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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2016 Aug 26;113(37):E5481–E5490. doi: 10.1073/pnas.1610994113

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

Sarah Anderson a, Kumud Raj Poudel a, Minna Roh-Johnson a, Thomas Brabletz b, Ming Yu c, Nofit Borenstein-Auerbach d, William N Grady c,e, Jihong Bai a, Cecilia B Moens a, Robert N Eisenman a,1, Maralice Conacci-Sorrell d,f,1
PMCID: PMC5027433  PMID: 27566402

Significance

The MYC family of transcription factors is deregulated in a broad range of cancers and drives the expression of genes that mediate biomass accumulation and promote cell proliferation and tumor initiation. We find that MYC can also trigger tumor cell migration and metastasis independently of its transcriptional activity, via its conversion to MYC-nick, a truncated form of MYC localized in the cytoplasm. MYC-nick promotes reorganization of the actin cytoskeleton by inducing expression of the actin-bundling protein fascin and by activating the Rho GTPase Cdc42, both of which lead to formation of filopodia, cellular structures known to drive cell migration. Our work links the repurposing of the MYC transcription factor to altered cytoskeletal structure and tumor cell metastatic behavior.

Keywords: MYC, MYC-nick, colon cancer, motility, fascin

Abstract

MYC-nick is a cytoplasmic, transcriptionally inactive member of the MYC oncoprotein family, generated by a proteolytic cleavage of full-length MYC. MYC-nick promotes migration and survival of cells in response to chemotherapeutic agents or withdrawal of glucose. Here we report that MYC-nick is abundant in colonic and intestinal tumors derived from mouse models with mutations in the Wnt, TGF-β, and PI3K pathways. Moreover, MYC-nick is elevated in colon cancer cells deleted for FBWX7, which encodes the major E3 ligase of full-length MYC frequently mutated in colorectal cancers. MYC-nick promotes the migration of colon cancer cells assayed in 3D cultures or grown as xenografts in a zebrafish metastasis model. MYC-nick accelerates migration by activating the Rho GTPase Cdc42 and inducing fascin expression. MYC-nick, fascin, and Cdc42 are frequently up-regulated in cells present at the invasive front of human colorectal tumors, suggesting a coordinated role for these proteins in tumor migration.

Members of the MYC proto-oncogene family (c-MYC, N-MYC, and L-MYC) are key regulators of tumor initiation and tumor maintenance in many types of cancer (1). MYC proteins initiate a transcriptional program of growth and proliferation, as well as suppression of cell-cycle arrest (2). Functionally, MYC proteins form dimers with Max and act broadly as transcriptional activators of a large number of genes (3–8). MYC binds Max and DNA via its C-terminal region comprising a basic helix–loop–helix leucine zipper (BHLH LZ) domain. The N terminus of MYC contains four highly conserved regions called MYC boxes (MB I–IV), involved in MYC’s function in transcriptional regulation (9). As one of the major determinants of MYC’s transcriptional function, MBII recruits coactivator complexes including histone acetyltransferases (HATs), such as GCN5 (10) and Tip60 (11). MYC is a very short-lived protein, and multiple E3 ligases have been implicated in regulating MYC protein turnover through the ubiquitin–proteasome system (12). Importantly, MYC levels have been demonstrated to 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 C terminus, which inactivates MYC’s transcriptional functions. Furthermore, the cleavage generates MYC-nick, a truncated product that retains MBI–MBIII (16). Although MYC-nick is expressed in most cultured cells and in mouse tissues, its levels are increased in cells cultured under conditions leading to stress, such as high cell density, nutrient deprivation, and hypoxia (15, 16, 18). Recently, we found that the conversion of MYC into MYC-nick occurs in the cytoplasm of colon cancer cells, where it promotes cell survival and motility (15). Here we demonstrate that MYC-nick promotes cell migration and invasion by inducing fascin expression and activating the Rho GTPase Cdc42 in distinct models of colon cancer.

