Functional analyses of a human vascular tumor FOS variant identify a novel degradation mechanism and a link to tumorigenesis

Epithelioid hemangioma is a locally aggressive vascular neoplasm, found in bones and soft tissue, whose cause is currently unknown, but may involve oncogene activation. FOS is one of the earliest viral oncogenes to be characterized, and normal cellular FOS forms part of the activator protein 1 (AP-1) transcription factor complex, which plays a pivotal role in cell growth, differentiation, and survival as well as the DNA damage response. Despite this, a causal link between aberrant FOS function and naturally occurring tumors has not yet been established. Here, we describe a thorough molecular and biochemical analysis of a mutant FOS protein we identified in these vascular tumors. The mutant protein lacks a highly conserved helix consisting of the C-terminal four amino acids of FOS, which we show is indispensable for fast, ubiquitin-independent FOS degradation via the 20S proteasome. Our work reveals that FOS stimulates endothelial sprouting and that perturbation of normal FOS degradation could account for the abnormal vessel growth typical of epithelioid hemangioma. To the best of our knowledge, this is the first functional characterization of mutant FOS proteins found in tumors.

Epithelioid hemangioma is a neoplasm composed of cells that are phenotypically endothelial, which form vascular lumina or grow as solid sheets (see Fig. 1A) (1). Until now, the molecular underpinnings of this disease have yet to be deciphered. A recent cytogenetic and karyotypic survey of the disease, by us (2) and others (3), aimed at refining diagnoses and tumor classification, unearthed a significant number of FOS translocations raising the possibility that disruption of FOS function could promote tumorigenesis. The immediate-early FOS proto-oncogene is activated rapidly and transiently in response to a wide spectrum of cell stimuli (4 -6), including serum, growth factors, cytokines, tumor-promoting agents, and DNA damage (7). The encoded FOS protein is a component of the crucial AP-1 transcription factor complex whose normal activity is regulated by controlled proteasome degradation (8,9), and corruption of this process can lead to cell transformation (10 -12). In this study, we have investigated the role of a novel mutant FOS protein we discovered in epithelioid hemangioma. We provide evidence that sustained expression of mutant FOS, due to loss of the C terminus, might drive the formation of vascular neoplasms by perturbing matrix metalloproteinase (MMP) 4 production and the Notch signaling pathway that are known to facilitate both physiological and pathological angiogenesis. Operationally, we found that the extreme C terminus of FOS renders it intrinsically susceptible to ubiquitin-independent degradation by the 20S proteasome, an essential mechanism bypassed by tumor FOS proteins. This is the first report of a module that directly mediates ubiquitinindependent proteasomal degradation (UIPD) and emphasizes the importance of UIPD in normal as well as tumor cells. Our work establishes the first demonstrable connection between mutations of FOS and the development of a naturally occurring tumor and unveils a potential, novel approach to treating epithelioid hemangioma by targeted inhibition of FOS or proteins whose expression is activated by FOS.

C-terminally truncated FOS mutant is expressed in epithelioid hemangioma
To determine whether mutations that disrupt the normal function of FOS might promote tumorigenesis, we investigated the role of a novel mutant FOS protein in epithelioid hemangioma (Fig. 1A). Fig. 1B depicts schematically a FOS deletion mutant (hereafter termed FOS⌬) that resulted from a FOS-MBNL1 translocation (2). The mutant transcript is predicted to encode a FOS isoform lacking the C-terminal 95 amino acids but including the bZIP domain. Western blot analysis of lysates prepared from patient tumor tissue revealed a truncated FOS protein of the expected size demonstrating that the mutant FOS

ACCELERATED COMMUNICATION: Human vascular tumor FOS analyses
gene is translated in vivo (Fig. 1C). Moreover, immunohistochemistry of tumor sections showed a significant enrichment of FOS in tumor blood vessel endothelial cells (Fig. 1C). In common with wild-type FOS, FOS⌬ is localized to the nucleus, can heterodimerize both with JUN and JUNB, and is efficiently associated with a consensus AP-1 DNA-binding site (Fig. 1D). However, FOS⌬ protein levels appear to be significantly higher than wild-type FOS protein levels in patient cells (see Fig. 1C) suggesting that the mutant protein may be aberrantly stable. To elucidate the mechanistic consequences of this deletion, we first assessed FOS protein stability in primary endothelial cells. Fig. 1E shows that wild-type FOS, as expected, has a relatively short half-life of ϳ1-2 h. By contrast, the deleted version of FOS is highly stable (half-life in excess of 8 h) suggesting that wild-type FOS harbors a destabilizing element in its C terminus, which is absent in the patient FOS⌬ protein. In support of this view, tethering the FOS C terminus to a GFP reporter construct, led to a striking destabilization of the GFP protein (Fig.  1F), whereas a truncated FOS C terminus did not substantially alter the stability of the GFP reporter (see Fig. 3G). This observation is consistent with previous reports relating to FOS stability (8,13,14). Additionally, blocking nuclear export had no effect either on rapid FOS degradation or the stability of FOS⌬ indicating that FOS is degraded in the nucleus and that FOS⌬ is resistant to this process (Fig. 1G). The above findings were confirmed in diploid HT1080 cell and HEK293T cells indicating that this mechanism is likely to be generic.

