MYC-nick promotes cell migration by inducing fascinexpression and Cdc42 activationSarah Andersona, Kumud Raj Poudela, Minna Roh-Johnsona, Thomas Brabletzb, Ming Yuc, Nofit Borenstein-Auerbachd,William N. Gradyc,e, Jihong Baia, Cecilia B. Moensa, Robert N. Eisenmana,1, and Maralice Conacci-Sorrelld,f,1

aDivision of Basic Sciences, Fred Hutchinson Cancer Research Center A2-025, Seattle, WA 98109; bNikolaus-Fiebiger-Center for Molecular Medicine,University Erlangen-Nuernberg, 91054 Erlangen, Germany; cClinical Research Division, Fred Hutchinson Cancer Research Center D4-100, Seattle, WA 98109;dDepartment of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9039; eDepartment of Medicine, University of WashingtonSchool of Medicine, Seattle, WA 98195; and fSimmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390-9039

Contributed by Robert N. Eisenman, July 12, 2016 (sent for review May 2, 2016; reviewed by Stephen R. Hann and Martine F. Roussel)

MYC-nick is a cytoplasmic, transcriptionally inactive member ofthe MYC oncoprotein family, generated by a proteolytic cleavageof full-length MYC. MYC-nick promotes migration and survival ofcells in response to chemotherapeutic agents or withdrawal ofglucose. Here we report that MYC-nick is abundant in colonic andintestinal tumors derived from mouse models with mutations inthe Wnt, TGF-β, and PI3K pathways. Moreover, MYC-nick is ele-vated in colon cancer cells deleted for FBWX7, which encodes themajor E3 ligase of full-length MYC frequently mutated in colorec-tal cancers. MYC-nick promotes the migration of colon cancer cellsassayed in 3D cultures or grown as xenografts in a zebrafish me-tastasis model. MYC-nick accelerates migration by activating theRho GTPase Cdc42 and inducing fascin expression. MYC-nick, fascin,and Cdc42 are frequently up-regulated in cells present at the inva-sive front of human colorectal tumors, suggesting a coordinatedrole for these proteins in tumor migration.

MYC | MYC-nick | colon cancer | motility | fascin

Members of the MYC proto-oncogene family (c-MYC,N-MYC, and L-MYC) are key regulators of tumor ini-

tiation and tumor maintenance in many types of cancer (1).MYC proteins initiate a transcriptional program of growthand proliferation, as well as suppression of cell-cycle arrest(2). Functionally, MYC proteins form dimers with Max andact broadly as transcriptional activators of a large number ofgenes (3–8). MYC binds Max and DNA via its C-terminal regioncomprising a basic helix–loop–helix leucine zipper (BHLH LZ)domain. The N terminus of MYC contains four highly conservedregions called MYC boxes (MB I–IV), involved in MYC’sfunction in transcriptional regulation (9). As one of the majordeterminants of MYC’s transcriptional function, MBII recruitscoactivator complexes including histone acetyltransferases (HATs),such as GCN5 (10) and Tip60 (11). MYC is a very short-livedprotein, and multiple E3 ligases have been implicated in regu-lating MYC protein turnover through the ubiquitin–proteasomesystem (12). Importantly, MYC levels have been demonstratedto be elevated in cancer cells because of prolonged protein half-life (13, 14).MYC is also targeted by calpain proteases in the cytoplasm

(15–17). Calpain-mediated scission of MYC degrades its Cterminus, which inactivates MYC’s transcriptional functions.Furthermore, the cleavage generates MYC-nick, a truncatedproduct that retains MBI–MBIII (16). Although MYC-nick isexpressed in most cultured cells and in mouse tissues, its levelsare increased in cells cultured under conditions leading tostress, such as high cell density, nutrient deprivation, andhypoxia (15, 16, 18). Recently, we found that the conversion ofMYC into MYC-nick occurs in the cytoplasm of colon cancercells, where it promotes cell survival and motility (15). Here wedemonstrate that MYC-nick promotes cell migration and in-vasion by inducing fascin expression and activating the RhoGTPase Cdc42 in distinct models of colon cancer.

ResultsMYC-Nick Is Expressed in Intestinal and Colon Lesions in MouseCancer Models Driven by Mutations in Apc, Tgfbr2, and Kras. Wehad previously shown that MYC-nick is expressed in cancer celllines and cancers arising from different primary tissues (15). Toextend those studies, we examined the expression of MYC variantsin mouse colon cancers derived from distinct models of intestinalcancer. Most colorectal carcinomas carry mutations that affectWnt, TGF-β, and PI3K signaling pathways (19, 20). We comparedthe expression of MYC in tumors arising from models containing(i) a truncation in one of the alleles of Apc (Apc1638/+; labeledATT); (ii) Pten and Tgfbr2 deletions combined (PPVcTT); (iii) Apctruncation in combination with Tgfbr2 deletion (AVcTT); and(iv) activated oncogenic KrasG13D and Tgfbr2 deletion (KVcTT).We found that both MYC and MYC-nick levels are frequentlyelevated in intestinal adenomas and adenocarcinomas, as well asin colon carcinomas in these mouse models (Fig. 1 A–C and TableS1). MYC-nick was shown to promote acetylation of cytoplasmicproteins (16, 21), and we found a correlation between MYC-nicklevel and acetylated α-tubulin in these samples (Fig. 1A).

Oncogenic Mutations Augment Stability of MYC and MYC-Nick. Mu-tations in MYC that prevent its binding to SCFFBW7 have beenreported to increase MYC levels and promote tumorigenesis(22). The Fbw7 binding site is also retained in MYC-nick (Fig.1E). To determine whether MYC-nick stability was also regulated

Significance

The MYC family of transcription factors is deregulated in abroad range of cancers and drives the expression of genes thatmediate biomass accumulation and promote cell proliferationand tumor initiation. We find that MYC can also trigger tumorcell migration and metastasis independently of its transcrip-tional activity, via its conversion to MYC-nick, a truncated formof MYC localized in the cytoplasm. MYC-nick promotes re-organization of the actin cytoskeleton by inducing expressionof the actin-bundling protein fascin and by activating the RhoGTPase Cdc42, both of which lead to formation of filopodia,cellular structures known to drive cell migration. Our worklinks the repurposing of the MYC transcription factor to alteredcytoskeletal structure and tumor cell metastatic behavior.

