Jelena Ivanovska

Death-Associated Protein Kinase Controls STAT3 Activity in Intestinal Epithelial Cells

The TNF-IL-6-STAT3 pathway plays a crucial role in promoting ulcerative colitis-associated carcinoma (UCC). To date, the negative regulation of STAT3 is poorly understood. Interestingly, intestinal epithelial cells of UCC in comparison to ulcerative colitis show high expression levels of anti-inflammatory death-associated protein kinase (DAPK) and low levels of pSTAT3. Accordingly, epithelial DAPK expression was enhanced in STAT3(IEC-KO) mice. To unravel a possible regulatory mechanism, we used an in vitro TNF-treated intestinal epithelial cell model. We identified a new function of DAPK in suppressing TNF-induced STAT3 activation as DAPK siRNA knockdown and treatment with a DAPK inhibitor potentiated STAT3 activation, IL-6 mRNA expression, and secretion. DAPK attenuated STAT3 activity directly by physical interaction shown in three-dimensional structural modeling. This model suggests that DAPK-induced conformational changes in the STAT3 dimer masked its nuclear localization signal. Alternatively, pharmacological inactivation of STAT3 led to an increase in DAPK mRNA and protein levels. Chromatin immunoprecipitation showed that STAT3 restricted DAPK expression by promoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negative regulation principle might balance TNF-induced inflammation and seems to play an important role in the inflammation-associated transformation process as confirmed in an AOM+DSS colon carcinogenesis mouse model. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatment of ulcerative colitis and UCC.

Identification of DAPK as a scaffold protein for the LIMK/cofilin complex in TNF-induced apoptosis

The role of cytoskeleton-associated proteins during TNF-induced apoptosis is not fully understood. A potential candidate kinase that might connect TNF signaling to actin reorganization is the death-associated protein kinase (DAPK). To identify new DAPK interaction partners in TNF-induced apoptosis, we performed a peptide array screen. We show that TNF-treatment enhanced the phosphorylation of LIMK at threonine508 and its downstream target cofilin at serine3 (p-cofilin(Ser3)). Modulation of DAPK activity and expression by DAPK inhibitor treatment, siRNA knockdown, and overexpression affected the phosphorylation of both proteins. We propose a 3D structural model where DAPK functions as a scaffold for the LIMK/cofilin complex and triggers a closer interaction of both proteins under TNF stimulation. Upon TNF a striking redistribution of LIMK, DAPK, and cofilin to the perinuclear compartment was observed. The pro-apoptotic DAPK/LIMK/cofilin multiprotein complex was abrogated in detached cells, indicating that its signaling was no longer needed if cells committed to apoptosis. P-cofilin(Ser3) was strongly accumulated in cells with condensed chromatin, pronounced membrane blebs and Annexin V up-regulation. From studying different cofilin(Ser3) mutants we suggest that p-cofilin(Ser3) is an indicator of TNF-induced apoptosis. Collectively, our findings identify a novel molecular cytoskeleton-associated mechanism in TNF-induced DAPK-dependent apoptosis.

DAPK and cytoskeleton-associated functions

Death-associated protein kinase (DAPK) undergoes activation in response to various death stimuli, and they have been associated with an increase in DAPK catalytic activity. One of the most prominent features of DAPK-induced cell death is the effect on the cytoskeleton, including loss of matrix attachment, and membrane blebbing. One known cytoskeletal-associated substrate of DAPK is the myosin-II light chain, phosphorylated by DAPK on Ser19, thus stabilizing actin stress fibres. Moreover, paxillin, a component of focal adhesions, was found to be localized in close proximity to the tips of the DAPK-positive filaments, indicating that stress fibres containing DAPK extend to focal contacts. Forced expression of DAPK in multiple cell types results in morphological changes such as cell rounding, membrane blebbing, shrinking and detachment. During directed migration, DAPK functions as a potent inhibitor of cell polarization, as evidenced by its perturbation of the formation of static protrusion at the leading edge. Furthermore, DAPK inhibits random migration by suppressing directional persistence. One of the studies considered DAPK as an anoikis inducer. Others showed that DAP-kinase inhibits the activities of cell surface integrins by converting them into an inactive conformation. Biochemical experiments have established the DAPK binding to Syntaxin1 and its subsequent phosphorylation at Ser188 in a Ca2+ dependent manner. This phosphorylation event has been shown to decrease the binding of Syntaxin to MUNC18-1, a protein critically involved in synaptic vesicle docking. Here, we have investigated the structural interactions that modulate DAPK phosphorylation with Syntaxin and its functional role in binding to the MUNC18-1 to regulate vesicle docking. This review will summarize our current knowledge of the role of DAPK on cytoskeleton reorganization and report the mechanisms that regulate these changes.

