Oncogene (2016) 35, 2075–2086 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE The EGF receptor ligand amphiregulin controls cell division via FoxM1 SW Stoll1, PE Stuart1, WR Swindell1, LC Tsoi2,BLi3, A Gandarillas4, S Lambert1, A Johnston1, RP Nair1 and JT Elder1,5 Epidermal growth factor receptor (EGFR) is central to epithelial cell physiology, and deregulated EGFR signaling has an important role in a variety of human carcinomas. Here we show that silencing of the EGF-related factor amphiregulin (AREG) markedly inhibits the expansion of human keratinocytes through mitotic failure and accumulation of cells with ⩾ 4n DNA content. RNA-sequencing- based transcriptome analysis revealed that tetracycline-mediated AREG silencing significantly altered the expression of 2331 genes, 623 of which were not normalized by treatment with EGF. Interestingly, genes irreversibly upregulated by suppression of AREG overlapped with genes involved in keratinocyte differentiation. Moreover, a significant proportion of the irreversibly downregulated genes featured upstream binding sites recognized by forkhead box protein M1 (FoxM1), a key transcription factor in the control of mitosis that is widely dysregulated in cancer. The downregulation of FoxM1 and its target genes preceded mitotic arrest. Constitutive expression of FoxM1 in AREG knockdown cells normalized cell proliferation, reduced the number of cells with ⩾ 4n DNA content and rescued expression of FoxM1 target genes. These results demonstrate that AREG controls G2/M progression and cytokinesis in keratinocytes via activation of a FoxM1-dependent transcriptional program, suggesting new avenues for treatment of epithelial cancer. Oncogene (2016) 35, 2075–2086; doi:10.1038/onc.2015.269; published online 3 August 2015 INTRODUCTION a variety of epithelial neoplasms and it may confer tumor cells 31–34 Epidermal homeostasis requires balance between keratinocyte with autonomous growth. proliferation and differentiation;1 however, the underlying regulatory The mechanisms by which AREG regulates proliferation remain network controlling this balance remains incompletely understood.2 to be elucidated. We recently reported that short hairpin RNA Epidermal growth factor receptor (EGFR) signaling is an important (shRNA)-mediated silencing of the AREG gene markedly inhibits regulator of epidermal development and homeostasis3–6 and has an keratinocyte growth in a manner that cannot be reversed by the addition of EGF or other EGFR ligands.9 Interestingly, AREG important role in the control of multiple keratinocyte functions 7,8 9 10–12 13,14 silencing results in the accumulation of flattened keratinocytes including migration, proliferation, differentiation, survival 9 15–17 that are often binucleated. In the present study, we set out to and activation of innate and adaptive immune responses. further elucidate the mechanism(s) by which proAREG controls Deregulated EGFR signaling is a hallmark of epithelial cancer,18 and 19–22 keratinocyte proliferation and differentiation. EGFR is a validated target in several types of carcinomas. EGFR Using conditional AREG knockdown keratinocytes9 in combina- becomes activated in response to binding members of the EGF tion with high-throughput RNA sequencing (RNA-seq) and 23 ligand family, several of which are expressed in human lentivirus-mediated gene expression, here we functionally impli- keratinocytes and skin including amphiregulin (AREG), betacellulin, cate forkhead box protein M1 (FoxM1), a winged helix transcrip- epigen, epiregulin, heparin-binding EGF-like growth factor tion factor,35 as a key mediator of AREG’s effects upon (HB-EGF) and transforming growth factor-α (TGF-α).7,24,25 keratinocyte proliferation and differentiation. Because FoxM1 is a Metalloproteinase-mediated cleavage of the transmembrane pre- major regulator of G2/M progression,36 which is widely over- cursors of these growth factors is thought to be required for major expressed in human cancer and implicated in cancer progression EGFR ligand functions including cell proliferation and migration.23 and metastasis,35 these findings have important mechanistic Different EGFR ligands mediate keratinocyte behavior in implications for the role of AREG in the pathogenesis and different cellular contexts.7 AREG participates in embryonic treatment of epithelial cancer. morphogenesis of mammary gland and bone.26,27 It is the predominant autocrine growth factor for cultured keratinocytes,7,9,28 and is markedly upregulated in hyperprolifera- RESULTS 17,29,30 tive skin conditions including psoriasis and wound healing. We subjected the inducible AREG knockdown keratinocyte cell line AREG is gaining increasing relevance in cancer research