BRCA1/BARD1-dependent ubiquitination ofNF2 regulates Hippo-YAP1 signalingSachin 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 normalcell physiology. Although the E3 ubiquitin ligase BRCA1 is a key playerin maintenance of genomic stability, its role in growth signalingremains elusive. Here, we show that BRCA1 facilitates stabilization ofYAP1 protein and turning “off” the Hippo pathway through ubiquiti-nation of NF2. In BRCA1-deficient cells Hippo pathway is “turned On.”Phosphorylation of YAP1 is crucial for this signaling process because aYAP1 mutant harboring alanine substitutions (Mt-YAP5SA) in LATS1kinase recognition sites not only resists degradation but also rescuesYAP1 transcriptional activity in BRCA1-deficient cells. Furthermore,an ectopic expression of the active Mt-YAP5SA, but not inactiveMt-YAP6SA, promotes EGF-independent proliferation and tumori-genesis in BRCA1−/− mammary epithelial cells. These findings es-tablish an important role of BRCA1 in regulating stability of YAP1protein that correlates positively with cell proliferation.

Hippo | BRCA1 | NF2 | ubiquitination | cancer

Inherited mutations in breast cancer early onset gene BRCA1predispose individuals to highly aggressive form of breast andovarian cancers (1, 2). Studies in a knockout mouse model haveshown that mouse BRCA1 plays a key role in cell proliferationduring embryonic development (3). Mouse BRCA1 function canbe compensated by replacing it with human BRCA1 gene despitepoor sequence homology (4, 5). BRCA1 is involved in multiplecellular functions, e.g., transcription, heterochromatin structureformation, replication fork stability, homologous recombinationrepair, centrosome regulation, and mitotic spindle formation (6–11).These diverse roles are specified by interaction of BRCA1 with itsheterodimeric partner BARD1 that greatly enhances the E3 ubiq-uitin ligase activity of the complex (12). How loss of BRCA1 activityin heterozygous carriers leads to tumorigenesis remains only par-tially understood (13–15).Ubiquitination is a highly regulated and reversible event in-

duced by various stimuli that not only regulate protein stabilitybut also functional interaction, localization, and signaling dy-namics (16). These changes in protein activity by ubiquitinationare governed by number of ubiquitin molecules attached andnature of linkage involved. The enzymatic activity of BRCA1/BARD1 ubiquitin ligase complex is unique because it can generatedifferent kinds (mono and poly) of atypical ubiquitin linkages (K6,K29, K48, and K63) depending on the substrate and interactingE2 subunit (17–19). Several cellular proteins have been identifiedas substrates of this ubiquitin ligase activity, e.g., NPM1, RPB1,CtiP, RPB8, PR-A, TFIIE, Histones H2A, H2B, H3, H4, Ƴ-Tubulin,ER-α, Aurora A/B, and BRCA1 itself (20–23). These proteins areknown to be involved in regulating key signaling steps in dividingcells, e.g., in regulating gene expression, proliferation, chromosomemaintenance, duplication, repair, and segregation. However, unlikesubstrates of K48 ubiquitination, which are directed for proteasomaldegradation, many substrates with other atypical ubiquitin linkagesare not degraded but involved in signaling transduction. Thus,multiple pathways are integrated by BRCA1/BARD1 activity toensure genomic stability in normal cells. Not only is BRCA1highly expressed in proliferating cells, it is transcriptionally regulated

by serum stimulation and changes in cell density (24). BRCA1 ac-tivity is further regulated via changes in its levels, phosphorylationstate, and subnuclear distribution, depending on different stages ofcell cycle and signaling (25–27). Furthermore, BRCA1/BARD1activity is also regulated by proteosomal degradation (28). There-fore, BRCA1/BARD1 activity is tightly controlled to coordinategrowth-factor-stimulated downstream signaling and genomic stabil-ity. Although BRCA1 is known to play key roles in maintenance ofgenomic stability, whether it cooperates with signaling pathwaysto regulate physiological processes such as cell growth is not fullyexplored.The Hippo pathway transducer, YAP1 protein, is a mitogen-

responsive transcriptional coactivator. YAP1 is stabilized in thepresence of serum which turns “Off” the Hippo pathway (29).Upon serum stimulation, nuclear YAP1 in association with TEADproteins promotes expression of genes involved in cell proliferation.In absence of serum, Hippo signaling kinases LATS1/2 are activatedwhich, in turn, phosphorylate YAP1 to cause its cytoplasmiclocalization and degradation (30), thereby enabling homeostaticgrowth-restrictive regulation. Although YAP1/TAZ activation

Significance

Normal cells harbor protective mechanisms to sense DNAdamage, halt cell growth, and repair chromatin lesions tomaintain genomic stability. How mitogen signaling in pro-liferating cells is affected upon BRCA1 loss as part of theseprotective checkpoints is an intriguing question. This studyreveals a unique finding of linking BRCA1 to Hippo signalingpathway to explain this conundrum. Our work shows thatserum-responsive expression of BRCA1 is required for YAP1stability. Ubiquitination of NF2 by BRCA1/BARD1 in proliferatingcells inhibits NF2/LATS association and Hippo signaling. Thesefindings suggest Hippo signaling activation as a protective bar-rier in BRCA1-deficient cells, which upon inactivation, promotescell proliferation and tumorigenesis.

Author contributions: S.V., K.-L.G., and I.M.V. designed research; S.V., N.Y., Y.S., J.M.,J.K.D., and A.H. performed research; Q.Z., G.P., J.M., J.K.D., S.P., and T.M. contributednew reagents/analytic tools; S.V., N.Y., Y.S., K.-L.G., and I.M.V. analyzed data; and S.V.,Q.Z., A.H., K.-L.G., and I.M.V. wrote the paper.

Reviewers: J.D., University of Nebraska Medical Center; and D.M.L., Dana FarberCancer Institute.

