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. 2014 Jul 14;26(1):48-60.
doi: 10.1016/j.ccr.2014.05.001.

Merlin/NF2 loss-driven tumorigenesis linked to CRL4(DCAF1)-mediated inhibition of the hippo pathway kinases Lats1 and 2 in the nucleus

Wei Li  1 , Jonathan Cooper  2 , Lu Zhou  3 , Chenyi Yang  4 , Hediye Erdjument-Bromage  5 , David Zagzag  6 , Matija Snuderl  6 , Marc Ladanyi  7 , C Oliver Hanemann  3 , Pengbo Zhou  4 , Matthias A Karajannis  8 , Filippo G Giancotti  9
Affiliations

Merlin/NF2 loss-driven tumorigenesis linked to CRL4(DCAF1)-mediated inhibition of the hippo pathway kinases Lats1 and 2 in the nucleus

Wei Li et al. Cancer Cell. .

Abstract

It is currently unclear whether Merlin/NF2 suppresses tumorigenesis by activating upstream components of the Hippo pathway at the plasma membrane or by inhibiting the E3 ubiquitin ligase CRL4(DCAF1) in the nucleus. We found that derepressed CRL4(DCAF1) promotes YAP- and TEAD-dependent transcription by ubiquitylating and, thereby, inhibiting Lats1 and 2 in the nucleus. Genetic epistasis experiments and analysis of tumor-derived missense mutations indicate that this signaling connection sustains the oncogenicity of Merlin-deficient tumor cells. Analysis of clinical samples confirms that this pathway operates in NF2-mutant tumors. We conclude that derepressed CRL4(DCAF1) promotes activation of YAP by inhibiting Lats1 and 2 in the nucleus.