Author contributions: R.N.E. and M.C.-S. designed research; S.A., K.R.P., M.R.-J., T.B., N.B.-A.,and M.C.-S. performed research; M.Y. contributed new reagents/analytic tools; K.R.P.,M.R.-J., W.N.G., J.B., C.B.M., R.N.E., and M.C.-S. analyzed data; and R.N.E. and M.C.-S.wrote the paper.

Reviewers: S.R.H., Vanderbilt University; and M.F.R., St. Jude Children’s Research Hospital.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1To whom correspondence may be addressed. Email: eisenman@fhcrc.org or maralice.conaccisorrell@utsouthwestern.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1610994113/-/DCSupplemental.

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Fig. 1. Immunoblotting of MYC and MYC-nick in tumors derived from several oncogenic mutations. (A and B) Normal mucosa (N) and adenoma/adeno-carcinoma lesions (T) were processed for Western blot for MYC and acetylated α-tubulin. (C) Genotypes of the mouse models used for Western blot in A and B.See Table S1 for detailed information. (D) Immunoblotting for MYC and MYC-nick in DLD1 cells lacking the FBXW7 gene. WT or FBXW7 knockout (FBXW7−/−)DLD1 cells were grown to confluency and incubated in the presence of CHX and calpain inhibitor XII for the indicated time points before nuclear and cy-toplasmic fractionation. (E) Schematic representation of MYC and MYC-nick displaying the binding regions for the E3 ligase SCFFBXW7. NLS, nuclear locali-zation sequence. (F) Expression levels of MYC and MYC-nick in HCT116 cells treated with 20 μM indirubin and kenpaulone, for 3 h before harvesting.(G) Phosphorylation status of T58 in MYC and MYC-nick. The 293T cells were transfected with MYC, T58A MYC, MYC-nick, and T58A MYC-nick, and 2 d laterwere processed for Western blot by using antibodies against total MYC and phosphorylated T58/S62 MYC.

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by FBXW7, we examined variants of the colon cancer cell linesDLD1 and HCT116, both of which have the FBXW7 gene deletedby gene targeting (23). We found that, compared with their WTcounterparts, both cell lines deleted for FBXW7 exhibited in-creased the stability of MYC and MYC-nick in the cytoplasm, asmeasured by cycloheximide (CHX) chase (Fig. 1 D and F and Fig.S1A). The increase in MYC-nick levels observed in FBXW7−/−

cells was not due to reduced calpain activity, because deletion ofFBXW7 had no effect on calpain-mediated cleavage of MYC (Fig.S1B). The reduction in the total levels of MYC observed inFBXW7−/− (Fig. 1D) was reported previously and is caused by areduction in MYC mRNA (23). Consistent with the increasedstability of MYC-nick upon FBXW7 deletion, we are able to detectboth endogenous MYC and MYC-nick associated with Fbw7α inthe cytoplasm of DLD1 cells (Fig. S1C). Fbw7α is the only Fbw7isoform endogenously expressed in these cells (23, 24).Inhibition of the proteasome with epoxomycin increases the

stability of overexpressed MYC-nick (Fig. S2A) and siRNAs againstcomponents of the proteasome (25) also augment the stability ofendogenous full-length MYC and MYC-nick in the cytoplasm (Fig.S2B). In nontransformed cells, we found that MYC-nick has a half-life of ∼30 min (Fig. S2 C–E), similar to the estimated half-life forfull-length MYC (26, 27). Mutations that affect the binding ofMYC to Fbw7, such as T58A, do not affect its targeting by calpainand conversion into MYC-nick. (Fig. S1D).The binding of SCFFbw7 to MYC requires phosphorylation of

MYC threonine 58 (T58), which is mediated by glycogen syn-thase kinase 3β [GSK3β (28)]. Several studies have shown thatGSK3β-mediated phosphorylation of MYC promotes its recog-nition by SCFFbw7, leading to proteasomal degradation (29, 30).As expected, treating cells with pharmacological inhibitors thatblock GSK3β activity, such as indirubin and kenpaulone, leads toincreased stability of both MYC and MYC-nick (Fig. 1F). Indeed,we found that a point mutation in MYC converting threonine 58to alanine (T58A) prevented its phosphorylation and caused ac-cumulation of both full-length MYC and MYC-nick (Fig. 1G). Ina CHX chase, we found that T58A MYC-nick was more stablethan WT MYC-nick when transfected in 293T (Fig. S2F).

MYC-Nick Induces Migration in 3D Substrates. We have demon-strated, using scratch healing and transwell migration assays, thatoverexpression of MYC-nick promotes the migration of coloncancer cells (ref. 15 and Fig. 2A). We found that MYC-nick in-duces migration and promotes filopodia formation, while up-regulating the actin-bundling protein fascin (15). Here we extendour studies to the 3D migration systems to better recapitulate theresistance provided by the environment during the migratoryprocess of metastatic cancer cells. We found that ectopic expres-sion of MYC-nick in DLD1 colon cancer cells also promoted theirmigration in 3D cultures (Fig. 2 B–F). Single cells or spheroidswere cultured in ECM such as 50% (vol/vol) Matrigel or collagen,and in both cases, MYC-nick expressing colonies displayed a moremigratory phenotype than control cells (Fig. 2 B–D). When platedin 20% (vol/vol) collagen, which is more permissive to migration,MYC-nick–expressing cells were still more migratory than controls(Fig. 2 E and F). Note that MYC-expressing cells were also moremigratory than control cells in 3D (Fig. 2 E and F), which isprobably caused by the constitutive cleavage of MYC into MYC-nick in these cells. These migratory cells displayed enhancedfilopodial protrusions (Figs. 2A and 3C and Fig. S3A), whichhave been strongly linked to the increased migratory behaviorand metastatic potential of cancer cells (31). In agreement withour previous observations, we found that cells expressing aMYC-nick mutant lacking MBII (ΔMBII) are unable to formfilopodia and to promote persistent migration in 3D cultures.We validated our results using a previously described human-

in-zebrafish xenotransplantation approach (32–36). We intro-duced DLD1 cells into 48-h postfertilization zebrafish larvae.