Results

MYC-Nick Is Expressed in Intestinal and Colon Lesions in Mouse Cancer Models Driven by Mutations in Apc, Tgfbr2, and Kras.

We had previously shown that MYC-nick is expressed in cancer cell lines and cancers arising from different primary tissues (15). To extend those studies, we examined the expression of MYC variants in mouse colon cancers derived from distinct models of intestinal cancer. Most colorectal carcinomas carry mutations that affect Wnt, TGF-β, and PI3K signaling pathways (19, 20). We compared the expression of MYC in tumors arising from models containing (i) a truncation in one of the alleles of Apc (Apc1638/+; labeled ATT); (ii) Pten and Tgfbr2 deletions combined (PPVcTT); (iii) Apc truncation 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 frequently elevated in intestinal adenomas and adenocarcinomas, as well as in colon carcinomas in these mouse models (Fig. 1 A–C and Table S1). MYC-nick was shown to promote acetylation of cytoplasmic proteins (16, 21), and we found a correlation between MYC-nick level and acetylated α-tubulin in these samples (Fig. 1A).

Fig. 1.

Fig. 1.

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Immunoblotting of MYC and MYC-nick in tumors derived from several oncogenic mutations. (A and B) Normal mucosa (N) and adenoma/adenocarcinoma 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 cytoplasmic fractionation. (E) Schematic representation of MYC and MYC-nick displaying the binding regions for the E3 ligase SCFFBXW7. NLS, nuclear localization 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.

Table S1.

Description of mouse colon and intestinal tissues used in Figs. 1 A and B, 2B, and 4C

Animal ID Genotype Pathology Location
3R1 pSI N AVcTT Normal Proximal small intestine
3R1 pSI T AVcTT Adenocarcinoma Proximal small intestine
4U2 pSI T AVcTT Adenocarcinoma Proximal small intestine
3Q3 pSI N AVcTT Normal Proximal small intestine
3Q3 pSI T AVcTT Adenocarcinoma Proximal small intestine
4J5 pSI N AVcTT Normal Proximal small intestine
4J5 pSI T AVcTT Adenocarcinoma Proximal small intestine
3m1 pSI N ATT Normal Proximal small intestine
3m1 pSI T ATT Adenoma Proximal small intestine
H4 pSI N ATT Normal Proximal small intestine
H4 pSI T ATT Adenoma Proximal small intestine
2M2 col N PPVcTT Normal Proximal small intestine
2M2 Col T PPVcTT Adenocarcinoma Colon
3Q2 pSI N KVcTT Normal Proximal small intestine
3Q2 pSI T KVcTT Adenocarcinoma Proximal small intestine
3R2 pSI N KVcTT Normal Proximal small intestine
3R2 pSI T KVcTT Adenocarcinoma Proximal small intestine
3R3 col N KVcTT Normal Colon
Q1 Col T KVcTT Adenocarcinoma Colon
5A1 Col T #1 KVcTT Adenocarcinoma Colon
4I3 Col N KVcTT Normal Colon
E1 dSI T #1 KVcTT Adenocarcinoma Distal small intestine
4G2 dSI N KVcTT Normal Distal small intestine
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Oncogenic Mutations Augment Stability of MYC and MYC-Nick.

Mutations in MYC that prevent its binding to SCFFBW7 have been reported 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 by FBXW7, we examined variants of the colon cancer cell lines DLD1 and HCT116, both of which have the FBXW7 gene deleted by gene targeting (23). We found that, compared with their WT counterparts, both cell lines deleted for FBXW7 exhibited increased the stability of MYC and MYC-nick in the cytoplasm, as measured 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 of FBXW7 had no effect on calpain-mediated cleavage of MYC (Fig. S1B). The reduction in the total levels of MYC observed in FBXW7−/− (Fig. 1D) was reported previously and is caused by a reduction in MYC mRNA (23). Consistent with the increased stability of MYC-nick upon FBXW7 deletion, we are able to detect both endogenous MYC and MYC-nick associated with Fbw7α in the cytoplasm of DLD1 cells (Fig. S1C). Fbw7α is the only Fbw7 isoform endogenously expressed in these cells (23, 24).

