Arginyltransferase Suppresses Cell Tumorigenic Potential and Inversely Correlates with Metastases in Human Cancers

Oncogene (2016) 35, 4058–4068 © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE Arginyltransferase suppresses cell tumorigenic potential and inversely correlates with metastases in human cancers RRai1,7, F Zhang1,2,7, K Colavita1, NA Leu1, S Kurosaka1, A Kumar2, MD Birnbaum2,BGyőrffy3,4, DW Dong5, M Shtutman6 and A Kashina1 Arginylation is an emerging post-translational modification mediated by arginyltransferase (ATE1) that is essential for mammalian embryogenesis and regulation of the cytoskeleton. Here, we discovered that Ate1-knockout (KO) embryonic fibroblasts exhibit tumorigenic properties, including abnormally rapid contact-independent growth, reduced ability to form cell–cell contacts and chromosomal aberrations. Ate1-KO fibroblasts can form large colonies in Matrigel and exhibit invasive behavior, unlike wild-type fibroblasts. Furthermore, Ate1-KO cells form tumors in subcutaneous xenograft assays in immunocompromised mice. Abnormal growth in these cells can be partially rescued by reintroduction of stably expressed specific Ate1 isoforms, which also reduce the ability of these cells to form tumors. Tumor array studies and bioinformatics analysis show that Ate1 is downregulated in several types of human cancer samples at the protein level, and that its transcription level inversely correlates with metastatic progression and patient survival. We conclude that Ate1-KO results in carcinogenic transformation of cultured fibroblasts, suggesting that in addition to its previously known activities Ate1 gene is essential for tumor suppression and also likely participates in suppression of metastatic growth. Oncogene (2016) 35, 4058–4068; doi:10.1038/onc.2015.473; published online 21 December 2015 INTRODUCTION RESULTS Protein arginylation is an emerging post-translational modification Ate1-KO cells exhibit density- and serum- independent growth mediated by arginyltransferase (ATE1).1 Arginylation was and chromosomal aberrations originally discovered in 1963,2 and was shown through recent Our previously published data show that immortalized Ate1-KO studies to have a global role in many biological processes, MEFs exhibit defects in cell spreading4 and cell–cell adhesion.9 including cardiovascular development, angiogenesis,3 cell Working with these cells, we observed that they generally grew to migration4 and tissue morphogenesis.5 Over 100 arginylated higher densities at confluency than the similarly treated wild-type proteins have been identified in vivo,5–8 and this list is growing by (WT) cells. To test whether Ate1-KO cells grow differently from WT, the day. we quantified their multiplication rates in comparison with Despite growing evidence of the importance of arginylation, its similarly derived and immortalized WT MEFs. In these assays, exact biological functions in normal physiology and disease WT cells typically reached confluency at 3–4 days post inoculation remain poorly understood. Ate1-knockout (KO) mouse embryonic and continued to survive in culture plates as a monolayer without fibroblasts (MEFs) exhibit pronounced defects in migration and undergoing further multiplication (Figure 1a). In contrast, Ate1-KO adhesion reminiscent of cancer cells.4,9 However, a disease MEFs continued to grow and multiply even after reaching confluency, eventually growing to the densities over 10-fold connection between arginylation and cancer has never been 10 higher than WT (Figure 1a). Notably, such contact-independent explored. growth is characteristic for many cancer cells and ultimately Here, we addressed the possibility that Ate1-KO confers underlies their ability to form tumors and metastases. cancerous phenotypes at the cellular level. We found that Ate1- To further test whether Ate1-KO cells exhibit behavior similar to KO in cultured cells leads to contact- and substrate-independent cancer cells in culture, we studied the ability of these cells to grow cell growth, formation of subcutaneous tumors in xenograft and multiply under low serum conditions, which inhibit the studies, and that reduction in Ate1 levels correlates with cancer growth of normal, but not highly malignant cells. To do this, we and is particularly associated with metastatic potential. Our study performed growth curves similar to those shown in Figure 1a, is the first direct demonstration of Ate1 role in cancer, identifying using immortalized WT and Ate1-KO cells grown in 0.5% serum. Ate1 as a potential novel tumor suppressor and a biomarker for While both cell types grew slower during serum deprivation, Ate1- metastatic cancers. KO cells were able to reach much higher densities compared with 