Four days after implantation, MYC-nick–expressing DLD1 cellsexhibited an increase in metastatic behavior, measured by thenumber of cells that migrate away from the site of injection (Fig.2 G and H). Deletion of MBII dramatically reduced MYC-nick’sability to drive migration (Fig. 2 G and H). Together, these re-sults further suggest that expression of MYC-nick induces tumorcell invasion in vivo.

MYC-Nick–Induced Migration Requires Fascin Expression.We reportedpreviously that MYC-nick promotes a dramatic increase in thelevels of fascin (15), which is a driver of metastatic behavior insolid tumors and is associated with tumor progression in the colonand stomach (31) (Fig. 3A). Fascin expression is induced by MYC-nick in a variety of cell lines, including colon cancer cell lines suchas DLD1 (Fig. 3A) and HCT116 (Fig. S3B) and human foreskinfibroblasts (HFFs) (Fig. S3C). Overexpression of full-length MYCin HFFs also induced fascin expression, probably due to consti-tutive generation of MYC-nick in these cells (Fig. S3C). More-over, we found a marked increase in fascin levels in mouseadenomas and adenocarcinomas expressing high levels of MYC-nick (Fig. 3B). Although fascin down-regulation by siRNA doesnot affect cell survival of MYC-nick–expressing cells (15), it pre-vented filopodia formation (Fig. 3C) and cell migration (Fig. 3D).This finding indicates that fascin is necessary for MYC-nick-induced cell migration, consistent with reports that filopodia forma-tion requires fascin (37–39). However, overexpression of GFP-taggedfascin in DLD1 cells did not mobilize actin to induce the formationof filopodia (Fig. 3 E and F), indicating that fascin is not sufficient forMYC-nick–induced filopodia formation and that additional changesin the actin cytoskeleton are required.

MYC-Nick–Induced Migration Requires Cdc42 Activation. The Rhofamily of GTPases plays an essential role in cellular motility byregulating the organization of the actin cytoskeleton (40, 41).Because MYC-nick promotes changes in the actin cytoskeletonand increases migratory properties, we asked whether RhoGTPases collaborate with fascin to mediate MYC-nick’s effectson filopodium formation and cell migration. We treated DLD1cells expressing either empty vector or MYC-nick with EGF,which activates Rho GTPases, or with the RAC1/Cdc42 inhibitorML141. Inhibiting RAC/Cdc42 activity for 10–16 h completelyablated filopodia in MYC-nick–expressing cells (Fig. 4 A and B).Conversely, activating GTPases with EGF promoted filopodiaformation in vector-expressing cells to the same extent as ex-pression of MYC-nick and further increased filopodium forma-tion in MYC-nick–expressing cells (Fig. 4 A and B). These resultsindicate that Cdc42 activity is required and sufficient to inducefilopodia formation in MYC-nick–expressing cells.Importantly, as with fascin siRNA, the viability of MYC-

nick–expressing cells was insensitive to treatment with Cdc42inhibitors. Even though the treated cells were not migratory,they survived for extended periods of time (4 d) in the pres-ence of these inhibitors. This finding is consistent with ourprevious observation that MYC-nick promotes survival by in-ducing acetylation of LC3BII and α-tubulin to accelerate auto-phagy, a function that appears not to require Cdc42 activity(Fig. S3D).Although cells overexpressing MYC-nick are resistant to

prolonged treatment with Cdc42 inhibitors, cells expressingempty vector, MYC-nick lacking the acetyltransferase bind-ing domain (ΔMBII), full-length MYC, or uncleavable MYC(Δ298–311) did not survive in the presence of Cdc42 inhib-itors (Fig. S3D).Because of their role as positive regulators of cellular motility and

invasion, Rho-GTPases have been linked to tumorigenic pheno-types in a variety of human cancers (42). Indeed, we observedthat the total levels of RAC1 and Cdc42 are up-regulatedin intestinal and colonic adenomas and adenocarcinomas

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Fig. 2. Effect of MYC-nick on migration of colon cancer cells in 3D systems. (A) Scratch assay: DLD1 cells expressing MYC-nick or empty vector were grown on Cytoselect24-Well plates for 48 h to confluency when the stopper was removed to allow migration. At 24 h later, cells were stained with phalloidin and photographed. (Magnification:63×.) (B and C) The 3D culture assay: a total of 100 cells expressingMYC-nick or empty vector were trypsinized, and single cells were embedded in 50% (vol/vol) Matrigel (B) orcollagen (C) matrix, grown for 3 d, and photographed. (D) Migration of colon cancer cells initially grown as spheroids for 3 d and then embedded in 50% (vol/vol) collagen forthe indicated time points. (Magnification: B–D, 20×.) (E and F) Migration of DLD1 colon cancer cells expressing empty vector or MYC-nick in soft collagen. Cells were grown asspheroids over agar for 2 d and then were embedded in 20% (vol/vol) collagen. The percentage of spheroids displaying at least one migratory cell was calculated 24-h afterseeding. Cultureswere photographed 3 d after seeding (F). (Magnification: 20×.) (G andH). A total of 25–50DLD1 cells expressing empty vector,MYC-nick, andMYC-nick lackingMYC box II (ΔMYC box II) were labeled with CellTracker Green, injected into the hindbrain of zebrafish embryos, and scored for migration (G) and photographed after 96 h.

