ARTICLE

Received 24 Sep 2016 | Accepted 21 Feb 2017 | Published 26 Apr 2017 DOI: 10.1038/ncomms15021 OPEN FAF1 phosphorylation by AKT accumulates TGF-b type II receptor and drives breast cancer metastasis

Feng Xie1,*, Ke Jin1,*, Li Shao2,*, Yao Fan1, Yifei Tu1, Yihao Li3, Bin Yang4, Hans van Dam3, Peter ten Dijke3, Honglei Weng5, Steven Dooley5, Shuai Wang6, Junling Jia1, Jin Jin1, Fangfang Zhou6 & Long Zhang1

TGF-b is pro-metastatic for the late-stage breast cancer cells. Despite recent progress, the regulation of TGF-b type II receptor remains uncertain. Here we report that FAF1 destabilizes TbRII on the cell surface by recruiting the VCP/E3 ligase complex, thereby limiting excessive TGF-b response. Importantly, activated AKTdirectly phosphorylates FAF1 at Ser 582, which disrupts the FAF1–VCP complex and reduces FAF1 at the plasma membrane. The latter results in an increase in TbRII at the cell surface that promotes both TGF-b-induced SMAD and non-SMAD signalling. We uncover a metastasis suppressing role for FAF1 through analyses of FAF1-knockout animals, various in vitro and in vivo models of epithelial-to- mesenchymal transition and metastasis, an MMTV-PyMT transgenic mouse model of mammary tumour progression and clinical breast cancer samples. These findings describe a previously uncharacterized mechanism by which TbRII is tightly controlled. Together, we reveal how SMAD and AKT pathways interact to confer pro-oncogenic responses to TGF-b.

1 Life Sciences Institute and Innovation Center for Cell Signalling Network, Hangzhou, Zhejiang 310058, China. 2 State Key Laboratory for Diagnostic and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, Hangzhou 310000, China. 3 Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Leiden University Medical Center, Postbus 9600 2300 RC Leiden, The Netherlands. 4 Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 94158, USA. 5 Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim 105760, Germany. 6 Institutes of Biology and Medical Science, Soochow University, Suzhou 215123, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to L.Z. (email: [email protected]) or to F.Z. (email: [email protected]).

NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021

ransforming growth factor-b (TGF-b) is a pro-metastatic expression and was enhanced by the depletion of endogenous factor in advanced cancer1–4. Upon ligand binding, the FAF1 (Fig. 1f). These data suggest that FAF1 inhibits TGF-b TTGF-b type II serine/threonine kinase receptor (TbRII) signalling by transiently binding to TbRII, which may result in activates the type I receptor (TbRI) to induce SMAD2/3 TbRII instability. phosphorylation. Activated SMAD2/3 forms hetero-oligomers To validate this hypothesis, we first examined whether with SMAD4, which accumulate in the nucleus to regulate FAF1 affects the immediate mediators of TGF-b receptor target genes1–3. In addition to the canonical SMAD pathway, signalling. The levels of TGF-b-induced SMAD2 phosphorylation TGF-b receptors can initiate other intracellular pathways via (P-SMAD2), SMAD2–SMAD4 complex formation, phosphory- either phosphorylation or direct interaction with signalling lated AKT (P-AKT) and phosphorylated p38 MAP kinase intermediates; these so-called non-SMAD signalling pathways (P-p38) were elevated in FAF1-depleted parental MDA-MB-231 include several branches that involve phosphatidylinositol kinase cells (Fig. 1g). These responses were severely inhibited by the (PI3K)/AKT, mitogen-activated protein kinases (MAPKs) and ectopic expression of FAF1-WT, but were not affected by a FAF1 Rho-like GTPase signalling intermediates5. mutant lacking the ubiquitin regulatory X (UBX) domain TGF-b cross-talks with other pathways6. Oncogenic PI3K/AKT (Fig. 1h). However, FAF1 did not affect the total levels of signalling antagonizes TGF-b-induced growth arrest and SMAD2, SMAD4, AKT or p38 MAP kinase. These data apoptotic responses7,8. Moreover, high TGF-b levels in tumours suggest that endogenous FAF1 is a critical antagonist for both correlate with overactive PI(3)K–AKT signalling, and poor TGF-b/SMAD and non-SMAD signalling. prognosis in breast cancer9–11. However, how AKT cross-reacts High-grade tumours and/or their cancer-associated fibroblasts with TGF-b-induced pro-invasive and pro-metastatic responses (CAFs) frequently express elevated levels of TGF-b, which in advanced tumours remains undefined. In the TGF-b/SMAD correlates with poor prognosis in cancer patients3,16. Given the canonical pathway, TbRI acts downstream of TbRII; the stability role of FAF1 in restricting TGF-b signalling activity, we and membrane localization of TbRII are therefore critical investigated the possibility that loss of FAF1 might be a determinants of both the sensitivity and duration of the TGF-b relevant prognostic factor in late-stage cancer. Oncomine17 response. Many previous studies have concluded that TbRII expression analysis revealed FAF1 downregulation in multiple mediates the cytostatic effects of TGF-b; loss of its function in human cancers in which TGF-b is pro-metastatic (Fig. 1i; many different cancer models promotes aggressive and metastatic Supplementary Fig. 2a). Using The Cancer Genome Atlas patient behaviour12,13. Whether a gain of function in TbRII can promote database, we observed that claudin-low breast cancer patients metastasis has not been thoroughly investigated. In this work, we show significant under-expressed FAF1 (Supplementary Fig. 2b). identify FAS-associated factor 1 (FAF1) as a key regulator of cell In the same database, individuals with breast carcinomas surface TbRII, in turn preventing the excessive activation of both exhibiting higher FAF1 expression had a longer life expectancy SMAD and non-SMAD TGF-b-induced signalling. During cancer than those with tumours exhibiting lower FAF1 expression progression, growth factor-induced (or oncogenic mutation) (Fig. 1j). These data suggest that FAF1 may play a tumour- activation of AKT mediates FAF1 phosphorylation and its suppressing role in multiple human cancer types by inhibiting dissociation from the plasma membrane and TbRII, thereby tumour-promoting pathways, such as TGF-b signals. reinforcing TbRII stability on the cell surface and activating the pro-metastatic functions induced by TGF-b in breast cancer cells. FAF1 promotes turnover of cell surface TbRII. The fact that depleting endogenous FAF1 enhances the half-life of exogenous C-terminal HA-tagged TbRII suggests that TbRII protein stability Results is highly regulated by FAF1 (Fig. 2a). To confirm this possibility, FAF1 associates with TbRII and inhibits TGF-b receptor we measured the stability of endogenous TbRII. Pulse-chase signalling. TGF-b can promote invasion and metastasis in labelling experiments showed that endogenous TbRII exhibited a advanced tumours3. Consistent with previous reports14,15,we shortened half-life upon the ectopic expression of FAF1-WT, observed that breast cancer cells with high metastatic potential but not upon the expression of a FAF1 mutant lacking the UBX appeared to have elevated TbRII protein levels (Supplementary domain (Fig. 2b). Conversely, TbRII displayed a prolonged Fig. 1a). Upon TbRII depletion, we observed a marked reduction half-life in FAF1-depleted cells (Supplementary Fig. 2c). We of both breast cancer and lung cancer metastasis in xenograft therefore investigated whether FAF1 misexpression affects TbRII mouse models (Fig. 1a; Supplementary Fig. 1b). Cells isolated levels at the plasma membrane, where signalling is initiated. from the metastatic nodules of mice showed a gain in TbRII Biotin-labelled cell surface TbRII displayed a severely decreased protein (but not messenger RNA) expression compared with their half-life upon ectopic FAF1 expression, but not upon expression parental cells, suggesting that TbRII protein is stabilized during of a FAF1 mutant lacking the UBX domain (Fig. 2c). As expected, cancer metastasis (Fig. 1b). We therefore sought to identify the FAF1 depletion in cells led to higher cell surface TbRII levels and critical regulators of TbRII. Treatment with lysosome inhibitors, an apparent decrease in protein turnover (Fig. 2d). These findings such as bafilomycin A1, NH4Cl or chloroquine (but not the suggest that FAF1 could promote TbRII instability by stimulating proteasome inhibitors MG132 or lactacystin), led to TbRII to TbRII turnover at the plasma membrane. accumulation (Fig. 1c), suggesting that TbRII is degraded via a lysosomal pathway. We therefore analysed proteins that FAF1 recruits VCP/bTRCP to target TbRII for polyubiquitylation. co-immunoprecipitated specifically with FLAG-tagged TbRII in FAF1 is not an E3 ubiquitin ligase but an adaptor for substrate the presence of lysosome inhibitor using mass spectrometry ubiquitylation and degradation18–20, and TbRII is regulated (Fig. 1d). FAF1, with 12 unique peptides detected, was identified by ubiquitination21. Analysis of the FAF1 interactome by as the strongest binding partner (Fig. 1d and Supplementary mass spectroscopy examination of the co-immunoprecipitants Table 1; Supplementary Data 1). By using limiting amounts of of Flag-tagged FAF1 revealed that FAF1 bound to TbRII antibody in immunoprecipitation, we pulled down equal valosin-containing protein (VCP) at a ratio near 1:1 (Fig. 3a,b; amounts of endogenous TbRII and verified that FAF1 bound to Supplementary Data 2). VCP has been shown to interact with the 18 endogenous TbRII in NH4Cl-treated non-transfected cells FAF1 UBX domain . We found that this UBX domain is (Fig. 1e). Furthermore, the TGF-b-induced CAGA12-Luc required for FAF1 to antagonize cell surface TbRII (Fig. 2c). This SMAD-dependent response was inhibited by FAF1 ectopic finding suggests that FAF1 might recruit VCP to regulate TbRII.

2 NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021 ARTICLE

ab MDA β Co.sh shTβRII 5 shT RII (n=5) Cells: -MB-231 A549 ×10 2.5 * 100 1.0

) 3 P M P M Mr (K) –1 2.0

) 0.5 sr

5 1.5 –2 2 50 β -100

T RII RII mRNA level 1.0 β

(×10 Co.sh

cm -75 0 T Radiance –1 0.5 (n=5) ** PM P M 1 Met BLI signals % Bone met-free 0 P: Parental cells (p s 0 Actin -35 Co. shTβRII 0 1020304050 M: Metastatic nodules Day after injection

c d 3.4e+004 Mod: 20,Oxidation[M] 100 3+ y 20 y 18 y 17 y 16 y 15 y 14 y 13 y 12 y 11 y 10 y 9 y 8 y 7 y 6 y 5 y 4 y 3 y13+ 1523.80 S S N E V L N V I Q G N T T V D E L M M R y11+ 1282.64 y12+ 1410.72 # Unique Sequence Spectral 90 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 b15 b16 b17 b18 b19 Cl Sample Proteins 80 4 peptides coverage (%) counts M (K) 70 y6+ 810.41 Treatment – MG132LactacystinBafilomycinNH ChloroquineA1 r ATβRII 28 38.8 390 60

β b9+ 956.45 IB:T RII -75 A FAF1 0 0 0 50 b8+ 843.49 β BTRII 27 39.5 425 40 b10+ 1084.67 b11+ 1141.71 y15+ 1736.98 y9+ 1111.58 IB:Actin B FAF1 12 20 22 30 y14++ 811.49 y8+ 1010.37 y10+ 1225.68 y5-Oxidation[M]+ 631.48 [M]-Oxidation[M]++ 1151.27