Mutant FOS is resistant to proteasomal degradation
To precisely delineate how FOS is degraded (and why FOS⌬ is not), we monitored FOS protein degradation by the proteasome. Fig. 2A shows that pharmacological inhibition of the proteasome, using either the specific inhibitor epoxomicin or MG132, markedly stabilized the wild-type FOS protein such that its half-life was comparable with that of the mutant FOS⌬ protein. The half-life of FOS⌬ was refractory to proteasome inhibition indicating that this deletion essentially lacks the motif(s) responsible for this degradative process ( Fig. 2A). It is established that degradation by the proteasome is either ubiquitin-dependent (15) or ubiquitin-independent (16 -18), and multiple different mechanisms have been reported to regulate FOS stability (19 -23). In agreement with others (17,18,20), Fig. 2B shows that wild-type FOS can be ubiquitinated and subsequently processed by the 26S proteasome (see also Fig.  3C). We found that patient FOS⌬ protein was not detectably ubiquitinated (Figs. 2B and 3C), and it fails to bind the E3 ligase, KDM2b, which has been demonstrated to stimulate FOS ubiquitination (see Fig. S1) (20). These observations could suggest that patient FOS⌬ stabilization results from the absence of FOS⌬ ubiquitin-dependent degradation. However, several lines of evidence support the view that the tumor FOS⌬ protein is intrinsically resistant to ubiquitin-independent proteasome degradation and that this is the principal cause of its substantially increased stability. First, pharmacological inhibition of ubiquitin-activating enzymes ablated FOS ubiquitination but had no detectable impact on FOS degradation (Fig. 2C). In the same experiment, ubiquitin-dependent degradation of an established substrate of the proteasome, MDM2, was com-pletely abrogated (see Fig. 2C). Second, wild-type non-ubiquitinated FOS but strikingly not FOS⌬ was efficiently degraded by the 20S proteasome in a cell-free in vitro system (Fig. 2D). By contrast, FOS was completely resistant to degradation by the 26S proteasome under identical conditions (Fig. 2E). Consistent with these findings, selective inhibition of the 26S proteasome, but not the 20S proteasome (through shRNA-mediated abolition of the 19S subunits PSMD2 and PSMD14), stabilized the ubiquitinated fraction of FOS but failed to conspicuously inhibit FOS protein degradation, in sharp contrast to inhibiting both the 20S and 26S proteasomes (see Fig. 2A), indicating that degradation is principally via the 20S and not the 26S proteasome (Fig. S2). These results show that the FOS C terminus is vital for both ubiquitin-independent and ubiquitin-dependent proteasomal degradation and that both of these processes are lost by the mutant FOS⌬ protein expressed in epithelioid hemangioma. These data show that the normal process of FOS degradation is severely corrupted in the tumor FOS⌬ mutant protein and substantiate previous studies (21,22) which suggest that FOS stability is governed chiefly by ubiquitin-independent proteasome degradation.