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derived from the mouse models of intestinal neoplasia com-pared with normal mucosa (Fig. 4C and Table S1). However,this increase in Cdc42 expression is probably not due to the

presence of MYC-nick because MYC-nick expression does notincrease either the total levels or the stability of Cdc42 protein(Fig. 4D).

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Fig. 3. Analysis of fascin expression in MYC-nick induced cell migration. (A) Effect of MYC-nick on abundance of endogenous fascin and exogenous GFP-fascin. (B) Fascin expression in murine intestinal and colonic lesions derived from different genetic backgrounds (see Fig. 1C for tumor genotypes and Table S1for details). Tissues were processed as in Fig. 1A. (C) DLD1 cells expressing MYC-nick were transfected with control or fascin siRNA and 48 h later were stainedwith phalloidin. (Magnification: 63×.) (D) DLD1 cell monolayers expressing MYC-nick were transfected with control or Fascin siRNA, scratched 48 h later inthe presence of mitomycin C, and photographed at 72 h. (Magnification: 10×.) (E and F) DLD1 cells expressing empty vector or MYC-nick were transfectedwith GFP-fascin and stained with phalloidin 48 h later. (Magnification: E, 20×; F, 100×.)

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Fig. 4. Analysis of expression and role of Rac, Rho, and Cdc42 in MYC-nick–induced migration. (A) Effect of Rac/Cdc42 inhibition and Cdc42 activation onMYC-nick–induced filopodia formation. (Magnification: 63×.) (B) Quantification of A. Cells surrounding individual colonies were scored for the presence of atleast one filopodium. n = 100. (C) Immunoblots of Cdc42, Rac in mouse colorectal cancer. (D) Effect of MYC-nick expression on Cdc42 levels and stability.(E) Determination of Rho, Rac, and Cdc42 activation in control and MYC-nick–expressing DLD1 cells. (F) Effect of MYC-nick on sustained activation of Cdc42,Rac, and Rho. (G) Cdc42 activation in cells expressing MYC-nick lacking MBII (MYC-nick ΔMBII) and in cells expressing the cleavage resistant form of full-lengthMYC (MYC Δ298–311). (H) Analyzes of fascin and Cdc42 expression in cells transfected with siRNA against these proteins. DLD1 cell expressing empty vector orMYC-nick were transfected with siRNA for fascin or Cdc42 and processed for Western blot 48 h later.

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MYC-Nick Promotes the Activation of Cdc42. Rho GTPases cyclebetween active (GTP-bound) and inactive (GDP-bound) states bybinding and hydrolyzing GTP. To determine whether MYC-nickexpression modulates Rho GTPase activity, we performed pull-downs of activated RhoA by using beads conjugated to its bindingpartner Rhotekin. We also pulled down active Rac1 and Cdc42using P21-activated kinase beads (PAK). We found that MYC-nickpromotes activation of Cdc42 when expressed in colon cancer cellssuch as DLD1 (Fig. 4E) and HCT116 (Fig. S3A). Rac1 and RhoAactivities were only modestly affected by MYC-nick: Rac1 activitywas elevated, whereas RhoA activity was reduced (Fig. 4E).Importantly, we found that MYC-nick promoted a sustained

activation of Cdc42 in MYC-nick–expressing cells as observed 16 hafter treatment with growth factors or EGF (Fig. 4F, compare lanes 3

and 4). In control cells, this activation was seen to the same extent, butwas transient (Fig. 4F, compare lanes 5 and 6). However, MYC-nickis not capable of promoting or sustaining the activation of Cdc42 inhighly confluent cultures (Fig. 4F). Thus, activation of Cdc42 byMYC-nick differs from fascin induction because MYC-nick ac-tivates fascin expression regardless of cellular density (15).

Cdc42 Activation by MYC-Nick Requires the MBII Region. The MBIIregion (amino acids 106–143) located within N-terminal segmentof MYC and MYC-nick constitutes a binding site for recruitmentof HATs to MYC. Deletion of MBII, although having no effecton MYC-nick expression levels, reduced MYC-nick’s ability topromote cell survival and migration (Fig. 2 E–G and Fig. S3D) (15).Although cells expressing MYC-nick ΔMBII appear more

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Fig. 5. Immunohistochemistry (IHC) of fascin and Cdc42 in human colon cancer biopsies. Representative IHC in normal mucosa, central area of the tumor, andthe invasive front of the same tumor is shown. (A–C) Cdc42 IHC (A), fascin IHC (B), and MYC IHC (antibody reactive against MYC N-terminal regions) (C).(D) Increased MYC, fascin, and Cdc42 IHC signal at the invasive front of colon cancer samples. (Magnification: A–C, 40×.)

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capable of initiating migration relative to control cells (Fig. 2E), theMYC-nick ΔMBII–expressing cells are not capable of completingmigration and forming new colonies in collagen (Fig. 2F). Todirectly address the relevance of MBII to Cdc42 activation, wecompared the activation of Cdc42 in cells expressing MYC-nickto cells expressing MYC-nick ΔMBII. We found that MBII isrequired for the activation of Cdc42 by MYC-nick (Fig. 4G). Asexpected, full-length MYC was also capable of inducing Cdc42activation, most likely through its ability to constitutively generateMYC-nick through calpain cleavage. Consistent with this finding,a mutation in MYC deleting the calpain cleavage region aminoacids 298–311 (MYC Δ298–311) is not capable of activating Cdc42(Fig. 4G). Note that all MYC constructs were expressed at similarlevels (Fig. S3E).