1School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA; 2 Department of Molecular & Cellular Pharmacology, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USA; 3MTA TTK Lendület Cancer Biomarker Research Group, Budapest, Hungary; 4Second Department of Pediatrics, Semmelweis University, Budapest, Hungary; 5Institute for Biomedical Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA and 6University of South Carolina, Columbia, SC, USA. Correspondence: Dr A Kashina, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. E-mail: [email protected] 7These authors contributed equally to this work. Received 7 May 2015; revised 9 November 2015; accepted 14 November 2015; published online 21 December 2015 ATE1 suppresses tumorigenesis R Rai et al 4059 Figure 1. Ate1-KO cells exhibit density- and serum-independent growth and early onset of chromosomal aberrations. (a) Growth curves of immortalized WT and Ate1-KO MEFs in normal culture media containing 10% serum (n = 4). (b) Growth curves of immortalized WT and Ate1-KO MEFs grown in low serum conditions (0.5% serum). A representative curve from duplicate experiments is shown (see also Supplementary Figure S1 for a repeat experiment using independently derived immortalized WT and Ate1-KO). (c) Growth curves of primary WT and Ate1-KO MEFs in normal culture media containing 10% serum (n = 4). (d) Karyotypes of primary WT and Ate1-KO MEFs freshly derived from E12.5 littermate embryos. Numbers on the y axis represent percentages of cells with each chromosome number shown on the x axis. Karyotypes in 20 WT cells and 27 KO cells were counted. In (a, c), error bars in all panels represent s.e.m. In (a), P-value was calculated by Student’s t-test using data at the final time point (day 8). WT (Figure 1b and Supplementary Figure S1), suggesting that varying densities and examined their multiplication rates under these cells can actively divide even in very low serum. these conditions. In scarce cultures, where cells largely remain Experiments showed that the contact-independent growth was isolated from each other, both Ate1-KO and WT cells multiplied at observed only in immortalized Ate1-KO MEFs, but not in primary nearly equal rates (Figure 2a), suggesting that in the active growth cultures freshly derived from Ate1-KO mouse embryos (Figure 1c), phase without proximity to their neighbors these cells have largely suggesting that this quality is acquired by these cells with similar doubling time. However, when plated in dense cultures, additional changes that occur during immortalization. Notably, Ate1-KO cells multiplied much faster than WT cells (Figure 2b). however, even in the primary fibroblasts freshly derived from This difference of growth in dense cultures can be explained E12.5 mouse embryos, the karyotypes were highly abnormal either by difference in apoptosis rates or by difference in contact (Figure 1d), suggesting that these cells have already accumulated inhibition. To test these possibilities, we first used Terminal genetic defects that may result in further abnormalities after deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) fl immortalization. assay to measure the rate of apoptosis in con uent WT and Ate1- KO cells. We found that both types of cells have negligible levels of apoptosis in such conditions, which cannot account for their The abnormal growth of Ate1-KO cells is associated with defects in difference in cell multiplication rates (Supplementary Figure S2A). contact inhibition We next tested whether Ate1-KO cells exhibit characteristic The abnormally high multiplication rates seen in Ate1-KO cells morphological changes that accompany defects in contact could be explained by either an overall reduction in cell doubling inhibition, including reduced cell–cell contacts and foci formation time that drives them to divide at a faster rate, or by in culture. Indeed, while WT MEFs formed normal cell monolayers compromising contact inhibition—a process commonly seen in in dense culture, the Ate1-KO cells tended to cluster and form foci, non-cancerous cells, which inhibits cell migration and proliferation with multiple layers of cells piling on top of each other (Figure 2c). upon sensing the proximity to their neighbors. To directly test Furthermore, consistent with our prior observations Zhang et al.,9 these two possibilities, we cultured the Ate1-KO and WT cells at Ate1-KO cells showed a greatly reduced ability to form © 2016 Macmillan Publishers Limited, part of Springer Nature. Oncogene (2016) 4058 – 4068 ATE1 suppresses tumorigenesis R Rai et al 4060 Figure 2. Ate1-KO cells grow differently from WT cells in dense cultures. (a) Growth curves of WT and Ate1-KO

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