NF2 knockout − − HEK293A cells (NF2 / ) were infected with lentiviral vectors expressing shRNA against BRCA1 for 72 h and harvested for immunoblotting. (G) HEK293T cells were cotransfected with plasmids encoding myc-tagged WT-YAP1 (Myc-YAP1) or Mt-YAP5SA (Myc-YAP5SA) and shRNA against BRCA1 followed by immunoblot analysis. Both WT- and Mt-YAP1s were detected by anti-Myc tag antibody. (H) HEK293T cells were cotransfected with WT-YAP1, and plasmids expressing two different shRNAs against BRCA1 (shBRCA1-1 and 2) and LATS1/2. (I) HEK293T cells were cotransfected with UAS-luciferase/TEAD-GAL4, renilla luciferase (normalization control), shBRCA1, and WT-YAP1 or Mt-YAP5SA followed by measurement of luciferase activity normalized to renilla luciferase reading. (J) A schematic model describing BRCA1-mediated NF2 ubiquitination, which promotes YAP1 stability in Hippo Off state via suppressing Hippo signaling. Results are expressed as means ± SEM. Only values of P < 0.05 were considered significant (****P < 0.0001), by ANOVA test.

Verma et al. PNAS | April 9, 2019 | vol. 116 | no. 15 | 7367 Downloaded by guest on September 23, 2021 (Fig. 4A). In comparison, NF2-null HEK293A cells exhibited partial shRNA was also not sufficient to directly initiate tumorigenesis in but significant rescue of growth inhibition caused by BRCA1 knock- BRCA1-deficient MCF10A cells under the observation period. In down. Similarly, serum stimulation of MCF10A cells expressing contrast, BRCA1-deficient cells expressing Mt-YAP5SA were shRNA-targeting BRCA1 resulted in delayed growth (Fig. 4B) able to produce tumors in all of the injected mice (Fig. 4E and SI in comparison with control cells. In contrast, cells with ectopic Appendix, Fig. S3C). We concluded that YAP activation but not expression of Mt-YAP5SA exhibited rescue of cell growth. These p53 inhibition alone can directly transform BRCA1-deficient observations suggest that YAP1 reactivation could confer growth mammary epithelial cells. advantage for BRCA1-deficient cells. Previously, YAP1 activation The BRCA1-deficient tumors also showed a significant re- was shown to promote growth of mammary epithelial cells in re- duction of tumor volume, compared with control BRCA1- duced growth factor conditions (44). We therefore evaluated proficient tumors (Fig. 3E). We examined the cellular levels of whether Mt-YAP5SA can promote EGF-independent growth of 53BP1, Mt-YAP5SA, and BRCA1 in tumor lysates (SI Appendix, BRCA1-deficient cells. As expected, MCF10A cells alone showed Fig. S3D). All of the samples showed comparable amount of Mt- no growth in reduced serum and EGF-free media (Fig. 4C). Cells YAP5SA, 53BP1 proteins. Intracellular expression of BRCA1 expressing null Mt-YAP6SA (with S94A mutation disrupting TEAD was monitored and as expected, tumor-cells–expressing shBRCA1 binding) behaved similarly with no growth advantage. However, we had efficient knockdown of BRCA1 protein. GFP protein observed significantly higher cell growth in active Mt-YAP5SA– expressed from lentivirus (Fig. 4F)andβ-Actin levels were moni- expressing cells. These results were further confirmed using 3D– tored as internal control, which were unchanged. Also, analysis of culture-based assays in Matrigel where Mt-YAP5SA but not tumor samples, revealed expression of Vimentin, basal markers Mt-YAP6SA promoted EGF-independent growth as well as in- Cytokeratin-5, and lack of E-cadherin expression in all of the tu- vasive structure formation in BRCA1-deficient mammary epithelial mors (SI Appendix,Fig.S3E). Moreover, cell proliferation was cells (Fig. 4D). We also observed loss of contact inhibition and in- analyzed by Ki-67 staining (Fig. 4F) and we observed no significant crease in pAKT levels in Mt-YAP5SA–expressing MCF10A cells, difference in proliferation index between the two groups of tumors. which was significantly up-regulated even in reduced growth factor We then wanted to determine whether higher sensitivity to replication conditions (SI Appendix,Fig.S3A