MYC-nick promotes cell migration by inducing fascin PNAS PLUS expression and Cdc42 activation

Sarah 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 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 Washington School 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 of Results the MYC oncoprotein family, generated by a proteolytic cleavage MYC-Nick Is Expressed in Intestinal and Colon Lesions in Mouse of full-length MYC. MYC-nick promotes migration and survival of Cancer Models Driven by Mutations in Apc, Tgfbr2, and Kras. We cells in response to chemotherapeutic agents or withdrawal of had previously shown that MYC-nick is expressed in cancer cell glucose. Here we report that MYC-nick is abundant in colonic and lines and arising from different primary tissues (15). To intestinal tumors derived from mouse models with mutations in extend those studies, we examined the expression of MYC variants the Wnt, TGF-β, and PI3K pathways. Moreover, MYC-nick is ele- in mouse colon cancers derived from distinct models of intestinal FBWX7, vated in colon cancer cells deleted for which encodes the cancer. Most colorectal carcinomas carry mutations that affect major E3 ligase of full-length MYC frequently mutated in colorec- Wnt, TGF-β, and PI3K signaling pathways (19, 20). We compared tal cancers. MYC-nick promotes the migration of colon cancer cells the expression of MYC in tumors arising from models containing + assayed in 3D cultures or grown as xenografts in a zebrafish me- (i) a truncation in one of the alleles of Apc (Apc1638/ ; labeled tastasis model. MYC-nick accelerates migration by activating the ATT); (ii) Pten and Tgfbr2 deletions combined (PPVcTT); (iii) Apc Rho GTPase Cdc42 and inducing fascin expression. MYC-nick, fascin, truncation in combination with Tgfbr2 deletion (AVcTT); and and Cdc42 are frequently up-regulated in cells present at the inva- (iv) activated oncogenic KrasG13D and Tgfbr2 deletion (KVcTT). sive front of human colorectal tumors, suggesting a coordinated We found that both MYC and MYC-nick levels are frequently role for these in tumor migration. elevated in intestinal adenomas and adenocarcinomas, as well as in colon carcinomas in these mouse models (Fig. 1 A–C and Table MYC | MYC-nick | colon cancer | motility | fascin S1). MYC-nick was shown to promote acetylation of cytoplasmic proteins (16, 21), and we found a correlation between MYC-nick embers of the MYC proto-oncogene family (c-MYC, level and acetylated α-tubulin in these samples (Fig. 1A). MN-MYC, and L-MYC) are key regulators of tumor ini- tiation and tumor maintenance in many types of cancer (1). Oncogenic Mutations Augment Stability of MYC and MYC-Nick. Mu- MYC proteins initiate a transcriptional program of growth tations in MYC that prevent its binding to SCFFBW7 have been and proliferation, as well as suppression of cell-cycle arrest reported to increase MYC levels and promote tumorigenesis (2). Functionally, MYC proteins form dimers with Max and (22). The Fbw7 binding site is also retained in MYC-nick (Fig. act broadly as transcriptional activators of a large number of 1E). To determine whether MYC-nick stability was also regulated genes (3–8). MYC binds Max and DNA via its C-terminal region – – comprising a basic helix loop helix leucine zipper (BHLH LZ) Significance domain. The N terminus of MYC contains four highly conserved

regions called MYC boxes (MB I–IV), involved in MYC’s MEDICAL SCIENCES The MYC family of transcription factors is deregulated in a function in transcriptional regulation (9). As one of the major broad range of cancers and drives the expression of genes that determinants of MYC’s transcriptional function, MBII recruits mediate biomass accumulation and promote cell proliferation coactivator complexes including histone acetyltransferases (HATs), and tumor initiation. We find that MYC can also trigger tumor such as GCN5 (10) and Tip60 (11). MYC is a very short-lived cell migration and metastasis independently of its transcrip- , and multiple E3 ligases have been implicated in regu- tional activity, via its conversion to MYC-nick, a truncated form lating MYC protein turnover through the ubiquitin–proteasome of MYC localized in the . MYC-nick