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 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 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 cancers 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 proteins 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- protein, 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 cytoplasm. MYC-nick promotes re- system (12). Importantly, MYC levels have been demonstrated organization of the actin cytoskeleton 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. www.pnas.org/cgi/doi/10.1073/pnas.1610994113 PNAS | Published online August 26, 2016 | E5481–E5490 Downloaded by guest on September 27, 2021 AE F B CG D 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 September 27, 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).