and B; lanes 9 and 10). Fur- stress could be involved in this phenomenon. As shown, BRCA1- thermore, we examined if YAP5A expression is sufficient to initiate deficient Mt-YAP5SA–expressing cells were highly sensitive to tumorigenesis in BRCA1-deficient MCF10A cells. MCF10A cells treatment with replication stress agent hydroxyurea (Fig. 4G). were injected into mammary fatpad of immunocompromised mice These observations highlight important implications of YAP1 and monitored for visible tumor growth for 45 d. As anticipated, reactivation in BRCA1-deficient cells. MCF10A cells which are nontumorigenic failed to generate tumors during these experimental periods (Fig. 4E). Similar observation was Discussion made with Mt-YAP6SA–expressing MCF10A cells, which also failed The cellular levels of oncogene YAP1 are regulated by phos- to originate tumors. It is important to note that p53 knockdown using phorylation through Hippo signaling. Growth factors inhibit

Fig. 4. Mt-YAP5SA expression in BRCA1-deficient mammary epithelial cells confers EGF-independent proliferation and tumorigenesis. (A–C) HEK293A or MCF10A cells were infected with lentivirus encoding shBRCA1 and indicated viruses. Then after 48 h, cells were seeded in 96-well plates, and treated with wst1 reagent to compare cell growth as described in Materials and Methods.(D) MCF10A cells were infected as described in A and then grown on matrigel in presence or absence of EGF. Fresh medium lacking EGF was added every 4 d. (E and F) Representative immunohistochemistry pictures and relative tumor volume. (G) MCF10A cells were treated with hydroxyurea as described in Materials and Methods, washed, and then incubated in six-well plates. Then after 10–12 d, cells were fixed, stained with crystal violet, and cell survival was determined by measuring absorbance at 590 nM. Results are expressed as means ± SEMs. Only values of P < 0.05 were considered significant (****P < 0.0001), by ANOVA test.

7368 | www.pnas.org/cgi/doi/10.1073/pnas.1822155116 Verma et al. Downloaded by guest on September 23, 2021 Hippo signaling and promote YAP1 stabilization. Although the 11.71%, 37 cases and shallow deletion: 66.46%, 210 cases; of 316 pa- interaction of NF2 with LATS1 and their recruitment to plasma tients, respectively) in patients with ovarian serous cystadenocarcinoma, membrane initiate Hippo-signaling activation, it remains unclear supporting its tumor suppressor function. Furthermore, TCGA what mechanisms dictate assembly of these signaling complexes. data analysis revealed coexistence of multiple alterations in Hippo The findings here establish an important role of ubiquitination pathway members (shallow deletion or deletion: NF2, LATS1, and signaling during Hippo pathway regulation. Ubiquitination profiling LATS2; gain of copy or amplification: YAP1 and TAZ) in these of core Hippo members revealed that NF2 is heavily ubiquitinated patients (SI Appendix, Fig. S3F). Former analysis performed from in proliferating cells (Fig. 1A). We found that ubiquitination of 33 cancer types (33) also highlighted multiple cooccurring alter- NF2 involves multiple lysine residues located in both FERM and ation in Hippo pathway components which may contribute to coiled-coil domain. Interestingly, point mutations in FERM domain YAP1/TAZ activation in human tumors. have been identified in human patients and shown to inactivate Both BRCA1 expression and YAP1 stabilization are known to NF2 protein by inhibiting LATS binding and its recruitment to be sensitive to serum stimulation (24, 29). We found that BRCA1 plasma membrane (35). We speculated that similar molecular expression is essential for YAP1 stabilization in multiple cell lines modification could be involved in reversible interconversion of with intact Hippo signaling. Furthermore, cytoplasmic localization NF2 protein to inactive state via ubiquitination switch. Indeed, of YAP1 protein in BRCA1 knockdown cells suggested activation ubiquitinated NF2 exhibits poor LATS1 