promotes re- system (12). Importantly, MYC levels have been demonstrated organization of the by inducing expression to be elevated in cancer cells because of prolonged protein half- of the actin-bundling protein fascin and by activating the Rho life (13, 14). GTPase Cdc42, both of which lead to formation of filopodia, MYC is also targeted by calpain proteases in the cytoplasm cellular structures known to drive cell migration. Our work (15–17). Calpain-mediated scission of MYC degrades its C ’ links the repurposing of the MYC transcription factor to altered terminus, which inactivates MYC s transcriptional functions. cytoskeletal structure and tumor cell metastatic behavior. Furthermore, the cleavage generates MYC-nick, a truncated product that retains MBI–MBIII (16). Although MYC-nick is Author contributions: R.N.E. and M.C.-S. designed research; S.A., K.R.P., M.R.-J., T.B., N.B.-A., expressed in most cultured cells and in mouse tissues, its levels and M.C.-S. performed research; M.Y. contributed new reagents/analytic tools; K.R.P., are increased in cells cultured under conditions leading to 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. stress, such as high cell density, nutrient deprivation, and Reviewers: S.R.H., Vanderbilt University; and M.F.R., St. Jude Children’s Research Hospital. hypoxia (15, 16, 18). Recently, we found that the conversion of The authors declare no conflict of interest. MYC into MYC-nick occurs in the cytoplasm of colon cancer Freely available online through the PNAS open access option. cells, where it promotes cell survival and motility (15). Here we 1To whom correspondence may be addressed. Email: [email protected] or maralice. demonstrate that MYC-nick promotes cell migration and in- [email protected]. vasion by inducing fascin expression and activating the Rho This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. GTPase Cdc42 in distinct models of colon cancer. 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 later were processed for Western blot by using antibodies against total MYC and phosphorylated T58/S62 MYC.

E5482 | www.pnas.org/cgi/doi/10.1073/pnas.1610994113 Anderson et al. Downloaded by guest on October 2, 2021 by FBXW7, we examined variants of the colon cancer cell lines Four days after implantation, MYC-nick–expressing DLD1 cells PNAS PLUS DLD1 and HCT116, both of which have the FBXW7 gene deleted exhibited an increase in metastatic behavior, measured by the by gene targeting (23). We found that, compared with their WT number of cells that migrate away from the site of injection (Fig. counterparts, both cell lines deleted for FBXW7 exhibited in- 2 G and H). Deletion of MBII dramatically reduced MYC-nick’s creased the stability of MYC and MYC-nick in the cytoplasm, as ability to drive migration (Fig. 2 G and H). Together, these re- measured by cycloheximide (CHX) chase (Fig. 1 D and F and Fig. sults further suggest that expression of MYC-nick induces tumor − − S1A). The increase in MYC-nick levels observed in FBXW7 / cell invasion in vivo. cells was not due to reduced calpain activity, because deletion of FBXW7 had no effect on calpain-mediated cleavage of MYC (Fig. MYC-Nick–Induced Migration Requires Fascin Expression. We reported S1B). The reduction in the total levels of MYC observed in previously that MYC-nick promotes a dramatic increase in the − − FBXW7 / (Fig. 1D) was reported previously and is caused by a levels of fascin (15), which is a driver of metastatic behavior in reduction in MYC mRNA (23). Consistent with the increased solid tumors and is associated with tumor progression in the colon stability of MYC-nick upon FBXW7 deletion, we are able to detect and stomach (31) (Fig. 3A). Fascin expression is induced by MYC- both endogenous MYC and MYC-nick associated with Fbw7α in nick in a variety of cell lines, including colon cancer cell lines such the cytoplasm of DLD1 cells (Fig. S1C). Fbw7α is the only Fbw7 as DLD1 (Fig. 3A) and HCT116 (Fig. S3B) and human foreskin isoform endogenously expressed in these cells (23, 24). fibroblasts (HFFs) (Fig. S3C). Overexpression of full-length MYC Inhibition of the proteasome with epoxomycin increases the in HFFs also induced fascin expression, probably