Dek overexpression in murine epithelia increases overt esophageal squamous cell carcinoma incidence

<div><p>Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression <i>in vivo</i>, we generated and validated a tetracycline responsive <i>Dek</i> transgenic mouse model referred to as <i>Bi-L-Dek</i>. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing <i>Bi-L-Dek</i> mice to keratin 5 tetracycline transactivator (<i>K5-tTA</i>) mice. Conditional transgene expression was validated in the resulting <i>Bi-L-Dek_K5-tTA</i> mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these data demonstrate that Dek overexpression in the cell of origin for SCC is sufficient to promote esophageal SCC development <i>in vivo</i>.</p></div

<i>Bi-L-Dek_K5-tTA</i> mice express luciferase and overexpress Dek in stratified squamous epithelium.

<p>(<b>A</b>) <i>In vivo</i> imaging system (IVIS) analysis depicts a single (<i>Bi-L-Dek</i>) and a bi-transgenic (<i>Bi-L-Dek_K5-tTA)</i> mouse after intraperitoneal injection of luciferin for luciferase detection in the skin of <i>Bi-L-Dek_K5-tTA</i> mice. (<b>B</b>) <i>Ex vivo</i> IVIS analysis of single transgenic (<i>K5-tTA</i>) versus bi-transgenic <i>(Bi-L-Dek_K5-tTA)</i> flank skin, ear, and esophagus following injection of luciferin, sacrifice, and dissection. (<b>C</b>) RT- qPCR of Dek mRNA levels in skin epithelium obtained from the flank of mice show a 3 fold induction of Dek transcript levels that is repressed to endogenous levels after seven days on dox chow. Primers detect endogenous and exogenous <i>Dek</i>. Error bars represent three mice for each genotype excluding the <i>Dek-/-</i> negative control which represents one mouse repeated in triplicate. (<b>D</b>) Representative western blot analysis for the detection of Dek protein levels in flank skin epithelium demonstrates increased levels of Dek protein in <i>Bi-L-Dek_K5-tTA</i> mice over those on dox and single transgenic controls. (<b>E</b>) Immunohistochemistry (IHC) with DEK antibodies (BD Biosciences, San Jose, CA, USA) in tongue epithelium confirms Dek protein overexpression in <i>Bi-L-Dek_K5-tTA</i> mice that is repressed within seven days of dox chow. (<b>F</b>) Immunofluorescence (IF) of cultured skin keratinocytes isolated from newborn <i>Bi-L-Dek_K5-tTA</i> pups with or without dox and their single transgenic littermates. Dox treated keratinocytes were cultured with 1ug/ml of dox for 48 hours before fixation. IF images of keratinocytes were taken at the same magnification and exposure after being probed for Dek, keratin 5 (K5), and stained with DAPI. (<b>G</b>) The mean fluorescent intensity of Dek staining in <b>2F</b> was quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007227#pgen.1007227.ref089" target="_blank">89</a>].</p

<i>Bi-L-Dek</i> transgene expression is detected in the context of Dek knockout mice.

<p>(<b>A</b>) <i>Bi-L-Dek_K5-tTA</i> mice were bred to Dek knockout (<i>Dek-/-)</i> mice to quantify Dek expression in the absence of endogenous Dek protein. (<b>B</b>) IVIS image of <i>Dek-/-</i> _<i>Bi-L-Dek_K5-tTA</i> mice with luciferase expression compared to <i>Dek-/-</i> and single transgenic <i>K5-tTA</i> mice after luciferin injection. (<b>C</b>) Western blot analysis detects Dek protein expression in murine flank skin from <i>Dek-/-</i> _<i>Bi-L-Dek_K5-tTA</i> mice.</p

Generation of a tetracycline off <i>Dek</i> transgenic mouse model.

<p>(<b>A</b>) <i>Bi-L-Dek</i> transgenic mice were engineered by micronuclear injection of linearized <i>Bi-L-Dek</i> DNA into the pronucleus of FVB/N fertilized eggs. <i>Bi-L-Dek</i> mice harbor a tetracycline response element (TRE) that controls two mini cytomegalovirus (CMV) promoters driving bi-directional transcription of <i>Dek</i> and <i>luciferase</i>. (<b>B</b>) Copy number analysis of the <i>Bi-L-Dek</i> transgene in founder #317 identified 2–4 insertions in the F2-F4 generation. Error bars represent differences between 2–3 mice for each generation excluding F0 for which only one mouse exists. F3 and subsequent generations from this founder line were used for the experiments. <b>(C)</b> <i>Bi-L-Dek</i> mice were bred to keratin 5 promoter driven tetracycline transactivator (<i>K5-tTA)</i> mice. (<b>D</b>) <i>Bi-L-Dek</i> and <i>K5-tTA</i> transgene presence in offspring was confirmed by genotyping along with identification of single transgenic and non-transgenic (Non Tg) littermates. FVB/N (WT) mice were negative controls (-) and the F2 parent carrying the transgene was the positive control (+). (<b>E</b>) Schematic of <i>Bi-L-Dek_K5-tTA</i> mice designed to express luciferase and to overexpress Dek in the K5-positive basal layer of stratified squamous epithelium (highlighted in blue). Transgene repression by dox in this tet-off system is indicated.</p

Dek overexpression increases the incidence of gross esophageal tumors.