DAPK-HSF1 interaction as a positive-feedback mechanism stimulating TNF-induced apoptosis in colorectal cancer cells.

ABSTRACT Death-associated protein kinase (DAPK) is a serine-threonine kinase with tumor suppressor function. Previously, we demonstrated that tumor necrosis factor (TNF) induced DAPK-mediated apoptosis in colorectal cancer. However, the protein–protein interaction network associated with TNF–DAPK signaling still remains unclear. We identified HSF1 as a new DAPK phosphorylation target in response to low concentrations of TNF and verified a physical interaction between DAPK and HSF1 both in vitro and in vivo. We show that HSF1 binds to the DAPK promoter. Transient overexpression of HSF1 protein led to an increase in DAPK mRNA level and consequently to an increase in the amount of apoptosis. By contrast, treatment with a DAPK-specific inhibitor as well as DAPK knockdown abolished the phosphorylation of HSF1 at Ser230 (pHSF1Ser230). Furthermore, translational studies demonstrated a positive correlation between DAPK and pHSF1Ser230 protein expression in human colorectal carcinoma tissues. Taken together, our data define a novel link between DAPK and HSF1 and highlight a positive-feedback loop in DAPK regulation under mild inflammatory stress conditions in colorectal tumors. For the first time, we show that under TNF the pro-survival HSF1 protein can be redirected to a pro-apoptotic program.

Apoptosis Signalling Activated by TNF in the Lower Gastrointestinal Tract-Review

The tumor necrosis factor (TNF) gene is an immediate early gene, rapidly transcribed in a variety of cell types following exposure to a broad range of pathogens and signals of inflammation and stress. Regulation of TNF gene expression at the transcriptional level is cell type- and stimulus-specific, involving epigenetic mechanisms or miRNAs. A better knowledge of the molecular mechanisms that control TNF gene regulation and TNF signalling will provide deeper understanding of the initiation and development of apoptotic and inflammatory processes triggered by TNF cytokine in the gut. The described efforts to embed TNF in clinical treatment regiments reflect its attractive effectiveness in killing tumor cells. Whether the described strategies will achieve the success of incorporating TNF in lower gastrointestinal tract therapy for inflammatory diseases and cancer remains to be determined.

Biofabrication of 3D Alginate-Based Hydrogel for Cancer Research: Comparison of Cell Spreading, Viability, and Adhesion Characteristics of Colorectal HCT116 Tumor Cells.

Hydrogels are an important class of biomaterials as they could mimic the extracellular matrix (ECM). Among the naturally occurring biopolymers, alginate and gelatin are extensively used for many biomedical applications. For developing biofabrication constructs as three-dimensional (3D) cell culture models, realistic imaging of cell spreading and proliferation inside the hydrogels represents a major challenge. Therefore, we aimed to establish a system that can mimic the structural architecture, composition, and biological functions of the ECM for cancer research approaches. For this, we compared the cell behavior of human colon cancer HCT116 cells in two biofabricated hydrogels as follows: pure alginate and cross-linked alginate-gelatin (ADA-GEL) matrixes. Our data indicate that cells from the ADA-GEL matrix showed highest proliferation and cellular networks through the material. Analyzing the mRNA expression of several integrins of cells cultured inside of the matrix, we showed that mRNA expression of integrin subunits differed based on the cell focal adhesion characteristics. Furthermore, we showed that recultured ADA-GEL immobilized cells do not differ from parental HCT116 cells regarding migration and proliferation capabilities. Comparing adhesion and other phenotypic characteristics of HCT116 tumor cells, we suggest that ADA-GEL hydrogel is a more suitable 3D system than pure alginate and seems to optimally mimic the physiological behavior of the tumor microenvironment. For the first time, we present a functional 3D hydrogel construct for colon cancer cells, which are supporting their physiological cell attachment, spreading, and viability. We strongly believe that it will be applicable as a suitable in vitro 3D tumor model to study different aspects of tumor cell behavior.