Induction of Fascin and Activation of Cdc42 by MYC-Nick AreIndependent Events. Fascin and Cdc42 function in coordination topromote filopodia formation and to drive cell migration (43–45).Moreover, Cdc42 was shown to regulate fascin localization andfunction. For example, constitutively active mutant forms of Rac andCdc42 were reported to trigger localization of fascin to lamellipodia,whereas fascin, in turn, is critical for cell migration driven by Rac andCdc42 (46). Cdc42 and fascin also cooperate to promote invado-podia formation in different model systems (47–50). To address theinvolvement of Cdc42 in the induction of fascin expression by MYC-nick, we silenced Cdc42 by siRNA in DLD1 cells expressing eitherempty vector or MYC-nick. Although silencing Cdc42 reduced fas-cin levels in control cells (Fig. 4H), it did not affect fascin levels inMYC-nick–expressing cells (Fig. 4H). Conversely, silencing fascin bysiRNA did not affect the levels of total Cdc42 (Fig. 4H).

Fascin and Cdc42 Are Highly Expressed at the Invasive Front of ColonCancers. We have shown previously that cytoplasmic staining withanti-MYC corresponding to MYC-nick is elevated in cells at theinvasive front of human colorectal cancers (Fig. 5C and ref. 15).Here we analyzed the expression of both Cdc42 (Fig. 5A) and fascin(Fig. 5B) in migratory cells at the invasive front of the same tumors(Table S2) (n = 19). We found that Cdc42 and fascin, similar to MYC-nick, are increased in tumor tissues and are often further elevated atthe invasive front of these tumors (Fig. 5D and Table S2). Theseobservations are in agreement with several studies indicating fascin up-regulation in cancer cells drives motility and invasiveness (31, 48, 51).

DiscussionOur earlier work showed that MYC-nick is generated by calpaincleavage of full-length MYC under conditions of stress, such asnutrient deprivation and hypoxia. Under these conditions, amutant form of MYC deficient in producing MYC-nick causesextensive apoptosis, which is attenuated by coexpression ofMYC-nick (15). This finding indicates that MYC-nick plays anactive role in cell survival, and its production is important foradaptation to stress (Fig. 6). MYC-nick–induced survival is me-diated, at least in part, through stimulation of an autophagicresponse. In addition, MYC-nick expression correlates with strikingchanges in cytoskeletal organization, including alterations in actindynamics, which lead to increased cell migration. These cellularchanges are abrogated by deletion of the MBII region in MYC-nick, a region known to recruit protein complexes involved in his-tone acetylation and chromatin remodeling. In MYC-nick, MBIIalso associates with the acetyltransferase GCN5 (and probablyothers), inducing the acetylation of cytoplasmic proteins andthereby modulating protein function (16).The abundance of full-length nuclear MYC has been long

known to be controlled posttranslationally by means of pro-teolytic degradation determined by the balanced actions of sev-eral ubiquitin ligases and ubiquitin-specific proteases (for review,see ref. 12). The conserved MBI and MBII regions in MYC serveas binding sites for the Fbw7 and Skp2 ubiquitin ligases, re-spectively. MBI is a phosphodegron in that phosphorylation ofserine 62 stabilizes MYC and primes threonine 58 (T58) forGSK3β phosphorylation, which, in turn, leads to Fbw7 binding,ubiquitination, and proteasome-mediated degradation. MYC-nick retains both the MBI and MBII regions, and we show herethat MYC-nick exhibits a similar rapid rate of proteasomal deg-radation, as does full-length nuclear MYC, which is also de-pendent in part on GSK3β phosphorylation and Fbw7 association.We find that deletion of the three Fbw7 isoforms, inhibition ofGSK3β or the proteasome, or mutation of the MYC-nick T58phosphorylation site each leads to the stabilization or increasedlevels of MYC-nick. Thus, the abundance of cytoplasmically lo-calized MYC-nick is subject to controls similar to those thatdetermine the abundance of full-length nuclear MYC. Interest-ingly, mutation of T58, or other residues within MBI that resultin MYC stabilization, is found in B-cell lymphomas and contributesto MYC’s oncogenic activity (52–54). Indeed, we observe that mu-rine intestinal tumors bearing different combinations of mutationsin the Wnt, TGF-β, and PI3K signaling pathways frequently have

Fig. 6. Model for MYC-nick function in cell migration. MYC-nick is highly expressed in the cytoplasm of migratory colon cancer cells, where it regulates theactin cytoskeleton. MYC-nick promotes activation of the Rho GTPase Cdc42 leading to an increase in actin polymerization into filopodia. MYC-nick alsoinduces the expression of the actin-bundling protein fascin, which promotes stabilization of actin filaments. MYC-nick is down-regulated by ubiquitin-mediated proteasomal degradation upon binding to the E3 ligase Fbw7. The interaction between MYC and Fbw7 is often impaired in tumors leading tostabilization of MYC and MYC-nick in cancer cells. MYC-nick also interacts with HATs via its MBII domain, leading to the acetylation of specific cytoplasmicproteins. The binding of MYC-nick to HATs is required for MYC-nick–induced cell migration.

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strikingly increased levels of both MYC and MYC-nick comparedwith normal mucosal tissue. We had previously shown that severaldistinct tumor types express high levels of MYC-nick (15, 16).Moreover, a recent study reported that MYC-nick expression isassociated with survival of neuroblastoma cells (55).An important property conferred by MYC-nick on cancer cells

is increased motility (15). That this is a robust effect is shown bythe results of 3D migration assays where MYC-nick promotescell migration of colon cancer cells embedded in Matrigel orcollagen, both of which provide resistance to movement. Fur-thermore, in vivo metastasis of colon cancer cells expressingMYC-nick, compared with vector controls, was significantly en-hanced after injection into zebrafish larvae. Our experiments im-plicate both fascin and Cdc42 in the mechanisms underlying theincreased invasiveness of MYC-nick–expressing cells. Both ofthese molecules have been shown to increase filopodia formationand to drive migration and metastasis. Although fascin is an actin-bundling protein critical for filopodia stability (31), Cdc42 drivesactin polymerization linked to the formation of filopodia (41, 43).We find that MYC-nick expression is accompanied by a