Conflict of interest statement: K.-L.G. is a cofounder and has equity interest in VivaceTherapeutics, Inc. The terms of this arrangement have been reviewed and approved bythe University of California, San Diego in accordance with its conflict of interest policies.

Published under the PNAS license.1Present address: Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037.2Present address: Project Division of ALA Advanced Medical Research, Institute of MedicalScience, University of Tokyo, 108-8639 Tokyo, Japan.

3Present address: Ludwig Institute for Cancer Research, La Jolla, CA 92093.4To whom correspondence may be addressed. Email: kuguan@ucsd.edu or imohanv1@gmail.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplemental.

Published online March 27, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1822155116 PNAS | April 9, 2019 | vol. 116 | no. 15 | 7363–7370

CELL

BIOLO

GY

Dow

nloa

ded

by g

uest

on

June

16,

202

1

http://crossmark.crossref.org/dialog/?doi=10.1073/pnas.1822155116&domain=pdfhttps://www.pnas.org/site/aboutpnas/licenses.xhtmlmailto:kuguan@ucsd.edumailto:imohanv1@gmail.commailto:imohanv1@gmail.comhttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/cgi/doi/10.1073/pnas.1822155116

has been reported in various cancers (31), mutations involvingcore Hippo signaling components are not frequent (32), suggestingpossible involvement of upstream regulators which are yet to beidentified. In a recent analysis performed using 9,125 tumorsprofiled by The Cancer Genome Atlas (TCGA) from 33 cancertypes, multiple alterations in Hippo pathway members were identi-fied per tumor samples (33), suggesting that cooccurring copynumber alteration events correlate with oncogenic activation ofYAP1. Among core pathway components (scaffold protein: NF2;kinases: MST1, MST2, LATS1, LATS2; accessory molecules:SAV1, MOB1, MOB2; transcriptional coactivators: YAP1, TAZ),NF2 is critical for Hippo signaling as its inactivation impairsLATS1/2 activation and subsequent phosphorylation of YAP1(34). The current model in the field suggests that NF2 activatesHippo signaling pathway via binding LATS1/2 to facilitate LATS1/2 activation by MST1/2 (35). Also, NF2 activity is regulated byintramolecular interactions between N-terminal Ferm domain andC-terminal coil domains, directing interconversion between opento closed conformation (36). What molecular mechanism restrictsNF2 activity during mitogen signaling and assembly of these sig-naling complexes however remains unclear. Recently reported in-teractions of Hippo pathway components with various ubiquitinligases and deubiquitinase enzymes (37–41) suggest existence ofubiquitin signaling switches during Hippo signaling regulation.In this article, we show an important role of BRCA1 expres-

sion in Hippo signaling pathways. During mitogen signaling,NF2 is inhibited by BRCA1/BARD1-mediated ubiquitination,leading to YAP1 stabilization. Our findings suggest a previouslyuncharacterized protective mechanism in normal cells for fine-tuning growth-factor signaling and genomic stability, which whenbypassed in BRCA1-deficient mammary epithelial cells, promotecell growth and tumorigenesis.

ResultsBRCA1/BARD1 Promote NF2 Ubiquitination and Inhibits HippoSignaling. We first tested if ubiquitin signaling could be involvedin Hippo signaling regulation in proliferating cells. We observedthat among all of the core Hippo components, only NF2 and to alesser extent LATS1 were heavily ubiquitinated in serum-stimulatedcells (Fig. 1A). LATS1/2 were previously shown to be ubiquitinatedby Itch and CRLA4(DCAF1) ubiquitin ligase complex that directLATS1/2 for degradation (38, 39). The nature of ubiquitinationand ligase(s) specific for NF2 as well as its possible implication inHippo signaling has not been previously explored. Enrichment ofubiquitinated NF2 in denaturing conditions followed by mass spec-trometry analyses revealed that NF2 ubiquitination is K63-linkedand involves multiple lysines (K159, K269, K274, K364, K387,K396, K439, and K449) residues located not only in unstructuredcoiled coil domain but also highly ordered N-terminus FERMdomain (Fig. 1B and SI Appendix, Fig. S1 A and B). Since FERMdomain is known to mediate interaction with LATS1 kinase (35),we examined binding of equal amounts of free and ubiquitinatedNF2 with LATS1 from cell extract. Compared with free NF2 thatis efficiently bound to LATS1 (Fig. 1C; lane 1), ubiquitinationof NF2 inhibited LATS1 binding (Fig. 1C; lane 2). Enrichmentof ubiquitinated proteins after cell fractionation revealed thatubiquitinated NF2 resided mainly in the nucleus (Fig. 1D). Usingmutants of ubiquitin with single lysine available for linkage (Ub-K6, Ub-K11, Ub-K27, Ub-K29, Ub-K33, Ub-K48, and Ub-K63),we found that NF2 ubiquitination preferentially incorporatesUb-K6, Ub-K27, Ub-K29, and Ub-K63 but not Ub-K48, Ub-K11,and Ub-K33 (Fig. 1E) ubiquitin mutants, suggesting its possibleinvolvement in nonproteosomal regulation. These results are inagreement with the long half-life (exceeding 24 h) time of NF2 incells (42). Expression of E3 ligase BRCA1 in cultured cell isserum responsive and known to catalyze formation of K6, K29,K48, and K63 linked ubiquitin chains (17–19, 24). To test involvementof BRCA1/BARD1 with NF2 ubiquitination, we inhibited BRCA1/