due to consti- stability of overexpressed MYC-nick (Fig. S2A) and siRNAs against tutive generation of MYC-nick in these cells (Fig. S3C). More- components of the proteasome (25) also augment the stability of over, we found a marked increase in fascin levels in mouse endogenous full-length MYC and MYC-nick in the cytoplasm (Fig. adenomas and adenocarcinomas expressing high levels of MYC- S2B). In nontransformed cells, we found that MYC-nick has a half- nick (Fig. 3B). Although fascin down-regulation by siRNA does life of ∼30 min (Fig. S2 C–E), similar to the estimated half-life for not affect cell survival of MYC-nick–expressing cells (15), it pre- full-length MYC (26, 27). Mutations that affect the binding of vented filopodia formation (Fig. 3C) and cell migration (Fig. 3D). MYC to Fbw7, such as T58A, do not affect its targeting by calpain This finding indicates that fascin is necessary for MYC-nick- and conversion into MYC-nick. (Fig. S1D). induced cell migration, consistent with reports that filopodia forma- The binding of SCFFbw7 to MYC requires phosphorylation of tion requires fascin (37–39). However, overexpression of GFP-tagged MYC threonine 58 (T58), which is mediated by glycogen syn- fascin in DLD1 cells did not mobilize actin to induce the formation thase kinase 3β [GSK3β (28)]. Several studies have shown that of filopodia (Fig. 3 E and F), indicating that fascin is not sufficient for GSK3β-mediated phosphorylation of MYC promotes its recog- MYC-nick–induced filopodia formation and that additional changes nition by SCFFbw7, leading to proteasomal degradation (29, 30). in the actin cytoskeleton are required. As expected, treating cells with pharmacological inhibitors that block GSK3β activity, such as indirubin and kenpaulone, leads to MYC-Nick–Induced Migration Requires Cdc42 Activation. The Rho increased stability of both MYC and MYC-nick (Fig. 1F). Indeed, family of GTPases plays an essential role in cellular motility by we found that a point mutation in MYC converting threonine 58 regulating the organization of the actin cytoskeleton (40, 41). to alanine (T58A) prevented its phosphorylation and caused ac- Because MYC-nick promotes changes in the actin cytoskeleton cumulation of both full-length MYC and MYC-nick (Fig. 1G). In and increases migratory properties, we asked whether Rho a CHX chase, we found that T58A MYC-nick was more stable GTPases collaborate with fascin to mediate MYC-nick’s effects than WT MYC-nick when transfected in 293T (Fig. S2F). on filopodium formation and cell migration. We treated DLD1 cells expressing either empty vector or MYC-nick with EGF, MYC-Nick Induces Migration in 3D Substrates. We have demon- which activates Rho GTPases, or with the RAC1/Cdc42 inhibitor strated, using scratch healing and transwell migration assays, that ML141. Inhibiting RAC/Cdc42 activity for 10–16 h completely overexpression of MYC-nick promotes the migration of colon ablated filopodia in MYC-nick–expressing cells (Fig. 4 A and B). cancer cells (ref. 15 and Fig. 2A). We found that MYC-nick in- Conversely, activating GTPases with EGF promoted filopodia

duces migration and promotes filopodia formation, while up- formation in vector-expressing cells to the same extent as ex- MEDICAL SCIENCES regulating the actin-bundling protein fascin (15). Here we extend pression of MYC-nick and further increased filopodium forma- our studies to the 3D migration systems to better recapitulate the tion in MYC-nick–expressing cells (Fig. 4 A and B). These results resistance provided by the environment during the migratory indicate that Cdc42 activity is required and sufficient to induce process of metastatic cancer cells. We found that ectopic expres- filopodia formation in MYC-nick–expressing cells. sion of MYC-nick in DLD1 colon cancer cells also promoted their Importantly, as with fascin siRNA, the viability of MYC- migration in 3D cultures (Fig. 2 B–F). Single cells or spheroids nick–expressing cells was insensitive to treatment with Cdc42 were cultured in ECM such as 50% (vol/vol) Matrigel or collagen, inhibitors. Even though the treated cells were not migratory, and in both cases, MYC-nick expressing colonies displayed a more they survived for extended periods