Death-associated Protein Research Articles

DAPK3 inhibits gastric cancer progression via activation of ULK1-dependent autophagy.

Dysregulation of the balance between cell proliferation and cell death is a central feature of malignances. Death-associated protein kinase 3 (DAPK3) regulates programmed cell death including apoptosis and autophagy. Our previous study showed that DAPK3 downregulation was detected in more than half of gastric cancers (GCs), which was related to tumor invasion, metastasis, and poor prognosis. However, the precise molecular mechanism underlying DAPK3-mediated tumor suppression remains unclear. Here, we showed that the tumor suppressive function of DAPK3 was dependent on autophagy process. Mass spectrometry, in vitro kinase assay, and immunoprecipitation revealed that DAPK3 increased ULK1 activity by direct ULK1 phosphorylation at Ser556. ULK1 phosphorylation by DAPK3 facilitates the ULK1 complex formation, the VPS34 complex activation, and autophagy induction upon starvation. The kinase activity of DAPK3 and ULK1 Ser556 phosphorylation were required for DAPK3-modulated tumor suppression. The coordinate expression of DAPK3 with ULK1 Ser556 phosphorylation was confirmed in clinical GC samples, and this co-expression was correlated with favorable survival outcomes in patients. Collectively, these findings indicate that the tumor-suppressor roles of DAPK3 in GC are associated with autophagy and that DAPK3 is a novel autophagy regulator, which can directly phosphorylate ULK1 and activate ULK1. Thus, DAPK3 might be a promising prognostic autophagy-associated marker.

Impairment of cytokinesis by cancer-associated DAPK3 mutations

Death-associated protein kinase 3 (DAPK3), a member of the DAPK family, contributes to cytokinesis by phosphorylating myosin II regulatory light chain (MRLC). Missense mutations in DAPK3, T112M, D161N, and P216S, were observed in the lung, colon, and cervical cancers, respectively, but the effects of these mutations on cytokinesis remain unclear. Here, we show that cells expressing EGFP-DAPK3-T112M, -D161N, or -P216S exhibited reduced rates of cytokinesis, with an increased ratio of multinucleated cells. In addition, these cells exhibited reduced levels of phosphorylated MRLC at the contractile ring. Collectively, our data demonstrates that cancer-associated DAPK3 mutations impair cytokinesis by reducing phosphorylated MRLC.

MiR-98-5p protects against cerebral ischemia/reperfusion injury through anti-apoptosis and anti-oxidative stress in mice.

Cerebral ischemia/reperfusion (I/R) injury is an obstacle in treating ischemic stroke effectively. miR-98-5p has been reported to have the ability of reducing myocardial I/R injury. To explore the function of miR-98-5p in cerebral I/R, we established mice model of middle cerebral artery occlusion and reperfusion (MCAO/R). The level of miR-98-5p was found to be downregulated in serum of stroke patients and brain tissues of MCAO/R mice. Examination of brain tissues indicated that upregulating miR-98-5p level alleviated the infarction in MCAO/R mice. Moreover, the upregulation of miR-98-5p reduced reactive oxygen species production and enhanced superoxide dismutase activity in brain tissues of MCAO/R mice. These results indicating that miR-98-5p could protect against oxidative stress. Further study showed that miR-98-5p inhibited apoptosis by reducing the levels of death-associated protein kinase 1, B cell lymphoma/leukaemia-2 associated x protein and cleaved caspase-3, as well as increasing the level of B cell lymphoma/leukaemia-2. In addition, miR-98-5p was found to protect against oxidative stress through downregulating the level of BTB domain and CNC homology 1 and upregulating the levels of NAD(P)H: quinone oxidoreductase 1 and heme oxygenase 1. Therefore, miR-98-5p might be a potential target to treat cerebral I/R injury.

Knockdown of Death-Associated Protein Expression Induces Global Transcriptome Changes in Proliferating and Differentiating Muscle Satellite Cells.