-35 y7-Oxidation[M]+ 845.58 y6-Oxidation[M]+ 746.48 y16+ 1850.15 b18++ 957.48 y14+ 1622.95 y7+ 909.54 20 y15-Oxidation[M]+ 1673.03 y4+ 566.26 b15+ 1556.79 y14-Oxidation[M]+ 1558.77 y5+ 695.44 y20++ 1140.48 y15-Oxidation[M]++ 836.66 y10-Oxidation[M]+ 1161.42 y13-Oxidation[M]+ 1459.79 b12+ 1255.66 b6+ 630.46 y11-Oxidation[M]+ 1218.61 y12-Oxidation[M]+ 1346.87 y10++ 613.48 Relative intensity (%) b13+ 1356.71 y3+ 453.33 y9-Oxidation[M]+ 1047.65 y20-Oxidation[M]++ 1108.43 b14+ 1457.87 b14++ 729.42 y4-Oxidation[M]+ 502.46 b16+ 1672.02 b17+ 1800.98 b5+ 517.16 b19++ 1022.59 b7+ 744.46 b17++ 900.48 y16++ 925.55

10 y14-Oxidation[M]++ 779.97 y13-Oxidation[M]++ 730.58 y17-Oxidation[M]++ 943.04 y18++ 1040.01 b11++ 571.35 [M]+++ 789.55 b13++ 678.50 y16-Oxidation[M]++ 893.57 y3-Oxidation[M]+ 389.38 y8++ 505.30 y9-Oxidation[M]++ 524.33 y17++ 974.70 y11++ 641.52 y19-Oxidation[M]++ 1064.52 b4+ 418.58 y8-Oxidation[M]+ 946.51 y7++ 455.31 y10-Oxidation[M]++ 581.13 y11-Oxidation[M]++ 609.50 y16-Oxidation[M]+ 1786.04 b9++ 478.33 0 b16++ 836.66 900 400 500 600 700 800 1,500 1,100 1,200 1,300 1,600 1,700 1,800 1,900 2,000 1,400 1,000 m/z e f i 45 CAGA-Luc

1.0 0.5 1.5 2.0 NH4Cl: –––++ –– IP antibody 30 1.0 β 0 –TGF- FAF1 0.5 TCL TβRII M (K) r +TGF-β 0 NS 0.5 –0.5 –0.5 IB:FAF1 -75 15 –1.0 (fold change) –1.0 –2.0

IB:TβRII Normalized expression units –1.5 -75 Relative luciferase activity 0 0 –1.5 =4) =5) =4)

FAF1: – wt UBX –– =37) =41) =27) =30) n n n =23) Brain ( PCC OEA Colon ( CRC Ovary ( GBM n n n n n ( ( ( ( (

shFAF1: –––++ Prostate P = 0.001 P = 1.01E-6 P = 6.83E-5 P = 0.005

g h j MDA-MB-231 cells MDA-MB-231 stable cells shRNA: Co.shshFAF1 #1 shFAF1 #2 Myc-constructs: Co. FAF1 fl UBX Mr (K) Mr (K) TGF-β (min): 0 45 90 180 0 45 90 180 0 45 90 180 TGF-β (min): -75 0 60 120 0 60 120 0 60 120 IB:SMAD4 -75 Breast cancer patient 100

IP: IB:SMAD4 IB:SMAD2/3 -75 Long (TCGA)

-50 IP:

SMAD2/3 exp. P-SMAD2 80 -50 SMAD2/3 IB:SMAD2/3 -50 FAF1 high SMAD4 -75 P-SMAD2 -50 (n = 171) SMAD2 -75 60 SMAD3 -50 SMAD4 P-AKT P-AKT -50 -50 40 FAF1 middle (n = 166) AKT AKT

TCL -50 -50 Percentage of distant -35 TCL -35 20

P-p38 metastasis-free survival P-p38 p38 -35 -35 Long P = 0.0092 FAF1 low (n = 166) exp. 0 FAF1 -75 p38 -35 0 50 100 150 200 IB: Myc -75 Actin -35 Time (months)

Figure 1 | FAF1 specifically associates with TbRII and inhibits TGF-b signalling. (a) Bioluminescent imaging (BLI) of representative mice from each group injected into the left heart ventricle with control (Co.) or MDA-MB-231 breast cancer cells stably depleted of TbRII (shTbRII). Images were captured at week 7. Dorsal images are shown. The BLI signal of every mouse in each experimental group is shown in the middle panel. The percentage of bone metastasis-free mice (right panel) in each experimental group is given. (b) Immunoblot (IB) analysis of TbRII protein levels in parental (P) and metastatic (M) variant of MDA-MB-231 and A549 cells (left panel). Quantitative PCR with reverse transcription analyses of TbRII in parental and metastatic cells are shown (right panel). Data are presented as the mean±s.d. (c) MDA-MB-231 cells were treated with MG132 (25 mM), lactacystin (30 mM), bafilomycin A1

(1 mM), NH4Cl (25 mM) or chloroquine (100 mM) for 5 h and collected for IB analysis. Actin IB is included as a loading control. (d) Mass spectrometry results of TbRII-Flag immunoprecipitants in the absence (sample A) and presence (sample B) of NH4Cl (25 mM) identified FAF1 as a binding partner; exemplified peptide is shown in the right panel. (e) IB of the input and immunoprecipitants (IPs) derived from MCF10A-RAS cells treated with NH4Cl (25 mM) as indicated; 5% of the total cell lysate was loaded as input. NS, non-specific antibody. (f) The CAGA12-Luc SMAD-dependent transcriptional response in HEK293T cells transfected with FAF1 wt/UBX-deleted mutant (mUBX) or shFAF1, as indicated, and treated with TGF-b (5 ng ml À 1) overnight. The data are presented as the mean±s.d. of three independent sets of experiments. (g,h) The IBs of total cell lysate (TCL) and anti-SMAD2/3 immunoprecipitants derived from control and FAF1 stable depletion (shFAF1#1 and shFAF1#2) (g) or Myc-FAF1 fl (full length)/UBX-deleted mutant stably expressing (h) MDA-MB-231 cells treated with ligand at the indicated time points. (i) Oncomine box plots of the FAF1 expression levels in multiple human advanced cancers versus normal tissues; Po0.01. (j) Kaplan–Meier curves of The Cancer Genome Atlas database showing that the metastasis-free survival of individuals positively correlated with FAF1 expression by the log-rank test (Po0.01).

NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021

ab TβRII-HA Co.sh Myc-FAF1 wt/ UBX: shRNA: Co.sh shFAF1 #1 120 – FAF1wt FAF1 UBX 120 shFAF1 #1 M (K) CHX (h): M (K) 100 Chase time (h): 0 1 2 4 0 1 2 4 0 1 2 4 r 100 0 2 4 6 8 0 2 4 6 8 r -100 -100 TβRII – IB:HA (Y11) -75 -75 autorad. -100 50 FAF1 wt 50 Long -75 FAF1 UBX IB:FAF1 -75 -75 exp. Intensity (%) IB:Myc TCL Percentage of time 0 0 IB:Actin -35 IB:Actin -35 0 0 12 4 024 6 8 Chase time (hrs) CHX (h)

cdβ Biotin-labelled TβRII Biotin-labelled T RII shFAF1 shFAF1 Co.vec shRNA: Co.sh Co.sh Co.vec FAF1 FAF1 UBX #1 #2 FAF1 shFAF1 #1 M (K) 120 TGF-β (h): M (K) 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 TGF-β (min): r FAF1 UBX 0 2 6 12 0 2 6 12 0 2 6 12 r 120 shFAF1 #2 -100 -75 100 -75 100 TβRII TβRII -100 Long Long -75 -75 exp. exp. 50 -75 50 IB: -50 IB:Myc Long Intensity (%) Intensity (%) P-SMAD2 -50

TCL IB:Actin -75 exp. 0 -35 TCL IB:FAF1 02 6 12 0 IB:Actin 0 -35 Biotin-labelled TβRII 60 30 90

120 turnover (h) Biotin-labelled TβRII turnover (min)

Figure 2 | FAF1 promotes TbRII turnover at the cell surface. (a) The immunoblot (IB) of cell lysate derived from HEK293T cells stably expressing TbRII-HA and depleted of FAF1 that were treated with CHX (20 mgmlÀ 1) at the indicated time points. The amount of protein after CHX treatment was expressed as a percentage of that present before treatment (time 0), and is shown in the right panel. The results are shown as the mean±s.d. of three independent sets of experiments. (b)[35S]-methionine labelling and pulse-chase analysis of TbRII in control and MDA-MB-231 cells stably overexpressing empty vector ( À ), Myc-FAF1 wt or FAF1mUBX (UBX domain-deleted mutants). The amount of labeled protein precipitated after the chase was expressed as the percentage of that at the beginning of the chase (time 0) and is shown in the right panel. The results are given as the mean±s.d. of three independent sets of experiments. (c,d) IB of biotinylated cell surface TbRII in MDA-MB-231 cells stably overexpressing FAF1 wt/FAF1 UBX-deleted mutant (mUBX) (c) or stably depleted of endogenous FAF1 by two independent shRNA (shFAF1 #1 and shFAF1 #2) (d) and treated with TGF-b (5 ng ml À 1) at the indicated time points. Quantification of the band intensities is shown in the right panel. Band intensity was normalized to the t ¼ 0 controls. The results are presented as the mean±s.d. of three independent sets of experiments.

VCP is an evolutionarily conserved hexameric AAA (ATPases (Fig. 3f). These results indicated that FAF1 recruits bTRCP to associated with diverse cellular activities) family member that can the VCP complex and subsequently recruits VCP/bTRCP to convert chemical energy from ATP hydrolysis into mechanical TbRII. This finding might be relevant for cell surface TbRII, force to remodel protein complexes22. One such complex—VCP– because we observed that FAF1 induced an increase in the levels ubiquitin fusion degradation 1 (UFD1)–nuclear protein of both VCP–UFD1–NPL4 and bTRCP in the membrane fraction localization 4 (NPL4)—plays a role in endoplasmic reticulum- (Supplementary Fig. 3c). To validate this hypothesis, we pulled associated degradation22. Other complexes, including VCP-p47 down biotin-labelled cell surface proteins and found that and VCP-UBXD1, have been proposed to catalyse proteasome- FAF1-WT but not the UBX-deleted mutant increased plasma independent membrane trafficking, including membrane protein membrane-associated VCP–UFD1–NPL4/bTRCP/CULLIN1 and segregation22. Interestingly, all of the important VCP-binding reduced the level of TbRII at the cell surface (Fig. 3g). In the partners were identified among FAF1 co-immunoprecipitants, same assay, FAF1 depletion reduced cell surface-bound including VAPA/VAPB, vesicle-associated membrane proteins VCP–UFD1–NPL4/bTRCP/CULLIN1 and increased the amount that function in trafficking and membrane fusion, and the E3 of TbRII at the plasma membrane (Fig. 3h). ubiquitin ligases b-transducin repeat containing E3 ubiquitin bTRCP is a F-box protein23. SGT1A, a homolog of S-phase protein ligase (bTRCP) and Cullin1/3 (Fig. 3a; Supplementary kinase-associated protein 1 (Skp1), was also identified as a Data 2). All of these proteins were confirmed to bind FAF1 FAF1-binding partner (Fig. 3a; Supplementary Data 2). Thus, (Supplementary Fig. 3a). FAF1 actually pulled down a SKP1-cullin1-F-box protein E3 As examined using a reporter assay, ectopic VCP expression ligase complex in which the substrate-recognition subunit bTRCP severely impaired TGF-b/SMAD signalling, whereas knocking plays an essential role24,25. In the ubiquitylation assay, FAF1 down endogenous VCP enhanced this response (Fig. 3c). promoted TbRII polyubiquitylation, an effect that was inhibited In cells ectopically expressing VCP, TbRII cell surface localization upon bTRCP depletion (Fig. 3i). Depletion of FAF1 together with was lost, and the protein appeared to localize in a dotted bTRCP therefore greatly decreased TbRII polyubiquitylation pattern in the cytosol (Fig. 3d). Furthermore, FAF1-mediated (Fig. 3j). These results indicate that TbRII polyubiquitylation inhibition of TbRII expression was partially rescued by N2, is primarily accomplished by the FAF1/bTRCP complex. N4-dibenzylquinazoline-2,4-diamine, an inhibitor of the ATPase Furthermore, ectopic expression of VCP also promoted activity of VCP (Supplementary Fig. 3b). TbRII polyubiquitylation and TbRII turnover on the cell VCP–UFD1–NPL4 as well as the E3 ligases bTRCP and surface—effects that were dependent on endogenous FAF1 CULLIN1 were found to associate with TbRII in control cells but expression (Fig. 3k). In summary, TbRII is primarily targeted not in FAF1-depleted cells (Fig. 3e). The loss of FAF1 had no for polyubiquitylation and turnover in the lysosome by the VCP effect on the VCP–UFD1–NPL4 complex formation but led to a and bTRCP E3 ubiquitin ligase complex, which is recruited by reduced association between VCP-TbRII and VCP-bTRCP FAF1 (Fig. 3l).

