EVI1 Promotes the Proliferation and Invasive Properties of Human Head and Neck Squamous Cell Carcinoma Cells

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor prognosis. So far, the EGFR inhibitor cetuximab is the only approved targeted therapy. A deeper understanding of the molecular and genetic basis of HNSCC is needed to identify additional targets for rationally designed, personalized therapeutics. The transcription factor EVI1, the major product of the MECOM locus, is an oncoprotein with roles in both hematological and solid tumors. In HNSCC, high EVI1 expression was associated with an increased propensity to form lymph node metastases, but its effects in this tumor entity have not yet been determined experimentally. We therefore overexpressed or knocked down EVI1 in several HNSCC cell lines and determined the impact of these manipulations on parameters relevant to tumor growth and invasiveness, and on gene expression patterns. Our results revealed that EVI1 promoted the proliferation and migration of HNSCC cells. Furthermore, it augmented tumor spheroid formation and the ability of tumor spheroids to displace an endothelial cell layer. Finally, EVI1 altered the expression of numerous genes in HNSCC cells, which were enriched for Gene Ontology terms related to its cellular functions. In summary, EVI1 represents a novel oncogene in HNSCC that contributes to cellular proliferation and invasiveness.

Keywords: EVI1; MECOM; PRDM3; cell proliferation; gene expression regulation; lymphatic metastasis; molecular targeted therapy; neoplasm metastasis; squamous cell carcinoma of head and neck.

Figure 1

Expression of EVI1 in native and transduced HNSCC cell lines. (A) EVI1 protein levels in seven HNSCC cell lines were determined by immunoblot analysis; GAPDH was used as a loading control. Left panel, representative immunoblot; right panel, quantification. EVI1 levels were normalized to GAPDH levels and to CAL-27 cells. Means + SEM, n = 3. (B) CAL-33 cells were transduced with a retroviral vector containing the human EVI1 cDNA (EVI1) or with an empty vector as a control (vec). Overexpression of EVI1 was confirmed by immunoblot analysis. (C) CAL-33 cells were transduced with lentiviral vectors containing shRNAs targeting human EVI1 (shEVI1-1, shEVI1-2) or a control shRNA targeting the renilla luciferase gene (shCtrl). Knock-down of EVI1 was confirmed by immunoblot analysis. (B,C) Left panels, representative immunoblots; right panels, quantification using GAPDH for normalization. Means + SEM, n = 3. * p < 0.05; one-sample t-test (B) or one-sample t-test with Bonferroni correction for multiple testing (C).

Figure 2

EVI1 promotes the proliferation of CAL-33 cells. (A,B) Proliferation of CAL-33_EVI1 and CAL-33_vec cells (A), or of CAL-33_shEVI1-1, CAL-33_shEVI1-2, and CAL-33_shCtrl cells (B) was monitored in real-time for 120 h. Impedance values determined by the xCELLigence system were normalized to the 24 h time point. Means ± SEM, n = 3. *** p < 0.001, according to the anova.lme function of the R package nlme. (C,D) Colony formation. CAL-33_EVI1 and CAL-33_vec cells (C), or CAL-33_shEVI1-1, CAL-33_shEVI1-2, and CAL-33_shCtrl cells (D) were seeded at low densities into six-well-plates and stained with trypan blue after 11 days. Top panels: quantification of colony numbers and sizes; bottom panels: representative well areas. Means + SEM, n = 3. * p < 0.05; Student’s two-sided t-test (C) or one-way ANOVA followed by Dunnett’s multiple comparison test (D).

Figure 3

EVI1 increases the size of CAL-33 spheroids. Tumor spheroids from CAL-33_EVI1 and CAL-33_vec cells (A), or from CAL-33_shEVI1-1, CAL-33_shEVI1-2, and CAL-33_shCtrl cells (B) were allowed to form over a period of 11 days. Top panels: quantification of spheroid sizes; bottom panels: representative spheroids. Means + SEM, n = 3. *, p < 0.05, ** p < 0.01; Student’s two-sided t-test (A) or one-way ANOVA followed by Dunnett’s multiple comparison test (B).

Figure 4

EVI1 enhances the migration of CAL-33 cells. (A,B) Scratch assay. CAL-33_EVI1 and CAL-33_vec cells (A), or CAL-33_shEVI1-1, CAL-33_shEVI1-2, and CAL-33_shCtrl cells (B) were grown to 90% confluence in a regular growth medium. The medium was changed to low serum conditions (0.2% fetal bovine serum (FBS)), scratches were introduced 24 h later, and gap closure was monitored at the indicated time points thereafter. Top panels: quantification; bottom panels: representative experiments. Means + SEM, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, two-way ANOVA followed by Bonferroni’s post-hoc test. (C,D) Transwell migration assay. Serum-starved CAL-33_EVI1 and CAL-33_vec cells (C), or CAL-33_shEVI1-1, CAL-33_shEVI1-2, and CAL-33_shCtrl cells (D) were seeded into transwell inserts and allowed to migrate towards a medium containing 10% FBS. After 24 h, cells at the bottom of the transwell membranes were stained with trypan blue. Top panels: quantification; bottom panels: images from representative experiments. Means + SEM, n = 3. * p < 0.05; one-sample t-test (C) or one-sample t-test with Bonferroni correction for multiple testing (D).

Figure 5

EVI1 enhances the displacement of endothelial cells by CAL-33-derived spheroids. (A,B) Spheroids were generated from CAL-33_EVI1 and CAL-33_vec cells (A), or from CAL-33_shEVI1-1, CAL-33_shEVI1-2, and CAL-33_shCtrl cells (B). They were placed onto confluent HUVECs that had been stained with Cell Tracker Dye, and HUVEC displacement was followed by live-cell imaging. Top panels: quantification; bottom panels: images of representative experiments. Means + SEM, n = 3. Differences in gap areas were tested for statistical significance after 15 h. * p < 0.05; Student’s two-sided t-test (A) or one-way ANOVA followed by Dunnett’s multiple comparison test (B).

Figure 6

EVI1 regulates genes implicated in epithelial development, adhesion, and proliferation in HNSCC cells. Genome-wide gene expression patterns in CAL-33_EVI1, CAL-33_vec, SCC-25_EVI1, and SCC-25_vec cells were determined by RNA-seq. (A) Venn diagram showing the numbers of genes differentially expressed at a false discovery rate (FDR) of <0.1 upon experimental expression of EVI1 in CAL-33 and/or SCC-25 cells. (B) Heatmap of 252 genes consistently regulated by EVI1 in CAL-33 and SCC-25 cells. The rows and columns represent genes and biological replicates, respectively. Blue, low expression; red, high expression. (C) Selected categories from the Gene Ontology enrichment analysis of the 252 commonly deregulated genes, performed using ShinyGO (v0.61).