of time (4 d) in the pres- migratory phenotype than control cells (Fig. 2 B–D). When plated ence of these inhibitors. This finding is consistent with our in 20% (vol/vol) collagen, which is more permissive to migration, previous observation that MYC-nick promotes survival by in- MYC-nick–expressing cells were still more migratory than controls ducing acetylation of LC3BII and α-tubulin to accelerate auto- (Fig. 2 E and F). Note that MYC-expressing cells were also more phagy, a function that appears not to require Cdc42 activity migratory than control cells in 3D (Fig. 2 E and F), which is (Fig. S3D). probably caused by the constitutive cleavage of MYC into MYC- Although cells overexpressing MYC-nick are resistant to nick in these cells. These migratory cells displayed enhanced prolonged treatment with Cdc42 inhibitors, cells expressing filopodial protrusions (Figs. 2A and 3C and Fig. S3A), which empty vector, MYC-nick lacking the acetyltransferase bind- have been strongly linked to the increased migratory behavior ing domain (ΔMBII), full-length MYC, or uncleavable MYC and metastatic potential of cancer cells (31). In agreement with (Δ298–311) did not survive in the presence of Cdc42 inhib- our previous observations, we found that cells expressing a itors (Fig. S3D). MYC-nick mutant lacking MBII (ΔMBII) are unable to form Because of their role as positive regulators of cellular motility and filopodia and to promote persistent migration in 3D cultures. invasion, Rho-GTPases have been linked to tumorigenic pheno- We validated our results using a previously described human- types in a variety of human cancers (42). Indeed, we observed in-zebrafish xenotransplantation approach (32–36). We intro- that the total levels of RAC1 and Cdc42 are up-regulated duced DLD1 cells into 48-h postfertilization zebrafish larvae. in 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 Cytoselect 24-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 expressing MYC-nick or empty vector were trypsinized, and single cells were embedded in 50% (vol/vol) Matrigel (B)or collagen (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 for the 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 as spheroids 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 after seeding. Cultures were photographed 3 d after seeding (F). (Magnification: 20×.) (G and H). A total of 25–50 DLD1 cells expressing empty vector, MYC-nick, and MYC-nick lacking MYC 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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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 S1 for 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 stained with phalloidin. (Magnification: 63×.) (D) DLD1 cell monolayers expressing MYC-nick were transfected with control or Fascin siRNA, scratched 48 h later in the presence of mitomycin C, and photographed at 72 h. (Magnification: 10×.) (E and F) DLD1 cells expressing empty vector or MYC-nick were transfected with GFP-fascin and stained with phalloidin 48 h later. (Magnification: E,20×; F, 100×.)

derived from the mouse models of intestinal neoplasia com- presence of MYC-nick because MYC-nick expression does not pared with normal mucosa (Fig. 4C and Table S1). However, increase either the total levels or the stability of Cdc42 protein this increase in Cdc42 expression is probably not due to the (Fig. 4D).

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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 on MYC-nick–induced filopodia formation. (Magnification: 63×.) (B) Quantification of A. Cells surrounding individual colonies were scored for the presence of at least one filopodium. n = 100. (C) Immunoblots of Cdc42, Rac in mouse . (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-length MYC (MYC Δ298–311). (H) Analyzes of fascin and Cdc42 expression in cells transfected with siRNA against these proteins. DLD1 cell expressing empty vector or MYC-nick were transfected with siRNA for fascin or Cdc42 and processed for Western blot 48 h later.

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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, and the 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×.)

