Saritha Chakilam

IL-33 regulates TNF-α dependent effects in synovial fibroblasts.

The recently described IL-33 acts as a pro-inflammatory cytokine, inducing the expression of multiple responses in the target cells. Although a nuclear localization of IL-33 has been described, its exact functional relevance is presently unknown. The present study was conducted to analyze the effects of IL-33 on the TNF-α induced synthesis of the pro-inflammatory mediators IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the pro-destructive molecules matrix metalloproteinase-1 (MMP-1), MMP-3, and TIMP-1 of rheumatoid arthritis synovial fibroblast (RA-SFs) using RNA overexpression and silencing. TNF-α significantly induced IL-33 mRNA expression and protein synthesis in RA-SFs. TNF-α-induced IL-33 protein expression was mediated via p38 signaling. Immunohistochemistry for IL-33 clearly showed that nuclear translocation of IL-33 was induced in TNF-α stimulated RA-SFs. IL-33 overexpression enhanced TNF-α-induced pro-inflammatory and pro-destructive functions in RA-SFs. IL-33 silencing significantly downregulated TNF-α-induced pro-inflammatory functions, whereas TNF-α-induced pro-destructive functions were less influenced by IL-33 silencing. This study identifies IL-33 as a critical regulator/enhancer of TNF-α-induced functions in RA-SFs, pointing to a central role of this cytokine in the perpetuation of pro-inflammatory and pro-destructive processes in rheumatoid arthritis (RA) and other inflammatory and degenerative diseases.

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-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.

Gallotannin is a DNA damaging compound that induces senescence independently of p53 and p21 in human colon cancer cells

The plant secondary metabolite gallotannin (GT) is the simplest hydrolyzable tannin shown to have anti‐carcinogenic properties in several cell lines and to inhibit tumor development in different animal models. Here, we determined if GT induces senescence and DNA damage and investigated the involvement of p53 and p21 in this response. Using HCT116 human colon cancer cells wildtype for p53+/+/p21+/+ and null for p53+/+/p21−/− or p53−/−/p21+/+, we found that GT induces senescence independently of p21 and p53. GT was found to increase the production of reactive oxygen species (ROS) by altering the redox balance in the cell, mainly by reducing the levels of glutathione and superoxide dismutase (SOD). Using the key antioxidants N‐acetyl cysteine, dithiothreitol, SOD, and catalase, we showed that ROS were partially involved in the senescence response. Furthermore, GT‐induced cell cycle arrest in S‐phase in all HCT116 cell lines. At later time points, we noticed that p53 and p21 null cells escaped complete arrest and re‐entered cell cycle provoking higher rates of multinucleation. The senescence induction by GT was irreversible and was accompanied by significant DNA damage as evidenced by p‐H2AX staining. Our findings indicate that GT is an interesting anti colon cancer agent which warrants further study.

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

Abstract 4779: TNF-induced Cofilin 1 phosphorylation by LIMK in colorectal tumor cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objectives: Recently, we have shown that the pro-inflammatory cytokine TNF induces apoptosis in HCT116 colorectal tumor cells. Among the first apoptotic signs is a drastic change in cell morphology through modulation of the cytoskeleton. In order to identify and to characterize phosphorylation events and kinase targets associated with early cytoskeletal reorganization after TNF-treatment, we performed a peptide array screen. Methods: HCT116 tumor cells were cultured for 6h to 72 hours in either normal or TNF- conditioned medium, and radioactive labelled lysates were hybridized to a PepChip kinase full slide array for screening the phosphorylation status of over 400 proteins at over 1400 potential phosphorylation sites. For verification, cell lysates were collected and co-immunoprecipitated. The precipitated proteins were analyzed by western blotting. We verified the subcellular localization of proteins by co-immunofluorescence. Results: Pepscan array and western blotting revealed that cofilin 1 is markedly phosphorylated on Serine 3 (pCofSer3) after 24 and 48 hours of TNF-treatment. A potential up-stream kinase of cofilin is LIMK1, a serine/threonine protein kinase known to be involved in actin cytoskeleton reorganization through phosphorylation and inactivation of cofilin 1. Interaction between LIMK/Cofilin1/pCofSer3 was verified by co-immunoprecipitation and co-immunofluorescence. F-actin, cofilin1 and LIMK colocalized in membrane ruffles and lamellipodia, consistent with a cofilin-dependent actin cytoskeletal remodelling after TNF treatment. The effects were reversed by ROCK 1 inhibition. Conclusion: Our data show that TNF-treatment in HCT116 tumor cells induces the formation of a LIMK/Cofilin1/pCofSer3 protein complex that is localized in membrane ruffles and lamellipodia, suggesting that TNF-induced cytoskeletal alterations early during apoptosis are regulated through cofilin signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4779. doi:10.1158/1538-7445.AM2011-4779