DAPK loss in colon cancer tumor buds: implications for migration capacity of disseminating tumor cells

Defining new therapeutic strategies to overcome therapy resistance due to tumor heterogeneity in colon cancer is challenging. One option is to explore the molecular profile of aggressive disseminating tumor cells. The cytoskeleton-associated Death-associated protein kinase (DAPK) is involved in the cross talk between tumor and immune cells at the invasion front of colorectal cancer. Here dedifferentiated tumor cells histologically defined as tumor budding are associated with a high risk of metastasis and poor prognosis. Analyzing samples from 144 colorectal cancer patients we investigated immunhistochemical DAPK expression in different tumor regions such as center, invasion front, and buds. Functional consequences for tumor aggressiveness were studied in a panel of colon tumor cell lines using different migration, wound healing, and invasion assays. DAPK levels were experimentally modified by siRNA transfection and overexpression as well as inhibitor treatments. We found that DAPK expression was reduced towards the invasion front and was nearly absent in tumor buds. Applying the ECIS system with HCT116 and HCT116 stable lentiviral DAPK knock down cells (HCTshDAPK) we identified an important role for DAPK in decreasing the migratory capacity whereas proliferation was not affected. Furthermore, the migration pattern differed with HCTshDAPK cells showing a cluster-like migration of tumor cell groups. DAPK inhibitor treatment revealed that the migration rate was independent of DAPKs catalytic activity. Modulation of DAPK expression level in SW480 and DLD1 colorectal cancer cells significantly influenced wound closure rate. DAPK seems to be a major player that influences the migratory capability of disseminating tumor cells and possibly affects the dynamic interface between pro- and anti-survival factors at the invasion front of colorectal cancer. This interesting and new finding requires further evaluation.

Abstract 3825: High DAPK expression is correlated with worse prognosis in colorectal cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objectives: Death-associated protein kinase (DAPK) is a cytoskeleton-associated serine/threonine kinase. It functions as a potent inhibitor of cell polarization and migration. DAPK loss has been shown to be associated with advanced tumor stages and unfavorable prognosis in many tumor types. However, the prognostic relevance of DAPK in patients with colorectal cancer is only poorly understood. Thus, we aimed to investigate the impact of DAPK protein expression on clinical outcome in patients with colorectal cancer. Methods: Samples from 220 colorectal cancer patients with a median follow-up of 60 months were analyzed for immunohistochemical DAPK protein expression on tissue microarrays. The effect of endogenous DAPK protein expression on cell migration was analyzed applying the ECIS system (electric cell-substrate impedance sensing) with stable DAPK knockdown (shRNA) and DAPK overexpressing cell lines generated from HCT116 wild type colon cancer cells. Colony formation ability was analyzed using soft agar assay. Results: In Kaplan-Meier-curve analysis DAPK loss was associated with longer survival time (p< 0.001). The 5-year survival rates were 52.3% and 25.6% in patients with DAPK negative tumors and DAPK positive tumors, respectively. 76.1% of DAPK positive tumors showed evidence of tumor budding. After adjusting for TNM stage in multivariate Cox regression analysis, DAPK retained its strong independent prognostic value. Patients with DAPK overexpressing tumors had a 1.9 times increased risk of death from the tumor than those with DAPK negative tumors. DAPK expression in tumor center showed a loss of expression toward the front (p=0.0126). This was in agreement with ECIS migration results indicating higher migration rate for DAPK shRNA cells. DAPK wild type cells showed an increased number of colonies in soft agar assay in comparison to other cell types. Colony formation ability showed time-dependent effects for DAPK overexpression. Conclusion: In contrast to the well-known function of DAPK as a tumor suppressor, our data showed DAPK overexpression to be associated with aggressiveness of tumors and an unfavorable outcome. This interesting and new finding has to be further evaluated. Citation Format: Jelena Ivanovska, Inti Zlobec, Eva Diamantis-Karamitopoulou, Heather Dawson, Viktor Hendrik Koelzer, Abbas Agaimy, Fabian Garreis, William Laqua, Alessandro Lugli, Arndt Hartmann, Regine Schneider-Stock. High DAPK expression is correlated with worse prognosis in colorectal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3825. doi:10.1158/1538-7445.AM2014-3825