BARD1 activity in cells by expressing shRNA against BRCA1 andBARD1. As shown (Fig. 1F; lane 3 compared with lane 2),ubiquitination of NF2 was greatly enhanced upon stimulationof cell with serum. On the other hand, NF2 ubiquitination wassignificantly reduced in BRCA1/BARD1-deficient cells (Fig. 1F; lane5 compared with 3). We further tested if BRCA1 overexpressioncan also augment NF2 ubiquitination. Overexpression of BRCA1greatly enhanced NF2 ubiquitination (Fig. 1G; lane 3 comparedwith 2). Also, to test involvement of BARD1 in this process wecoexpressed shRNA against BARD1 in the same cells, which re-versed enhanced NF2 ubiquitination (Fig. 1G; lane 4 compared with3). Similarly, ubiquitination of endogenous NF2 was confirmed to besensitive to shRNA-mediated inhibition of BRCA1/BARD1 (Fig.1H). We also inhibited BRCA1 expression in HEK293T-Cas9 cells(stably transduced with Cas9 gene) by transient transfection of singleguide RNA (sgRNA) targeting BRCA1 and observed inhibition ofNF2 ubiquitination (SI Appendix, Fig. S1C). Mutations in BRCA1N-terminus ring finger and C-terminus BRCT domains have beenidentified in human patients (43). We therefore tested the patho-genic mutant defective in BARD1 interaction and ubiquitin ligaseactivity (BRCA1 C61G) or BRCT domain mutants (BRCA1M1755R) for their ability to promote NF2 ubiquitination. Both ofthe pathogenic mutants BRCA1 C61G or BRCA1 M1755R failedto enhance NF2 ubiquitination (Fig. 1I, lanes 5 and 6). On thecontrary, expression of ring-finger domain mutant, BRCA1-I31Mthat does not alter ubiquitin ligase activity, was found to be equallypotent as wild-type protein in promoting NF2 ubiquitination (Fig.1I, lanes 3 and 4). Based on these observations, we speculated thatBRCA1-dependent ubiquitination of NF2 in proliferating cellsmay inhibit Hippo signaling.To dissect molecular consequences of BRCA1-mediated in-

hibition of Hippo signaling, we examined effects of BRCA1 ex-pression or knockdown on YAP1 transcriptional activity. Measuredby YAP1-responsive UAS-luciferase/TEAD-Gal4 reporter (30), weobserved a profound increase in YAP1 transcriptional output uponBRCA1 coexpression (Fig. 1J). On the other hand, we observedstrong inhibition of reporter activity in BRCA1 knockdown cells.These data suggest that BRCA1 directly regulates Hippo signalingthrough NF2 ubiquitination and promotes YAP1 activation.

BRCA1 Binds NF2 Through FERM and C-Terminal Domains.We furtheranalyzed direct role of BRCA1 protein in suppressing Hipposignaling through protein interactions. We tested interaction ofBRCA1 with core Hippo pathway components by pull-down as-says. HA-tagged NF2, but not any other core component (MOB,SAV, MST1, LATS, YAP1, and TAZ), was able to interact withGST-BRCA1 fusion protein (Fig. 2A). The intracellular interac-tion of BRCA1 with NF2 was further confirmed by immunopre-cipitation of endogenous BRCA1, which also recovered NF2protein in eluted fractions (Fig. 2B). Also, HA-NF2 was able tospecifically bind to GST-BRCA1 fusion protein but not GSTnegative control in vitro (Fig. 2C), confirming direct interaction be-tween the two proteins. We tested pathogenic mutants in BRCT-domain (P1744R and M1775R) or ring-finger domain (I31M,C39Y, T37R, C61G, V11A, and ring-domain deleted dRing), fortheir ability to interact with NF2. As shown, both wild-type andall ring-finger mutants were equally potent in NF2 binding (Fig.2D). However, interaction with NF2 was abolished in BRCT-domain mutants. We also compared patient-derived BRCA1-mutant HCC1937 cell line (harboring mutation in BRCT domainand loss of WT allele) with HCC1937-WTBRCA1 cells (recon-stituted with WTBRCA1), for their ability to bind NF2. Consistentwith our observations, endogenous NF2 binds BRCA1 in WT-BRCA1 reconstituted HCC1937 cells, but not Mt-BRCA1–expressing HCC1937 cells (SI Appendix, Fig. S2A). We nextmapped the protein domain involved in BRCA1/NF2 interaction.The N-terminal half of NF2, which possesses FERM domain, is re-quired for BRCA1 binding (Fig. 2E). Reciprocally, the C-terminal

7364 | www.pnas.org/cgi/doi/10.1073/pnas.1822155116 Verma et al.

Dow

nloa

ded

by g

uest

on

June

16,

202

1

https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/cgi/doi/10.1073/pnas.1822155116

fragment of BRCA1, but not its N-terminal ring-finger domain orcentral intrinsically disordered region, is required for NF2 binding(Fig. 2F). These results suggested that BRCA1 could regulateHippo signaling through direct NF2 interaction.

Serum-Responsive Expression of BRCA1 Contributes to YAP1 StabilizationThrough Regulation of Hippo Signaling. Because NF2-mediated Hipposignaling is involved in regulation of YAP1 stability, we furtherprobed a possible relationship between BRCA1 expression andYAP1 protein. As shown, stimulation of various cell lines with serumled to significant increase in BRCA1 expression (Fig. 3 A and B; lane2 compared with lane 1). As expected, protein levels of YAP1, pAKT,and pEGFR, which are growth signaling markers, were also elevatedin these cells. In contrast, BRCA1 knockdown by shRNA suppressedYAP1 levels in multiple cell types (Fig. 3 C and D; lane 2 comparedwith lane 1). As anticipated, BRCA1 knockdown activated DNAdamage signaling as judged by levels of ɣH2AX. All of the otherserum-responsive markers tested using specific antibodies, e.g.,pAKT, pEGFR, and pERK1/2, remained unchanged. We exam-