4 NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021 ARTICLE

a b c d # Unique Sequence Proteins Score β peptides coverage (%) –TGF- TβRII-HA TβRII-HA+V5-VCP FAF1 18,935.37 55 64.8 +TGF-β VCP 14604.47 60 75.2 NPL4 1,851.23 30 44.6 90 CAGA-Luc VAPB 1,411.79 14 61.7 – M (K) VAPA 1,061.75 15 56.1 Flag-FAF1Flag-VCPr 60 UFD1 664.88 20 54.7 -250 TβRII TβRII VCP Ubiquitin 466.67 9 72.1 -150 UBXD1 421.29 15 42.2 VCP -100 FAF1 -75 30 BTRCP 415.63 14 31.5 -50 CULLIN1 197.25 16 23.2 Relative luciferase

p47 103.06 10 31.6 -35 activity (fold changed) 0 CULLN3 59.81 3 4.6 -25 VCP: – + –– DAPI DAPI Merge SGT1A 42.3 1 3.6 μ shVCP: ––#1 #2 Scale bar 10 m

e fghBiotin- TCL labelled Biotin- TCL labelled

TCL IP:Flag TCL IP:Flag UBX UBX –– Myc-FAF1: wt wt M (K) TβRII-Flag: –++–++ r Flag-VCP: –++–++ shRNA: Mr (K) Mr (K) M (K) Long Co. FAF1 Co. FAF1 shFAF1: – – + – – + shFAF1: – – + – – + r -75 -100 IB:TβRII exp. Long β -75 IB:T RII -75 IB:TβRII -75 β exp. -75 IB:T RII -75 IB:FAF1 IB:FAF1 -75 -75 -75 -100 IB:VCP -100 IB:Myc IB:Flag IB:FAF1 -75 IB:VCP -100 IB:UFD1 -35 IB:UFD1 IB:VCP -100 -75 -35 IB:UFD1 -35 IB:NPL4 IB:NPL4 -75 IB:UFD1 -35 IB:NPL4 -75 -75 IB:βTRCP -50 β IB:NPL4 IB: TRCP -50 IB:βTRCP β IB:Cul1 -50 IB: TRCP -50 -75 IB:Cul1 -75 IB:Cul1 -75 IB:Cul1 -75

ijHA-Ub HA-Ub k l HA-Ub TβRII-Flag: ++ + + TβRII-Flag: +++++ TβRII-Flag: ++++ Myc-FAF1: ––++ shFAF1: – ++– – M (K) V5-VCP: ––++ Co.sh sh-βTRCP1/2 Mr (K) shβTRCP1/2: ––++– r Co.sh shFAF1 M (K) TGF-β -250 -250 r -150 -250 -150 III IB: IB: RII-(Ub)n RII-(Ub)n

-100 β -150 HA-Ub -100 β HA-Ub T

T IB: RII-(Ub)n HA-Ub -100 β -75 T IP:Flag VCP -75 IP:Flag FAF1 IP:HA -75 E3 IB:Flag -75 IB:Flag -75 IB:Flag -75 IB:Myc -75 -75 IB:FAF1 -100 IB:V5 Internalization IB:βTRCP -50 β -75 TCL

TCL IB: TRCP -50 IB:FAF1 TCL degradation IB:Actin Ubiquitin -35 IB:Actin -35 IB:Actin -35 (lysosome)

Figure 3 | FAF1 recruits VCP/bTRCP to target TbRII for polyubiquitination. (a) Mass spectrometry studies of anti-Flag-FAF1 immunoprecipitants revealed FAF1-binding partners. (b) FAF1 bound to VCP at a ratio close to 1:1. (c) The CAGA12-Luc SMAD-dependent transcriptional response of HEK293T cells expressing VCP or VCP shRNA (#1 and #2) and treated with TGF-b (2.5 ng ml À 1) overnight is shown. The data are presented as the mean±s.d. of three independent sets of experiments. (d) Immunofluorescence and 4, 6-diamidino-2-phenylindole (DAPI) staining of HeLa cells transfected with TbRII-HA or TbRII-HA with V5-VCP plasmids. Scale bar, 10 mm. (e,f) HEK293T cells were transfected with TbRII-Flag (e) or Flag-VCP (f) and with FAF1 depletion or not, as indicated. Cells were collected for immunoprecipitation (IP) and immunoblot (IB) analysis; 5% of the total cell lysate was loadedasan input. (g,h) MDA-MB-231 cells expressing Myc-FAF1 wt/UBX-deletion mutant (mUBX) (g) or infected with FAF1 shRNA as indicated (h) were harvested for biotinylation and immunoblot (IB) analysis. Co., control non-targeting shRNA. (i) HA-Ub stably expressing HEK293T cells were transfected with TbRII- Flag along with Myc-FAF1 and were infected with control (Co.sh) or bTRCP shRNA lentivirus as indicated. The cells were then collected for IP and IB analysis. (j) HA-Ub stably expressing HEK293Tcells were transfected with TbRII-Flag and infected with bTRCP1/2 shRNA and/or FAF1 shRNA lentivirus as indicated. Cells were then collected for IP and IB analysis. (k) HA-Ub stably expressed HEK293T cells were transfected with TbRII-Flag and VCP, and were infected with FAF1 shRNA lentivirus as indicated. Cells were then collected for IP and IB analysis. (l) Working model of how FAF1 mediates TbRII polyubiquitylation and turnover via lysosomal mediated degradation.

FAF1 correlates with good prognosis in breast cancer patients. (n ¼ 103) compared to those with lower expression of this gene Epithelial-like breast cancer cells can undergo epithelial-to- (n ¼ 104), strongly suggesting that FAF1 correlates with a good mesenchymal transition (EMT), upon which they become more prognosis (Fig. 4a). migratory, invasive and metastatic26–28. The upregulation of Using immunofluorescence, we found that FAF1-expressing mesenchymal markers N-cadherin, fibronectin, smooth muscle cells exhibited an epithelial-like phenotype similar to those actin and vimentin, and the downregulation of epithelial marker of control cells and cells treated with the TbRI kinase inhibitor E-cadherin are typical characteristics of EMT. Gene set SB431542, whereas FAF1-depleted cells exhibited mesenchymal- enrichment analysis (GSEA) of the NKI breast cancer patients like characteristics. The latter phenotype was partially inhibited data set29 demonstrated that gene signatures representing a good by SB431542 (Supplementary Fig. 4a). Upon ectopic FAF1 prognosis or less invasiveness of the tumour were significantly expression, the changes in EMT marker expression induced by enriched in patients with higher levels of FAF1 expression TGF-b were attenuated, whereas FAF1 depletion had the reverse

NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021 c Co. FAF1 Co. #1 #2 TGF-β: –+–+ –+–+–+ ab Twist1 MCF10A (Ras) Twist2 Snail1 Snail2 Co. FAF1 shFAF1 Snail3 Breast Cancer_Prognosis_Up Tnfsf10 β Id1 NES=1.999 TGF- (48 h): ––+ –– +–– + Zeb1 0.6 M (K) Zeb2 P =0 SB431542: + ––++–– –– r RhoA 0.4 -150 Plaur PThrP 0.2 FN1 IB:N-cadherin -150 Runx1 Long Runx2 0 Runx3 exp. Acta2 CDH2 IB:fibronectin -250 VIM MMP2 MMP3 Tumor_Less invasiveness IB:SMA MMP9 -35 MMP14 0.5 NES=1.979 Col1a1 0.4 Co1a2 P=0.001 ITGA5 Enrichment score (ES) 0.3 IB:vimentin MYH9 0.2 -50 CTGF -150 CXCR4 0.1 IB:E-cadherin CCL22 0 HES1 IL11 SERPINE1 IB:FAF1 -75 VEGFA ELF5 CDH1 FAF1-high versus FAF1-low patients IB:Actin -35 0 15 30 01326

de Breast cancer_metastasis_DN Breast cancer_metastasis_up Breast cancer relapse in bone 0.8 0 0 NES=–1.718 NES= –1.716 0.6 NES=1.99 P = 0.002 P = 0.012 P = 0.0084 0.4 –0.3 –0.3 0.2 –0.6 0 –0.6 Enrichment score (ES) Enrichment score (ES) Ranked gene list in FAF1-high Enrichment score (ES) versus FAF1-low patients Ranked gene list in FAF1-low versus FAF1-high patients

fg Dorsal Ventral Dorsal Ventral

) * ) Co.sh #1#2 #3 #1 #2 #3 7 #1 #2 #3 #1 #2 #3 –1 4

sr * shFAF1 8 –2 8 3 Co.sh cm +SB 431542 6 –1 6 2 shFAF1

Control vector +SB 431542 4 4 1 FAF1

2 Radiance (p s 2

Bone met BLI signals (×10 0

Co.vec ) *** –1 100 3 +SB shFAF1 Co.shRNA )

FAF1 sr

7 100 –2 2 Co.shRNA 50 cm 50 –1 * 1 ** Bone met BLI signals (×10 0 *

% Bone met-free * 0 % Bone met-free 05010 20 30 40 0

Co. FAF1 shFAF1+SB Days after injection 05010 20 30 40

Radiance (p s Days after injection Figure 4 | FAF1 inhibits bone metastasis in transplantable mouse model of metastasis and correlates with good prognosis in breast cancer patients. (a) Pre-ranked gene set enrichment analysis (GSEA) in FAF1-high versus FAF1-low patients. The consensus signature of the genes correlated with good prognosis (upper panel) or lower invasiveness (lower panel) was enriched in FAF1-high compared with FAF1-low patients. (b) Immunoblot (IB) of control and MCF10A-RAS cells stably expressing FAF1 or depleted of endogenous FAF1 (shFAF1) and treated with TGF-b (5 ng ml À 1) and SB431542 (10 mM) as indicated for 48 h. Epithelial-to-mesenchymal transition (EMT) marker proteins were analysed as indicated. (c) Heat map of TGF-b target and EMT-related genes in control or MCF10A-RAS cells stably expressing FAF1 or depleted of FAF1 (#1 and #2) and treated with or without TGF-b (1 ng ml À 1) for 8 h. (d) Consensus signature of genes significantly and negatively correlated with breast cancer metastasis are enriched in FAF1-high versus FAF1-low patients, as shown by pre-ranked GSEA. (e) Consensus signature of genes significantly and positively correlated with breast cancer metastasis (left panel), especially bone metastasis (right panel), are enriched in FAF1-low versus FAF1-high patients, as shown by GSEA (right panel). (f) Bioluminescent imaging (BLI) of three representative mice from each group at week 6 following injection with control or MDA-MB-231 cells stably expressing FAF1. Both ventral and dorsal images are shown (upper panel). The BLI signal of every mouse (lower left panel) and the percentage of bone metastasis-free mice (lower right panel) in each experimental group were followed over time. (g) The BLI of three representative mice from each group at week 6 following injection with control or MDA-MB-231 cells stably depleted of FAF1 and pretreated with or without SB431542 (SB, 10 mM) overnight. Both ventral and dorsal images are shown (left panel). The BLI signal of every mouse (right upper panel) and the percentage of bone metastasis-free mice (right lower panel) in each experimental group are shown over time. Data are presented as the mean±s.d.