Fig. S1.

Fig. S1.

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Analysis of MYC and MYC-nick proteolysis. (A) Densitometric quantification of MYC and MYC-nick bands in a CHX chase experiment. (B) Deletion of FBXW7 in DLD1 and HCT116 cells does not affect calpain-mediated cleavage of MYC. Cytoplasmic extracts of WT or FBXW7−/− DLD1 and HCT116 cells were prepared in the absence of protease inhibitors and used to cleave 35S-methionine labeled–IVT MYC in vitro for 2 h. (C) Analysis of interaction between Fbw7 and MYC or MYC-nick in the cytoplasm of DLD1 cells. A total of 5 mg of cytoplasmic extracts were immunoprecipitated for Fbw7 and immunoblotted for MYC (C, Upper) or immunoprecipitated for MYC and immunoblotted for Fbw7 (C, Lower). (D) 35S-methionine labeled–IVT WT MYC and T58A MYC mutant were incubated with cytoplasmic extracts of Rat1 fibroblasts for 1 h. A concentration of 10 μM MG132 was used to inhibit calpain activity.

Inhibition of the proteasome with epoxomycin increases the stability of overexpressed MYC-nick (Fig. S2A) and siRNAs against components of the proteasome (25) also augment the stability of endogenous 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 for full-length MYC (26, 27). Mutations that affect the binding of MYC to Fbw7, such as T58A, do not affect its targeting by calpain and conversion into MYC-nick. (Fig. S1D).

Fig. S2.

Fig. S2.

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MYC-nick is degraded by the proteasome. (A) Inhibition of the proteasome stabilizes both MYC and MYC-nick. Rat1 MYC-null fibroblasts expressing MYC were treated with epoxomycin for 3 h before cell fractionation. (B) Silencing components of the proteasome stabilizes MYC and MYC-nick. HFF cells expressing MYC were transfected with the indicated amounts of siRNAs against components of the proteasome. (C) MYC-nick has a half-life of ∼30 min when expressed in MYC-null fibroblasts. Rat1 MYC-null fibroblasts expressing MYC-nick cells were incubated with CHX for the indicated time points. (D) The bands obtained in C were quantified and normalized to the levels of α-tubulin to determine the half-life of MYC-nick. (E) MYC and MYC-nick display similar pattern of decay. Proliferating cultures of Rat1 MYC-null fibroblasts infected with MYC-expressing retrovirus were incubated with CHX and calpeptin for the indicated time points. (F) WT MYC-nick or T58A MYC-nick were transfected into 293T cells and 48 h later incubated with CHX and calpeptin for the indicated time points.

The binding of SCFFbw7 to MYC requires phosphorylation of MYC threonine 58 (T58), which is mediated by glycogen synthase kinase 3β [GSK3β (28)]. Several studies have shown that GSK3β-mediated phosphorylation of MYC promotes its recognition by SCFFbw7, leading to proteasomal degradation (29, 30). As expected, treating cells with pharmacological inhibitors that block GSK3β activity, such as indirubin and kenpaulone, leads to increased stability of both MYC and MYC-nick (Fig. 1F). Indeed, we found that a point mutation in MYC converting threonine 58 to alanine (T58A) prevented its phosphorylation and caused accumulation of both full-length MYC and MYC-nick (Fig. 1G). In a CHX chase, we found that T58A MYC-nick was more stable than WT MYC-nick when transfected in 293T (Fig. S2F).

MYC-Nick Induces Migration in 3D Substrates.