Death-associated protein (DAP) undergoes substantial changes in expression during turkey skeletal muscle development, decreasing from the 18 day embryonic stage to 1 day posthatch, and again from 1 day posthatch to 16 weeks of age. These changes suggest that DAP plays an important role at critical stages of the developmental process. The objective of this study was to elucidate the role of DAP in muscle development by examining the effect of reduced DAP expression on global gene expression in proliferating and differentiating turkey pectoralis major muscle satellite cells. Small interfering RNA was used to knock down expression of DAP and the transcriptome was subsequently profiled using a turkey skeletal muscle long oligonucleotide microarray. Microarray data were corroborated using quantitative real-time PCR. In proliferating cells, 458 loci, resulting in 378 uniquely annotated genes, showed differential expression (false discovery rate, FDR < 0.05). Pathway analysis highlighted altered eukaryotic translational initiation factors (eIFs) signaling, protein ubiquitination, sirtuin signaling, and mechanistic target of rapamycin (mTOR) signaling as the primary pathways affected in the knockdown proliferating cells. The findings underpinned the potential DAP involvement in cell proliferation of turkey satellite cells through the coordination between protein synthesis and cell cycle. In differentiating cells, 270 loci, accounting for 189 unique genes, showed differential expression (FDR < 0.05). Decreased expression of genes encoding various myofibrillar proteins and proteins involved in sarcoplasmic reticulum calcium flux suggests that DAP may affect regulation of calcium homeostasis and cytoskeleton signaling. This study provides the first evidence that reduced expression of DAP significantly alters the transcriptome profile of pectoralis major muscle satellite cells, thereby reducing proliferation and differentiation.

Beyond Solvent Exclusion: i-Motif Detecting Capability and an Alternative DNA Light-Switching Mechanism in a Ruthenium(II) Polypyridyl Complex.

Cytosine-rich DNA can fold into secondary structures known as i-motifs. Mounting experimental evidence suggests that these non-canonical nucleic acid structures form in vivo and play biological roles. However, to date, there are no optical probes able to identify i-motif in the presence of other types of DNA. Herein, we report for the first time the interactions between the three isomers of [Ru(bqp)2]2+ with i-motif, G-quadruplex, and double-stranded DNA. Each isomer has vastly different light-switching properties: mer is "on", trans is "off", and cis switches from "off" to "on" in the presence of all types of DNA. Using emission lifetime measurements, we show the potential of cis to light up and identify i-motif, even when other DNA structures are present using a sequence from the promoter region of the death-associated protein (DAP). Moreover, separated cis enantiomers revealed Λ-cis to have a preference for the i-motif, whereas Δ-cis has a preference for double-helical DNA. Finally, we propose a previously unreported light-switching mechanism that originates from steric compression and electronic effects in a tight binding site, as opposed to solvent exclusion. Our work suggests that many published non-emissive Ru complexes could potentially switch on in the presence biological targets with suitable binding sites, opening up a plethora of opportunity in the detection of biological molecules.

5′-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5' UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5' UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5' UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

DAP1 negatively regulates autophagy induced by cardamonin in SKOV3 cells.

Autophagy is closely related to the formation and development of multiple human tumors including ovarian cancer. As a major regulator of this process, the role of mTOR (mammalian target of rapamycin) has been well proven. Cardamonin, a kind of flavonoid from plants, has effects on induction of autophagy and thus antiproliferation of cancer cells. However, the detailed mechanism remains unclear. DAP1 (death-associated protein 1) is a proline-rich protein, which is involved in the regulation of cellular growth and programmed cell death including autophagy and apoptosis. The aim of this study was to investigate whether DAP1 is involved in proliferation inhibition and autophagy induced by cardamonin in tumor cells. Using online bioinformatics tools, we found that DAP1 expression is closely related to the survival of patients with ovarian cancer. Our study showed that autophagy induced by cardamonin was associated with mTOR inhibition, and DAP1 was involved in this process. Silence of DAP1 decreased cell proliferation but enhanced the antiproliferative effect of cardamonin in SKOV3 cells. The level of autophagy was elevated by DAP1 silencing in SKOV3 cells. Notably, cardamonin showed higher autophagy flux in the DAP1 small interfering RNA group. Taken together, our results implied that DAP1 negatively regulates autophagy induced by cardamonin, and it may be a potential target for ovarian cancer therapy.