MYC-Nick Promotes the Activation of Cdc42. Rho GTPases cycle and 4). In control cells, this activation was seen to the same extent, but between active (GTP-bound) and inactive (GDP-bound) states by was transient (Fig. 4F, compare lanes 5 and 6). However, MYC-nick binding and hydrolyzing GTP. To determine whether MYC-nick is not capable of promoting or sustaining the activation of Cdc42 in expression modulates Rho GTPase activity, we performed pull- highly confluent cultures (Fig. 4F). Thus, activation of Cdc42 by downs of activated RhoA by using beads conjugated to its binding MYC-nick differs from fascin induction because MYC-nick ac- partner Rhotekin. We also pulled down active Rac1 and Cdc42 tivates fascin expression regardless of cellular density (15). using P21-activated kinase beads (PAK). We found that MYC-nick promotes activation of Cdc42 when expressed in colon cancer cells Cdc42 Activation by MYC-Nick Requires the MBII Region. The MBII such as DLD1 (Fig. 4E)andHCT116(Fig. S3A). Rac1 and RhoA region (amino acids 106–143) located within N-terminal segment activities were only modestly affected by MYC-nick: Rac1 activity of MYC and MYC-nick constitutes a binding site for recruitment was elevated, whereas RhoA activity was reduced (Fig. 4E). of HATs to MYC. Deletion of MBII, although having no effect Importantly, we found that MYC-nick promoted a sustained on MYC-nick expression levels, reduced MYC-nick’s ability to activation of Cdc42 in MYC-nick–expressing cells as observed 16 h promote cell survival and migration (Fig. 2 E–G and Fig. S3D)(15). after treatment with growth factors or EGF (Fig. 4F, compare lanes 3 Although cells expressing MYC-nick ΔMBII appear more

Anderson et al. PNAS | Published online August 26, 2016 | E5487 Downloaded by guest on October 2, 2021 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 the actin cytoskeleton. MYC-nick promotes activation of the Rho GTPase Cdc42 leading to an increase in actin polymerization into filopodia. MYC-nick also induces 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 to stabilization of MYC and MYC-nick in cancer cells. MYC-nick also interacts with HATs via its MBII domain, leading to the acetylation of specific cytoplasmic proteins. The binding of MYC-nick to HATs is required for MYC-nick–induced cell migration.

capable of initiating migration relative to control cells (Fig. 2E), the Discussion MYC-nick ΔMBII–expressing cells are not capable of completing Our earlier work showed that MYC-nick is generated by calpain migration and forming new colonies in collagen (Fig. 2F). To cleavage of full-length MYC under conditions of stress, such as directly address the relevance of MBII to Cdc42 activation, we nutrient deprivation and hypoxia. Under these conditions, a compared the activation of Cdc42 in cells expressing MYC-nick mutant form of MYC deficient in producing MYC-nick causes to cells expressing MYC-nick ΔMBII. We found that MBII is extensive apoptosis, which is attenuated by coexpression of required for the activation of Cdc42 by MYC-nick (Fig. 4G). As MYC-nick (15). This finding indicates that MYC-nick plays an expected, full-length MYC was also capable of inducing Cdc42 active role in cell survival, and its production is important for – activation, most likely through its ability to constitutively generate adaptation to stress (Fig. 6). MYC-nick induced survival is me- MYC-nick through calpain cleavage. Consistent with this finding, diated, at least in part, through stimulation of an autophagic response. In addition, MYC-nick expression correlates with striking a mutation in MYC deleting the calpain cleavage region amino changes in cytoskeletal organization, including alterations in actin acids 298–311 (MYC Δ298–311) is not capable of activating Cdc42 dynamics, which lead to increased cell migration. These cellular (Fig. 4G). Note that all MYC constructs were expressed at similar changes are abrogated by deletion of the MBII region in MYC- levels (Fig. S3E). nick, a region known to recruit protein complexes involved in his- tone acetylation and chromatin remodeling. In MYC-nick, MBII Induction of Fascin and Activation of Cdc42 by MYC-Nick Are also associates with the acetyltransferase GCN5 (and probably Independent Events. Fascin and