We have demonstrated, using scratch healing and transwell migration assays, that overexpression of MYC-nick promotes the migration of colon cancer cells (ref. 15 and Fig. 2A). We found that MYC-nick induces migration and promotes filopodia formation, while up-regulating the actin-bundling protein fascin (15). Here we extend our studies to the 3D migration systems to better recapitulate the resistance provided by the environment during the migratory process of metastatic cancer cells. We found that ectopic expression of MYC-nick in DLD1 colon cancer cells also promoted their migration in 3D cultures (Fig. 2 B–F). Single cells or spheroids were cultured in ECM such as 50% (vol/vol) Matrigel or collagen, and in both cases, MYC-nick expressing colonies displayed a more migratory phenotype than control cells (Fig. 2 B–D). When plated in 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 more migratory than control cells in 3D (Fig. 2 E and F), which is probably caused by the constitutive cleavage of MYC into MYC-nick in these cells. These migratory cells displayed enhanced filopodial protrusions (Figs. 2A and 3C and Fig. S3A), which have been strongly linked to the increased migratory behavior and metastatic potential of cancer cells (31). In agreement with our previous observations, we found that cells expressing a MYC-nick mutant lacking MBII (ΔMBII) are unable to form filopodia and to promote persistent migration in 3D cultures.

Fig. 2.

Fig. 2.

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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.

Fig. 3.

Fig. 3.

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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×.)

Fig. S3.

Fig. S3.

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Analysis of Cdc42 and fascin in MYC-nick–expressing cells. (A) MYC-nick–overexpressing HFF cells show increased filopodia formation that is further enhanced when Cdc42 is activated. (Magnification: 63×.) (B) HCT116 cells overexpressing MYC-nick show increased fascin levels, as well as increased activation of Cdc42. (C) HFF cells overexpressing both MYC and MYC-nick have increased fascin levels. (D) DLD1 treated with Cdc42 inhibitor display increased survival when overexpressing MYC-nick. Cells were treated with the Rac/Cdc42 inhibitor ML1 for 48 h and then stained with crystal violet and photographed. Plates shown are replicates. (E) Expression of MYC, MYC-nick, and mutants in DLD1. DLD1 cells were infected with retroviruses-expressing empty vector and either MYC, MYC Δ298–311, MYC-nick, or MYC-nick ΔMBII.

We validated our results using a previously described human-in-zebrafish xenotransplantation approach (32–36). We introduced DLD1 cells into 48-h postfertilization zebrafish larvae. Four days after implantation, MYC-nick–expressing DLD1 cells exhibited an increase in metastatic behavior, measured by the number of cells that migrate away from the site of injection (Fig. 2 G and H). Deletion of MBII dramatically reduced MYC-nick’s ability to drive migration (Fig. 2 G and H). Together, these results further suggest that expression of MYC-nick induces tumor cell invasion in vivo.

MYC-Nick–Induced Migration Requires Fascin Expression.

We reported previously that MYC-nick promotes a dramatic increase in the levels of fascin (15), which is a driver of metastatic behavior in solid tumors and is associated with tumor progression in the colon and stomach (31) (Fig. 3A). Fascin expression is induced by MYC-nick in a variety of cell lines, including colon cancer cell lines such as DLD1 (Fig. 3A) and HCT116 (Fig. S3B) and human foreskin fibroblasts (HFFs) (Fig. S3C). Overexpression of full-length MYC in HFFs also induced fascin expression, probably due to constitutive generation of MYC-nick in these cells (Fig. S3C). Moreover, we found a marked increase in fascin levels in mouse adenomas and adenocarcinomas expressing high levels of MYC-nick (Fig. 3B). Although fascin down-regulation by siRNA does not affect cell survival of MYC-nick–expressing cells (15), it prevented 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 formation requires fascin (37–39). However, overexpression of GFP-tagged fascin in DLD1 cells did not mobilize actin to induce the formation of filopodia (Fig. 3 E and F), indicating that fascin is not sufficient for MYC-nick–induced filopodia formation and that additional changes in the actin cytoskeleton are required.

MYC-Nick–Induced Migration Requires Cdc42 Activation.