ined the cellular localization of YAP1, which is regulated uponphosphorylation through Hippo signaling. As expected, control cellswith Hippo signaling suppressed, show strong nuclear localization ofYAP1 protein (Fig. 3E). On the other hand, BRCA1 knockdownpromoted cytoplasmic localization of YAP1 protein. In contrast, theYAP1 protein level remained unaffected in NF2-null HEK293Acells (Fig. 3F; lane 4 compared with lane 2), suggesting that NF2downstream Hippo signaling is required for suppression of YAP1levels in BRCA1-deficient cells. Suppression of YAP1 protein levelsby Hippo signaling requires phosphorylation of YAP1 at multipleserine residues by LATS1/2 kinases. We therefore compared cel-lular levels of WT-YAP1 with Mt-YAP5SA protein that has mu-tations in all five LATS phosphorylation sites. As expected, expressionof shBRCA1 strongly suppressed total levels of WT-YAP1 (Fig. 3G;lane 2 compared with lane 1). Also, the pYAP1 levels in WT-YAP1–expressing cell remained comparable to control cells, suggestingaccumulation of phosphorylated WT-YAP1 in BRCA1 knockdowncells. However, BRCA1 knockdown had little or no effect onMt-YAP5SA levels (Fig. 3G; lane 5 compared with lane 4).

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 totalubiquitinated proteins were pulled down with the use of Ni-NTA beads, and ubiquitination was checked by immunoblotting with anti-HA antibody. (B) Aschematic 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-bindingbeads. The HA-bead–bound proteins were then incubated with cell lysate for LATS1 binding as described in Materials and Methods. (D) NF2 ubiquitinationwas 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 fromtransfected cells were then analyzed by immunoblotting using NF2 antibody. (F) HEK293T cells transfected with ubiquitin and HA-NF2 constructs were firststarved 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 indicatedplasmids, 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 areexpressed as means ± SEMs. 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 | 7365

CELL

BIOLO

GY

Dow

nloa

ded

by g

uest

on

June

16,

202

1

Correspondingly, shRNA-mediated inhibition of LATS1/2 kinasesrescued WT-YAP1 protein degradation in BRCA1 knockdowncells (Fig. 3H; lane 6 compared with lane 3). YAP1 degradationwas also rescued by treatment of cells with proteasome inhibitorMG132 or by coexpression of dominant negative mutant of ubiquitin(SI Appendix, Fig. S2 B and C). Furthermore, we tested rescue ofYAP1 transactivation activity by Mt-YAP5SA using UAS-Luciferase/TEAD-Gal4/YAP1 reporter system. Expression of WT-YAP1 andMt-YAP5SA protein promoted luciferase output from UAS-drivenluciferase/Gal-4-TEAD reporter. As expected, BRCA1 knockdownsuppressed reporter output in cells expressing WT-YAP1 (Fig. 3I).

However, cells expressing Mt-YAP5SA exhibited significantly higherreporter activity compared with WT-YAP1–expressing cells. These re-sults show that BRCA1 deficiency activatesHippo signaling and leads tosubsequent proteosomal degradation of WT-YAP1 protein (Fig. 3J).

Mt-YAP5SA Expression in BRCA1-Deficient Mammary Epithelial CellsConfers EGF-Independent Proliferation and Tumorigenesis. Havingestablished that BRCA1 deficiency activates Hippo signaling, wenext examined whether Hippo signaling contributes to growth in-hibition observed in BRCA1-deficient cells. As expected, knockdownof BRCA1 expression suppressed cell growth in HEK293A cells

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 weresubjected 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 inMaterials 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-bindingbeads. The resulting eluent was used to measure GST-BRCA1–bound NF2 by immunoblotting with anti-NF2 antibody. (F) HEK293T cells expressing the in-dicated BRCA1 Myc-tag fragments (1–303, 303–772, 772–1314, and 1314–1863) were tested for interaction with HA-NF2.

7366 | www.pnas.org/cgi/doi/10.1073/pnas.1822155116 Verma et al.

Dow

nloa

ded

by g

uest

on

June

16,

202

1

https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/cgi/doi/10.1073/pnas.1822155116

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 mediumfor 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 knockoutHEK293A cells (NF2−/−) were infected with lentiviral vectors expressing shRNA against BRCA1 for 72 h and harvested for immunoblotting. (G) HEK293T cellswere cotransfected with plasmids encoding myc-tagged WT-YAP1 (Myc-YAP1) or Mt-YAP5SA (Myc-YAP5SA) and shRNA against BRCA1 followed by immu-noblot analysis. Both WT- and Mt-YAP1s were detected by anti-Myc tag antibody. (H) HEK293T cells were cotransfected with WT-YAP1, and plasmidsexpressing two different shRNAs against BRCA1 (shBRCA1-1 and 2) and LATS1/2. (I) HEK293T cells were cotransfected with UAS-luciferase/TEAD-GAL4, renillaluciferase (normalization control), shBRCA1, and WT-YAP1 or Mt-YAP5SA followed by measurement of luciferase activity normalized to renilla luciferasereading. (J) A schematic model describing BRCA1-mediated NF2 ubiquitination, which promotes YAP1 stability in Hippo Off state via suppressing Hipposignaling. 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