Abstract 3649: DAPK-dependent apoptosis induction after treatment of colorectal tumor cells with the histone deacetylase inhibitor panobinostat (LBH589)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Objectives: Among Histone deacetylase inhibitors (HDACi), panobinostat is a relatively new compound showing potent antitumor activities. Although the signal transduction after panobinostat treatment has been studied in various tumor cell lines, the role of the death associated protein kinase (DAPK) in panobinostat-induced apoptosis has not been investigated to date. Dependent on the stimuli, a dual role for DAPK has been shown as apoptosis inducer or pro-survival factor. The aim of this study was to characterize the role of DAPK in panobinostat-induced apoptosis using HCT116-wildtype (DAPK-wt) and HCT116-DAPK knockdown (DAPK-ko) cells. Methods: DAPK-ko stable cell line was generated using DAPK shRNA lentiviral particles (Santa Cruz, USA). DAPK-ko cells expressed only 30% of the original DAPK protein level. DAPK-wt and DAPK-ko cells were stimulated with 0.05µM panobinostat for 6, 24 or 48 h, respectively. Long term survival was studied by clonogenic assay. Anti-tumoral activity of the HDACi was assessed by immunoblotting for p21WAF-1, Ac-H3, and Ac-H4. Apoptosis induction was verified by PARP cleavage, BrdU proliferation assay, M30 cytodeath FACS, and caspase 3/7 ELISA. In vivo relevance of our findings was confirmed in a xenograft mouse model by Western Bloting and immunohistochemical analysis. Results: As expected the acetylation of histones H3 and H4 was significantly induced in both cell types. Panobinostat significantly reduced the colony formation ability (long term survival) in DAPK-wt as well as in the DAPK-ko cells. Interestingly panobinostat exposure resulted in up-regulation of DAPK in both cell lines, whereas the protein level of the cell cycle regulator p21WAF1 was increased only in the DAPK-ko cells. DAPK-ko cells were more sensitive to panobinostat treatment than the DAPK-wt cells. They showed a higher PARP cleavage, an increase in caspase 3/7 activity, and a higher number of apoptotic cells in M30 cytodeath FACS. In a subcutaneous xenograft mouse model, DAPK-ko tumors were growing slower than DAPK-wt cells. Panobinostat treatment yielded a more effective size reduction in DAPK-ko tumors. In accordance with the in vitro data, DAPK-ko tumors showed a significantly higher level of p21WAF1 protein and a reduced Ki67-proliferation index in immunohistochemistry. DAPK protein expression and Ki-67 index were significantly correlated (p=0.001). Conclusions: We have generated a suitable model for understanding DAPK-signaling as a new target for HDACi treatment in colorectal cancer cells. Our results show a clear involvement of DAPK in panobinostat-induced apoptosis. From these preliminary data we assume a pro-survival function of DAPK upon drug treatment. The association between DAPK, p21WAF1 and DNA damage response has to be further elucidated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3649.