6 NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021 ARTICLE effect (Fig. 4b). To evaluate whether FAF1 regulates global was strongly increased in FAF1 À / À /PyMT lesions compared EMT-related changes, we performed gene expression profiling of with FAF1 þ / þ /PyMT metastasis lesions (Fig. 5h). Finally, these RAS-transformed MCF10A cells. PCR array analyses indicated results were confirmed by independent experiments in which that the loss of FAF1 led to several molecular features of EMT, FAF1-overexpressing primary tumour cells derived from MMTV- including the upregulation of key transcriptional inducers such as PyMT transgenic mice (Fig. 5i) exhibited a significant decrease in Twist1, Twist2, Snail1, Snail2, Snail3, Zeb1 and Zeb2. Importantly, lung metastasis without detectable changes in primary tumour all of these genes were suppressed by an increase of FAF1 either growth following mammary fat pad injection in Friend Virus B in the basal or TGF-b-induced level (Fig. 4c), indicating that (FVB) mice (Fig. 5j–m). Overall, our data from transgenic mice FAF1 could promote mesenchymal-to-epithelial transition. In experiments complement our findings from cell line models and line with this finding, FAF1 attenuated TGF-b-induced migration clinical breast cancer samples, strongly supporting the role of in two-dimensional transwell assays (Supplementary Fig. 4b) as FAF1 as a metastasis-suppressing gene via the direct targeting of well as invasion in three-dimensional spheroid-collagen culture TbRII. systems (Supplementary Fig. 4c). In addition, knockdown of endogenous FAF1 promoted breast cancer migration and AKT phosphorylates FAF1 to inhibit its membrane localiza- invasion (Supplementary Fig. 4b,d). Moreover, GSEA analysis tion. In FAF1-expressing epithelial-like cells, the initiation of an showed that genes correlating with decreased metastasis were oncogenic TGF-b signal requires the dissociation of FAF1 from significantly enriched in FAF1-high patients (Fig. 4d), whereas TbRII. We next investigated how this process is achieved. In our gene signatures linked to increased metastasis, especially in the mass spectrometry analysis, we found that FAF1 was phos- bone, were significantly enriched in FAF1-low patients (Fig. 4e). phorylated on residues Ser 270, Ser 320 and Ser 582 We therefore directly investigated whether FAF1 suppresses bone (Supplementary Fig. 6a; Supplementary Table 2). Ser 582 is an metastasis in a transplant mouse model. AKT consensus RxRxxS(T) phosphorylation motif conserved in We used the highly bone metastatic subline MDA-MB-231 to FAF1 orthologs (Fig. 6a,b); the other conserved residue, Ser 426, evaluate the effect of FAF1 on metastasis30–32. Mice that had been was not phosphorylated (Supplementary Fig. 6b). FAF1 was intracardially injected with control MDA-MB-231 cells began to associated with AKT in vitro and in vivo (Supplementary develop detectable bone metastases after 35 days, and the number Fig. 6c,d). We used a phospho-specific antibody that recognizes of metastatic nodules and the area covered by them expanded in the optimal AKT phosphorylation consensus motif and found the following 2 weeks. Mice injected with FAF1-expressing cells that endogenous FAF1 was indeed phosphorylated by AKT; developed fewer bone metastases and had significantly longer this phosphorylation was elevated by an activated allele of AKT bone metastases-free survival periods (Fig. 4f). Furthermore, the (Myr-AKT1) and was blocked by LY294002, a selective inhibitor intracardial injection of cells expressing FAF1 short hairpin RNA of PI3K (Fig. 6c). Using in vitro and in vivo assays, we found that (shRNA) revealed potentiated bone colonization of circulating AKT phosphorylated FAF1 (Fig. 6d; Supplementary Fig. 6e). MDA-MB-231 cells, which was partially blocked by pretreatment Phosphorylation was not detected for the FAF1 S582A mutant of the cells with SB431542 (Fig. 4g). This finding confirms that but was present in FAF1 protein with other sites mutated FAF1 inhibits breast cancer bone metastasis, at least in part, via (Fig. 6d). The reactivity of FAF1 with the p-AKT substrate the suppression of TGF-b receptor signalling. antibody was reversed when the cell lysates were incubated with lambda phosphatase (Supplementary Fig. 6f). These data strongly FAF1 depletion promotes lung metastasis in the MMTV-PyMT suggest that AKT specifically phosphorylates FAF1 at Ser 582. mouse model. To determine the in vivo physiological functions Under physiological conditions, both insulin-like growth factor of FAF1, we generated FAF1-knockout mice by utilizing gene- (IGF)-1 and TGF-b-induced phosphorylation of endogenous targeted ES cells in which exon 3 of the FAF1 gene had been FAF1 were detected by the AKT substrate antibody and were replaced with the neomycin-resistance gene to generate a frame abolished when the cells were treated with LY294002 (Fig. 6e), shift in the remaining FAF1 sequence (Fig. 5a; Supplementary indicating that FAF1 is regulated by various cytokines and Fig. 5). FAF1 protein expression was greatly reduced in FAF1 oncogenic stimuli that activate AKT. heterozygous embryos and was absent in FAF1 homozygous The phosphorylation of certain AKT substrates controls their embryos (Fig. 5b), confirming gene knockout. FAF1 À / À female subcellular localization35. Cell fractionation analysis showed mice were viable and fertile and displayed no obvious abnorm- that activated Myr-AKT inhibits the membrane localization of alities when monitored for up to 1 year. In FAF1 þ / À and FAF1-WT, but not of the phospho-resistant FAF1-S582A FAF1 À / À mouse embryonic fibroblast cells, we observed (Fig. 6f). Compared with FAF1-WT, the basal level of FAF1- stabilized cell surface localization of TbRII, which led to a S582A in the membrane fraction was increased, whereas the more sensitive TGF-b response as indicated by increased phospho-mimetic FAF1-S582D was undetectable (Supplementary TGF-b-induced phosphorylation of both SMAD2 and AKT Fig. 7a). Upon LY294002 treatment or the expression of activated (Fig. 5c). The expression of downstream TGF-b target genes, such AKT, endogenous FAF1 showed increased or reduced as plasminogen activator inhibitor 1 (PAI1), interleukin 11 (IL11) localization, respectively, at the membrane, leaving the FAF1 and N-Cadherin (CDH2), were also elevated in FAF1 þ / À and level unchanged in both the cytoplasmic and nuclear fractions FAF1 À / À mouse embryonic fibroblasts (Fig. 5d). (Fig. 6g). Similarly, TGF-b reduced the membrane association of To complement our xenograft metastasis models, we examined FAF1, which was rescued by the selective inhibition of either the effects of FAF1 on metastasis using the well-established PI3K (LY294002 or BKM120) or AKT (GDC0068 or MK2206), MMTV-PyMT transgenic mouse model. These mice develop suggesting that the effect was dependent on PI3K/AKT activity luminal adenocarcinoma with a high incidence of lung metastasis, (Fig. 6h; Supplementary Fig. 7b). Therefore, Ser 582 phosphory- which relies on AKT activity33,34. Similar to the results obtained lation by AKT inhibited the membrane localization of FAF1. We from the xenograft models, we observed that FAF1 À / À /PyMT further hypothesized that this phosphorylation might also affect and FAF1 þ / À /PyMT mice exhibited an increase in the incidence the capacity of FAF1 to recruit the VCP/E3 complex (Fig. 6i). of lung metastasis (Fig. 5e), as well as a significant increase in the number of lung metastasis nodules (Fig. 5f) and greater lung AKT phosphorylates FAF1 thus disrupts FAF1-VCP/bTRCP lesion surface area (Fig. 5g,h) compared with their FAF1 þ / þ / complex. Ser 582 is located in the FAF1 UBX domain, a region PyMT counterparts. Furthermore, the level of TbRII expression that is required for FAF1’s association with VCP and bTRCP18,20.

NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021

d a b c +/+ +/– –/– FAF1FAF1FAF1 +/+ +/– –/– PAL1 MEF cells: FAF1 FAF1 FAF1 IL11 –/– +/+ +/– β CDH2 TGF- : – + – + – + Mr (K) +/+ +/– –/– FAF1 FAF1 FAF1 Biotin- β ZEB2 M (K) IB:T RII - 75 FAF1 FAF1 FAF1 r labelled CTGF 4 - 100 IB:p-SMAD2 - 50 SMAD7 IB:FAF1 - 75 IB:SMAD2 - 50 SLUG - 50 CXCR4 IB:P-AKT - 50 ZEB1 2 * IB:AKT - 50 SNAIL IB:FAF1 - 75 Ponceau IGFBP1 IB:Actin - 35 CDH1 0

e fgh TβRII 100 8 * 4 * H&E * ) * 2 75 6 /PyMT +/+

50 4 2

incidence (%) 25 2 Number of lung Lung metastasis /PyMT FAF1 surface area (log metastasis nodules Average lung lesion –/– 0 0 0 FAF1 FAF1+/+/PyMT FAF1+/–/PyMT FAF1–/–/PyMT

i j k l m Control P=0.84

40 *** 600 120 8 **

400 80 20 4 FAF1 200 40 incidence (%) Tumour volume Lung metastasis FAF1 mRNA level

0 0 0 Lung metastasis nodules 0 Co. FAF1 Co. FAF1 Co. FAF1 Co. FAF1 MMTV-PyMT tumour cells