CELL

BIOLO

GY

Dow

nloa

ded

by g

uest

on

June

16,

202

1

(Fig. 4A). In comparison, NF2-null HEK293A cells exhibited partialbut significant rescue of growth inhibition caused by BRCA1 knock-down. Similarly, serum stimulation of MCF10A cells expressingshRNA-targeting BRCA1 resulted in delayed growth (Fig. 4B)in comparison with control cells. In contrast, cells with ectopicexpression of Mt-YAP5SA exhibited rescue of cell growth. Theseobservations suggest that YAP1 reactivation could confer growthadvantage for BRCA1-deficient cells. Previously, YAP1 activationwas shown to promote growth of mammary epithelial cells in re-duced growth factor conditions (44). We therefore evaluatedwhether Mt-YAP5SA can promote EGF-independent growth ofBRCA1-deficient cells. As expected, MCF10A cells alone showedno growth in reduced serum and EGF-free media (Fig. 4C). Cellsexpressing null Mt-YAP6SA (with S94Amutation disrupting TEADbinding) behaved similarly with no growth advantage. However, weobserved significantly higher cell growth in active Mt-YAP5SA–expressing cells. These results were further confirmed using 3D–culture-based assays in Matrigel where Mt-YAP5SA but notMt-YAP6SA promoted EGF-independent growth as well as in-vasive structure formation in BRCA1-deficient mammary epithelialcells (Fig. 4D). We also observed loss of contact inhibition and in-crease in pAKT levels in Mt-YAP5SA–expressing MCF10A cells,which was significantly up-regulated even in reduced growth factorconditions (SI Appendix, Fig. S3 A and B; lanes 9 and 10). Fur-thermore, we examined if YAP5A expression is sufficient to initiatetumorigenesis in BRCA1-deficient MCF10A cells. MCF10A cellswere injected into mammary fatpad of immunocompromised miceand monitored for visible tumor growth for 45 d. As anticipated,MCF10A cells which are nontumorigenic failed to generate tumorsduring these experimental periods (Fig. 4E). Similar observation wasmade withMt-YAP6SA–expressing MCF10A cells, which also failedto originate tumors. It is important to note that p53 knockdown using

shRNA was also not sufficient to directly initiate tumorigenesis inBRCA1-deficient MCF10A cells under the observation period. Incontrast, BRCA1-deficient cells expressing Mt-YAP5SA wereable to produce tumors in all of the injected mice (Fig. 4E and SIAppendix, Fig. S3C). We concluded that YAP activation but notp53 inhibition alone can directly transform BRCA1-deficientmammary epithelial cells.The BRCA1-deficient tumors also showed a significant re-

duction of tumor volume, compared with control BRCA1-proficient tumors (Fig. 3E). We examined the cellular levels of53BP1, Mt-YAP5SA, and BRCA1 in tumor lysates (SI Appendix,Fig. S3D). All of the samples showed comparable amount of Mt-YAP5SA, 53BP1 proteins. Intracellular expression of BRCA1was monitored and as expected, tumor-cells–expressing shBRCA1had efficient knockdown of BRCA1 protein. GFP proteinexpressed from lentivirus (Fig. 4F) and β-Actin levels were moni-tored as internal control, which were unchanged. Also, analysis oftumor samples, revealed expression of Vimentin, basal markersCytokeratin-5, and lack of E-cadherin expression in all of the tu-mors (SI Appendix, Fig. S3E). Moreover, cell proliferation wasanalyzed by Ki-67 staining (Fig. 4F) and we observed no significantdifference in proliferation index between the two groups of tumors.We then wanted to determine whether higher sensitivity to replicationstress could be involved in this phenomenon. As shown, BRCA1-deficient Mt-YAP5SA–expressing cells were highly sensitive totreatment with replication stress agent hydroxyurea (Fig. 4G).These observations highlight important implications of YAP1reactivation in BRCA1-deficient cells.

DiscussionThe cellular levels of oncogene YAP1 are regulated by phos-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 orMCF10A cells were infected with lentivirus encoding shBRCA1 and indicated viruses. Then after 48 h, cells were seeded in 96-well plates, and treated withwst1 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 inpresence or absence of EGF. Fresh medium lacking EGF was added every 4 d. (E and F) Representative immunohistochemistry pictures and relative tumorvolume. (G) MCF10A cells were treated with hydroxyurea as described in Materials and Methods, washed, and then incubated in six-well plates. Then after10–12 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.

Dow

nloa

ded

by g

uest

on

June

16,

202

1

https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/cgi/doi/10.1073/pnas.1822155116

Hippo signaling and promote YAP1 stabilization. Although theinteraction of NF2 with LATS1 and their recruitment to plasmamembrane initiate Hippo-signaling activation, it remains unclearwhat mechanisms dictate assembly of these signaling complexes.The findings here establish an important role of ubiquitinationsignaling during Hippo pathway regulation. Ubiquitination profilingof core Hippo members revealed that NF2 is heavily ubiquitinatedin proliferating cells (Fig. 1A). We found that ubiquitination ofNF2 involves multiple lysine residues located in both FERM andcoiled-coil domain. Interestingly, point mutations in FERM domainhave been identified in human patients and shown to inactivateNF2 protein by inhibiting LATS binding and its recruitment toplasma membrane (35). We speculated that similar molecularmodification could be involved in reversible interconversion ofNF2 protein to inactive state via ubiquitination switch. Indeed,ubiquitinated NF2 exhibits poor LATS1 binding and residespredominately in nucleus. NF2 ubiquitination was found to begreatly enhanced in cells upon serum stimulation. However, changesin NF2 ubiquitination profile do not alter total NF2 protein levels,suggesting a mechanism of regulation that does not involve degra-dation. Furthermore, NF2 was found to be ubiquitinated by ubiquitinmutants with only K-6, K-27, K-29, and K-63 available. It is to benoted that expression of BRCA1 ubiquitin ligase is also serumresponsive and known to generate ubiquitin chains with K-6, K-29,K-48, and K-63 (17–19, 24). We have shown that BRCA1/BARD1ubiquitin ligase complex contributes to NF2 ubiquitination (Fig. 1).Furthermore, BRCA1/BARD1 knockdown using shRNA was foundto inhibit endogenous NF2 ubiquitination. We also tested whetherBRCA1 overexpression promotes NF2 ubiquitination by coex-pressing NF2 and BRCA1 protein. Expression of only WT butnot ring-finger-domain mutant of BRCA1 was found to enhanceNF2 ubiquitination, which was also reversed by BARD1 knock-down. Reduced NF2 ubiquitination observed with BRCT-domainmutant M1755R could be attributed to its ability to compete withendogenous BRCA1 and heterodimerize BARD1 and sub-sequently inhibit NF2 binding by complex. Among core Hippo-signaling components tested, NF2 was found to interact withBRCA1. Furthermore, interaction between the two proteins wasfound to be direct in in vitro binding assays, which further supportsthe hypothesis that NF2 activity is regulated by BRCA1 interaction.Inherited mutations or reduced BRCA1 activity are known to