Abstract 2861: A novel negative regulation principle between DAPK and STAT3 in inflammation-associated carcinogenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC It has been shown that the TNF-IL-6-STAT3 pathway plays a crucial role in promoting ulcerative colitis associated carcinoma (UCC). As inflammation compromises gut homeostasis and is associated with cancer progression, it is important to understand the role of key signalling molecules that are involved in the activation of the inflammatory cascade. Interestingly, we found that intestinal epithelial cells (IEC) of UCC in comparison to ulcerative colitis (UC) show high expression level of the anti-inflammatory Death associated protein kinase (DAPK) but only low levels of pSTAT3. Accordingly, epithelial DAPK expression was enhanced in STAT3IEC-KO mice. To unravel a possible regulatory mechanism we used an in vitro model of TNF-treated IEC. We identified a new function of DAPK in suppressing TNF-induced STAT3 activation as DAPK si knockdown and treatment with a DAPK inhibitor potentiated STAT3 activation and IL-6 secretion. DAPK attenuated STAT3 activity directly by physical interaction shown in a 3D structural model. This model suggests that DAPK induced conformational changes in the STAT3 dimer masked its nuclear localization signal. Vice versa, pharmacologic inactivation of STAT3 led to an increase in DAPK mRNA and protein levels. Chromatin immunoprecipitation data showed that STAT3 restricted DAPK expression by promoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negative regulation principle was confirmed in an AOM-DSS-colon carcinogenesis mouse model. It might balance TNF-induced inflammation and seems to play an important role in the inflammation-associated transformation process. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatment of UC and UCC. Citation Format: Saritha Chakilam, Muktheswar Gandesiri, Tilman Rau, Clemens Neufert, Abbas Agaimy, Vijayalakshmi Mahadevan, Jelena Ivanovska, Ralph Wirtz, Natalya Benderska, Jan Schulze-Luhrmann, Nadine Wittkopf, Ajithavalli Chellappan, Petra Ruemmele, Michael Vieth, Margret Rave-Frank, Hans Christiansen, Arndt Hartmann, Raja Atreya, Christoph Becker, Pablo Steinberg, Regine Schneider-Stock. A novel negative regulation principle between DAPK and STAT3 in inflammation-associated carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2861. doi:10.1158/1538-7445.AM2013-2861 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 4673A: DAPK-mediated phosphorylation of HSF1 enhances apoptosis level upon TNF in colorectal carcinoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objectives: Tumor necrosis factor ≤ (TNF) is an inflammatory cytokine, which is released upon different stimuli, including irradiation. Recently it has been shown, that the Death-associated protein kinase (DAPK) mediates TNF-induced apoptosis in colon cancer cells [1]. Here, we aimed to identify new DAPK binding partners and to characterize the functional role of novel protein interaction complexes during TNF-induced apoptosis in colon cancer cells. Methods/Results: HCT116 colorectal cancer cells were cultured for 6 to 48 hours in either normal or TNF-conditioned medium. For phosphopeptide microarray (PPM) whole cell lysates were incubated on peptide platforms with radioactive-labeled P33. Apoptosis was detected by Annexin V staining and caspase 3 cleavage in Western Blotting. PPM analysis revealed heat shock transcription factor 1 (HSF1) as a new potential substrate of DAPK phosphorylation under TNF-stimulation. DAPK Co-IP, co-immunofluorescence (Co-IF), and mass spectrometry showed that DAPK interacts with HSF1 after TNF-treatment. Phosphorylation on serine residues of HSF1 is necessary for HSF1 nuclear translocation and the initiation of transcription of its target genes. Among them, Ser 230 is part of a consensus phosphorylation motif for DAPK. Maximal level of pHSF1Ser230 was observed at the time point where massive apoptosis was induced. Co-IF microscopy confirmed enrichment of pHSF1Ser230 in the nucleus already after 24 hours of TNF-stimulation. EMSA and Chromatin-IP revealed that pHSF1Ser230 binds to the heat shock response element in the DAPK promoter region and enhances its transcriptional activity. Exogenous over-expression of HSF1 protein led to a significant increase in mRNA DAPK levels and consequently to an enforcement of apoptosis. As expected, DAPK knockdown cells did not show any variation in pHSF1Ser230 level, supporting again that DAPK is an important mediator of the TNF-driven signaling pathway in colorectal cancer cells. The significance of the DAPK/pHSF1Ser230 interaction for response prediction was evaluated by immunohistochemical staining on tissue microarrays of colorectal cancer before and after radiotherapy. Conclusion: Our data show a novel functional interaction between HSF1 and DAPK under TNF-stress and highlight a positive feedback mechanism in DAPK-regulation. These results help to understand cell death pathways in response to radiotherapy. [1].Bajbouj K.et al., Am J Pathol. (2009) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4673A. doi:1538-7445.AM2012-4673A