Lisa M. Privette Vinnedge

The human DEK oncogene stimulates β-catenin signaling, invasion and mammosphere formation in breast cancer.

Breast cancer is a major cause of cancer-related deaths in American women; therefore, the identification of novel breast cancer-related molecules for the discovery of new markers and drug targets remains essential. The human DEK gene, which encodes a chromatin-binding protein and DNA topology regulator, is upregulated in many types of cancer. DEK has been implicated as an oncogene in breast cancer based on mRNA expression studies, but its functional significance in breast cancer growth and progression has not yet been tested directly. We demonstrate that DEK is highly expressed in breast cancer cells compared with normal tissue, and functionally important for cellular growth, invasion and mammosphere formation. DEK overexpression in non-tumorigenic MCF10A cells resulted in increased growth and motility, with a concomitant downregulation of E-cadherin. Conversely, DEK knockdown in MCF7 and MDA-MB-468 breast cancer cells resulted in decreased growth and motility with upregulation of E-cadherin. The use of DEK-proficient and -deficient breast cancer cells in orthotopic xenografts provided further in vivo evidence that DEK contributes to tumor growth. Activation of the β-catenin signaling pathway is important for normal and cancer stem cell character, growth and metastasis. We show that DEK expression stimulated, and DEK knockdown repressed β-catenin nuclear translocation and activity. Importantly, the expression of constitutively active β-catenin rescued breast cancer invasion defects of DEK knockdown cells. Together, our data indicate that DEK expression stimulates the growth, stem cell character and motility of breast cancer cells, and that DEK-dependent cellular invasion occurs at least in part via β-catenin activation.

Stacking the DEK: From chromatin topology to cancer stem cells

Stem cells are essential for development and tissue maintenance and display molecular markers and functions distinct from those of differentiated cell types in a given tissue. Malignant cells that exhibit stem cell-like activities have been detected in many types of cancers and have been implicated in cancer recurrence and drug resistance. Normal stem cells and cancer stem cells have striking commonalities, including shared cell surface markers and signal transduction pathways responsible for regulating quiescence vs. proliferation, self-renewal, pluripotency and differentiation. As the search continues for markers that distinguish between stem cells, progenitor cells and cancer stem cells, growing evidence suggests that a unique chromatin-associated protein called DEK may confer stem cell-like qualities. Here, we briefly describe current knowledge regarding stem and progenitor cells. We then focus on new findings that implicate DEK as a regulator of stem and progenitor cell qualities, potentially through its unusual functions in the regulation of local or global chromatin organization.

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

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 in vitro following exposure to the hormones estrogen, progesterone, and androgen. Chromatin immunoprecipitation experiments identify DEK 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.

Dissecting the Potential Interplay of DEK Functions in Inflammation and Cancer

There is a long-standing correlation between inflammation, inflammatory cell signaling pathways, and tumor formation. Understanding the mechanisms behind inflammation-driven tumorigenesis is of great research and clinical importance. Although not entirely understood, these mechanisms include a complex interaction between the immune system and the damaged epithelium that is mediated by an array of molecular signals of inflammation—including reactive oxygen species (ROS), cytokines, and NFκB signaling—that are also oncogenic. Here, we discuss the association of the unique DEK protein with these processes. Specifically, we address the role of DEK in chronic inflammation via viral infections and autoimmune diseases, the overexpression and oncogenic activity of DEK in cancers, and DEK-mediated regulation of NFκB signaling. Combined, evidence suggests that DEK may play a complex, multidimensional role in chronic inflammation and subsequent tumorigenesis.

Defects in the Fanconi anemia pathway in head and neck cancer cells stimulate tumor cell invasion through DNA-PK and Rac1 signaling

Purpose: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells. Experimental Design: Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors. Results: We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity. Conclusions: These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes. Clin Cancer Res; 22(8); 2062–73. ©2015 AACR.

DEK is required for homologous recombination repair of DNA breaks

DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.

Decreased plasma DEK Oncogene Levels Correlate with p16-Negative Disease and Advanced Tumor Stage in a Case–Control Study of Patients with Head and Neck Squamous Cell Carcinoma

Head and neck cancer (HNC) remains the sixth most common malignancy worldwide and survival upon recurrence and/or metastasis remains poor. HNSCC has traditionally been associated with alcohol and nicotine use, but more recently the Human Papilloma Virus (HPV) has emerged as a favorable prognostic risk factor for oropharyngeal HNSCC. However, further stratification with additional biomarkers to predict patient outcome continues to be essential. One candidate biomarker is the DEK oncogenic protein, which was previously detected in the urine of patients with bladder cancer and is known to be secreted by immune cells such as macrophages. Here, we investigated if DEK could be detected in human plasma and if DEK levels correlated with clinical and pathological variables of HNSCC. Plasma was separated from the peripheral blood of newly diagnosed, untreated HNSCC patients or age-matched normal healthy controls and analyzed for DEK protein using ELISA. Plasma concentrations of DEK protein were lower in p16-negative tumors compared to both normal controls and patients with p16-positive tumors. Patients with lower plasma concentrations of DEK were also more likely to have late stage tumors and a lower white blood cell count. Contrary to previously published work demonstrating a poor prognosis with high intratumoral DEK levels, we show for the first time that decreased concentrations of DEK in patient plasma correlates with poor prognostic factors, including HPV-negative status as determined by negative p16 expression and advanced tumor stage.