increase the risk of human breast and ovarian cancers. Pre-viously, we demonstrated aberrant expression of satellite RNAsin BRCA1-deficient tumors, which itself alone can lead to tu-morigenesis by promoting genomic instability (13, 14). Inagreement with mutational theory, genomic instability can fostertumorigenesis by selecting cells with growth advantage, oncogeneactivation, or tumor suppressor inactivation. As a protectivemechanism, normal cells have multiple checkpoints involvingATM/ATR, CHK1/2, p53, p21, Bax, and 53BP1 activation torestrict growth in the absence of BRCA1 (45, 46). Evolution ofsignaling adaptation(s) that allow BRCA1-deficient cells tooverride these and similar protective checkpoints may thus becomerate-limiting steps during the process of cell transformation. In sup-port of this notion, development of mammary tumors is acceleratedby introduction of p53 null allele in conditional mouse model withmammary-specific deletion of BRCA1. These tumors also exhibitednumerous other somatic alterations because of genomic instability(46, 47). Not all BRCA1-deficient human tumors however havep53 mutations, suggesting existence of alternative checkpoint(s).Previous genetic screen using tissue-specific BRCA1 deletion in ap53 heterozygous mouse model identified amplification of YAP1encoding locus in resulting tumors (44). A recent report alsoidentified YAP1/TAZ activation as a frequent event in humanbreast cancer, which also correlates with higher histological gradeof tumors (ref. 48 and Fig. 1; analysis on 993 primary tumorsamples). Additionally, TCGA data indicated that mutation andshallow deletions of BRCA1 are also highly frequent (mutations:

11.71%, 37 cases and shallow deletion: 66.46%, 210 cases; of 316 pa-tients, respectively) in patients with ovarian serous cystadenocarcinoma,supporting its tumor suppressor function. Furthermore, TCGAdata analysis revealed coexistence of multiple alterations in Hippopathway members (shallow deletion or deletion: NF2, LATS1, andLATS2; gain of copy or amplification: YAP1 and TAZ) in thesepatients (SI Appendix, Fig. S3F). Former analysis performed from33 cancer types (33) also highlighted multiple cooccurring alter-ation in Hippo pathway components which may contribute toYAP1/TAZ activation in human tumors.Both BRCA1 expression and YAP1 stabilization are known to

be sensitive to serum stimulation (24, 29). We found that BRCA1expression is essential for YAP1 stabilization in multiple cell lineswith intact Hippo signaling. Furthermore, cytoplasmic localizationof YAP1 protein in BRCA1 knockdown cells suggested activationof Hippo signaling, which was then found to degrade YAP1through proteosomal degradation. The transcriptional programcontrol by YAP1/TEAD1–4 is involved in leading cell-cycle pro-gression. Critical among the known transcriptional target are proteininvolved in replication licensing, DNA synthesis and damage repair(CDC6, GINS1, MCM3, MCM7, POLA2, POLE3, TOP2A, andRAD18), transcriptional regulators (ETS1, MYC, and MYBL1),cyclins and their activators (CCNA2 and CDC25A), as well asprotein involved in mitosis (CENPF, CDCA5, and KIF23) (49).These observations are in agreement with the known role ofBRCA1 in maintaining growth and genomic stability. The specificdegradation of WT-YAP1 but not phosphorylation defective Mt-YAP5SA mutant upon BRCA1 knockdown clearly established theinhibitory role of BRCA1 in Hippo signaling. We also investigatedthe physiological consequences of YAP1 degradation in BRCA1-deficient cells. We compared cell growth in cells constituted withMt-YAP5SA or with NF2 deletion. Compared with WT cells, cellswith NF2 deletion or Mt-YAP5SA expression were found to beless sensitive to BRCA1 knockdown with respect to growth in-hibition. Since YAP1-mediated cell proliferation also involvesAKT activation (44), we also tested AKT phosphorylation in activeMt-YAP5SA or inactive Mt-YAP6SA–expressing MCF10A cells.We found that AKT phosphorylation was significantly up-regulated in MCF10A cells expressing Mt-YAP5SA. The pAKTlevels remained up-regulated even in reduced growth-factor con-ditions, which also correlated with EGF-independent cell growth inboth 2D and 3D culture assay. Furthermore, we demonstrated therole of YAP1 activation in promoting tumorigenesis in BRCA1-deficient MCF10A cells. It is important to note that we observesmaller tumors formed in BRCA1-deficient cells and, on treatmentwith hydroxyurea, the same cells exhibited higher sensitivity towardreplication stress compared with BRCA1-proficient cells. BRCA1-deficient cells are known to be highly sensitive to replication stress(50), which is experienced among other stresses by cells in growingtumors. Thus, we concluded that tumor size might correlate withreplication stress in these tumors. Higher sensitivity of Mt-YAP5SA–transformed BRCA1-deficient cells to replication stressagent presented here also confirms that these cells are still sensitiveto genotoxic agents and combination with YAP1 inhibitors mayenhance the therapeutic outcome.Taken together, here we describe a homeostatic mechanism

where expression of chromosomal custodian protein, BRCA1,inhibits Hippo signaling via NF2 ubiquitination to stabilizeYAP1. These results identified a ubiquitination switch involvingBRCA1-NF2/Hippo-YAP signaling axis, which possibly engagescell growth and genomic stability in normal cells.