The Rho family of GTPases plays an essential role in cellular motility by regulating the organization of the actin cytoskeleton (40, 41). Because MYC-nick promotes changes in the actin cytoskeleton and increases migratory properties, we asked whether Rho GTPases collaborate with fascin to mediate MYC-nick’s effects on filopodium formation and cell migration. We treated DLD1 cells expressing either empty vector or MYC-nick with EGF, which activates Rho GTPases, or with the RAC1/Cdc42 inhibitor ML141. Inhibiting RAC/Cdc42 activity for 10–16 h completely ablated filopodia in MYC-nick–expressing cells (Fig. 4 A and B). Conversely, activating GTPases with EGF promoted filopodia formation in vector-expressing cells to the same extent as expression of MYC-nick and further increased filopodium formation in MYC-nick–expressing cells (Fig. 4 A and B). These results indicate that Cdc42 activity is required and sufficient to induce filopodia formation in MYC-nick–expressing cells.

Fig. 4.

Fig. 4.

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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 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-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.

Importantly, as with fascin siRNA, the viability of MYC-nick–expressing cells was insensitive to treatment with Cdc42 inhibitors. Even though the treated cells were not migratory, they survived for extended periods of time (4 d) in the presence of these inhibitors. This finding is consistent with our previous observation that MYC-nick promotes survival by inducing acetylation of LC3BII and α-tubulin to accelerate autophagy, 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 expressing empty vector, MYC-nick lacking the acetyltransferase binding domain (ΔMBII), full-length MYC, or uncleavable MYC (Δ298–311) did not survive in the presence of Cdc42 inhibitors (Fig. S3D).

Because of their role as positive regulators of cellular motility and invasion, Rho-GTPases have been linked to tumorigenic phenotypes in a variety of human cancers (42). Indeed, we observed that the total levels of RAC1 and Cdc42 are up-regulated in intestinal and colonic adenomas and adenocarcinomas derived from the mouse models of intestinal neoplasia compared 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 not increase either the total levels or the stability of Cdc42 protein (Fig. 4D).

MYC-Nick Promotes the Activation of Cdc42.

Rho GTPases cycle between active (GTP-bound) and inactive (GDP-bound) states by binding and hydrolyzing GTP. To determine whether MYC-nick expression modulates Rho GTPase activity, we performed pull-downs of activated RhoA by using beads conjugated to its binding partner Rhotekin. We also pulled down active Rac1 and Cdc42 using P21-activated kinase beads (PAK). We found that MYC-nick promotes activation of Cdc42 when expressed in colon cancer cells such as DLD1 (Fig. 4E) and HCT116 (Fig. S3A). Rac1 and RhoA activities were only modestly affected by MYC-nick: Rac1 activity was 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 h after treatment with growth factors or EGF (Fig. 4F, compare lanes 3 and 4). In control cells, this activation was seen to the same extent, but was transient (Fig. 4F, compare lanes 5 and 6). However, MYC-nick is not capable of promoting or sustaining the activation of Cdc42 in highly confluent cultures (Fig. 4F). Thus, activation of Cdc42 by MYC-nick differs from fascin induction because MYC-nick activates fascin expression regardless of cellular density (15).

Cdc42 Activation by MYC-Nick Requires the MBII Region.

The MBII region (amino acids 106–143) located within N-terminal segment of MYC and MYC-nick constitutes a binding site for recruitment of HATs to MYC. Deletion of MBII, although having no effect on MYC-nick expression levels, reduced MYC-nick’s ability to promote cell survival and migration (Fig. 2 E–G and Fig. S3D) (15). Although cells expressing MYC-nick ΔMBII appear more capable of initiating migration relative to control cells (Fig. 2E), the MYC-nick ΔMBII–expressing cells are not capable of completing migration and forming new colonies in collagen (Fig. 2F). To directly address the relevance of MBII to Cdc42 activation, we compared the activation of Cdc42 in cells expressing MYC-nick to cells expressing MYC-nick ΔMBII. We found that MBII is required for the activation of Cdc42 by MYC-nick (Fig. 4G). As expected, full-length MYC was also capable of inducing Cdc42 activation, most likely through its ability to constitutively generate MYC-nick through calpain cleavage. Consistent with this finding, a mutation in MYC deleting the calpain cleavage region amino acids 298–311 (MYC Δ298–311) is not capable of activating Cdc42 (Fig. 4G). Note that all MYC constructs were expressed at similar levels (Fig. S3E).