striking increase in fascin mRNA and protein (15). For example,our panel of murine intestinal and colonic lesions expressingendogenous MYC-nick also exhibited a higher abundance offascin relative to normal mucosal cells. Moreover, induction offascin, although required for MYC-nick–induced filopodia for-mation and cell migration, is not sufficient for filopodia forma-tion in the absence of MYC-nick. This finding prompted us toexamine the potential involvement of MYC-nick in other pathwaysthat control incorporation of the actin cytoskeleton into filopodia.Rac1, RhoA, and Cdc42 have been extensively studied in the con-text of cell invasion and actin cytoskeleton remodeling (41, 44). Rhopromotes stress fiber formation, whereas Rac1 regulates lamelli-podia, and Cdc42 promotes actin assembly into filopodia (43). Bymeans of pull-down experiments, we determined that Cdc42 un-dergoes sustained activation in cells expressing MYC-nick. AlthoughRac1 activity was increased, and Rho activity was reduced, thesechanges were modest relative to the activation of Cdc42. Moreover,when we blocked Rac/Cdc42 activity, we observed a complete loss offilopodia from MYC-nick–expressing cells, whereas activation ofGTPases with EGF enhanced filopodia formation.Our data suggest that MYC-nick plays a role in filopodia

formation and cell migration through its capacity to induce fascinexpression and modulate the activity of Rho-GTPases, particu-larly Cdc42. Indeed, in tissue sections, we observed an associa-tion between MYC-nick expression (indicated by the presence ofcytoplasmic MYC staining) and high levels of fascin and Cdc42in the invasive front of human colon cancers. Although themechanisms by which MYC-nick induces fascin expression andCdc42 activation are still unclear, an important clue to the mo-lecular regulation of cell migration by MYC-nick comes from aloss-of-function mutation. Deletion of MBII abrogates MYC-nick–induced fascin expression, cdc42 activation, and metastasisin zebrafish embryos, indicating that the binding of MYC-nick toacetyltransferases and protein acetylation mediates these effects.MYC-nick associates with acetyltransferase and induces acety-lation of several cytoplasmic proteins, among which we charac-terized α-tubulin and ATG3 (15, 16). We speculate that MYC-nick binding and acetylation of cytoplasmic factors, such asspecific GAPs or GEFs, could account for modulation of Cdc42activity by MYC-nick (45). Similarly, MYC-nick may influencecytoplasmic signaling to the nucleus, or perhaps itself serve as atranscriptional cofactor, leading to the expression of fascin orother genes. Further studies directed at unbiased identification ofMYC-nick–binding proteins and acetylation targets will define themolecular pathways linking MYC-nick production to increasedcell migration and invasiveness. Our data showing that MYC-nick,Cdc42, and fascin are highly expressed at the invasive front ofhuman colon cancers is consistent with the idea that MYC-nick’s

capacity to trigger cell motility is relevant to the biology of primarycancers (Fig. 6).

Materials and MethodsCell Lines andMouse Tissues. Cell lines weremaintained in DMEMwith 4.5 g ofglucose, 10% (vol/vol) FCS, and 100 U/mL penicillin/streptomycin. For CHXchase experiments, cells were cultured to confluency, and the culturemediumwas replaced 24 h before lysis. CHX (50 μg/mL) was added to cells for theindicated time points. All CHX chase experiments were performed in thepresence of calpeptin to ensure that the decline in MYC and MYC-nick levelswere due to proteasomal turnover, not from calpain-mediated cleavage.

Immunofluorescence and retroviral infections were performed as de-scribed by ref. 24. For overexpression experiments in 293T cells, Lipofect-amine 2000 (Thermo Fisher)-transfected cells were harvested 3 d aftertransfection, with a change in culture medium 24 h before harvesting.

Mouse tissues were handled as described (27). Normal mouse tissue andtumors were snap-frozen upon dissection, and total extracts were preparedin radioimmunoprecipitation assay buffer (pH 7.6) by sonication. Beforeloading on a gel, every sample was diluted in sample buffer [4% (wt/vol),SDS, 20% glycerol, 10% (vol/vol) β-mercaptoethanol, and 0.125 M Tris·HCl,pH 6.8] and boiled for 10 min. Then, 30 μg of total extracts were probedovernight with indicated antibodies. All animal studies were performedaccording to the guidelines of the Fred Hutchinson Cancer Research Center.

GTPase Assay. GTPase assay was performed with the RhoA/Rac1/Cdc42 Ac-tivation Assay kit from Cytoskeleton, Inc. Cells were split into 10-cm dishes toachieve 40% confluence 48 h later. Fresh growth medium was added 24 hbefore harvest. Pull-downs were performed according to manufacturer’sinstructions, with the following exceptions: no activator was used, lysateswere used fresh, and 500 μg of lysate was added to 0.2 μg of PAK-PBD beads.

Migration Assays.Wound healing was performed by using Cytoselect 24-WellWound Healing Assay from Cell Biolabs Inc., according to manufacturer’s in-structions. siRNA treatment of cells was performed with LipofectamineRNAiMAX (Thermo Fisher) according to the manufacturer’s recommendations.

Spheroid cultures were prepared as described (28). Briefly, 5,000 cellsresuspended in 500 μL of medium were plated over solid 1.5% (wt/vol) agarand grown for 4 d until spheroids were formed. Spheres were resuspended in50% (vol/vol) collagen or Matrigel and cultured for the indicated time points.

For zebrafish migration assays, cells were split to be 70% confluentin 10-cm dishes the next day. WT zebrafish embryos were collected,dechorionated, and treated with 0.2 mM phenylthiourea (PTU) to preventmelanization. Cells were labeled with CellTracker Green CMFDA (Invi-trogen), resuspended in HBSS (pH 7.3) at 106 cells per milliliter and 25–50cells were injected into the hindbrain ventricle of anesthetized 48-h-postfertilization larvae by using an a micromanipulator. Injected zebrafishlarvae were incubated for 4 d in 0.2 mM PTU at 31 °C. Metastasis wasscored for the percentage of zebrafish in which one or more cells hadmigrated away from the primary injection site.