Death-associated Protein Kinase 1 Mediates Ventilator-induced Lung Injury in Mice by Promoting Alveolar Epithelial Cell Apoptosis

Alveolar epithelial cell apoptosis is implicated in the onset of ventilator-induced lung injury. Death-associated protein kinase 1 (DAPK1) is associated with cell apoptosis. The hypothesis was that DAPK1 participates in ventilator-induced lung injury through promoting alveolar epithelial cell apoptosis. Apoptosis of mouse alveolar epithelial cell was induced by cyclic stretch. DAPK1 expression was altered (knockdown or overexpressed) in vitro by using a small interfering RNA or a plasmid, respectively. C57/BL6 male mice (n = 6) received high tidal volume ventilation to establish a lung injury model. Adeno-associated virus transfection of short hairpin RNA and DAPK1 inhibitor repressed DAPK1 expression and activation in lungs, respectively. The primary outcomes were alveolar epithelial cell apoptosis and lung injury. Compared with the control group, the 24-h cyclic stretch group showed significantly higher alveolar epithelial cell apoptotic percentage (45 ± 4% fold vs. 6 ± 1% fold; P < 0.0001) and relative DAPK1 expression, and this group also demonstrated a reduced apoptotic percentage after DAPK1 knockdown (27 ± 5% fold vs. 53 ± 8% fold; P < 0.0001). A promoted apoptotic percentage in DAPK1 overexpression was observed without stretching (49 ± 6% fold vs. 14 ± 3% fold; P < 0.0001). Alterations in B-cell lymphoma 2 and B-cell lymphoma 2-associated X are associated with DAPK1 expression. The mice subjected to high tidal volume had higher DAPK1 expression and alveolar epithelial cell apoptotic percentage in lungs compared with the low tidal volume group (43 ± 6% fold vs. 4 ± 2% fold; P < 0.0001). Inhibition of DAPK1 through adeno-associated virus infection or DAPK1 inhibitor treatment appeared to be protective against lung injury with reduced lung injury score, resolved pulmonary inflammation, and repressed alveolar epithelial cell apoptotic percentage (47 ± 4% fold and 48 ± 6% fold; 35 ± 5% fold and 34 ± 4% fold; P < 0.0001, respectively). DAPK1 promotes the onset of ventilator-induced lung injury by triggering alveolar epithelial cell apoptosis through intrinsic apoptosis pathway in mice.

2,3′,4,4′,5-Pentachlorobiphenyl Induced Thyrocyte Autophagy by Promoting Calcium Influx via Store-Operated Ca2+ Entry

PCB118, a 2,3',4,4',5-pentachlorobiphenyl, has been shown to destroy thyroidal ultrastructure and induce thyrocyte autophagy. Previously, we reported that PCB118 promoted autophagosome formation in vivo and in vitro, but more details remain to be revealed. To explore the underlying mechanism by which PCB118 regulates thyrocyte autophagy, Fischer rat thyroid cell line-5 (FRTL-5) cells were exposed to different doses of PCB118 at 0, 0.25, 2.5, and 25 nM for 0-48 h. Western blot analysis of autophagy-related proteins P62, BECLIN1, and LC3 demonstrated that PCB118 induced autophagy formation in dose- and time-dependent manner. Moreover, laser scanning confocal microscopy and flow cytometry showed PCB118 treatment led to time- and dose-dependent increase in intracellular calcium concentration ([Ca2+]i). Additionally, PCB118 promoted store-operated Ca2+ entry (SOCE) channel followed by significant increase of ORAI1 and STIM1 protein levels. Onthe other hand, PCB118 induced thyroidal autophagy via class III β-tubulin (TUBB3)/death-associated protein kinase 2(DAPK2)/myosin regulatory light chain (MRLC)/autophagy-related 9A (ATG9A) pathway in FRTL-5 cells. Pretreatment with SOCE inhibitor SKF96365 reduced cytosolic Ca2+, ORAI1, STIM1, and BECLIN1 levels as well as LC3 II/LC3 I ratio, while increased P62 expression. SKF96365 also inhibited TUBB3/DAPK2/MRLC/ATG9A pathway in FRTL-5 cells treated by PCB118. Our results provide evidence that PCB118 may induce thyroidal autophagy through TUBB3-related signaling pathway, and these effects are likely to be regulated by calcium influx via SOCE channel.

Death-associated Protein Kinase 1 Phosphorylates α-Synuclein at Ser129 and Exacerbates Rotenone-induced Toxic Aggregation of α-Synuclein in Dopaminergic SH-SY5Y Cells.