and N/TERT-TR-shAREG to autocrine growth assays as described7 and treatment, as it is overexpressed in a wide variety of tumors, is a determined cell counts, proliferation and cell cycle distribution by marker of poor prognosis and resistance to anti-EGFR therapies in flow cytometry (Figure 1a). Tetracycline (Tet)-mediated AREG 1Department of Dermatology, University of Michigan, Ann Arbor, MI, USA; 2Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA; 3Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA; 4Cell Cycle, Stem Cell Fate and Cancer Laboratory, Fundación Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain and 5Ann Arbor Veterans Affairs Health System, Ann Arbor, MI, USA. Correspondence: Dr SW Stoll, Department of Dermatology, University of Michigan, 7415 Medical Sciences Building I, 1301 East Catherine, Ann Arbor, MI 48109-5675, USA. E-mail: [email protected] Received 13 December 2014; revised 4 June 2015; accepted 13 June 2015; published online 3 August 2015 AREG regulates G2/M progression via FoxM1 SW Stoll et al 2076 Figure 1. AREG silencing profoundly alters keratinocyte morphology and cell cycle distribution in human keratinocytes. (a) Overview of the keratinocyte cell growth assay with and without 1 μg/ml Tet with and without 100 ng/ml rhAREG. (b) Parental N/TERT-TR-shAREG keratinocytes were subjected to growth assays and photographed at the end of the 6-day assay. Note that Tet-induced AREG silencing leads to the appearance of binucleated cells (arrows). (c) Quantification of cell numbers as assessed by flow cytometry-based cell counting using fluorescent reference beads. Data are expressed as cell numbers and represent 10% of the total cells per well. Data are mean ± s.e.m. (n = 4 with three biological replicates per experiment and condition). Significant pairwise contrasts (mean ratio; corrected P): control vs Tet (12.8; 0.0097), control vs Tet/AREG (12.2; 0.0051), AREG vs Tet (13.1; 0.011), AREG vs Tet/AREG (12.5; 0.0041). (d and e) CFSE-labeled keratinocytes were used in 6-day growth assays followed by DAPI labeling and analysis by flow cytometry. ‘Day 0’ denotes keratinocytes at the time of plating. Note that Tet-induced AREG silencing causes a marked decrease in cell division (~8-fold reduction relative to control keratinocytes, as assessed by CFSE dye dilution) (d) accompanied by a large increase in cells with ⩾ 4n DNA content (e) even in the presence of exogenous AREG. Oncogene (2016) 2075 – 2086 © 2016 Macmillan Publishers Limited AREG regulates G2/M progression via FoxM1 SW Stoll et al 2077 silencing led to the appearance of flattened, binucleated cells that were enriched for processes of immune response and regulation differed markedly in appearance from the ‘cobblestone’ mor- of cell communication (Supplementary Figure S3). Notably, the phology of these cells grown in the absence of Tet (Figure 1b). DEGs engendered by AREG silencing (up- or downregulated, Although addition of recombinant human (rh)AREG (or rhEGF or irrespective of normalization by EGF) overlapped significantly rhTGF-α, data not shown) had little or no effect in the absence of (Po0.001 by Wilcoxon's rank-sum test, see Materials and Tet (Figure 1b, upper panels), prominent cell enlargement was Methods) with genes whose expression is altered in response to observed in its presence (lower panels). As assessed by flow 6 h of Ca2+-mediated keratinocyte differentiation (Figure 2c) and cytometry-based cell counting (Figure 1c) and by carboxyfluor- in response to EGFR inhibitor treatment (Figure 2d). escein succinimidyl ester (CFSE) dye dilution (Figure 1d), AREG We confirmed the RNA-seq results for a subset of genes on the silencing resulted in a 12.5-fold decrease in cell counts and an same samples by quantitative real-time–polymerase chain reac- 8-fold reduction in proliferation, respectively, irrespective of the tion (QRT–PCR) using prevalidated TaqMan assays (Figure 3), addition of 100 ng/ml exogenous rhAREG. Relative to logarithmic- revealing excellent agreement with the RNA-seq results shown in phase N/TERT-TR-shAREG keratinocytes grown in complete Figure 2. These QRT–PCR data also confirm the lack of normal- medium (Figure 1e, upper panel), 6-day growth under autocrine ization for these genes by exogenous EGF. conditions led to a modest increase of cells with 44n DNA To determine the temporal relationship between AREG silen- content in the absence of Tet (middle panels). However, addition cing, downregulation of FOXM1 and its targets and the cellular of Tet caused a massive increase in cells with ⩾ 4n DNA content response, we performed time-course experiments in the presence (lower panels). Notably, neither the decreased proliferation nor the or absence