Immunohistochemistry. Sections (4 mm)were deparaffinized, rehydrated, andpretreated for 10 min in a microwave (or in a pressure cooker) in Dako buffer(pH 6). They were then incubated overnight at 4 °C, with the primary anti-bodies diluted in RPMI 1640 plus 10% (wt/vol) bovine serum. Slides werewashed twice with TBST and developed with the EnVision System (Dako) andAEC for visualization according to the manufacturer’s instructions.

Antibodies, Inhibitors, siRNAs, and Constructs. Antibodies against c-MYC (9E10),Sin3, and fascin were from Santa Cruz Biotechnology. Anti-MYC 274 and 143were gifts from N. Ikegaki, University of Illinois, Chicago. Anti-tubulin (α andacetylated), GFP, and actin were from Sigma-Aldrich. P-T58 MYC and LC3IIBwere from Cell Signaling. Anti-Rac1, RhoA, and Cdc42 were from Cytoskele-ton, Inc. Anti-Fbw7 was from Bethyl Laboratories. Alexa Fluor 594 Phalloidinwas from Invitrogen. Anti-E-Cadherin was from Abcam. Fascin and Cdc42siRNAs were from Santa Cruz Biotechnology. Cdc42 activator (EGF) wasfrom Cytoskeleton, Inc., and the Cdc42/Rac1 inhibitor ML141 was fromCalbiochem.

MYC, MYC-nick, MYC-nick ΔMBII, and MYC Δ298–311 were cloned by PCRinto BamHI and EcoRI sites of pBabe puro and pBabe hygro and used toprepare retrovirus. WTMYC, T58AMYC,WTMYC-nick, and T58AMYC-nick werecloned into PCS2+.

ACKNOWLEDGMENTS. We thank Nao Ikegaki, John Sedivy, JonathanGrim, Bruce Clurman, and Markus Welcker for essential reagents. This

Anderson et al. PNAS | Published online August 26, 2016 | E5489

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research was supported by Grants R01 CA20525 (to R.N.E.), R21 CA195126(to C.B.M.); K99 CA190836 (to M.R.-J.); T32CA080416 and 14POST18230006(to K.R.P.); 2T32DK007742-16 (to M.Y.); R01CA194663, P30CA15704,

U01CA152756, and 5R00CA151672 (to W.N.G.); and CPRIT RR150059(to M.C.-S.). M.C.-S. is a Virginia Murchison Linthicum Scholar in MedicalResearch.

1. Vita M, Henriksson M (2006) The Myc oncoprotein as a therapeutic target for humancancer. Semin Cancer Biol 16(4):318–330.

2. Dang CV (2012) MYC on the path to cancer. Cell 149(1):22–35.3. Orian A, et al. (2003) Genomic binding by the Drosophila Myc, Max, Mad/Mnt tran-

scription factor network. Genes Dev 17(9):1101–1114.4. Ji H, et al. (2011) Cell-type independent MYC target genes reveal a primordial sig-

nature involved in biomass accumulation. PLoS One 6(10):e26057.5. Guccione E, et al. (2006) Myc-binding-site recognition in the human genome is de-

termined by chromatin context. Nat Cell Biol 8(7):764–770.6. Knoepfler PS, et al. (2006) Myc influences global chromatin structure. EMBO J 25(12):

2723–2734.7. Rahl PB, et al. (2010) c-Myc regulates transcriptional pause release. Cell 141(3):

432–445.8. Nie Z, et al. (2012) c-Myc is a universal amplifier of expressed genes in lymphocytes

and embryonic stem cells. Cell 151(1):68–79.9. Chakraborty AA, et al. (2014) A common functional consequence of tumor-derived

mutations within c-MYC. Oncogene 34(18):2406–2409.10. McMahon SB, Wood MA, Cole MD (2000) The essential cofactor TRRAP recruits the

histone acetyltransferase hGCN5 to c-Myc. Mol Cell Biol 20(2):556–562.11. Frank SR, et al. (2003) MYC recruits the TIP60 histone acetyltransferase complex to

chromatin. EMBO Rep 4(6):575–580.12. Farrell A, Sears RC (2014) MYC degradation. Cold Spring Harb Perspect Med 4(3):

a014365.13. Clark HM, et al. (1994) Mutations in the coding region of c-MYC in AIDS-associated

and other aggressive lymphomas. Cancer Res 54(13):3383–3386.14. Salghetti SE, Kim SY, Tansey WP (1999) Destruction of Myc by ubiquitin-mediated

proteolysis: Cancer-associated and transforming mutations stabilize Myc. EMBO J18(3):717–726.

15. Conacci-Sorrell M, Ngouenet C, Anderson S, Brabletz T, Eisenman RN (2014) Stress-induced cleavage of Myc promotes cancer cell survival. Genes Dev 28(7):689–707.

16. Conacci-Sorrell M, Ngouenet C, Eisenman RN (2010) Myc-nick: A cytoplasmic cleavageproduct of Myc that promotes alpha-tubulin acetylation and cell differentiation. Cell142(3):480–493.

17. Small GW, Chou TY, Dang CV, Orlowski RZ (2002) Evidence for involvement of calpainin c-Myc proteolysis in vivo. Arch Biochem Biophys 400(2):151–161.

18. Conacci-Sorrell M, Eisenman RN (2011) Post-translational control of Myc functionduring differentiation. Cell Cycle 10(4):604–610.

19. Sjöblom T, et al. (2006) The consensus coding sequences of human breast and co-lorectal cancers. Science 314(5797):268–274.

20. Chittenden TW, et al. (2008) Functional classification analysis of somatically mutatedgenes in human breast and colorectal cancers. Genomics 91(6):508–511.

21. Conacci-Sorrell M, McFerrin L, Eisenman RN (2014) An overview of MYC and its in-teractome. Cold Spring Harb Perspect Med 4(1):a014357.

22. Grim JE, et al. (2012) Fbw7 and p53 cooperatively suppress advanced and chromo-somally unstable intestinal cancer. Mol Cell Biol 32(11):2160–2167.