The formation of Lewy bodies (LBs), intracellular filamentous inclusions, is one of the hallmarks of Parkinson's disease (PD). α-Synuclein is the main component of LBs and its abnormal accumulation contributes to the pathogenesis of PD. Direct phosphorylation of α-synuclein at multiple Ser/Tyr residues is known to induce its aggregation, consequently promoting LB formation. Death-associated protein kinase 1 (DAPK1), originally identified as a positive mediator of γ-interferon-induced programmed cell death, possesses tumor-suppressive activity and mediates a wide range of cellular processes, including apoptosis and autophagy. Accumulating evidence suggests that DAPK1 is also associated with neuronal cell death and neurodegeneration. For example, DAPK1 phosphorylates tau and amyloid precursor protein, and induces tau aggregation and amyloid β production, respectively, in Alzheimer’s disease. DAPK1 is also accumulated to a larger extent in a mouse model of PD, causing synucleinopathy and dopaminergic neuron degeneration. In this study, we attempted to determine whether DAPK1 phosphorylates α-synuclein and affects cell viability in human dopaminergic neuroblastoma SH-SY5Y cells. We demonstrated that DAPK1 directly phosphorylates α-synuclein at Ser129, and induces the formation of insoluble α-synuclein aggregates. We also showed that DAPK1 enhances rotenone-induced aggregation of α-synuclein, potentiating neuronal cell death. Taken together, these findings suggest that DAPK1 acts as a novel regulator of toxic α-synuclein aggregation, possibly affecting and playing a role in the development of PD.

Death-associated protein kinase 1 suppresses hepatocellular carcinoma cell migration and invasion by upregulation of DEAD-box helicase 20.

Death‐associated protein kinase 1 (DAPK) is a calcium/calmodulin kinase that plays a vital role as a suppressor gene in various cancers. Yet its role and target gene independent of p53 is still unknown in hepatocellular carcinoma (HCC). In this study, we discovered that DAPK suppressed HCC cell migration and invasion instead of proliferation or colony formation. Using a proteomics approach, we identified DEAD‐box helicase 20 (DDX20) as an important downstream target of DAPK in HCC cells and critical for DAPK‐mediated inhibition of HCC cell migration and invasion. Using integrin inhibitor RGD and GTPase activity assays, we discovered that DDX20 suppressed HCC cell migration and invasion through the CDC42‐integrin pathway, which was previously reported as an important downstream pathway of DAPK in cancer. Further research using cycloheximide found that DAPK attenuates the proteasomal degradation of DDX20 protein, which is dependent on the kinase activity of DAPK. Our results shed light on new functions and regulation for both DAPK and DDX20 in carcinogenesis and identifies new potential therapeutic targets for HCC.

Correction: Elbadawy, M., et al. Novel Functions of Death-Associated Protein Kinases through Mitogen-Activated Protein Kinase-Related Signals. Int. J. Mol. Sci. 2018, 19, 3031.

The authors wish to make the following corrections to this paper [...].

Suppression of adenosine-to-inosine (A-to-I) RNA editome by death associated protein 3 (DAP3) promotes cancer progression.

RNA editing introduces nucleotide changes in RNA sequences. Recent studies have reported that aberrant A-to-I RNA editing profiles are implicated in cancers. Albeit changes in expression and activity of ADAR genes are thought to have been responsible for the dysregulated RNA editome in diseases, they are not always correlated, indicating the involvement of secondary regulators. Here, we uncover DAP3 as a potent repressor of editing and a strong oncogene in cancer. DAP3 mainly interacts with the deaminase domain of ADAR2 and represses editing via disrupting association of ADAR2 with its target transcripts. PDZD7, an exemplary DAP3-repressed editing target, undergoes a protein recoding editing at stop codon [Stop →Trp (W)]. Because of editing suppression by DAP3, the unedited PDZD7WT, which is more tumorigenic than edited PDZD7Stop518W, is accumulated in tumors. In sum, cancer cells may acquire malignant properties for their survival advantage through suppressing RNA editome by DAP3.

Destablilization of TRAF6 by DRAK1 Suppresses Tumor Growth and Metastasis in Cervical Cancer Cells.