Figure 5 | Loss of Faf1 in mice results in the accumulation of TbRII and promotes lung metastasis in the MMTV-PyMT transgenic mouse model. (a) Genotyping of the generated FAF1 þ / þ , FAF1 þ / À and FAF1 À / À mouse lines. PCR analysis of genomic DNA obtained from the tails of E13.5 mouse embryos. The asterisk indicates a FAF1 knockout-specific band. (b) IB of FAF1 protein derived from mouse embryonic fibroblasts (MEFs) from E13.5 FAF1 þ / þ , FAF1 þ / À and FAF1 À / À embryos. (c) FAF1 þ / þ , FAF1 þ / À and FAF1 À / À primary MEFs from E13.5 embryos were treated with TGF-b (5 ng ml À 1) for 45 min and then collected for biotinylation and IB analysis for cell surface TbRII expression. Expression levels of (phosphorylated) SMAD2 and AKT are also shown by IB with indicated antibodies. (d) FAF1 þ / þ , FAF1 þ / À and FAF1 À / À primary MEFs from E13.5 embryos were collected for quantitative PCR with reverse transcription (qRT–PCR) analysis of TGF-b-targeted and invasion-related genes. Relative mRNA levels are shown as a heat map. (e–g) The incidence of spontaneous lung metastasis (e), the number of lung metastasis lesions (f) and the average lung lesion surface area (g) (arbitrary units based on pixel quantification from digital images, the data represented are shown as the mean±s.d.) from FAF1 þ / þ /MMTV-PyMT þ (n ¼ 9), FAF1 þ / À /MMTV-PyMT þ (n ¼ 8) and FAF1 À / À /MMTV-PyMT þ animals (n ¼ 10). *Po0.05 by Mann–Whitney U-test in f and Student’s t-test in g.(h)TbRII staining in lung lesions collected from FAF1 þ / þ /MMTV-PyMT þ or FAF1 À / À /MMTV-PyMT þ mice. Scale bars, 1 mm and 20 mm for lung haematoxylin and eosin (H&E) and TbRII immunohistochemistry images, respectively. (i–m) Overexpression of FAF1 in MMTV-PyMT tumour cells inhibits lung metastasis. (i) qRT–PCR analysis of FAF1 expression in control or FAF1-overexpressing MMTV-PyMT primary tumour cells. Experiments were performed three times, each with qRT–PCR in technical duplicates. The data are presented as the mean±s.d. (j) Volumes of primary mammary fat pad tumours; n ¼ 6 per experimental group. P value computed by Mann–Whitney U-test. (k,l) The incidence of spontaneous lung metastasis (k) and the number of lung metastasis lesions (l) in control and FAF1-overexpressing primary tumours; n ¼ 6 per experimental group, **Po0.01 by Mann–Whitney U-test in l.(m) Representative lung nodules from mice injected with control and FAF1-overexpressing MMTV-PyMT tumour cells. Black arrowheads indicate lung metastasis nodules. Scale bar, 2 mm.

This fact suggests that the binding of FAF1 to VCP might be finding, FAF1-S582A potentiated TbRII ubiquitylation more influenced by AKT. In Myr-AKT-expressing cells, FAF1 barely efficiently (Fig. 7c). In cells expressing FAF1-WT/S582A/S582D bound to TbRII and inefficiently recruited VCP-UFD1-NPL4 or mutants, we isolated cell surface biotin-labelled proteins and bTRCP/CULLIN1 (Fig. 7a). To verify whether this finding is due found that FAF1-S582A significantly increased VCP-UFD1- to Ser 582 phosphorylation, we compared interactions using NPL4 and bTRCP/Cullin1 in the cell membrane fraction, FAF1-WT/S582A/S582D constructs. FAF1-S582A showed a thereby reducing the cell surface expression of TbRII (Fig. 7d). higher binding affinity for TbRII, VCP-UFD1-NPL4 and This suggests that the negative effects of FAF1 on TbRII could be bTRCP/Cullin1, whereas FAF1-S582D exhibited decreased directly inhibited by the AKT-mediated FAF1 phosphorylation of interactions with these proteins (Fig. 7b). In line with this Ser 582.

8 NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021 ARTICLE

a b Human (574-587) Mod: 3,Phospho[S] Macaca mulatta (574-587) 2.4e+005

8 7 6 5 4 3 2 1 Horse (569-582) 100 3+ y y y y y y y y 90 T P S G E F L E R c HA-Myr-AKT: – + – – – FAF1 Cow (574-587) b2 b3 b5 b6 b7 b8 80 [M]-Phospho[S]++ 509.41 LY294002: – –– Rat (573-586) 70 y8++ 507.99 IP Antibody: NS FAF1 Mr (K) Mouse (573-586) 60 50 p-AKT substrate - 75 Zebrafish (579-592) 40 y8-Phospho[S]++ 459.02 IP (RXRXXpS/pT) 30 y7-Phospho[S]+ 819.48

AKT consensus y7-Phospho[S]++ 410.52 y4+ 564.38

20 y6+ 750.46

Relative intensity (%) - 75 y7++ 459.02 y7+ 917.42 IB:FAF1 b2+ 199.18

Human p27 (148-158) y3+ 417.35 y2+ 304.31 10 b5-Phospho[S]+ 454.29 y1+ 175.21 y5+ 693.41 b5+ 552.26 b6+ 699.35 b8-Phospho[S]+ 843.45 b7-Phospho[S]+ 714.43 b4-Phospho[S]+ 325.32 b8+ 941.40 b6-Phospho[S]+ 601.26 b7+ 812.39 Human p21 (137-150) 0 b3+ 366.27 TCL 200 300 400 500 600 700 800 900 1,000 1,100 IB:HA - 50 Human Skp2 (64-76) m/z

d ef FAF1 wt FAF1 S582A Flag-FAF1: wt S582A T426A S270A S320A IGF-1: –+––– + HA-Myr-AKT: –++ –+–+–+– TGF-β: –+––+ – MemCytoNuc Mem Cyto Nuc Mr (K) p-AKT substrate LY294002: –++ –+– Mr (K) HA-Myr-AKT: – + – + – + – + – + – + M (K) - 75 p-AKT substrate r (RXRXXpS/pT) - 75 IB:Flag - 75 (RXRXXpS/pT) IP:Flag IB:HA IP:FAF1 - 150 - 50 IB:FAF1 - 75 IB:N-cadherin IB:p-AKT(Ser473) IB:Tubulin IB:Flag - 75 - 50 - 50 TCL IB:HDAC1 - 50 IB:AKT - 50 TCL IB:HA - 50

g h i Mem Cyto Nuc Mem Cyto Nuc Mem Cyto Nuc TGF-β: –++++ – ++ –+ ++ HA-Myr-AKT: –––––– –++ –+– p LY294002: ––––+ – + – – – + – p LY294002: –++ –+– ––––––M (K) BKM120: –+– ––– – + – – – + M (K) AKT r r E3 FAF1 - 75 IB:FAF1 - 75 p IB:FAF1 Long VCP - 75 IB:N-cadherin - 150 exp. FAF1 IB:N-cadherin - 100 IB:Tubulin - 50 &tubulin VCP - 50 - 50 E3 IB:HDAC1 IB:HDAC1

Figure 6 | AKT phosphorylates FAF1 at Ser 582 and inhibits its membrane localization. (a) Sequence alignment of the AKT phosphorylation site within FAF1 orthologs from different species and the known AKT-phosphorylating proteins p27, p21 and Skp2. (b) AKT-phosphorylated FAF1 peptide identified by mass spectrometry analysis. (c) Immunoblot (IB) of total cell lysate (TCL) and immunoprecipitants derived from HeLa cells transfected with Myr-HA-AKT and treated with increasing doses of LY294002 (25 and 50 mM) for 12 h as indicated. NS, non-specific antibody. (d) HEK293T cells were transfected with Flag-FAF1 wt or S582A, T426A, S270A or S320A mutants along with Myr-HA-AKT expression plasmids as indicated. Cells were then collected for immunoprecipitation (IP) and IB analysis. (e) IB analysis of TCL and anti-FAF1 immunoprecipitates derived from serum-starved HeLa cells treated with IGF-1 (200 ng ml À 1), TGF-b (5 ng ml À 1) and LY294002 (50 mM) as indicated for 8 h. (f) HeLa cells stably overexpressing FAF1 wt or FAF1 S582A mutant constructs were transfected with or without HA-Myr-AKT as indicated. Cells were then collected for membrane (Mem), cytoplasm (Cyto) and nuclear (Nuc) extraction and IB analysis. (g) IB of Mem, Cyto and Nuc extraction derived from HeLa cells transfected with or without HA-Myr-Akt and treated with LY294002 (50 mM for 12 h) as indicated. (h) HeLa cells were treated with TGF-b (5 ng ml À 1) and selective PI3K inhibitors LY294002 (50 mM) or BKM120 (1 mM) as indicated for 8 h. Cells were then collected for Mem, Cyto and Nuc extraction and IB analysis. (i) Hypothetical working model of AKT-mediated FAF1 phosphorylation: phosphorylated FAF1 does not efficiently attach to the cell surface or associate with the VCP/E3 complex.

These data suggest that the initial activation of AKT 4 weeks after intracardial injection (Fig. 7h). The immediate (for example, by oncogenic mutation or the activation of receptor expression of FAF1 wt significantly delayed metastatic outgrowth, tyrosine kinases) dissociates FAF1 from TbRII. TbRII may in whereas the expression of FAF1 S582A nearly abolished it turn lead to the further enhancement of SMAD and non-SMAD (Fig. 7h). This result suggests that the induction of FAF1 AKT activation downstream of TbRII. This process is a self- expression could inhibit metastasis, but FAF1 protein was enforcing loop. If this mechanism indeed occurs during cancer functionally suppressed by endogenous AKT kinase. In summary, progression, PI3K/AKT activity should correlate with TbRII AKT antagonizes FAF1 to maintain TbRII stability in metastatic levels in malignant cells. Indeed, blocking PI3K/AKT activity (malignant) breast cancer cells (Fig. 7i). enhanced TGF-b-induced and FAF1-dependent TbRII ubiquity- The non-SMAD signals downstream of TbRII incorporates lation (Fig. 7e). AKT inhibited TbRII polyubiquitylation induced AKT, leading to the positive amplification loop of AKT-TGF-b by FAF1-WT but had no effect in the case of FAF1-S582A signalling (Fig. 7i). (Supplementary Fig. 7c). TbRII was reduced to a low level by LY294002 in bone metastatic MDA-MB-231 cells, which impaired SMAD phosphorylation by TGF-b (Fig. 7f; Loss of FAF1 correlates with gain of TbRII function in Supplementary Fig. 7d). This process is apparently related to patients. Our findings suggest that pre-activated AKT phos- FAF1 recruitment of VCP; first, LY294002-induced loss of phorylates and therefore antagonizes FAF1, which subsequently TbRII was rescued by N2, N4-dibenzylquinazoline-2,4-diamine, strengthens cell surface TbRII and results in the reinforced co- a selective VCP inhibitor (Fig. 7f). Second, LY294002-induced activation of SMAD and non-SMAD signalling (for example, decrease of cell surface TbRII was not observed in FAF1-depleted AKT), which promote metastasis. To verify this model, we used cells (Fig. 7g). an orthotopic mouse model. Pre-treatment with epidermal To directly implicate FAF1 and its regulation by growth factor (EGF) at a low dose promoted the lung metastasis AKT-mediated phosphorylation in metastasis, we used a of 4T1 cells without significantly altering primary tumour growth doxycycline-regulated promoter to express FAF1 wt or the under the nipple; this effect on metastasis was further promoted phosphorylation-resistant S582A mutant in MDA-MB-231 cells by FAF1 depletion (Fig. 8a,b).