23. Grim JE, et al. (2008) Isoform- and cell cycle-dependent substrate degradation by theFbw7 ubiquitin ligase. J Cell Biol 181(6):913–920.

24. Welcker M, Clurman BE (2008) FBW7 ubiquitin ligase: A tumour suppressor at thecrossroads of cell division, growth and differentiation. Nat Rev Cancer 8(2):83–93.

25. Chen X, Barton LF, Chi Y, Clurman BE, Roberts JM (2007) Ubiquitin-independentdegradation of cell-cycle inhibitors by the REGgamma proteasome. Mol Cell 26(6):843–852.

26. Hann SR, Eisenman RN (1984) Proteins encoded by the human c-myc oncogene: Dif-ferential expression in neoplastic cells. Mol Cell Biol 4(11):2486–2497.

27. Gregory MA, Hann SR (2000) c-Myc proteolysis by the ubiquitin-proteasome pathway:Stabilization of c-Myc in Burkitt’s lymphoma cells. Mol Cell Biol 20(7):2423–2435.

28. Gregory MA, Qi Y, Hann SR (2003) Phosphorylation by glycogen synthase kinase-3controls c-MYC proteolysis and subnuclear localization. J Biol Chem.

29. Welcker M, et al. (2004) The Fbw7 tumor suppressor regulates glycogen synthasekinase 3 phosphorylation-dependent c-Myc protein degradation. Proc Natl Acad SciUSA 101(24):9085–9090.

30. Yada M, et al. (2004) Phosphorylation-dependent degradation of c-Myc is mediatedby the F-box protein Fbw7. EMBO J 23(10):2116–2125.

31. Machesky LM, Li A (2010) Fascin: Invasive filopodia promoting metastasis. CommunIntegr Biol 3(3):263–270.

32. Stoletov K, et al. (2010) Visualizing extravasation dynamics of metastatic tumor cells.J Cell Sci 123(Pt 13):2332–2341.

33. Stoletov K, Montel V, Lester RD, Gonias SL, Klemke R (2007) High-resolution imagingof the dynamic tumor cell vascular interface in transparent zebrafish. Proc Natl AcadSci USA 104(44):17406–17411.

34. Chapman A, et al. (2014) Heterogeneous tumor subpopulations cooperate to driveinvasion. Cell Reports 8(3):688–695.

35. Lee LM, Seftor EA, Bonde G, Cornell RA, Hendrix MJ (2005) The fate of human ma-lignant melanoma cells transplanted into zebrafish embryos: Assessment of migrationand cell division in the absence of tumor formation. Dev Dyn 233(4):1560–1570.

36. White RM, et al. (2008) Transparent adult zebrafish as a tool for in vivo trans-plantation analysis. Cell Stem Cell 2(2):183–189.

37. Zanet J, et al. (2012) Fascin promotes filopodia formation independent of its role inactin bundling. J Cell Biol 197(4):477–486.

38. Aratyn YS, Schaus TE, Taylor EW, Borisy GG (2007) Intrinsic dynamic behavior of fascinin filopodia. Mol Biol Cell 18(10):3928–3940.

39. Vignjevic D, et al. (2006) Role of fascin in filopodial protrusion. J Cell Biol 174(6):863–875.

40. Hall A (1998) Rho GTPases and the actin cytoskeleton. Science 279(5350):509–514.41. Raftopoulou M, Hall A (2004) Cell migration: Rho GTPases lead the way. Dev Biol

265(1):23–32.42. Sahai E, Marshall CJ (2002) RHO-GTPases and cancer. Nat Rev Cancer 2(2):133–142.43. Nobes CD, Hall A (1995) Rho, rac, and cdc42 GTPases regulate the assembly of mul-

timolecular focal complexes associated with actin stress fibers, lamellipodia, and fi-lopodia. Cell 81(1):53–62.

44. Sit ST, Manser E (2011) Rho GTPases and their role in organizing the actin cytoskel-eton. J Cell Sci 124(Pt 5):679–683.

45. Song EH, et al. (2015) Acetylation of the RhoA GEF Net1A controls its subcellularlocalization and activity. J Cell Sci 128(5):913–922.

46. Adams JC, Schwartz MA (2000) Stimulation of fascin spikes by thrombospondin-1 ismediated by the GTPases Rac and Cdc42. J Cell Biol 150(4):807–822.

47. Desmarais V, et al. (2009) N-WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells. Cell Motil Cytoskeleton 66(6):303–316.

48. Li A, et al. (2010) The actin-bundling protein fascin stabilizes actin in invadopodia andpotentiates protrusive invasion. Curr Biol 20(4):339–345.

49. Schoumacher M, Goldman RD, Louvard D, Vignjevic DM (2010) Actin, microtubules,and vimentin intermediate filaments cooperate for elongation of invadopodia. J CellBiol 189(3):541–556.

50. Yamaguchi H, et al. (2005) Molecular mechanisms of invadopodium formation: Therole of the N-WASP-Arp2/3 complex pathway and cofilin. J Cell Biol 168(3):441–452.

51. Vignjevic D, et al. (2007) Fascin, a novel target of beta-catenin-TCF signaling, is ex-pressed at the invasive front of human colon cancer. Cancer Res 67(14):6844–6853.

52. Bhatia K, et al. (1995) Mutations in the coding region of c-myc occur independently ofmutations in the regulatory regions and are predominantly associated with myc/Igtranslocation. Curr Top Microbiol Immunol 194:389–398.

53. Hemann MT, et al. (2005) Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants. Nature 436(7052):807–811.

54. Wang X, et al. (2011) Phosphorylation regulates c-Myc’s oncogenic activity in themammary gland. Cancer Res 71(3):925–936.

55. Shoji W, et al. (2015) NCYM promotes calpain-mediated Myc-nick production in hu-man MYCN-amplified neuroblastoma cells. Biochem Biophys Res Commun 461(3):501–506.

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