The adaptor protein TNF receptor-associated factor 6 (TRAF6) is a key mediator in inflammation. However, the molecular mechanisms controlling its activity and stability in cancer progression remain unclear. Here we show that death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) inhibits the proinflammatory signaling pathway by targeting TRAF6 for degradation, thereby suppressing inflammatory signaling-mediated tumor growth and metastasis in advanced cervical cancer cells. DRAK1 bound directly to the TRAF domain of TRAF6, preventing its autoubiquitination by interfering with homo-oligomerization, eventually leading to autophagy-mediated degradation of TRAF6. Depletion of DRAK1 in cervical cancer cells resulted in markedly increased levels of TRAF6 protein, promoting activation of the IL1β signaling-associated pathway and proinflammatory cytokine production. DRAK1 was specifically underexpressed in metastatic cervical cancers and inversely correlated with TRAF6 expression in mouse xenograft model tumor tissues and human cervical tumor tissues. Collectively, our findings highlight DRAK1 as a novel antagonist of inflammation targeting TRAF6 for degradation that limits inflammatory signaling-mediated progression of advanced cervical cancer. SIGNIFICANCE: Serine/threonine kinase DRAK1 serves a unique role as a novel negative regulator of the inflammatory signaling mediator TRAF6 in cervical cancer progression.

Meta-analysis: Assocation between promoter hypermethylation of DAPK (Death-Associated Protein Kinase) and cervical cancer

Purpose: Death-associated protein kinase (DAPK or DAPK1) is an important tumor suppressor protein that involved in the regulation of cell activities. The aberrant methylation of DAPK promoter has been reported in patients with cervical cancer. However, the association between of DAPK1 and cervical cancer was not always unification, in previous studies. Therefore, in current study, a meta-analysis was performed for association of between DAPK gene’s promoter hypermethylated and cervical cancer.&#x0D; Methods: A systematic literature analysis was conducted based on the previous studies published in PubMed, PubMed Central (NCBI), Google by using following keywords: cervical cancer, cervical carcinoma, Methylation, by the end of January, 2018. The association between DAPK promoter methylation and cervical cancer was evaluated by odds ratio (ORs) and 95% confidence intervals (CI). To evaluate the potential sources of heterogeneity, the meta-regression analysis and subgroup analysis were conducted.&#x0D; Results: A total of 21 case-control studies which relevant to the association between DAPK1 gene’s promoter methylation frequency and cervical cancer, that including 1600 cancer cases and 1011 control cases (non-cancerous cases). The analysis results indicated that the characteristic of candidate gene’s promoter methylation increased the cervical cancer risk through the calculation of OR value (OR = 21.25; 95% CI = 8.73 – 52.97; p &lt; 0.001; Random effect model). The association between DAPK1 gene’s promoter hypermethylation was confirmed in all the subgroups analyses, including materials and assays methods, ethnicity. Furthermore, this association is higher in cervical squamous cell carcinoma than cervical adenocarcinoma and is a characteristic of late-stage disease.&#x0D; Conclusion: The hypermethylated DAPK1 gene’s promoter was also one of etiological factor, lead to the cervical tumorigenesis.

Diagnostic significance of DNA methylation of PTEN and DAPK in thyroid tumors.

DNA Methylation of the tumour suppressor gene leading to gene silencing plays an important role in thyroid tumour development. The purpose was to determine the DNA methylation status of phosphatase and tensin homolog (PTEN) and death-associated protein kinase (DAPK) in patients with thyroid nodules and to explore whether they can be used as molecular diagnostic tools to differentiate benign and malignant thyroid nodules. Thyroid tissue and blood samples were obtained from normal healthy individuals (controls) and patients suffering from clinically diagnosed thyroid nodular disease [papillary thyroid carcinoma (PTC), adenoma and nodular goitre]. DNA methylation level, mRNA expression and protein expression of PTEN and DAPK in the thyroid tissues and peripheral blood were detected using methylation-specific PCR, semi-quantitative reverse transcription PCR and Western blot, respectively. Diagnostic sensitivity, specific and accuracy of detection were evaluated between blood and thyroid tissue. There was a significant increase in the level of DNA methylation of PTEN and DAPK in PTC (P<.05) compared with controls and other types of thyroid nodules. Levels of the mRNA of both PTEN and DAPK were lower in PTC in both peripheral blood and tissue samples compared with controls, while there was concomitant decrease of both PTEN and DAPK protein expression in PTC tissues (P<.05). There was no significant difference in diagnostic specificity, sensitivity and accuracy between blood sample and thyroid tissues. Hypermethylated status of both PTEN and DAPK in peripheral blood and tissue samples can be useful biomarkers for clinical diagnosis and, distinguishing of benign and malignant thyroid nodules.