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To determine the clinical relevance and validity of our findings, normal breast tissue samples (11 cases) as controls. TbRII, we examined the expression of TbRII, P-SMAD2 and P-AKT 473, P-SMAD2 and P-AKT 473 levels were significantly higher in and their relationship with FAF1 in patient-derived tissue breast carcinomas, whereas the FAF1 level was significantly lower samples. We performed immunohistochemistry analysis on in cancer tissues compared with normal breast tissues (Fig. 8c). invasive breast carcinomas (110 cases), with tumour-adjacent Consistent with its role in mitigating TbRII protein levels, we

abcd TCL IP:Flag TCL IP:Flag Biotin- TCL labelled Flag-FAF1: – ++ –++ Myc-Ub Myr-HA-Akt: – – +––+Mr (K) Flag-FAF1: M (K) β * wt S582A S582D wt S582A S582D r T RII-HA: ++++ Long Flag-FAF1: – – β Flag-FAF1: –wtSASD M (K) wt S582A S582D wt S582A S582D M (K) -75 exp. T RII r r * IB:TβRII (Long Long -75 exp.) -150 IB:TβRII RII- β -75 exp. IB: β IB:T RII (Ub)n

-75 -100 T Myc-Ub -100 * IP:HA -75 IB:VCP -75 -75 -100 IB:Flag -75 IB:UFD1 -35 IB:HA -75 IB:VCP -75 IB:VCP -100 IB:NPL4 -100 Long exp. β IB:Flag -75 IB:UFD1 IB: TRCP -50 IB:UFD1 -35 -35 TCL -75 -100 -75 IB:NPL4 IB:CUL1 IB:NPL4 IB:actin -35 IB:βTRCP IB:HA IB:βTRCP -50 -50 -50 -100 -100 IB:CUL1 IB:Flag -75 IB:CUL1 IB:Flag -75

efMyc-Ub g β TβRII: Biotin-labelled T RII +++++++++ DBeQ shFAF1: ––––––+++ DMSO (VCP inhibitor) TGF-β (h): 024024024 Co.sh shFAF1 Mr (K) DMSO LY LY LY294002 (h): 0 36120 3612Mr (K) DMSO +LY +LY Mr (K) Treatment (h): 036 9036 9036 9 -150 -75 IB: RII- IB:TβRII β

(Ub)n β -75 Myc-Ub -100 T Long IB:T RII IP:TβRII -75 exp. -75 IB:FAF1 -75 IB:Actin & tubulin -50 -35 IB:TβRII -75 IB:FAF1 -75 TCL IB:Actin -35 Day: 21 28 35 42

(×105) h 10 10 Co.vector Co. vector (n =5) ) 5

FAF1 wt (n =5) ) 8 8 –1

FAF1 S582A (n =5) sr * –2

6 cm –1

FAF1 wt 6 * 4

Norm. photon flux (×10 4

2 Radiance (p s 0 20 40 60 2 FAF1 S582A Dox

EGF/IGF... i TGF-β TGF-β Oncogenic mutation

p p AKT p FAF1 VCP E3 FAF1 AKT SMAD response SMAD Non-SMAD (for example AKT activation)

Cytostatic effect Invasion/metastasis

β TGF- as tumour suppressor TGF-β as tumour promoter

10 NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021 ARTICLE observed a significant inverse correlation between high FAF1 level activated AKT and TGF-b cooperate to antagonize the inhibitory and high levels of TbRII (Pearson w2 ¼ 38.03, P ¼ 3.5E À 08), effect of FAF1 on TGF-b signalling. When oncogenic AKT is P-SMAD2 (Pearson w2 ¼ 7.85, P ¼ 0.001) and P-AKT 473 aberrantly activated in cells (for example, by oncogenic mutations (Pearson w2 ¼ 1.88, P ¼ 0.05; Fig. 8d,e; Supplementary or excessive growth factors39), AKT can directly phosphorylate Fig. 8a,b). As shown in Fig. 8d, representative staining of the FAF1 at Ser 582, which dissociates FAF1 from the cell surface and serial sections showed that the relative low FAF1 staining disrupts its ability to complex with VCP/bTRCP, which is correlates with high levels of TbRII, P-SMAD2 and P-AKT 473, required for the polyubiquitylation of TbRII. This process which were all found to be low in high FAF1 samples apparently allows for the activation of both SMAD and (Supplementary Fig. 8a). As a control, we confirmed a significant non-SMAD pathways. In this context, the tumour-suppressive and positive correlation between TbRII and its downstream target effects of the SMAD pathway are abrogated by AKT4; thus, P-SMAD2 (Pearson w2 ¼ 27.44, P ¼ 3.37E À 06; Supplementary SMAD-dependent gene responses are advantageous for cancer Fig. 8c,e); more importantly, activation of AKT (P-AKT 473) migration and invasion. The subverted TGF-b/SMAD signalling positively correlated with TbRII (Fig. 8f; Supplementary Fig. 8c), pathway then actively drives tumour cell progression. Tumour confirming our finding that AKT activity supports TbRII cells with this signature fail to execute TGF-b/SMAD-mediated function and collaborates with TGF-b-induced pro-invasive and growth arrest; rather, they undergo EMT in which cells lose cell pro-metastatic responses in advanced tumours. Our data also polarity and cell–cell contacts, become more motile, and acquire highlight the frequent co-activation of AKT and SMAD in breast fibroblast-like properties. AKT has been shown to reduce SMAD3 cancer (Supplementary Fig. 8d,f), suggesting that AKT could function in Hep3B cells in which TGF-b induces an apoptotic cooperate with SMAD to drive malignancy. response7,8. However, SMAD3 levels are often reduced in advanced human tumours, and low SMAD3 levels are sufficient for tumour promotion40,41. Aberrant AKT over-activation may Discussion therefore redirect TGF-b intracellular signalling, thereby TGF-b signalling is hyperactivated in advanced cancers36.We contributing to its switch from tumour suppressor to tumour found that TbRII protein levels are increased in metastasis, and promoter. Thus, AKT-mediated inactivation of FAF1 protein that TbRII levels determine metastasis severity, raising a novel confirms the high level of cell surface TbRII, which in turn mechanism by which TGF-b signalling is activated. As the first further strengthens both SMAD and AKT (one of the non-SMAD signalling molecule engaged by the TGF-b ligand, cell surface pathways) signals. Thus, a self-enforcing loop directing the TbRII is thought to be required for all TGF-b signalling tumour-promotive TGF-b pathway is triggered by AKT through responses. As a low dose of TbRII is sufficient for cytostatic the inactivation of FAF1, via which cancer cells are stimulated to SMAD activation, an increase in TbRII levels contributes to invade and metastasize. cancer and drug resistance37, and TbRII expression in CAFs FAF1 gene loss was observed to be significantly and inversely supports cancer growth and survival38. Here we provide correlated with TGF-b3 gene expression. In light of the FAF1 important new insights into how cell surface TbRII is downregulation and the loss of epithelial differentiation that we dynamically regulated in stability and subcellular localization clearly observed in different breast cancer models, we found the through FAF1-mediated polyubiquitination. We generated FAF1- clinical relevance of FAF1 in the percentage of distant metastasis- knockout mice and found that cell membrane TbRII accumulates free survival of breast cancer patients where the cutoff by FAF1 in FAF1-deficient cells from mouse embryos, confirming that expression correlates with prognosis (Fig. 1j). EMT is only clearly FAF1 restrains TbRII function under physiological conditions. detectable in low frequent breast carcinosarcomas such as the Pathologically, we identified FAF1 as a strong suppressor of molecular subtype of claudin-low breast cancers. We found that TGF-b signalling and showed that it recruits the VCP/bTRCP the claudin-low clinical samples have reduced expression of complex to promote turnover of cell surface TbRII, thereby FAF1, further supporting our analysis. Importantly, FAF1 gene inhibiting tumorigenic TGF-b function in EMT, invasion and loss has been reported in many types of human cancers, metastasis. This was confirmed by multiple in vivo models of suggesting that the tumour-suppressive role of FAF1 is not metastasis, an MMTV-PyMT transgenic model of mammary limited to breast cancer. Moreover, both TbRII and P-SMAD2 tumour progression and clinical breast cancer samples. levels correlated inversely with the FAF1 level in our breast cancer FAF1 is highly expressed in normal epithelial cells or tissue microarray analysis, further corroborating that FAF1 plays pre-malignant cells (such as MCF7 or MCF10A cells) but is an essential role in controlling TbRII and TGF-b signals. In vivo attenuated in the well-established mesenchymal-like MDA-MB- cancer models using cell lines or transgenic mice consistently 231 cells; thus, it limits the amount of cell surface TbRII to showed that loss of FAF1 expression is closely associated with prevent excessive TGF-b responses. Our study revealed that breast cancer metastasis. Either loss of FAF1 or disassembly of the

Figure 7 | AKT-mediated phosphorylation of FAF1 at Ser 582 functionally disrupts the FAF1-VCP/E3 complex and dismisses FAF1 from targeting cell surface TbRII. (a,b) Immunoblot (IB) of total cell lysate (TCL) and immunoprecipitants derived from HEK293T cells transfected with Flag-FAF1 and Myr-HA-AKT (a) or Flag-FAF1 wt/S582A/S582D expression plasmids (b) as indicated. *Indicates a non-specific band. (c) IB of TCL and immunoprecipitants derived from Myc-Ub stably expressed HEK293T cells transfected with TbRII-HA and Flag-FAF1 wt/SA/SD expression plasmids as indicated. (d) IB of TCL and biotinylated cell surface TbRII in MDA-MB-231 cells infected with Flag-FAF1 wt/S582A/S582D as indicated. (e) IB of TCL and immunoprecipitants derived from Myc-Ub stably expressed HEK293T cells transfected with TbRII and shFAF1 plasmids and treated with or without LY 294002 (LY, 50 mM for 6 h) and TGF-b (5 ng ml À 1) at the indicated time points. (f) IB of total TbRII in bone metastatic MDA-MB-231 cells treated with dimethylsulphoxide (DMSO) or N2, N4-dibenzylquinazoline-2,4-diamine (DBeQ; 10 mM) and LY294002 (50 mM) at the indicated time points. (g)IBof TCL and biotinylated cell surface TbRII in bone metastatic MDA-MB-231 cells infected with control (Co.sh) or shFAF1 lentivirus and treated with LY294002 (50 mM) at the indicated time points. (h) MDA-MB-231 cells expressing control vector (Co.vector) or DOX-inducible FAF1 wt/FAF S582A were injected intracardially into mice. DOX was administered 28 days after the inoculation of the cells (as indicated). Metastasis was measured by BLI. Normalized photon flux for the indicated time is presented; error bars, mean±s.e. P values; Student’s t-test (left panel). Representative images (right panel). (i) In our working model, AKT phosphorylates FAF1 to disassociate the membrane FAF1–VCP/E3 complex, thereby stabilizing cell surface TbRII for oncogenic invasion or metastasis (in which AKT activation has already blunted cytostatic TGF-b/SMAD to support oncogenic growth).

NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021

ab Primary tumour

NS Metastatic nodules in the lung 4 * 4 NS ×10

×10 4 10 NS 4 4 * ) ) –1 ) ×10 –1 8 * ) ×10 –1 sr Co.sh sr 8 –1 –2 sr 8 –2 3 Co.sh sr –2

6 –2 cm cm Co.sh+EGF –1 cm –1 6

6 cm –1 2 4 –1 4 4 2 1

Primary tumour BLI signals 2 Radiance (p s 2 Radiance (p s shFAF1 shFAF1 Lung metastasis BLI signals Radiance (p s 0

Radiance (p s 0 shFAF1+EGF shFAF1 + EGF Co.sh + EGF shRNA: Co. Co. FAF1 FAF1 shRNA: Co. Co. FAF1 FAF1 EGF: ––++ EGF: ––++

cdOverview Detail * *** 300 300

200 200 FAF1 score score H H RII

100 β 100 FAF1 T

0 0 RII β Normal BC Normal BC T

300 *** *** 300

200 200 score score H H p-SMAD2

100 100 p-SMAD2 p-AKT 473

0 0 Normal BC Normal BC p-AKT 473

e TβRII p-SMAD2 p-AKT f staining index staining index staining index r =0.237 P <0.0001 Low High LowHigh Low High 300 P < 0.001 P < 0.001 P < 0.001

100% 100% 100% 200 score 75% 75% 75% H

50% 50% 50% 100 pAKT 473 25% 25% % of specimens 25% % of specimens % of specimens 0 0 0 0 0 100 200 300 Low High Low High Low High TβRII H score FAF1 FAF1 FAF1

Figure 8 | Loss of FAF1 correlates with increased TbRII as well as downstream p-SMAD2 and p-AKTexpression in patients. (a) Bioluminescent imaging (BLI) of representative mice from each group at week 3 following injection with empty vector (PLKO.1) or 4T1 breast cancer cells stably depleted of FAF1 and pre-treated with or without EGF (5 ng ml À 1) for 8 h. Ventral images of the mice are shown (left panel). The BLI signal from primary tumours obtained from every mouse in each experimental group is shown. NS, not significant (right panel). (b) BLI of representative metastatic nodules in the lung from each experimental group is shown (left panel). Lung metastasis BLI signal for every mouse in each experimental group was analysed and is shown in the right panel (right panel). Data are presented as the mean±s.d. (c–f) Immunohistochemistry analysis of FAF1, TbRII, p-SMAD2 and P-AKT 473 in breast cancer tissue microarrays; expression of FAF1 and TbRII, P-SMAD2 and P-AKT 473 in normal (n ¼ 11) and breast cancer (n ¼ 110) tissues. The data are the mean±s.e.m. P values obtained using Student’s t-tests are indicated (c). Representative images of each antibody staining are shown. Left panel: objective, Â 5; right panel: objective, Â 25 (d). Percentage of specimens displaying low or high FAF1 expression compared with the expression levels of TbRII, P-SMAD2 and P-AKT (e). Scatterplot showing the positive correlation between TbRII and P-AKT 473 expression in patients. Pearson’s coefficient tests were performed to assess significance (f).

12 NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021 ARTICLE

FAF1/VCP/bTRCP complex via AKT phosphorylation can lead Mouse metastasis assay. MDA-MB-231-Luc and A549-Luc cells32 were to increased breast cancer metastatic potential. We evaluated in used to stably overexpress or were depleted of target genes by lentivirus the breast cancer tissue microarray specimens and found the infection and puromycin selection for 3 days. A single-cell suspension of these cells (1  105/100 ml of PBS) was inoculated into the left heart ventricle (n45) according status of AKT activation (p-AKT 473 staining) and FAF1 to the method described by Arguello et al.48. The development of metastases was expression (FAF1 staining) were not correlated, suggesting that monitored weekly by bioluminescent reporter imaging. After 6 weeks (or according these two events might be independent of each other. to the description in the figure legend), the mice were killed, and the metastasis In metastatic breast cancer cells, blocking PI3K/AKT activity nodules were dissected. For the induced model, doxycycline was administered to mice at indicated time point after injection of the cancer cells, through the diet reverses TbRII expression to a very low level (Fig. 7f,g; (625 mg kg À 1 of food) as well as by intraperitoneal injection (25 mg kg À 1 of body Supplementary Fig. 7d), demonstrating that TbRII-mediated weight; three times a week). For the MMTV-PyMT models, FAF1 KO mice were oncogenic TGF-b signalling is supported by AKT activity. backcrossed to FVB background for more than six generations before breeding Therapeutic targeting of PI3K/AKT holds significant promise as with MMTV-PyMT transgenic mice (Jackson Laboratory) in FVB background. 42 For spontaneous tumorigenesis and metastasis studies, female mice carrying the a treatment for cancer patients . Our results suggest that targeted specific oncogenes were examined weekly for mammary tumours. Tumours were therapy of the PI3K/AKT pathway may already, in part, target considered established when they became palpable for 2 consecutive weeks, and TGF-b signalling. Moreover, combined therapeutics against tumours were measured by calipers for calculation of tumour volumes PI3K/AKT and TbRII might serve as an efficient method for (p  length  width2/6). Lung nodules were counted directly after fixation or after 4 sectioning and staining of the lungs. For orthotopic primary tumour formation, advanced breast cancer patients in the future . female FVB (for PyMT tumour cells experiments) mice at 6 weeks old were In human patients, 70% of breast cancers are oestrogen anaesthetized and a small incision was made to reveal the mammary gland. receptor a (ER) positive and are thus treated with endocrine MMTV-PyMT tumour cells (2.5  104) resuspended in 10 ml PBS were injected therapies. However, 25% of these cancers eventually develop directly into the mammary fat pad. The primary tumour growth was monitored weekly by measurement of the tumour size. Experimental metastasis to the lungs endocrine resistance due to ESR1 gain-of-function mutations, 6 43–45 was induced by injecting 0.2  10 cells in 100 ml of PBS in the tail vein of female with ligand-independent ER activity driving metastasis .In athymic nude mice. Mouse nipple implantation of 4T1 cells was based on a an analysis of 4500 breast cancer patients, ESR1 apparently previously published method49. Female BALB/c mice were anaesthetized and used correlated with the loss of FAF1. It is therefore likely that ESR1 for this assay. A total of 1  105 4T1-Luc cells were injected through the nipple area correlates with a gain of TbRII function. Thus, endocrine into the mammary fat pad. At 21 days after injection, luciferin was injected and the primary tumours were analysed, then the mice were killed and analysed for resistance by ESR1 could be the result of oncogenic TGF-b acquisition of secondary tumour(s). All primary/metastatic tumours were detected activation. Although this hypothesis awaits further investigation, by bioluminescent imaging with the IVIS 100 (Caliper Life Sciences, Hopkinton, it raises the possibility that combined treatment with a TGF-b MA, USA). The bioluminescent imaging signal intensity was quantified as the sum antagonist could counteract the development of tolerance to of photons within a region of interest given as the total flux (photons per second). endocrine therapy in ER-positive breast cancer patients. In closing, our study proposes models of TbRII control and GSEA. We used GSEA v2.0 to perform GSEA on various functional and/or TGF-b functional switch triggered by activation of AKT. It will be characteristic gene signatures. Gene sets were obtained from the MSigDB database v3.0 (September 2010 release). Statistical significance was assessed by comparing interesting to see in the future whether the mechanisms we have the enrichment score to enrichment results generated from 1,000 random unravelled will extend to other cancers. If so, these hitherto permutations of the gene set to obtain P values (nominal P value). Data from underappreciated roles of TGF-b in malignancy and drug NKI 295, a well-annotated human breast cancer database, were analysed for the resistance could serve as a foundation for improving therapeutics enriched gene signature, and FAF1-high (FAF1Z0.28, n ¼ 103) versus FAF1-low against devastating cancers. (FAF1r À 0.34, n ¼ 104) samples were compared.

Plasmids and reagents. FAF1 and related expression constructs were cloned and Methods verified by DNA sequencing. FAF1 wt and FAF1 UBX domain-deleted mutant Animal studies. All mouse experiments were approved by and performed fragments were subcloned into the pLv-bc-puro lentivirus construct. FAF1 S582A following the guidelines of the Institutional Animal Care and Use Committee at and other mutants were generated by site-directed mutagenesis and confirmed by Zhejiang University. DNA sequencing. Myr-AKT1 constructs were kindly provided by P. Coffer (University Medical Center Utrecht, The Netherlands). The reagents used were IGF-1 (R&D 291-G1), LY294002 (Cell Signalling), MG132 (Selleck, catalog no. Nude mice and cell culture. Nude mice were purchased from the animal S2619), CHX (Sigma, C104450), SB431542 (Millipore, 616461) and l-phosphatase husbandry centre of the Shanghai Institute of Cell Biology, Academia Sinica, (Biolabs). BKM120, MK2206 and GDC0068 were purchased from Selleck. The Shanghai, China. HEK293T cells, HeLa cervical cancer cells, MCF7, MDA-MB-231, recombinant proteins used were human active AKT1 protein (R&D, 1775-KS), MDA-MB-435 breast cancer cells and A549 lung cancer cells from Leiden Uni- TbRII-ICD (Sino Biological Inc., 10358) and TGF-b (Sino Biological Inc., 10804). versity Medical Center46,47 were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U ml À 1 penicillin–streptomycin. MCF10A (M1), MCF10A- RAS (M2), M3 and M4 cells were cultured in DMEM/F12 supplemented with 5% Immunoprecipitation and immunoblotting. Cells were lysed with 1 ml of lysis horse serum, 20 ng ml À 1 EGF, 0.5 mgmlÀ 1 hydrocortisone, 100 ng ml À 1 cholera buffer (20 mM Tris-HCl pH 7.4, 2 mM EDTA, 25 mM NaF and 1% Triton X-100) toxin and 100 U ml À 1 penicillin–streptomycin. containing protease inhibitors (Sigma) for 10 min at 4 °C. After centrifugation at 12 Â 103g for 15 min, the protein concentrations were measured, and equal amounts of lysate were used for immunoprecipitation. Immunoprecipitation was Generation of Faf1 À / À mice. Faf1flox/ þ mice (C57BL/6J) were generated by performed with different antibodies and protein A-Sepharose (GE Healthcare standard homologous recombination at Shanghai Biomodel Organism, Shanghai, Bio-Sciences AB) for 3 h at 4 °C. Thereafter, the precipitants were washed three China. In these mice, Faf1 exon 3 was flanked by loxP sequences. Faf1flox/ þ mice times with washing buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet were then mated to EIIa-Cre transgenic mice (FVB/N; Jackson Laboratory), in P-40, 0.5% sodium deoxycholate and 0.1% SDS), and the immune complexes were which the adenovirus EIIa promoter directs the expression of Cre enzyme in early eluted with sample buffer containing 1% SDS for 5 min at 95 °C. The immuno- mouse embryos (two- to eight-cell stage) to achieve homologous recombination precipitated proteins were then separated by SDS–polyacrylamide gel electro- between LoxP sites, thereby triggering the deletion of exon 3 in all cells of the phoresis (SDS–PAGE). Western blotting was performed with specific antibodies developing animal, including the germ cells that transmit the genetic alteration to and secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish progeny. Deletion of exon 3 results in loss of the signal peptide and disrupts its peroxidase (Amersham Biosciences). Visualization was achieved with chemilumi- ORF, leading to the loss of Faf1 expression. The first generation of EIIa-Cre; nescence. For proteins that migrated close to the IgG heavy chain, protein Faf1flox/ þ mice might be chimeric due to the mosaic activity of Cre recombinase. A-horseradish peroxidase was used. Biotinylation analysis of cell surface receptors Therefore, chimeric offspring were backcrossed with C57BL/6J to generate was performed as previously described46,47, the details were shown below. The cells Faf1 þ / À mice, which were then intercrossed for the production of Faf1-deficient were biotinylated for 40 min at 4 °C and then incubated at 37 °C for the indicated (Faf1 À / À ) mice. Mouse genotyping was performed using genomic DNA isolated times. The biotinylated cell surface receptors were precipitated with streptavidin from mouse tails by PCR with the following primers: 50-CAGCCCACAACT beads and analysed by immunoblotting. The antibodies used for immunopreci- CACCTTTT-30 (faf1-KO-P1); 50-AATTGAAGGCCAGACGTAGC-30 (faf1-KO- pitation, immunoblotting and immunofluorescence were as follows: AKT at 1:5,000 P3-W686); 50-CTGAGCCCAGAAAGCGAAGGA-30 (Neo-R). A 1,086-bp and (IB) and 1:250 (IP; no. 2938, Cell Signalling); phospho-AKT substrate (RXRXXS*- B686-bp fragment were produced for the WT and null alleles, respectively. T*) at 1:1,000 (IB; no. 10001, Cell Signalling); phospho-AKT (Ser 473) at 1:1,000