Induction of Fascin and Activation of Cdc42 by MYC-Nick Are Independent Events.

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

Fascin and Cdc42 Are Highly Expressed at the Invasive Front of Colon Cancers.

We have shown previously that cytoplasmic staining with anti-MYC corresponding to MYC-nick is elevated in cells at the invasive 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 at the invasive front of these tumors (Fig. 5D and Table S2). These observations are in agreement with several studies indicating fascin up-regulation in cancer cells drives motility and invasiveness (31, 48, 51).

Fig. 5.

Fig. 5.

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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×.)

Table S2.

Immunohistochemistry results obtained for 19 colorectal cancer tumor biopsies stained for cdc42 and fascin

Case no. CDC42* Acetylated tubulin† Fascin‡ Myc N-terminal antibody (274)
Central Invasive Central Invasive Central Invasive Central Invasive
1. 435 ++ ++ (+) (+) + +++ − cp ++
2. 473 + ++ + + + + n (+) cp ++ cp ++
3. 636 + + (+) (+) + + n + cp + cp +
4. 683 + ++ + ++ − + cp (+) cp +
5. 705 (+) + (+) + + ++ tu ++ n (+) cp + cp ++
6. 713 + + (+) + + + cp + cp +
7. 730 + ++ (+) ++ + ++ tu ++ n (+) cp + cp +
8. 800 + + + + + ++ n (+) cp + cp +
9. 803 + ++ (+) + + ++ tu ++ n + cp (+) cp ++
11. 2,078 + ++ + + (+) + cp (+) cp +
12. 2,085 + ++ (+) ++ + tu + ++ tu ++ n (+) cp (+) cp +
13. 2,802 + ++ + ++ + tu + + tu + n + cp + cp +
14. 3,485 ++ ++ ++ ++ ++ ++ cp + cp +
15. 3,798 + ++ (+) (+) + + n (+) cp (+) cp +
16. 4,055 ++ ++ + + + + n + cp + n (+) cp +
17. 4,112 + + + + + + cp + cp +
18. 4,429 + ++ − (+) + ++ tu + n + cp (+) cp ++
19. 29,646 + ++ (+) + + ++ tu + − cp ++
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These tumors had previously been stained for MYC using 9E10 and 274 antibodies (15). −, negative; (+), weakly positive; +, positive; ++, strongly positive. All cases were moderately-differentiated (G2) colorectal adenocarcinomas; only case 18 was poorly differentiated (G3).

*

Normal mucosa: −/(+) tumor cells cytoplasmic staining.

†

Normal mucosa: −/(+).

‡

Normal mucosa: −/(+) only stroma tumor always up in stroma, if additional cytoplasmic staining in tumor cells = tu.

Discussion

Our earlier work showed that MYC-nick is generated by calpain cleavage of full-length MYC under conditions of stress, such as nutrient deprivation and hypoxia. Under these conditions, a mutant form of MYC deficient in producing MYC-nick causes extensive apoptosis, which is attenuated by coexpression of MYC-nick (15). This finding indicates that MYC-nick plays an active role in cell survival, and its production is important for adaptation to stress (Fig. 6). MYC-nick–induced survival is mediated, at least in part, through stimulation of an autophagic response. In addition, MYC-nick expression correlates with striking changes in cytoskeletal organization, including alterations in actin dynamics, which lead to increased cell migration. These cellular changes are abrogated by deletion of the MBII region in MYC-nick, a region known to recruit protein complexes involved in histone acetylation and chromatin remodeling. In MYC-nick, MBII also associates with the acetyltransferase GCN5 (and probably others), inducing the acetylation of cytoplasmic proteins and thereby modulating protein function (16).

