The interferon family of cytokines is critical for promoting several physiologic processes, such as antiviral, antitumor, and immune responses. They are in clinical use for the therapy of a number of cancers, viral diseases and neurodegenerative disorders. By interacting with other cytokines IFNs form a large network of intercellular signaling molecules that control neoplastic cell growth and host defenses against pathogens. Previously, we have identified a novel IFN-regulated element and its cognate transcription factors. One such protein is CCAAT/Enhancer Binding protein-beta (C/EBP-2), a transcription factor known to regulate cell differentiation, energy metabolism, immune response, tumor growth and apoptosis. A gene expression micro- array analysis in our lab identified several IFN regulated genes, whose expression required C/EBP-2. One of them is the death associated protein kinase1 (DAPK1), an important regulator of apoptosis, cell cycle, and metastasis. The expression of dapk1 gene is frequently lost in several human cancers. DAPK1 also regulates autophagy (a novel form of death), which is critical for the removal of damaged organelle, fighting intracellular pathogens, antigen presentation and tumor suppression. Interestingly, the loss of C/EBP-2 gene in mice causes many of these defects. During the last funding period, we have shown a central role for C/EBP-2 in regulating DAPK1. In preliminary studies, we show that the expression of DAPK1 requires other transacting factors. Notably, Activating transcription factor 6 (ATF6), a key regulator of endoplasmic reticulum-dependent stress responses, appears to regulate DAPK1 expression. We propose that a direct interaction between ATF6 and C/EBP-2 leads to DAPK1 expression and growth suppression via autophagy, wherein the MAP Kinase, apoptosis- stimulating kinase 1(ASK1), provides critical signal inputs.
In specific aim 1 of this proposal we will investigate how C/EBP-2 and ATF6 collaborate to upregulate DAPK1 expression.
In specific aim 2, we will investigate how ASK1 controls DAPK1 expression via ATF6. Enhancer bound transcription factors (TFs) promote transcription using transcriptional co-activators. One of them the Mediator, a molecular bridge comprised of multiple proteins, communicates the transcriptional signals from the TFs to general transcription factor complex at the TATA-box. Studies during the last funding period also identified Med1, a major subunit of Mediator, an IFN-induced binding partner of C/EBP- 2. We present preliminary evidence for the involvement of ZIP-kinase(ZIPK), a member of the DAPK family, in regulating DAPK1 expression in response to IFNs. We hypothesize that ZIPK regulates DAPK1 expression by modulating the phosphorylation of C/EBP-binding domain of Med1. This aspect will be investigated in specific aim 3. We will evaluate the critical relevance of these factors to DAPK1 and autophagy using RNAi, knockout mice, protein-interactions, ChIP assays, mutagenesis and transcriptional analyses. Knowledge gained from an understanding these pathways, will not only define the critical regulators of DAPK1, but also will provide indicators into how a loss of DAPK1 can occur. We will investigate the relevance of these pathways to human CLL, a disease in which dapk1 appears to play an important tumor suppressive role. These in turn will allow a better design of therapeutics to combat tumor progression and metastasis.
Tumor metastasis occurs due to a loss of certain critical genes and a suppression of corresponding biological processes. Studies proposed in this application will investigate the regulation of an anti-metastatic gene, DAPK1, which is critical for tumor suppression and autophagic responses.
|Gade, Padmaja; Kimball, Amy S; DiNardo, Angela C et al. (2016) Death-associated Protein Kinase-1 Expression and Autophagy in Chronic Lymphocytic Leukemia Are Dependent on Activating Transcription Factor-6 and CCAAT/Enhancer-binding Protein-Î². J Biol Chem 291:22030-22042|
|Gade, Padmaja; Manjegowda, Srikanta B; Nallar, Shreeram C et al. (2014) Regulation of the death-associated protein kinase 1 expression and autophagy via ATF6 requires apoptosis signal-regulating kinase 1. Mol Cell Biol 34:4033-48|
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|Shanmugam, Rajasubramaniam; Gade, Padmaja; Wilson-Weekes, Annique et al. (2012) A noncanonical Flt3ITD/NF-Ã½Ã½B signaling pathway represses DAPK1 in acute myeloid leukemia. Clin Cancer Res 18:360-9|
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