NATURE COMMUNICATIONS | 8:15021 | DOI: 10.1038/ncomms15021 | www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15021

(IB; no. 9271, Cell Signalling); TbRII at 1:1,000 (IB) and 1:50 (IP; L-21, Santa Mass spectrometry. SDS–PAGE gels were minimally stained with Coomassie Cruz); N-cadherin at 1:50,000 (IB; 610920, BD); tubulin at 1:1,000 (IB; no. 2146, brilliant blue, cut into six molecular weight ranges based on heavy-chain IgG Cell Signalling); SMAD4 at 1:1,000 (IB; B8, Santa Cruz); SMAD2-3 at 1:2,500 (IB) bands, and digested with trypsin. Immunocomplexes were identified on a Thermo and 1:500 (IP; 610842 BD); phospho-SMAD2 at 1:5,000 (IB; no. 3101, Cell Fisher LTQ (majority) or Velos-Orbitrap mass spectrometer. Spectral data were Signalling); p38 at 1:3,000 (IB; no. 535, Santa Cruz); p-p38 at 1:2,000 (IB; no. 4511, then searched against the human protein RefSeq database in BioWorks or the Cell Signalling); Ub at 1:1,000 (IB; P4D1 Santa Cruz); fibronectin at 1:1,000 (IB; Proteome Discoverer Suites using either SeQuest (for LTQ data) or Mascot Sigma); SMA (Sigma); vimentin at 1:1,000 (IB; no. 5741 Cell Signalling); (Orbitrap data) software. The immunoprecipitation–mass spectrometry results E-cadherin at 1:10,000 (IB; BD 610181); b-actin at 1:10,000 (IB; A5441, Sigma); were transferred into a FileMaker-based relational database generated in-house, c-Myc at 1:1,000 (IB; a-14, sc-789, Santa Cruz Biotechnology); HA at 1:1,000 (IB; where protein identification numbers (protein GIs) were converted to GeneID Y-11, sc-805, Santa Cruz Biotechnology); HA at 1:10,000 (IB; 12CA5, home-made); identifiers according to the NCBI ‘gene accession’ table31. Flag at 1:10,000 (IB; M2, Sigma); FAF1 (Bethyl Laboratories); VCP at 1:2,000 (IB; no. 2648, Cell Signalling); Ufd1 at 1:1,000 (IB; no. 13789, Cell Signalling); Npl4 (IB; no. 13489, Cell Signalling); Cul1 at 1:1,000 (IB; no.4995 Cell Signalling); and Immunofluorescence. For the EMT assay, HaCaT cells were plated onto collagen I-coated coverslips. After adhesion, the cells were serum starved overnight and bTRCP at 1:1,000 (IB; no. 4394, Cell Signalling). All the uncropped scans of the À 1 western blots are shown in Supplementary Fig. 9. either left untreated or treated 36 h with TGF-b (2 ng ml ) in 2% fetal bovine serum. Cells were fixed for 10 min in 4% paraformaldehyde in PBS, permeabilized with 0.2% TritonX100-PBS and then blocked with 3% bovine serum albumin Lentiviral transduction and the generation of stable cell lines. Lentiviruses (BSA) in PBS for 30 min at room temperature. Anti-E-cadherin (BD Transduction were produced by transfecting HEK293T cells with shRNA-targeting plasmids and Laboratories) was used at a dilution of 1:1,000 in 3% BSA-PBS and incubated for the helper plasmids pCMV-VSVG, pMDLg-RRE (gag/pol) and pRSV-REV. The 3 h at room temperature. Secondary, AlexaFluor488-labelled anti-mouse antibody cell supernatants were collected 48 h after transfection and were either used to (Molecular Probes) was used at a dilution of 1:200 in 3% BSA-PBS, together with Phalloidin (Molecular Probes) and incubated for 1 h at room temperature. infect cells or stored at À 80 °C. To obtain stable cell lines, cells were infected at 0 low confluence (20%) for 24 h with lentiviral supernatants diluted 1:1 in normal Coverslips were mounted with 4 ,6-diamidino-2-phenylindole-containing culture medium in the presence of 5 ng ml À 1 of polybrene (Sigma). At 48 h after VECTASHIELD mounting medium (Vector Laboratories Inc.). Fluorescence was infection, the cells were placed under puromycin selection for 1 week and then recorded using a Zeiss Axioplan microscope. passaged before use. Puromycin was used at 1 mgmlÀ 1 to maintain MDA-MB-231, For localization, HeLa cells were transfected with target plasmids and then MCF10A and HaCaT cells. Lentiviral shRNAs were obtained from Sigma processed as described above with a specific primary antibody and secondary (MISSION shRNA). Typically, five shRNAs were identified and tested, and the AlexaFluor488-labelled and AlexaFluor593-labelled anti-rabbit antibodies two most effective shRNAs were used for the experiment. We used the following (Molecular Probes). shRNAs: TRCN000000424 (1#) and TRCN000000424 (#2) for human FAF1 knockdown; Three-dimensional spheroid invasion assays. Semi-confluent MCF10A-RAS TRCN0000004 250 (1#) and TRCN0000004 252 (#2) for human VCP knockdown; (MII) cells were trypsinized, counted and re-suspended in medium containing TRCN0000006541 þ TRCN0000315200 mixture for Human bTRCP 2.4 mg ml À 1 methylcellulose (Sigma) at a concentration of 104 cells per ml. A total knockdown; TRCN000039793 þ TRCN000039797 þ TRCN000010162 of 100 ml of suspension was added into each well of a U-bottom 96-well-plate, þ TRCN0000010174 mixture for Human AKT1 knockdown; and allowing for the formation of one spheroid per well. All spheroids consisted of TRCN0000191433 for mouse FAF1 knockdown. 103 cells. Two days after plating, the spheroids were collected and embedded into collagen. A flat-bottom 96-well-plate was coated with neutralized bovine collagen-I (PureCol, Advanced BioMatrix) according to the manufacturer’s protocol. Transcription reporter assay. HEK293T cells were seeded in 24-well plates and Single spheroids were embedded in a 1:1 mix of neutralized collagen and medium transfected with the indicated plasmids using calcium phosphate. At 24 h after supplemented with 12 mg ml À 1 of methylcellulose. TGF-b (2 ng l À 1) was directly transfection, the cells were treated with TGF-b overnight or left untreated and then added to the embedding solution. Invasion was monitored over the subsequent 2 collected. Luciferase activity was measured with a PerkinElmer luminometer. days and quantified by measuring the area occupied by the cells using ImageJ The internal transfection control Renilla expression plasmid (10 ng) was used to software. Images were captured at days 0, 1 and 1.5 after embedding. normalize luciferase activity. Each experiment was performed in triplicate, and the data represent the mean±s.d. of three independent experiments. Migration assays. Transwell assays were performed in 24-well PET inserts (Falcon 8.0-mm pore size) for migration assays. MDA-MB-231 cells with targeted 4 4 Quantitative real-time PCR. Total RNA samples were prepared using a gene manipulations were serum starved overnight. Then, 5 Â 10 or 10 Â 10 cells NucleoSpin RNA II kit (Biospin). A total of 1 mg of RNA was reverse-transcribed were plated in transwell inserts (at least three replicas for each sample) and left À 1 using the RevertAid First Strand cDNA Synthesis kit (Fermentas). Real-time treated with or without TGF-b (5 ng ml ) for 8 h. Cells in the upper part of the PCR was conducted with SYBR Green (Applied Bioscience) using a StepOne Plus transwells were removed with a cotton swab; migrated cells were fixed in 4% real-time PCR system (Applied Bioscience). Target gene expression values were paraformaldehyde and stained with 0.5% crystal violet. The filters were imaged, normalized to 18S RNA levels. All primers used in quantitative PCR with reverse and the total number of cells was counted. Every experiment was repeated at least transcription are listed in Supplementary Table 3. three times independently.

Purification of bacterially expressed recombinant FAF1 proteins. GST-FAF1 wt Ubiquitination assay Cells were washed with PBS and lysed in two pellet volumes . and mutant expression constructs were generated by sub-cloning into pGEX-4T1 of RIPA buffer (20 mM NAP, pH7.4, 150 mM NaCl, 1% Triton, 0.5% sodium- vectors. Plasmids were used to transform the Escherichia coli strains BL21 and deoxycholate and 1% SDS) supplemented with protease inhibitors and 10 mM Rosetta, respectively. Cultures were grown overnight at 37 °C. The next day, the ° N-ethylmaleimide. The lysates were sonicated, boiled at 95 C for 5 min, diluted in cultures were diluted 1:50 in fresh Luria Bertani medium and grown at 37 °Ctoan RIPA buffer containing 0.1% SDS and centrifuged at 4 °C (16 Â 103g for 15 min). OD600 of 0.6. The cells were then induced overnight at 24 °C in the presence of The supernatant was incubated with specific antibodies and protein A-Sepharose 0.5 mM isopropyl-b-D-thiogalactopyranoside, 20 mM HEPES pH 7.5, 1 mM MgCl ° Â 2 for 3 h at 4 C. After extensive washing, bound proteins were eluted with 2 SDS and 0.05% glucose. For purification, the washed pellets were resuspended in lysis sample buffer and separated on SDS–PAGE followed by western blotting47. For the buffer (PBS, 0.5 M NaCl, complete protease inhibitors (Roche), 1 mM detection of TbRII ubiquitination, cells were treated with the lysosome inhibitor phenylmethanesulfonyl fluoride, 1% Triton X-100). After sonication and a m bafilomycin A1 (1 M) for 6 h before they were collected for the ubiquitination freeze–thaw step, the supernatants of the cell lysates were incubated with assay. glutathione Sepharose beads (GE Healthcare). The beads were washed twice with lysis buffer and three times with 50 mM Tris-HCl pH 7.5, 0.5 M NaCl. Purified proteins were eluted in 50 mM Tris-HCl pH 7.5, 0.5 M NaCl and 20 mM glu- Cellular fractions . Cytosolic, membrane and nuclear fractions were prepared tathione. For protein purification, GST was removed using biotin-tagged thrombin. using the ProteoExtract kit (Calbiochem) according to the manufacturer’s instructions. Immunohistochemical staining and evaluation. Formalin-fixed paraffin- embedded microarrays of breast cancer tissues were obtained from US Biomax Pulse chase. As previously described46, cells were plated in six-well plates. (BC081120). Primary antibodies specific to p-SMAD2 (1:50; Cell Signalling 3108), Cells were starved for 3 h in Met/Cys-free medium and pulsed for 40 min with FAF1 (1:200; Bethyl), TbRII (1:100; Santa Cruz) and p-Akt Ser 473 (1:200; Cell 200 mCi ml À 1 of 35SMet/Cys. After two washes, the cells were chased in medium Signalling #9271) were used for immunohistochemical staining. The quantification supplemented with cold (unlabelled) Met and Cys (100 mgmlÀ 1) before collecting. of staining was expressed as an H score. The H score was determined by the Endogenous proteins were collected from the extracts by immunoprecipitation, formula 3 Â the percentage of strongly staining cells þ 2 Â the percentage of then were resolved by SDS–PAGE and visualized by autoradiography. Each moderately staining cells þ the percentage of weakly staining cells, yielding a range experiment was performed in duplicate. of 0 to 300.

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