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Figures

Figure 1
Figure 1
CRL4DCAF1 controls YAP activation and TEAD-dependent transcription in Merlin-deficient cells. (A) Schematics depict wild type (WT) and Merlin-resistant (1417X) DCAF1 (top). Meso-33 cells were transfected with HA-Merlin alone or in combination with HA-DCAF1 WT or 1417X and subjected to immunoblotting. (B) Meso-33 cells were transfected with the indicated si-RNAs and examined by immunoblotting. (C) Nf2 mutant FC-1801 cells were transduced with the indicated sh-RNAs and subjected to immunofluorescent staining with anti-YAP/TAZ. Nuclei were stained with DAPI. Scale bar, 10μm. (D) FC-1801 cells were transduced with either a control sh-RNA, a sh-RNA targeting DCAF1, Merlin, or dominant negative TEAD2 followed by a TEAD reporter, and subjected to luciferase assay. Error bar, +/−SEM. (E) and (G) Meso-33 cells were transfected with the indicated si-RNAs and immunoblotted as indicated. (F) and (H) Meso-33 cells were transfected with the indicated si-RNAs and then with empty vector or Merlin and immunoblotted as indicated. See also Figure S1.
Figure 2
Figure 2
DCAF1 interacts with Lats1/2, which are ubiquitylated in vivo. (A) and (B) 293T cells transfected with empty vector or Flag-HA (FH)-tagged DCAF1 were immunoprecipitated with anti-Flag and immunoblotted as indicated. (C) and (D) Nickel precipitates and total lysates from 293T cells expressing HA-Lats1 or HA-Lats2 alone or in combination with His-Ubiquitin were immunoblotted with anti-HA. Asterisk points to a band that may correspond to Lats1/2 non-specifically bound to nickel beads. (E) Summary of ubiquitylation sites identified on Lats1/2 by mass spectrometry. See also Figure S2 and Table S1.
Figure 3
Figure 3
CRL4DCAF1 ubiquitylates and inhibits Lats. (A) Meso-33 cells were transfected with His-Ubiquitin and the indicated recombinant proteins. Ubiquitylated proteins were nickel purified and immunoblotted with anti-HA. Total lysates were immunoblotted as indicated. (B) CRL4DCAF1 was reconstituted in vitro using purified recombinant Cul4A/Rbx1, DDB1, and DCAF1 and incubated with recombinant FH-Lats1 as indicated. Reaction mixtures were immunoblotted as indicated. (C) Meso-33 cells were transfected with the indicated si-RNAs and subjected to a cycloheximide chase assay. Lysates were immunoblotted as indicated. (D) The results in (C) and an additional replicate experiment were quantified by densitometry. Error bar, +/−SEM. (E) 293T cells expressing FH-DCAF1 in combination with increasing quantities of HA-Merlin were immunoprecipitated with anti-Flag and immunoblotted as indicated. (F) Diagram illustrates a model based on the definition of the sequence requirements for binding of DCAF1 to Lats and of Merlin to DCAF1. The WD40 domain of DCAF1, which is implicated in substrate selection, binds directly to the kinase domain of Lats (right). Merlin binds through its FERM domain to the C-terminal segment of DCAF1, disrupting the association of DCAF1 with Lats (left). See also Figure S3.
Figure 4
Figure 4
Nuclear Merlin promotes YAP phosphorylation and inhibits proliferation. (A) Summary of biochemical subcellular fractionation experiments. Cytoplasmic/membrane (CM) and soluble nuclear fractions (N) from Meso-33 cells expressing Merlin or indicated mutants were immunoblotted for Merlin. (B) Subcellular fractions from Meso-33 cells expressing wild-type Merlin or the indicated mutants were immunoblotted as indicated. (C) Meso-33 cells expressing wild-type Merlin or Merlin Δ24–27 were treated or not with leptomycin B to block nuclear export. Fixed cells were immunostained as indicated. Scale bar, 20μm. Meso-33 cells expressing Merlin or its mutants were subjected to immunoblotting as indicated (D) or BrdU assay (E). (F) FC-1801 cells transduced with wild-type Merlin or its mutants were subjected to soft agar assay. Error bar, +/−SEM. See also Figure S4.
Figure 5
Figure 5
CRL4DCAF1 inhibits Lats in the nucleus. (A) Total lysates, cytosolic and crude membrane fractions (CM), and nuclear fractions (N) from sparse (Sp) or confluent (Con) Met-5A cells were immunoblotted as indicated. (B) and (C) Sparse Met-5A cells and Meso-33 cells were stained with an anti-Lats1 antibody and DAPI. Scale bar, 20 μm. (D) Endogenous Lats1 was immunoprecipitated with an anti-Lats1 antibody from cytosolic and crude membrane fractions and nuclear fractions from sparse or confluent Met5-A cells and were immunoblotted as indicated. (E) 293T cells were transfected with empty vector (HA Ctrl) or Flag-HA-tagged Merlin (wild type or its mutants) and were lysed 24 hours later in RIPA buffer without SDS. Flag immunoprecipitates were washed using RIPA buffer without SDS. Flag immunoprecipitates and input were immunoblotted as indicated. (F) Fold enrichment of immunoprecipitated DCAF1 and Lats1 was estimated by densitometry of blots in (E), where enrichment is expressed as the total density of the immunoprecipitated bands normalized to their respective inputs. (G) Meso-33 cells expressing Merlin or its mutants were subjected to immunoblotting as indicated. See also Figure S5.
Figure 6
Figure 6
CRL4DCAF1-mediated inhibition of Lats and de-regulated YAP signaling sustain the oncogenic properties of Merlin-deficient tumor cells. (A) Meso-33 cells expressing GFP, YAP, or YAP-5SA were transfected with a SMART pool control si-RNA or one targeting DCAF1 and subjected to BrdU incorporation assay. (B) FC-1801 cells expressing empty vector or YAP-5SA were transduced with the indicated sh-RNAs and subjected to soft agar assay. The graph illustrates the results (±SEM) normalized to relative control in the vector or YAP-5SA group. (C) FC-1801 cells treated as in (B) were injected subcutaneously in nude mice. (D) Meso-33 cells transduced with a control sh-RNA or sh-RNAs targeting Lats1/2 were transduced with a control sh-RNA or two different sh-RNAs targeting DCAF1 and subjected to BrdU incorporation assay. (E) Meso-33 cells treated as in (D) were subjected to soft agar assay. Typical cell colonies in individual culture wells in the 24-well plate are shown (F). (G) FC-1801 cells transduced with a control sh-RNA (top) or sh-RNAs targeting Lats1/2 (bottom) were subsequently transduced with a control sh-RNA or a sh-RNA targeting DCAF1 and were injected subcutaneously in nude mice. Error bar, +/− SEM. See also Figure S6.
Figure 7
Figure 7
CRL4DCAF1 controls YAP/TAZ-dependent gene expression in NF2 mutant tumors. (A) A panel of human mesothelioma cell lines, including Merlin-wild-type (WT) and Merlin-mutant, were cultured under the same condition. Lysates were immunoblotted as indicated. (B) Meso-33 cells transfected with the indicated SMART pool si-RNAs were subjected to BrdU incorporation assay. (C) Meso-33 cells transfected with the indicated SMART pool si-RNAs were subjected to DNA microarray analysis and unsupervised hierarchical clustering. (D) Venn diagram analysis of the genes downregulated or upregulated following depletion of DCAF1 or both YAP and TAZ. P-values were obtained by Fisher Exact Test. (E) Genes whose expression depend on DCAF1 (orange), on both DCAF1 and YAP/TAZ (green), or on YAP/TAZ alone (blue), were subjected to GSEA in the gene expression profiles of human malignant mesothelioma classified by NF2 mutation status (NF2−, mutant; NF2+, wild-type). (F) Merlin-deficient primary human Schwannoma cells were transduced with a vector encoding GFP, Merlin, and control or DCAF1 sh-RNAs and subjected to immunoblotting. (G) Human meningioma and schwannoma samples classified by NF2 mutation status were analyzed for YAP phosphorylation (arbitrary unit, A.U.), which was normalized against total YAP expression. (H) Representative formalin-fixed and paraffin-embedded NF2 wild-type or NF2-deficient meningioma sections immunostained for YAP/TAZ and phospho-YAP and counterstained with hematoxylin. Scale bar, 100 μm. Error bar, +/−SEM. See also Figure S7 and Table S2–6.
Figure 8
Figure 8
The model illustrates the mechanism by which CRL4DCAF1 promotes oncogenesis. In Merlin-deficient cells, CRL4DCAF1 promotes ubiquitylation of Lats1/2, and suppresses phosphorylation and inactivation of YAP. YAP promotes TEAD-dependent expression of pro-proliferative genes. It is likely that CRL4DCAF1 contributes to oncogenesis by ubiquitylating additional targets. In normal cells, anti-mitogenic signals promote the accumulation of the de-phosphorylated, active form of Merlin. Upon translocation in the nucleus, this form of Merlin binds to DCAF1 and suppresses CRL4DCAF1 activity. This model does not exclude that Merlin activates the core Hippo kinase cascade by a distinct mechanism.
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