Fig. 6.

Fig. 6.

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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.

The abundance of full-length nuclear MYC has been long known to be controlled posttranslationally by means of proteolytic degradation determined by the balanced actions of several ubiquitin ligases and ubiquitin-specific proteases (for review, see ref. 12). The conserved MBI and MBII regions in MYC serve as binding sites for the Fbw7 and Skp2 ubiquitin ligases, respectively. MBI is a phosphodegron in that phosphorylation of serine 62 stabilizes MYC and primes threonine 58 (T58) for GSK3β 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 here that MYC-nick exhibits a similar rapid rate of proteasomal degradation, as does full-length nuclear MYC, which is also dependent in part on GSK3β phosphorylation and Fbw7 association. We find that deletion of the three Fbw7 isoforms, inhibition of GSK3β or the proteasome, or mutation of the MYC-nick T58 phosphorylation site each leads to the stabilization or increased levels of MYC-nick. Thus, the abundance of cytoplasmically localized MYC-nick is subject to controls similar to those that determine the abundance of full-length nuclear MYC. Interestingly, mutation of T58, or other residues within MBI that result in MYC stabilization, is found in B-cell lymphomas and contributes to MYC’s oncogenic activity (52–54). Indeed, we observe that murine intestinal tumors bearing different combinations of mutations in the Wnt, TGF-β, and PI3K signaling pathways frequently have strikingly increased levels of both MYC and MYC-nick compared with normal mucosal tissue. We had previously shown that several distinct tumor types express high levels of MYC-nick (15, 16). Moreover, a recent study reported that MYC-nick expression is associated 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 by the results of 3D migration assays where MYC-nick promotes cell migration of colon cancer cells embedded in Matrigel or collagen, both of which provide resistance to movement. Furthermore, in vivo metastasis of colon cancer cells expressing MYC-nick, compared with vector controls, was significantly enhanced after injection into zebrafish larvae. Our experiments implicate both fascin and Cdc42 in the mechanisms underlying the increased invasiveness of MYC-nick–expressing cells. Both of these molecules have been shown to increase filopodia formation and to drive migration and metastasis. Although fascin is an actin-bundling protein critical for filopodia stability (31), Cdc42 drives actin 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 expressing endogenous MYC-nick also exhibited a higher abundance of fascin relative to normal mucosal cells. Moreover, induction of fascin, although required for MYC-nick–induced filopodia formation and cell migration, is not sufficient for filopodia formation in the absence of MYC-nick. This finding prompted us to examine the potential involvement of MYC-nick in other pathways that control incorporation of the actin cytoskeleton into filopodia. Rac1, RhoA, and Cdc42 have been extensively studied in the context of cell invasion and actin cytoskeleton remodeling (41, 44). Rho promotes stress fiber formation, whereas Rac1 regulates lamellipodia, and Cdc42 promotes actin assembly into filopodia (43). By means of pull-down experiments, we determined that Cdc42 undergoes sustained activation in cells expressing MYC-nick. Although Rac1 activity was increased, and Rho activity was reduced, these changes were modest relative to the activation of Cdc42. Moreover, when we blocked Rac/Cdc42 activity, we observed a complete loss of filopodia from MYC-nick–expressing cells, whereas activation of GTPases 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 fascin expression and modulate the activity of Rho-GTPases, particularly Cdc42. Indeed, in tissue sections, we observed an association between MYC-nick expression (indicated by the presence of cytoplasmic MYC staining) and high levels of fascin and Cdc42 in the invasive front of human colon cancers. Although the mechanisms by which MYC-nick induces fascin expression and Cdc42 activation are still unclear, an important clue to the molecular regulation of cell migration by MYC-nick comes from a loss-of-function mutation. Deletion of MBII abrogates MYC-nick–induced fascin expression, cdc42 activation, and metastasis in zebrafish embryos, indicating that the binding of MYC-nick to acetyltransferases and protein acetylation mediates these effects. MYC-nick associates with acetyltransferase and induces acetylation of several cytoplasmic proteins, among which we characterized α-tubulin and ATG3 (15, 16). We speculate that MYC-nick binding and acetylation of cytoplasmic factors, such as specific GAPs or GEFs, could account for modulation of Cdc42 activity by MYC-nick (45). Similarly, MYC-nick may influence cytoplasmic signaling to the nucleus, or perhaps itself serve as a transcriptional cofactor, leading to the expression of fascin or other genes. Further studies directed at unbiased identification of MYC-nick–binding proteins and acetylation targets will define the molecular pathways linking MYC-nick production to increased cell migration and invasiveness. Our data showing that MYC-nick, Cdc42, and fascin are highly expressed at the invasive front of human colon cancers is consistent with the idea that MYC-nick’s capacity to trigger cell motility is relevant to the biology of primary cancers (Fig. 6).