Materials and MethodsAll protocols involving animal experiments were approved by the InstitutionalAnimal Care and Use, Committee of the Salk institute for Biological studies.HEK293T, HEK293A (human embryonic kidney cell), U2OS (human osteosarcomacell), and H-1299 (human non–small-cell lung carcinoma cell) were maintained inDMEM (Gibco, Invitrogen) supplemented with glutamine, 10% FCS, 100 U/mL

Verma et al. PNAS | April 9, 2019 | vol. 116 | no. 15 | 7369

CELL

BIOLO

GY

Dow

nloa

ded

by g

uest

on

June

16,

202

1

https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplemental

penicillin, and 100 μg/mL streptomycin (Invitrogen) at 37 °C with 5% CO2. MCF10Acells (human mammary epithelial cell) were cultured in DMEM/F12 (Invitrogen)supplemented with 5% horse serum (Invitrogen), 20 ng/mL EGF, 0.5 μg/mL hy-drocortisone, 10 μg/mL insulin, 100 ng/mL cholera toxin, and 100 μg/mL strepto-mycin (Invitrogen) at 37 °C with 5% CO2. Generation of HEK293A cells knockoutfor NF2 gene has been previously described (34). The constructs expressing fulllength BRCA1 and its deletion fragments were described previously (51). Details ofmaterials and methods including antibodies, plasmids, ubiquitination (52), and allcell-based assays are described in SI Appendix, Material and Methods.

ACKNOWLEDGMENTS. We thank Duojia Pan, Department of MolecularBiology and Genetics, Johns Hopkins University School of Medicine, forpGal4-TEAD4 and UAS-Luc construct. We also thank Mark Schmitt, BethCoyne, Mie Soda, and I.M.V. Laboratory members for their help. S.V. wassupported by the “The George E Hewitt Foundation for Medical Research”Newport Beach, CA, USA (Salk Institute, La Jolla, CA). This work was supportedby the Mass Spectrometry Core of the Salk Institute with funding from NIH-NCICCSG: P30 014195 and the Helmsley Center for Genomic Medicine. K.-L.G. issupported by grants from NIH (CA196878 and GM51586).

1. Miki Y, et al. (1994) A strong candidate for the breast and ovarian cancer susceptibilitygene BRCA1. Science 266:66–71.

2. King MC, Marks JH, Mandell JB; New York Breast Cancer Study Group (2003) Breastand ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 302:643–646.

3. Hakem R, et al. (1996) The tumor suppressor gene Brca1 is required for embryoniccellular proliferation in the mouse. Cell 85:1009–1023.

4. Chandler J, Hohenstein P, Swing DA, Tessarollo L, Sharan SK (2001) HumanBRCA1 gene rescues the embryonic lethality of Brca1 mutant mice. Genesis 29:72–77.

5. Szabo CI, et al. (1996) Human, canine and murine BRCA1 genes: Sequence comparisonamong species. Hum Mol Genet 5:1289–1298.

6. Chapman MS, Verma IM (1996) Transcriptional activation by BRCA1. Nature 382:678–679.

7. Scully R, et al. (1997) BRCA1 is a component of the RNA polymerase II holoenzyme.Proc Natl Acad Sci USA 94:5605–5610.

8. Willis NA, et al. (2014) BRCA1 controls homologous recombination at Tus/Ter-stalledmammalian replication forks. Nature 510:556–559.

9. Bunting SF, et al. (2010) 53BP1 inhibits homologous recombination in Brca1-deficientcells by blocking resection of DNA breaks. Cell 141:243–254.

10. Sankaran S, Starita LM, Groen AC, Ko MJ, Parvin JD (2005) Centrosomal microtubulenucleation activity is inhibited by BRCA1-dependent ubiquitination. Mol Cell Biol 25:8656–8668.

11. Joukov V, et al. (2006) The BRCA1/BARD1 heterodimer modulates ran-dependentmitotic spindle assembly. Cell 127:539–552.

12. Xia Y, Pao GM, Chen HW, Verma IM, Hunter T (2003) Enhancement of BRCA1E3 ubiquitin ligase activity through direct interaction with the BARD1 protein. J BiolChem 278:5255–5263.

13. Zhu Q, et al. (2011) BRCA1 tumour suppression occurs via heterochromatin-mediatedsilencing. Nature 477:179–184.

14. Zhu Q, et al. (2018) Heterochromatin-encoded satellite RNAs induce breast cancer.Mol Cell 70:842–853.

15. Venkitaraman AR (2014) Cancer suppression by the chromosome custodians,BRCA1 and BRCA2. Science 343:1470–1475.

16. Kerscher O, Felberbaum R, Hochstrasser M (2006) Modification of proteins by ubiquitinand ubiquitin-like proteins. Annu Rev Cell Dev Biol 22:159–180.

17. Wu W, Koike A, Takeshita T, Ohta T (2008) The ubiquitin E3 ligase activity ofBRCA1 and its biological functions. Cell Div 3:1.

18. Christensen DE, Brzovic PS, Klevit RE (2007) E2-BRCA1 RING interactions dictate syn-thesis of mono- or specific polyubiquitin chain linkages. Nat Struct Mol Biol 14:941–948.

19. Nishikawa H, et al. (2004) Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase. J Biol Chem279:3916–3924.

20. Starita LM, et al. (2004) BRCA1-dependent ubiquitination of gamma-tubulin regu-lates centrosome number. Mol Cell Biol 24:8457–8466.

21. Thakar A, Parvin J, Zlatanova J (2010) BRCA1/BARD1 E3 ubiquitin ligase can modifyhistones H2A and H2B in the nucleosome particle. J Biomol Struct Dyn 27:399–406.

22. Eakin CM, Maccoss MJ, Finney GL, Klevit RE (2007) Estrogen receptor alpha is a pu-tative substrate for the BRCA1 ubiquitin ligase. Proc Natl Acad Sci USA 104:5794–5799.