Cdc42 function in coordination to others), inducing the acetylation of cytoplasmic proteins and – promote filopodia formation and to drive cell migration (43 45). thereby modulating protein function (16). Moreover, Cdc42 was shown to regulate fascin localization and The abundance of full-length nuclear MYC has been long function. For example, constitutively active mutant forms of Rac and known to be controlled posttranslationally by means of pro- Cdc42 were reported to trigger localization of fascin to lamellipodia, teolytic degradation determined by the balanced actions of sev- whereas fascin, in turn, is critical for cell migration driven by Rac and eral ubiquitin ligases and ubiquitin-specific proteases (for review, Cdc42 (46). Cdc42 and fascin also cooperate to promote invado- see ref. 12). The conserved MBI and MBII regions in MYC serve podia formation in different model systems (47–50). To address the as binding sites for the Fbw7 and Skp2 ubiquitin ligases, re- involvement of Cdc42 in the induction of fascin expression by MYC- spectively. MBI is a phosphodegron in that phosphorylation of nick, we silenced Cdc42 by siRNA in DLD1 cells expressing either serine 62 stabilizes MYC and primes threonine 58 (T58) for β empty vector or MYC-nick. Although silencing Cdc42 reduced fas- GSK3 phosphorylation, which, in turn, leads to Fbw7 binding, cin levels in control cells (Fig. 4H), it did not affect fascin levels in ubiquitination, and proteasome-mediated degradation. MYC- MYC-nick–expressing cells (Fig. 4H). Conversely, silencing fascin by nick retains both the MBI and MBII regions, and we show here that MYC-nick exhibits a similar rapid rate of proteasomal deg- siRNA did not affect the levels of total Cdc42 (Fig. 4H). radation, as does full-length nuclear MYC, which is also de- β Fascin and Cdc42 Are Highly Expressed at the Invasive Front of Colon pendent in part on GSK3 phosphorylation and Fbw7 association. Cancers. We have shown previously that cytoplasmic staining with We find that deletion of the three Fbw7 isoforms, inhibition of GSK3β or the proteasome, or mutation of the MYC-nick T58 anti-MYC corresponding to MYC-nick is elevated in cells at the phosphorylation site each leads to the stabilization or increased invasive front of human colorectal cancers (Fig. 5C and ref. 15). levels of MYC-nick. Thus, the abundance of cytoplasmically lo- Here we analyzed the expression of both Cdc42 (Fig. 5A)andfascin calized MYC-nick is subject to controls similar to those that (Fig. 5B) in migratory cells at the invasive front of the same tumors determine the abundance of full-length nuclear MYC. Interest- = (Table S2)(n 19). We found that Cdc42 and fascin, similar to MYC- ingly, mutation of T58, or other residues within MBI that result nick, are increased in tumor tissues and are often further elevated at in MYC stabilization, is found in B-cell lymphomas and contributes the invasive front of these tumors (Fig. 5D and Table S2). These to MYC’s oncogenic activity (52–54). Indeed, we observe that mu- observations are in agreement with several studies indicating fascin up- rine intestinal tumors bearing different combinations of mutations regulation in cancer cells drives motility and invasiveness (31, 48, 51). in the Wnt, TGF-β, and PI3K signaling pathways frequently have

E5488 | www.pnas.org/cgi/doi/10.1073/pnas.1610994113 Anderson et al. Downloaded by guest on October 2, 2021 strikingly increased levels of both MYC and MYC-nick compared capacity to trigger cell motility is relevant to the biology of primary PNAS PLUS with normal mucosal tissue. We had previously shown that several cancers (Fig. 6). distinct tumor types express high levels of MYC-nick (15, 16). Moreover, a recent study reported that MYC-nick expression is Materials and Methods associated with survival of neuroblastoma cells (55). Cell Lines and Mouse Tissues. Cell lines were maintained in DMEM with 4.5 g of An important property conferred by MYC-nick on cancer cells glucose, 10% (vol/vol) FCS, and 100 U/mL penicillin/streptomycin. For CHX is increased motility (15). That this is a robust effect is shown by chase experiments, cells were cultured to confluency, and the culture medium μ the results of 3D migration assays where MYC-nick promotes was replaced 