Materials and Methods

Cell Lines and Mouse Tissues.

Cell lines were maintained in DMEM with 4.5 g of glucose, 10% (vol/vol) FCS, and 100 U/mL penicillin/streptomycin. For CHX chase experiments, cells were cultured to confluency, and the culture medium was replaced 24 h before lysis. CHX (50 μg/mL) was added to cells for the 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 were due to proteasomal turnover, not from calpain-mediated cleavage.

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

Mouse tissues were handled as described (27). Normal mouse tissue and tumors were snap-frozen upon dissection, and total extracts were prepared in radioimmunoprecipitation assay buffer (pH 7.6) by sonication. Before 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, pH 6.8] and boiled for 10 min. Then, 30 μg of total extracts were probed overnight with indicated antibodies. All animal studies were performed according to the guidelines of the Fred Hutchinson Cancer Research Center.

GTPase Assay.

GTPase assay was performed with the RhoA/Rac1/Cdc42 Activation Assay kit from Cytoskeleton, Inc. Cells were split into 10-cm dishes to achieve 40% confluence 48 h later. Fresh growth medium was added 24 h before harvest. Pull-downs were performed according to manufacturer’s instructions, with the following exceptions: no activator was used, lysates were 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-Well Wound Healing Assay from Cell Biolabs Inc., according to manufacturer’s instructions. siRNA treatment of cells was performed with Lipofectamine RNAiMAX (Thermo Fisher) according to the manufacturer’s recommendations.

Spheroid cultures were prepared as described (28). Briefly, 5,000 cells resuspended in 500 μL of medium were plated over solid 1.5% (wt/vol) agar and grown for 4 d until spheroids were formed. Spheres were resuspended in 50% (vol/vol) collagen or Matrigel and cultured for the indicated time points.

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

Immunohistochemistry.

Sections (4 mm) were deparaffinized, rehydrated, and pretreated 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 antibodies diluted in RPMI 1640 plus 10% (wt/vol) bovine serum. Slides were washed twice with TBST and developed with the EnVision System (Dako) and AEC 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 143 were gifts from N. Ikegaki, University of Illinois, Chicago. Anti-tubulin (α and acetylated), GFP, and actin were from Sigma-Aldrich. P-T58 MYC and LC3IIB were from Cell Signaling. Anti-Rac1, RhoA, and Cdc42 were from Cytoskeleton, Inc. Anti-Fbw7 was from Bethyl Laboratories. Alexa Fluor 594 Phalloidin was from Invitrogen. Anti-E-Cadherin was from Abcam. Fascin and Cdc42 siRNAs were from Santa Cruz Biotechnology. Cdc42 activator (EGF) was from Cytoskeleton, Inc., and the Cdc42/Rac1 inhibitor ML141 was from Calbiochem.

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

Acknowledgments

We thank Nao Ikegaki, John Sedivy, Jonathan Grim, Bruce Clurman, and Markus Welcker for essential reagents. This 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 Medical Research.

Footnotes

The authors declare no conflict of interest.

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

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