Mutant FOS lacks a conserved motif essential for ubiquitin-independent degradation by the 20S proteasome
To identify the motif(s) in the C terminus of FOS, which mediates FOS degradation (and is absent in FOS⌬), we performed a thorough mutational analysis of the FOS C terminus. Fig. 3A shows that deleting the C-terminal four amino acids was sufficient to strongly stabilize FOS and that lack of these amino acids might therefore cause the aberrant stability of the FOS⌬ tumor protein. Ab initio modeling (24) of the FOS tail revealed that the C terminus is composed of an intrinsically unstructured region terminating in a helix composed of the C-terminal four amino acids (LLAL), which is conserved in all metazoans sequenced to date (Fig. 3B). Unlike FOS⌬, eliminating this helical region, either through point mutation or deletion, had no effect upon FOS ubiquitination (Fig. 3C). The same mutations did, however, efficiently block FOS degradation to the same degree as the tumor FOS⌬ protein (see Fig. 3, D and E). The integrity of the four C-terminal amino acids, but not residues immediately adjacent to this motif, is absolutely required for priming FOS labileness (Fig. 3F). Deletions or point mutations of adjacent amino acids, which include consensus phosphorylation sites for ERK and GSK, failed to augment FOS stability. Indeed, a subset of these, in agreement with others (25), served to enhance FOS instability suggesting they play a role in stabilizing but not destabilizing the FOS protein (correspondingly, chemical inhibitors of MEK or deletion of the consensus ERKdocking site had a comparable effect; data not shown). Three additional experiments further validated the importance of the C-terminal motif. One, deletion of the C-terminal four amino acids strongly attenuated the capacity of the FOS C terminus to destabilize GFP (Fig. 3G). Two, a deletion mutant lacking the intrinsically disordered region (IDR) (which is highly stable) but retaining the C-terminal four amino acids was as unstable as wild-type FOS (Fig. 3H). Three, in common with tumor FOS⌬, a mutant FOS lacking the C-terminal four amino acids, was highly resistant to 20S proteasomal degradation in a cell-ACCELERATED COMMUNICATION: Human vascular tumor FOS analyses free in vitro assay (Fig. 3I). These data highlight a short helical region at the extreme C terminus of FOS as a crucial determinant of FOS stability and that perturbation of this motif leads to pronounced FOS stabilization. A block in ubiquitin-independent degradation, due to loss of the extreme C terminus, is sufficient to explain mutant FOS⌬ stability. Experiments in cell-free systems indicate that this motif can orchestrate direct proteasomal degradation of FOS independently of accessory proteins. IDRs have been reported to strongly influence proteasomal degradation (26), including the IDR found in the C ter-   (27). Our data show that the FOS IDR, by itself, does not stimulate FOS degradation. Rather, a highly conserved helical motif at the extreme C terminus of FOS is essential for triggering ubiquitin-independent degradation.

FOS potently stimulates endothelial sprouting
Vascular neoplasms result from the dysregulated growth of endothelial cells or their precursors (1) and represent a unique model for gaining insights into pathological as well as normal angiogenesis. To recapitulate the cell biological consequences of the mutant FOS stabilization observed in epithelioid hemangioma, we ectopically expressed wild-type and mutant FOS proteins in primary HUVECs and assessed their ability to sprout. Fig. 4A shows that whereas loss of FOS abolished sprouting, sustained expression of FOS strongly promoted endothelial sprouting and the formation of stable endothelial cell networks. Similarly, expression of tumor FOS⌬ or FOS lacking an intact C-terminal four amino acids strongly stimulated endothelial sprouting of HUVECs (Fig. 4B). This phenomenon was independent of marked changes in cell proliferation (Fig. 4B). Fig. S3 shows that FOS and FOS⌬ also stimulated sprouting of human lung microvascular endothelial cells. The endothelial cell networks produced by cells expressing FOS and FOS⌬ were stable and persistent. In this assay, ordinarily the sprouting network is relatively short-lived and collapses after ϳ24 h. By contrast, endothelial cell networks expressing elevated levels of FOS and FOS⌬ were sustained for at least 2 weeks of culture (sprouting networks expressing FOS⌬ were noticeably more robust than the wild-type FOS-expressing networks), which resembles the illicit vessel growth observed in human epithelioid hemangioma.

Mutant FOS-driven sprouting is dependent on MMPs and Notch signaling
To understand the mechanistic basis of FOS-driven sprouting, we performed global transcriptome analyses of sprouts formed by FOS or patient FOS⌬-expressing primary endothelial cells. Fig. 4C shows a confirmatory qPCR of a selection of angiogenesis-control genes, which were up-regulated, including MMPs and components of the Notch-signaling pathway that are known to facilitate both physiological and pathological angiogenesis (28 -33). ChIP analyses showed that endogenous FOS bound to these promoters (Fig. S4), and complementary ChIP studies showed that FOS⌬ directly interacts with these promoters (see Fig. 4C). Our experiments uncover a previously unreported role for FOS as an activator of endothelial sprouting and show that patient FOS⌬ could stimulate illicit endothelial sprouting by activating the Notch signaling pathway and increasing the production of MMPs. In this regard, it is notable that inhibitors of either MMPs or Notch signaling significantly inhibited the sprouting of FOS⌬-expressing endothelial cells (Fig. 4D). The same inhibitors had relatively little effect on cells expressing wild-type FOS under these assay conditions. This could reflect the fact that both MMP production and Notch signaling (as well as other FOS target pathways, see under "Transcriptome profiling) were significantly more augmented in cells expressing wild-type FOS compared with cells expressing FOS⌬ (presumably because FOS⌬ lacks the C-terminal TAD). Accordingly, a recently reported small molecule inhibitor of FOS (34), which has advanced to human Phase II clinical trials for the treatment of rheumatoid arthritis, efficiently inhibited FOS-driven endothelial sprouting (Fig. S5).
In summary, our data have uncovered a previously unreported role for FOS in stimulating endothelial cell sprouting. We show that sustained expression of FOS, due to loss of the C terminus, could drive the formation of vascular neoplasms. By analyzing the C-terminal region of FOS, which is deleted in epithelioid hemangioma, we have discovered a highly conserved motif at the extreme C terminus of FOS that is critical for controlling its stability by rendering it intrinsically susceptible to ubiquitin-independent degradation by the 20S proteasome. Our work suggests that targeted inhibition of FOS or proteins whose expression is activated by FOS might represent a legitimate novel approach to treating these locally aggressive tumors.