Loss of death-associated protein kinase 1 in human bone marrow mesenchymal stem cells decreases immunosuppression of CD4+ T cells.

ObjectiveTo explore the roles of human mesenchymal stem cell (hMSC) death-associated protein kinase 1 (DAPK1) in modulating CD4+ T lymphocyte proliferation.MethodsHuman MSCs and peripheral blood mononuclear cells were isolated and cocultured in vitro for 3 days. Lentiviral-mediated RNA interference (LV-sh-DAPK1) was used to silence DAPK1 expression in hMSCs. Expression of DAPK1 was assessed by western blotting. Transcriptional levels of DAPK1, transforming growth factor-β1, indoleamine 2,3-dioxygenase, inducible nitric oxide synthase, interleukin (IL)-6, suppressor of cytokine signaling 1, IL-10 and cyclooxygenase-2 were investigated by quantitative PCR. Levels of IL-10 were assessed by ELISA. Proliferation of CD4+ T cells was assessed by flow cytometry.ResultsDAPK1 was abundantly expressed in ex vivo-expanded hMSCs and expression was positively correlated with hMSC suppression of CD4+ T cell proliferation. Silencing of DAPK1 in hMSCs reduced the ability of these cells to inhibit CD4+ T cell proliferation and resulted in decreased IL-10 levels compared with untreated controls. Exogenous supplementation with recombinant human IL-10 in DAPK1-silenced hMSCs restored immunosuppression of CD4+ T cells.ConclusionsThe DAPK1-IL-10 axis mediates a novel immunoregulatory function of hMSCs toward CD4+ T cells.

Melatonin directly binds and inhibits death-associated protein kinase 1 function in Alzheimer's disease.

Death-associated protein kinase 1 (DAPK1) is upregulated in the brains of human Alzheimer's disease (AD) patients compared with normal subjects, and aberrant DAPK1 regulation is implicated in the development of AD. However,little is known about whether and how DAPK1 function is regulated in AD. Here, we identified melatonin as a critical regulator of DAPK1 levels and function. Melatonin significantly decreases DAPK1 expression in a post-transcriptional manner in neuronal cell lines and mouse primary cortical neurons. Moreover, melatonin directly binds to DAPK1 and promotes its ubiquitination, resulting in increased DAPK1 protein degradation through a proteasome-dependent pathway. Furthermore, in tau-overexpressing mouse brain slices, melatonin treatment and the inhibition of DAPK1 kinase activity synergistically decrease tau phosphorylation at multiple sites related to AD. In addition, melatonin and DAPK1 inhibitor dramatically accelerate neurite outgrowth and increase the assembly of microtubules. Mechanistically, melatonin-mediated DAPK1 degradation increases the activity ofPin1,aprolyl isomerase known to play a protective role against tau hyperphosphorylation and tau-related pathologies. Finally, elevated DAPK1 expression shows a strong correlation with the decrease in melatonin levels in human AD brains. Combined, these results suggest that DAPK1 regulation by melatonin is a novel mechanism that controls tau phosphorylation and function and offers new therapeutic options for treating human AD.

Prospecting for Breast Cancer Blood Biomarkers: Death-Associated Protein Kinase 1 (DAPK1) as a Potential Candidate.