Neuroanatomical Distribution of DEK Protein in Corticolimbic Circuits Associated with Learning and Memory in Adult Male and Female Mice

DEK, a chromatin-remodeling gene expressed in most human tissues, is known for its role in cancer biology and autoimmune diseases. DEK depletion in vitro reduces cellular proliferation, induces DNA damage subsequently leading to apoptosis, and down-regulates canonical Wnt/β-catenin signaling, a molecular pathway essential for learning and memory. Despite a recognized role in cancer (non-neuronal) cells, DEK expression and function is not well characterized in the central nervous system. We conducted a gene ontology analysis (ToppGene), using a cancer database to identify genes associated with DEK deficiency, which pinpointed several genes associated with cognitive-related diseases (i.e., Alzheimers disease, presenile dementia). Based on this information, we examined DEK expression in corticolimbic structures associated with learning and memory in adult male and female mice using immunohistochemistry. DEK was expressed throughout the brain in both sexes, including the medial prefrontal cortex (prelimbic, infralimbic and dorsal peduncular). DEK was also abundant in all amygdalar subdivisions (basolateral, central and medial) and in the hippocampus including the CA1, CA2, CA3, dentate gyrus (DG), ventral subiculum and entorhinal cortex. Of note, compared to males, females had significantly higher DEK immunoreactivity in the CA1, indicating a sex difference in this region. DEK was co-expressed with neuronal and microglial markers in the CA1 and DG, whereas only a small percentage of DEK cells were in apposition to astrocytes in these areas. Given the reported inverse cellular and molecular profiles (e.g., cell survival, Wnt pathway) between cancer and Alzheimers disease, these findings suggest a potentially important role of DEK in cognition.

Sex differences in DEK expression in the anterior cingulate cortex and its association with dementia severity in schizophrenia

DEK is a chromatin-remodeling phosphoprotein found in most human tissues, but its expression and function in the human brain is largely unknown. DEK depletion in vitro induces cellular and molecular anomalies associated with cognitive impairment, including down-regulation of the canonical Wnt/β-catenin signaling pathway. ToppGene analyses link DEK loss to genes associated with various dementias and age-related cognitive decline. To examine the role of DEK in cognitive impairment in severe mental illness, DEK protein expression was assayed by immunoblot in the anterior cingulate cortex (ACC) of subjects with schizophrenia. Cognitive impairment is a core feature of schizophrenia and cognitive function in subjects was assessed antemortem using the clinical dementia rating (CDR) scale. DEK protein expression was not significantly altered in schizophrenia (n = 20) compared to control subjects (n = 20). Further analysis revealed significant reduction in DEK protein expression in women with schizophrenia, and a significant increase in expression in men with schizophrenia, relative to their same-sex controls. DEK protein expression levels were inversely correlated with dementia severity in women. Conversely, in men, DEK protein expression and dementia severity were positively correlated. Notably, there was no sex difference in DEK protein expression in the control group, suggesting that this sex difference is specific to schizophrenia and not due to inherent differences in DEK expression between males and females. These results suggest a novel, sex-specific role for DEK in cognitive performance and highlight a putative sex-specific link between central nervous system DEK protein expression and a neuropsychiatric disease that is commonly associated with cognitive impairment.

Loss of DEK induces radioresistance of murine restricted hematopoietic progenitors

Self-renewing hematopoietic stem cells and multipotent progenitor cells are responsible for maintaining hematopoiesis throughout an individuals lifetime. For overall health and survival, it is critical that the genome stability of these cells is maintained and that the cell population is not exhausted. Previous reports have indicated that the DEK protein, a chromatin structural protein that functions in numerous nuclear processes, is required for DNA damage repair in vitro and long-term engraftment of hematopoietic stem cells in vivo. Therefore, we investigated the role of DEK in normal hematopoiesis and response to DNA damaging agents in vivo. Here, we report that hematopoiesis is largely unperturbed in DEK knockout mice compared with wild-type (WT) controls. However, DEK knockout mice have fewer radioprotective units, but increased capacity to survive repeated sublethal doses of radiation exposure compared with WT mice. Furthermore, this increased survival correlated with a sustained quiescent state in which DEK knockout restricted hematopoietic progenitor cells (HPC-1) were nearly three times more likely to be quiescent following irradiation compared with WT cells and were significantly more radioresistant during the early phases of myeloid reconstitution. Together, our studies indicate that DEK functions in the normal hematopoietic stress response to recurrent radiation exposure.