Cell culture, biochemistry, and molecular biology
Primary HUVECs (Lonza) were cultured in EGM2 medium (Lonza). Chondrosarcoma HT1080 and human embryonic kidney 293T cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco). Transfections, lentivirus production and cell infections, Western blotting, and co-immunoprecipitations have been described previously (35). FOS stability assays were performed by incubating cells in the presence or absence of cycloheximide for a defined time course (hours). Protein levels were determined by Western blotting.

Plasmid and shRNA construction
Human FOS cDNAs fused in-frame with a FLAG or an HA epitope tag were cloned into the pLV lentiviral vector and pCS2  ACCELERATED COMMUNICATION: Human vascular tumor FOS analyses expression plasmid. Gene-specific shRNA-expressing lentiviruses were generated using the TRC2-pLKO lentiviral vector system.

Transcriptome profiling
RNA was isolated from HUVECs stably expressing FOS or FOS⌬ by treatment with TRIzol (Invitrogen) column purification (Direct-zol RNA isolation kit-Zymo Research). RNA quality was verified with a Bioanalyzer (Agilent), and sequencing was performed on the Illumina HiSeq 2500 (Genome Scan). HUVEC transcript sequencing data have been deposited under GenBank TM accession no. PRJNA390521.

Analysis of mRNA expression
RNA isolation, first strand cDNA synthesis, and analysis of expression of transcripts by quantitative PCR were performed as described previously (33).

Ubiquitination assay
293T cells were transfected with the appropriate plasmids. Proteasome degradation was blocked for 8 h with 10 M MG132 (Sigma). HIS pulldowns were performed as described previously (36).

HUVEC sprouting assay
96-well plates were coated with 60 l of Matrigel/well 30 min prior to seeding HUVECs. EGM-2 medium was supplemented with 50 ng/ml recombinant human VEGF 165 (R & D Systems). Images were taken at multiple time points. Analysis of the sprouting was performed with Stacks (in-house software, Department of Molecular Cell Biology, LUMC).

Immunohistochemistry/Immunofluorescence
Staining was performed on 4-m tissue sections. Paraffin was removed with xylene, and sections were rehydrated in a gradient of ethanol. Exogenous peroxidase was blocked using 0.3% H 2 O 2 . Microwave antigen retrieval was performed in Tris-EDTA (pH 9.0). FOS antibody was used at a 1:400 concentration. Antibody was detected with 3,3Ј-diaminobenzidine, and counterstaining was performed with hematoxylin. Immunostaining was performed as described previously (37).

Protein-DNA interaction assays
In vitro-translated protein was made as above. 50 pmol of biotinylated double-stranded oligonucleotides harboring three contiguous AP-1 DNA-binding sites were coupled to MyOne streptavidin C1 beads (Invitrogen). Reactions were incubated at 4°C with vigorous shaking for 30 min in the presence of 1 g of poly(dI/dC), 4 mM spermidine, 50 mM KCl, 10 mM HEPES (pH 7.6), 5 mM MgCl 2 , 10 mM Tris (pH 8), 0.05 mM EDTA (pH 8), 0.1% Triton X-100, and 20% glycerol. Beads were successively washed three times with the aforementioned buffer. Associated proteins were eluted in Laemmli buffer, and protein-DNA interactions were determined by Western blotting.

ChIP
ChIP analyses were performed on confluent 10-cm tissue culture dishes of HUVECs as described previously (35).

Bioinformatics
Rosetta (RosettaCommons) was used for structure prediction of the FOS C terminus (24). Secondary structure was predicted using Psipred (version 4.01, UCL). Degree of disorder was predicted using Disopred (version 3.16, UCL).