The DEK Oncogene Is a Target of Steroid Hormone Receptor Signaling in Breast Cancer

<div><p>Expression of estrogen and progesterone hormone receptors indicates a favorable prognosis due to the successful use of hormonal therapies such as tamoxifen and aromatase inhibitors. Unfortunately, 15–20% of patients will experience breast cancer recurrence despite continued use of tamoxifen. Drug resistance to hormonal therapies is of great clinical concern so it is imperative to identify novel molecular factors that contribute to tumorigenesis in hormone receptor positive cancers and/or mediate drug sensitivity. The hope is that targeted therapies, in combination with hormonal therapies, will improve survival and prevent recurrence. We have previously shown that the DEK oncogene, which is a chromatin remodeling protein, supports breast cancer cell proliferation, invasion and the maintenance of the breast cancer stem cell population. In this report, we demonstrate that DEK expression is associated with positive hormone receptor status in primary breast cancers and is up-regulated <em>in vitro</em> following exposure to the hormones estrogen, progesterone, and androgen. Chromatin immunoprecipitation experiments identify <em>DEK</em> as a novel estrogen receptor α (ERα) target gene whose expression promotes estrogen-induced proliferation. Finally, we report for the first time that DEK depletion enhances tamoxifen-induced cell death in ER+ breast cancer cell lines. Together, our data suggest that DEK promotes the pathogenesis of ER+ breast cancer and that the targeted inhibition of DEK may enhance the efficacy of conventional hormone therapies.</p> </div

Association of DEK expression with clinical and pathological variables in invasive adenocarcinomas of the breast.

*<p>Chi-Squared statistic.</p

Model for DEK transcriptional up-regulation following 17β-estradiol exposure.

<p>Upon 17β-estradiol exposure, ERα is activated and binds to the <i>DEK</i> promoter at least at two locations – an ERE half site at −716 bp and at ERα/Sp1 binding sites more proximal to the transcriptional start site. A second potential mechanism of up-regulation is the ERα-mediated up-regulation of E2F proteins (particularly E2F3) that also increase <i>DEK</i> transcription. Increased levels of DEK then promote proliferation. DEK expression can be targeted with the anti-estrogen tamoxifen to inhibit cell proliferation. The knockdown of DEK by RNAi can increase tamoxifen sensitivity of ER+ cell lines by synergistically inducing an apoptotic response.</p

DEK expression is associated with positive hormone receptor status in human primary and cultured breast cancers.

<p>(A) Estrogen receptor (ER) negative tumors were often negative for DEK staining (right) while ER positive tumors were often positive for DEK staining (left). Immunohistochemical staining for DEK in two invasive ductal carcincomas showing positive DEK staining (DEK+) in a hormone receptor positive (ER+/PR+) tumor and lack of DEK expression (DEK-) in a hormone receptor negative tumor (ER−/PR−). Low power images are at 200× and all high power images are at 1000× magnification. (B) Western blotting for DEK showed increased expression following exposure to 17β-estradiol for 48 hours in ER+ MCF7 and T47D cells but not in ER- BT20 cells. (C) Cell morphology of T47D cells in CS-FBS (left), 48 hours of 17β-estradiol treatment (middle), and under normal culture conditions (right). Bright field images of cultured cells were obtained at 100× total magnification. (D) <i>DEK</i> expression increases in 17β-estradiol and R1881 treated cells. Quantitative RT-PCR was performed to detect <i>DEK</i> expression in hormone starved MCF7 cells treated with 10 nM 17β-estradiol or 1 µM methyltrienolone (R1881) for six hours. <i>GAPDH</i> was used as a control and values are normalized to the untreated sample. (E) Western blotting for DEK shows increased protein levels after treatment of hormone starved MCF7 cells with 10 nM 17β-estradiol, 10 nM progesterone, or 1 µM methyltrienolone (R1881) over the course of 48 hours.</p

DEK is necessary for 17β-estradiol stimulated cell proliferation and modulates sensitivity to tamoxifen.

<p>(A) DEK expression is required for 17β-estradiol stimulated cellular proliferation. Hormone starved MCF7 cells transduced with non-targeting shRNA (NTsh) or DEK shRNA (DEKsh2) were untreated (CS-FBS) or exposed to 10 nM 17β-estradiol, then cultured in BrdU. The percentage of BrdU positive cells was determined by flow cytometry. Asterisk (*) denotes p<0.05 using Student’s t-test. (B and C) DEK depletion by shRNA (DEKsh2) works synergistically with tamoxifen to induce apoptosis in breast cancer cell lines. (B) Bright field images (100× magnification) of MCF7 cells expressing either NTsh or DEKsh2 were cultured in low serum media and either untreated or treated with tamoxifen for 18 hours. (C) DEK depletion by shRNA (DEKsh2) enhances the cytotoxic effect of tamoxifen. DEK proficient and deficient MCF7 (left) and T47D (right) cells were grown in low serum media then treated with 3 µg/ml tamoxifen for 22 hours. Cells were labeled with 7AAD then analyzed for sub-G1 content by flow cytometry as a measure of apoptosis. Results shown are the average of triplicate experiments. Two asterisks (**) indicate p<0.01 as determined using a 2-way ANOVA test for significance. For MCF7 cells, p = 0.08. (A and B insets) DEK shRNA knockdown is shown by western blot analysis for normally cultured cells that were transduced with lentivirus carrying either non-targeting shRNA (NTsh) or DEK specific shRNA (DEKsh2).</p

<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

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</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