binding and resides of Hippo signaling, which was then found to degrade YAP1 predominately in nucleus. NF2 ubiquitination was found to be through proteosomal degradation. The transcriptional program greatly enhanced in cells upon serum stimulation. However, changes control by YAP1/TEAD1–4 is involved in leading cell-cycle pro- in NF2 ubiquitination profile do not alter total NF2 protein levels, gression. Critical among the known transcriptional target are protein suggesting a mechanism of regulation that does not involve degra- involved in replication licensing, DNA synthesis and damage repair dation. Furthermore, NF2 was found to be ubiquitinated by ubiquitin (CDC6, GINS1, MCM3, MCM7, POLA2, POLE3, TOP2A, and mutants with only K-6, K-27, K-29, and K-63 available. It is to be RAD18), transcriptional regulators (ETS1, MYC, and MYBL1), noted that expression of BRCA1 ubiquitin ligase is also serum cyclins and their activators (CCNA2 and CDC25A), as well as responsive and known to generate ubiquitin chains with K-6, K-29, protein involved in mitosis (CENPF, CDCA5, and KIF23) (49). K-48, and K-63 (17–19, 24). We have shown that BRCA1/BARD1 These observations are in agreement with the known role of ubiquitin ligase complex contributes to NF2 ubiquitination (Fig. 1). BRCA1 in maintaining growth and genomic stability. The specific Furthermore, BRCA1/BARD1 knockdown using shRNA was found degradation of WT-YAP1 but not phosphorylation defective Mt- to inhibit endogenous NF2 ubiquitination. We also tested whether YAP5SA mutant upon BRCA1 knockdown clearly established the CELL BIOLOGY BRCA1 overexpression promotes NF2 ubiquitination by coex- inhibitory role of BRCA1 in Hippo signaling. We also investigated pressing NF2 and BRCA1 protein. Expression of only WT but the physiological consequences of YAP1 degradation in BRCA1- not ring-finger-domain mutant of BRCA1 was found to enhance deficient cells. We compared cell growth in cells constituted with NF2 ubiquitination, which was also reversed by BARD1 knock- Mt-YAP5SA or with NF2 deletion. Compared with WT cells, cells down. Reduced NF2 ubiquitination observed with BRCT-domain with NF2 deletion or Mt-YAP5SA expression were found to be mutant M1755R could be attributed to its ability to compete with less sensitive to BRCA1 knockdown with respect to growth in- endogenous BRCA1 and heterodimerize BARD1 and sub- hibition. Since YAP1-mediated cell proliferation also involves sequently inhibit NF2 binding by complex. Among core Hippo- AKT activation (44), we also tested AKT phosphorylation in active signaling components tested, NF2 was found to interact with Mt-YAP5SA or inactive Mt-YAP6SA–expressing MCF10A cells. BRCA1. Furthermore, interaction between the two proteins was We found that AKT phosphorylation was significantly up- found to be direct in in vitro binding assays, which further supports regulated in MCF10A cells expressing Mt-YAP5SA. The pAKT the hypothesis that NF2 activity is regulated by BRCA1 interaction. levels remained up-regulated even in reduced growth-factor con- Inherited mutations or reduced BRCA1 activity are known to ditions, which also correlated with EGF-independent cell growth in increase the risk of human breast and ovarian cancers. Pre- both 2D and 3D culture assay. Furthermore, we demonstrated the viously, we demonstrated aberrant expression of satellite RNAs role of YAP1 activation in promoting tumorigenesis in BRCA1- in BRCA1-deficient tumors, which itself alone can lead to tu- deficient MCF10A cells. It is important to note that we observe morigenesis by promoting genomic instability (13, 14). In smaller tumors formed in BRCA1-deficient cells and, on treatment agreement with mutational theory, genomic instability can foster with hydroxyurea, the same cells exhibited higher sensitivity toward tumorigenesis by selecting cells with growth advantage, oncogene replication stress compared with BRCA1-proficient cells. BRCA1- activation, or tumor suppressor inactivation. As a protective