23. Starita LM, et al. (2005) BRCA1/BARD1 ubiquitinate phosphorylated RNA polymeraseII. J Biol Chem 280:24498–24505.

24. Rajan JV, Wang M, Marquis ST, Chodosh LA (1996) Brca2 is coordinately regulatedwith Brca1 during proliferation and differentiation in mammary epithelial cells. ProcNatl Acad Sci USA 93:13078–13083.

25. Ruffner H, Verma IM (1997) BRCA1 is a cell cycle-regulated nuclear phosphoprotein.Proc Natl Acad Sci USA 94:7138–7143.

26. Ruffner H, Jiang W, Craig AG, Hunter T, Verma IM (1999) BRCA1 is phosphorylated atserine 1497 in vivo at a cyclin-dependent kinase 2 phosphorylation site. Mol Cell Biol19:4843–4854.

27. Scully R, et al. (1997) Dynamic changes of BRCA1 subnuclear location and phos-phorylation state are initiated by DNA damage. Cell 90:425–435.

28. Choudhury AD, Xu H, Baer R (2004) Ubiquitination and proteasomal degradation ofthe BRCA1 tumor suppressor is regulated during cell cycle progression. J Biol Chem279:33909–33918.

29. Yu FX, Zhao B, Guan KL (2015) Hippo pathway in organ size control, tissue homeo-stasis, and cancer. Cell 163:811–828.

30. Zhao B, Li L, Tumaneng K, Wang CY, Guan KL (2010) A coordinated phosphorylationby Lats and CK1 regulates YAP stability through SCF(beta-TRCP). Genes Dev 24:72–85.

31. Plouffe SW, Hong AW, Guan KL (2015) Disease implications of the Hippo/YAP path-way. Trends Mol Med 21:212–222.

32. Harvey KF, Zhang X, Thomas DM (2013) The Hippo pathway and human cancer. NatRev Cancer 13:246–257.

33. Sanchez-Vega F, et al.; Cancer Genome Atlas Research Network (2018) Oncogenicsignaling pathways in The Cancer Genome Atlas. Cell 173:321–337.e10.

34. Plouffe SW, et al. (2016) Characterization of Hippo pathway components by geneinactivation. Mol Cell 64:993–1008.

35. Yin F, et al. (2013) Spatial organization of Hippo signaling at the plasma membranemediated by the tumor suppressor Merlin/NF2. Cell 154:1342–1355.

36. Sher I, Hanemann CO, Karplus PA, Bretscher A (2012) The tumor suppressor merlincontrols growth in its open state, and phosphorylation converts it to a less-activemore-closed state. Dev Cell 22:703–705.

37. Ma B, et al. (2015) Hypoxia regulates Hippo signalling through the SIAH2 ubiquitinE3 ligase. Nat Cell Biol 17:95–103.

38. Li W, et al. (2014) Merlin/NF2 loss-driven tumorigenesis linked to CRL4(DCAF1)-mediated inhibition of the hippo pathway kinases Lats1 and 2 in the nucleus.Cancer Cell 26:48–60.

39. Ho KC, et al. (2011) Itch E3 ubiquitin ligase regulates large tumor suppressor 1 sta-bility. Proc Natl Acad Sci USA 108:4870–4875, and erratum (2016) 113:E5776.

40. Kim Y, et al. (2017) Deubiquitinase YOD1 potentiates YAP/TAZ activities throughenhancing ITCH stability. Proc Natl Acad Sci USA 114:4691–4696.

41. Kim Y, Jho EH (2018) Regulation of the Hippo signaling pathway by ubiquitin mod-ification. BMB Rep 51:143–150.

42. Li W, et al. (2010) Merlin/NF2 suppresses tumorigenesis by inhibiting the E3 ubiquitinligase CRL4(DCAF1) in the nucleus. Cell 140:477–490.

43. Ruffner H, Joazeiro CA, Hemmati D, Hunter T, Verma IM (2001) Cancer-predisposingmutations within the RING domain of BRCA1: Loss of ubiquitin protein ligase activityand protection from radiation hypersensitivity. Proc Natl Acad Sci USA 98:5134–5139.

44. Overholtzer M, et al. (2006) Transforming properties of YAP, a candidate oncogeneon the chromosome 11q22 amplicon. Proc Natl Acad Sci USA 103:12405–12410.

45. Deng CX (2006) BRCA1: Cell cycle checkpoint, genetic instability, DNA damage re-sponse and cancer evolution. Nucleic Acids Res 34:1416–1426.

46. Dine J, Deng CX (2013) Mouse models of BRCA1 and their application to breast cancerresearch. Cancer Metastasis Rev 32:25–37.

47. Brodie SG, et al. (2001) Multiple genetic changes are associated with mammary tu-morigenesis in Brca1 conditional knockout mice. Oncogene 20:7514–7523.

48. Cordenonsi M, et al. (2011) The Hippo transducer TAZ confers cancer stem cell-relatedtraits on breast cancer cells. Cell 147:759–772.

49. Zanconato F, et al. (2015) Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth. Nat Cell Biol 17:1218–1227.

50. Densham RM, et al. (2016) Human BRCA1-BARD1 ubiquitin ligase activity counteractschromatin barriers to DNA resection. Nat Struct Mol Biol 23:647–655.

51. Pao GM, Janknecht R, Ruffner H, Hunter T, Verma IM (2000) CBP/p300 interact withand function as transcriptional coactivators of BRCA1. Proc Natl Acad Sci USA 97:1020–1025.

52. Verma S, Ali A, Arora S, Banerjea AC (2011) Inhibition of β-TrcP-dependent ubiquitinationof p53 by HIV-1 Vpu promotes p53-mediated apoptosis in human T cells. Blood 117:6600–6607.

7370 | www.pnas.org/cgi/doi/10.1073/pnas.1822155116 Verma et al.

Dow

nloa

ded

by g

uest

on

June

16,

202

1

https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1822155116/-/DCSupplementalhttps://www.pnas.org/cgi/doi/10.1073/pnas.1822155116