Background Breast cancer is the commonest malignancy in women worldwide. It is estimated to affect approximately 1.5 million women annually and responsible for the greatest number of cancer-related mortalities among women. In 2018, breast cancer mortalities stood at 627,000 women representing approximately 15% of all cancer deaths among women. In Ghana, breast cancer is the second leading cause of cancer deaths, with an incidence of 2,900 cases annually; one of eight women with the disease die. This gives impetus to the fight for improved early detection, treatment, and/management. In this light, we investigated the potential of death-associated protein kinase 1 (DAPK1) as a biomarker for breast cancer. As a tumour suppressor, its expression is activated by several carcinogens to influence cellular pathways that result in apoptosis, autophagy, immune response, and proliferation. Aim To investigate DAPK1 as a blood biomarker for breast cancer. Methods Blood samples of participants diagnosed with breast cancer and healthy controls were collected and processed to obtain serum. Information on age, treatment, diagnosis, and pathology numbers was retrieved from folders. Pathology numbers were used to retrieve breast tissue blocks of patients at the Department of Pathology of the KBTH. Tissue blocks were sectioned and immunohistochemically stained with anti-DAPK1 and counterstained with hematoxylin to determine the DAPK1 expression levels. DAKP1 levels in blood sera were quantified using a commercial anti-DAPK1 ELISA kit. Case and control group means were compared using one-way ANOVA and Chi-square test. Statistical significance was set at p ≤ 0.05. Results and Discussion. DAPK1 levels were higher in sera and breast tissues of breast cancer patients than controls. The augmented DAPK1 expression can be interpreted as a stress response survival mechanism to remediate ongoing deleterious events in the cells orchestrated by carcinogenesis. In the presence of abundant DAPK1, the proliferative power of cells (both cancerous and noncancerous) is increased. This may explain why high DAPK1 expression strongly associates with aggressive breast cancer phenotypes like the ER-negative breast cancers, especially the triple-negative breast cancers (TNBC) which are the most aggressive, fast-growing, and highly metastatic. Conclusion DAPK1 is highly expressed in sera and breast tissues of breast cancer patients than nonbreast cancer participants. The elevated expression of DAKP1 in circulation rather than in breast tissues makes it a candidate for use as a blood biomarker and potential use as therapeutic target in drug development.

SAT-179 Increased Telomere Length in Adrenocortical Tumors Is Associated with Abnormal Expression of Chromatin Remodelling Factors

Background: The pathogenesis of adrenocortical tumors (ACTs) in the pediatric population is partially known, and few prognostic factors have been identified in this age group. Recently, ATRX and DAXX have been implicated in the pathogenesis and prognosis of a variety of cancers. Their altered function has been shown to affect telomere length through a telomerase-independent mechanism. Objective: To investigate ATRX and DAXX gene expression, ATRX and DAXX protein expression, and telomere length, as well as their clinical significance, in ACT samples from pediatric patients. Methods: The records of 110 pediatric patients with available ACT samples were reviewed. ATRX, DAXX, TERT and TERC gene expression was assessed by qPCR (n = 100 ACTs; n = 12 normal adrenals). ATRX and DAXX protein expression was assessed by IHC (n = 45 ACTs). Telomere length was assessed by qPCR (n = 64 ACTs). For survival analysis, Kaplan-Meier curves were obtained. For association analysis, simple linear regression models were adjusted. Results: Most patients were female (70.9%) and harbored germline TP53 mutations (90.2%). Median age at diagnosis was 21.1 months (2.1 – 199). Younger patients (< 3 years) had better survival (p < 0.01), while those with metastasis at diagnosis and carcinomas (classified by the Wieneke score) had worse survival (p < 0.01). ATRX gene expression was decreased (p < 0.01), while DAXX gene expression was increased (p < 0.01) in ACTs, compared to normal adrenals. ATRX gene expression was even lower in the context of the germline TP53 (R337H) mutation (p < 0.01). TERT expression was not detected in ACTs or normal adrenals, and TERC expression was not altered (p = 0.69). ATRX protein expression was lost in the majority of ACTs (95.6%), while DAXX was lost in a minority (21.1%). There was no association between gene or protein expression and disease-free or overall survival. There was a significant association between decreased ATRX and DAXX gene expression and increased telomere length (p < 0.01 and p = 0.03, respectively). Conclusion: In pediatric ACTs, decreased ATRX and DAXX gene expression was associated with increased telomere length, independently of TERT or TERC expression. In these tumors, ATRX gene expression was decreased and ATRX protein expression was overall lost, while DAXX gene expression was increased and DAXX protein expression was overall retained. No significant association between these alterations and prognosis was found in this cohort. These findings suggest that ATRX and DAXX altered function may be more involved in the pathogenesis of pediatric ACTs than in the prognosis of the affected patients.