24 h before lysis. CHX (50 g/mL) was added to cells for the cell migration of colon cancer cells embedded in Matrigel or indicated time points. All CHX chase experiments were performed in the presence of calpeptin to ensure that the decline in MYC and MYC-nick levels collagen, both of which provide resistance to movement. Fur- were due to proteasomal turnover, not from calpain-mediated cleavage. thermore, in vivo metastasis of colon cancer cells expressing Immunofluorescence and retroviral infections were performed as de- MYC-nick, compared with vector controls, was significantly en- scribed by ref. 24. For overexpression experiments in 293T cells, Lipofect- hanced after injection into zebrafish larvae. Our experiments im- amine 2000 (Thermo Fisher)-transfected cells were harvested 3 d after plicate both fascin and Cdc42 in the mechanisms underlying the transfection, with a change in culture medium 24 h before harvesting. increased invasiveness of MYC-nick–expressing cells. Both of Mouse tissues were handled as described (27). Normal mouse tissue and these molecules have been shown to increase filopodia formation tumors were snap-frozen upon dissection, and total extracts were prepared and to drive migration and metastasis. Although fascin is an actin- in radioimmunoprecipitation assay buffer (pH 7.6) by sonication. Before bundling protein critical for filopodia stability (31), Cdc42 drives loading 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, actin polymerization linked to the formation of filopodia (41, 43). μ We find that MYC-nick expression is accompanied by a pH 6.8] and boiled for 10 min. Then, 30 g of total extracts were probed overnight with indicated antibodies. All animal studies were performed striking increase in fascin mRNA and protein (15). For example, according to the guidelines of the Fred Hutchinson Cancer Research Center. our panel of murine intestinal and colonic lesions expressing endogenous MYC-nick also exhibited a higher abundance of GTPase Assay. GTPase assay was performed with the RhoA/Rac1/Cdc42 Ac- fascin relative to normal mucosal cells. Moreover, induction of tivation Assay kit from Cytoskeleton, Inc. Cells were split into 10-cm dishes to fascin, although required for MYC-nick–induced filopodia for- achieve 40% confluence 48 h later. Fresh growth medium was added 24 h mation and cell migration, is not sufficient for filopodia forma- before harvest. Pull-downs were performed according to manufacturer’s tion in the absence of MYC-nick. This finding prompted us to instructions, with the following exceptions: no activator was used, lysates examine the potential involvement of MYC-nick in other pathways were used fresh, and 500 μg of lysate was added to 0.2 μg of PAK-PBD beads. that control incorporation of the actin cytoskeleton into filopodia. Migration Assays. Wound healing was performed by using Cytoselect 24-Well Rac1, RhoA, and Cdc42 have been extensively studied in the con- ’ text of cell invasion and actin cytoskeleton remodeling (41, 44). Rho Wound Healing Assay from Cell Biolabs Inc., according to manufacturer sin- structions. siRNA treatment of cells was performed with Lipofectamine promotes stress fiber formation, whereas Rac1 regulates lamelli- RNAiMAX (Thermo Fisher) according to the manufacturer’s recommendations. podia, and Cdc42 promotes actin assembly into filopodia (43). By Spheroid cultures were prepared as described (28). Briefly, 5,000 cells means of pull-down experiments, we determined that Cdc42 un- resuspended in 500 μL of medium were plated over solid 1.5% (wt/vol) agar dergoes sustained activation in cells expressing MYC-nick. Although and grown for 4 d until spheroids were formed. Spheres were resuspended in Rac1 activity was increased, and Rho activity was reduced, these 50% (vol/vol) collagen or Matrigel and cultured for the indicated time points. changes were modest relative to the activation of Cdc42. Moreover, For zebrafish migration assays, cells were split to be 70% confluent when we blocked Rac/Cdc42 activity, we observed a complete loss of in 10-cm dishes the next day. WT zebrafish embryos were collected, filopodia from MYC-nick–expressing cells, whereas activation of dechorionated, and treated with 0.2 mM phenylthiourea (PTU) to prevent GTPases with EGF enhanced filopodia formation. melanization. Cells were labeled with CellTracker Green CMFDA (Invi- 6 – Our data suggest that MYC-nick plays a role in filopodia trogen), resuspended in HBSS (pH 7.3) at 10 cells per milliliter and 25 50 formation and cell migration through its capacity to induce fascin cells were injected into the hindbrain ventricle of anesthetized 48-h- postfertilization larvae by using an a micromanipulator. Injected zebrafish expression and modulate the activity of Rho-GTPases, particu- larvae were incubated for 4 d in 0.2 mM PTU at 31 °C. Metastasis was