<p>(<b>A</b>) Details on mice and pathologies including esophageal tumor volumes in the 4NQO-treated mice. (<b>B</b>) Representative IHC images for Dek protein overexpression in the esophagus of <i>Bi-L-Dek_K5-tTA</i> mice treated with 4NQO compared to mice on dox (Dek antibody: Cusabio, Balitmore, MD, USA; magnification: 40x). (<b>C</b>) Percent incidence of gross, microscopic, invasive, and multifocal tumors within the two groups of mice. Statistics is indicated when significantly different between the no dox/dox treated groups as determined by a Fisher Exact test. (<b>D</b>) Gross tumor volumes within the two groups. Each dot represents total gross tumor volume per mouse (no statistics due to an n = 1 for the no dox group). (<b>E</b>) Survival of the <i>Bi-L-Dek_K5-tTA</i> Dek overexpressing mice +/- dox treatment. Tissue from a seventh <i>Bi-L-Dek_K5-tTA</i> mouse that died at 27 weeks could not be evaluated for tumors at necropsy (not included in Fig 5A). (<b>F-G</b>) Images of esophagi at the time of dissection (top), and the corresponding H&E stained histologic sections of esophagus (middle), and Dek staining by IHC in the corresponding tumor (bottom) from <i>Bi-L-Dek_K5-tTA</i> mice in the absence (<b>F</b>) or presence (<b>G</b>) of dox (H&E magnification: 2x; Dek IHC magnification: 120x; Dek antibody: Cusabio, Baltimore, MD, USA). (<b>H-I</b>) Images of H&E stained esophageal sections illustrate morphological features of tumors in (<b>H</b>) Dek overexpressing <i>Bi-L-Dek_K5-tTA</i> mice and (<b>I</b>) normal esophagus and tumors in dox treated <i>Bi-L-Dek_K5-tTA</i> mice. Extensive necrosis in a poorly differentiated invasive squamous cell carcinoma (H, left panel arrows), and dyskeratotic cells (H, middle panel arrows) along with cellular dysplasia and intercellular bridges (H, middle panel inset), and extensive stromal invasion (H, right panel arrows) with focal squamous differentiation (H, right panel arrowhead) in papillary squamous cell carcinoma in Dek overexpressing mice are shown. Esophageal images from dox treated <i>Bi-L-Dek_K5-tTA</i> mice illustrate the normal esophagus from mouse lacking tumors (I, left panel), a microscopic papillary squamous cell carcinoma with minimal superficial stroma invasion (I, middle panel, arrows), and the single grossly apparent tumor characterized as a well differentiated invasive squamous cell carcinoma with abundant keratin production (I, right panel, arrows and inset). (Original magnifications: 40x, inserts 100x).</p

Dek overexpression in 4NQO-treated mice leads to trends in increased cellular proliferation.

<p>(<b>A</b>) The water soluble carcinogen 4-nitroquinoline 1-oxide (4NQO) confers susceptibility to head and neck squamous cell carcinoma (HNSCC). At six weeks of age, mice were given 4NQO drinking water for 16 weeks in the presence or absence of dox chow. After 16 weeks, the mice were returned to normal water and were monitored until death or sacrifice at 45 weeks of age, or earlier if moribund. Prior to sacrifice, mice were injected with BrdU to measure proliferative differences in tissues. Abbreviations: time (T), weeks (wks). (<b>B-C</b>) Quantification of BrdU positive cells per millimeter of squamous epithelium in (<b>B</b>) normal and (<b>C</b>) 4NQO treated tongue and esophagus of <i>Bi-L-Dek_K5-tTA</i> mice in the absence or presence of dox. Three to six mice were examined for each tissue in the no dox/dox treatment groups and a student’s t-test was used to determine statistical significance. A minimum of 100mm of epithelium was quantified per mouse and epithelial distance was measured using ImageJ [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007227#pgen.1007227.ref089" target="_blank">89</a>]. (<b>D</b>) Representative IHC images of increased BrdU incorporation in the tongue and esophagus of <i>Bi-L-Dek_K5-tTA</i> mice treated with 4NQO compared to those on dox.</p