deficient cells are known to be highly sensitive to replication stress mechanism, normal cells have multiple checkpoints involving (50), which is experienced among other stresses by cells in growing ATM/ATR, CHK1/2, p53, p21, Bax, and 53BP1 activation to tumors. Thus, we concluded that tumor size might correlate with restrict growth in the absence of BRCA1 (45, 46). Evolution of replication stress in these tumors. Higher sensitivity of Mt- signaling adaptation(s) that allow BRCA1-deficient cells to YAP5SA–transformed BRCA1-deficient cells to replication stress override these and similar protective checkpoints may thus become agent presented here also confirms that these cells are still sensitive rate-limiting steps during the process of cell transformation. In sup- to genotoxic agents and combination with YAP1 inhibitors may port of this notion, development of mammary tumors is accelerated enhance the therapeutic outcome. by introduction of p53 null allele in conditional mouse model with Taken together, here we describe a homeostatic mechanism mammary-specific deletion of BRCA1. These tumors also exhibited where expression of chromosomal custodian protein, BRCA1, numerous other somatic alterations because of genomic instability inhibits Hippo signaling via NF2 ubiquitination to stabilize (46, 47). Not all BRCA1-deficient human tumors however have YAP1. These results identified a ubiquitination switch involving p53 mutations, suggesting existence of alternative checkpoint(s). BRCA1-NF2/Hippo-YAP signaling axis, which possibly engages Previous genetic screen using tissue-specific BRCA1 deletion in a cell growth and genomic stability in normal cells. p53 heterozygous mouse model identified amplification of YAP1 encoding locus in resulting tumors (44). A recent report also Materials and Methods identified YAP1/TAZ activation as a frequent event in human All protocols involving animal experiments were approved by the Institutional breast cancer, which also correlates with higher histological grade Animal Care and Use, Committee of the Salk institute for Biological studies. of tumors (ref. 48 and Fig. 1; analysis on 993 primary tumor HEK293T, HEK293A (human embryonic kidney cell), U2OS (human osteosarcoma samples). Additionally, TCGA data indicated that mutation and cell), and H-1299 (human non–small-cell lung carcinoma cell) were maintained in shallow deletions of BRCA1 are also highly frequent (mutations: DMEM (Gibco, Invitrogen) supplemented with glutamine, 10% FCS, 100 U/mL

Verma et al. PNAS | April 9, 2019 | vol. 116 | no. 15 | 7369 Downloaded by guest on September 23, 2021 penicillin, and 100 μg/mL streptomycin (Invitrogen) at 37 °C with 5% CO2.MCF10A ACKNOWLEDGMENTS. We thank Duojia Pan, Department of Molecular cells (human mammary epithelial cell) were cultured in DMEM/F12 (Invitrogen) Biology and Genetics, Johns Hopkins University School of Medicine, for supplemented with 5% horse serum (Invitrogen), 20 ng/mL EGF, 0.5 μg/mL hy- pGal4-TEAD4 and UAS-Luc construct. We also thank Mark Schmitt, Beth drocortisone, 10 μg/mL insulin, 100 ng/mL cholera toxin, and 100 μg/mL strepto- Coyne, Mie Soda, and I.M.V. Laboratory members for their help. S.V. was

mycin (Invitrogen) at 37 °C with 5% CO2. Generation of HEK293A cells knockout supported by the “The George E Hewitt Foundation for Medical Research” for NF2 gene has been previously described (34). The constructs expressing full Newport Beach, CA, USA (Salk Institute, La Jolla, CA). This work was supported length BRCA1 and its deletion fragments were described previously (51). Details of by the Mass Spectrometry Core of the Salk Institute with funding from NIH-NCI materials and methods including antibodies, plasmids, ubiquitination (52), and all CCSG: P30 014195 and the Helmsley Center for Genomic Medicine. K.-L.G. is cell-based assays are described in SI Appendix, Material and Methods. supported by grants from NIH (CA196878 and GM51586).

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