larly Cdc42. Indeed, in tissue sections, we observed an associa- scored for the percentage of zebrafish in which one or more cells had MEDICAL SCIENCES tion between MYC-nick expression (indicated by the presence of migrated away from the primary injection site. cytoplasmic MYC staining) and high levels of fascin and Cdc42 in the invasive front of human colon cancers. Although the Immunohistochemistry. Sections (4 mm) were deparaffinized, rehydrated, and mechanisms by which MYC-nick induces fascin expression and pretreated for 10 min in a microwave (or in a pressure cooker) in Dako buffer Cdc42 activation are still unclear, an important clue to the mo- (pH 6). They were then incubated overnight at 4 °C, with the primary anti- lecular regulation of cell migration by MYC-nick comes from a bodies diluted in RPMI 1640 plus 10% (wt/vol) bovine serum. Slides were loss-of-function mutation. Deletion of MBII abrogates MYC- washed twice with TBST and developed with the EnVision System (Dako) and ’ nick–induced fascin expression, cdc42 activation, and metastasis AEC for visualization according to the manufacturer s instructions. in zebrafish embryos, indicating that the binding of MYC-nick to Antibodies, Inhibitors, siRNAs, and Constructs. Antibodies against c-MYC (9E10), acetyltransferases and protein acetylation mediates these effects. Sin3, and fascin were from Santa Cruz Biotechnology. Anti-MYC 274 and 143 MYC-nick associates with acetyltransferase and induces acety- were gifts from N. Ikegaki, University of Illinois, Chicago. Anti-tubulin (α and lation of several cytoplasmic proteins, among which we charac- acetylated), GFP, and actin were from Sigma-Aldrich. P-T58 MYC and LC3IIB terized α-tubulin and ATG3 (15, 16). We speculate that MYC- were from . Anti-Rac1, RhoA, and Cdc42 were from Cytoskele- nick binding and acetylation of cytoplasmic factors, such as ton, Inc. Anti-Fbw7 was from Bethyl Laboratories. Alexa Fluor 594 Phalloidin specific GAPs or GEFs, could account for modulation of Cdc42 was from Invitrogen. Anti-E-Cadherin was from Abcam. Fascin and Cdc42 activity by MYC-nick (45). Similarly, MYC-nick may influence siRNAs were from Santa Cruz Biotechnology. Cdc42 activator (EGF) was cytoplasmic signaling to the nucleus, or perhaps itself serve as a from Cytoskeleton, Inc., and the Cdc42/Rac1 inhibitor ML141 was from transcriptional cofactor, leading to the expression of fascin or Calbiochem. Δ Δ – other genes. Further studies directed at unbiased identification of MYC, MYC-nick, MYC-nick MBII, and MYC 298 311 were cloned by PCR – into BamHI and EcoRI sites of pBabe puro and pBabe hygro and used to MYC-nick binding proteins and acetylation targets will define the prepare retrovirus. WTMYC, T58AMYC, WTMYC-nick, and T58AMYC-nick were molecular pathways linking MYC-nick production to increased cloned into PCS2+. cell migration and invasiveness. Our data showing that MYC-nick, Cdc42, and fascin are highly expressed at the invasive front of ACKNOWLEDGMENTS. We thank Nao Ikegaki, John Sedivy, Jonathan human colon cancers is consistent with the idea that MYC-nick’s Grim, Bruce Clurman, and Markus Welcker for essential reagents. This

Anderson et al. PNAS | Published online August 26, 2016 | E5489 Downloaded by guest on October 2, 2021 research was supported by Grants R01 CA20525 (to R.N.E.), R21 CA195126 U01CA152756, and 5R00CA151672 (to W.N.G.); and CPRIT RR150059 (to C.B.M.); K99 CA190836 (to M.R.-J.); T32CA080416 and 14POST18230006 (to M.C.-S.). M.C.-S. is a Virginia Murchison Linthicum Scholar in Medical (to K.R.P.); 2T32DK007742-16 (to M.Y.); R01CA194663, P30CA15704, Research.

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