Expression Of DEC1 Research Articles

Abstract B057: The circadian master regulator BMAL1 blocks immune cell recognition of pancreatic ductal adenocarcinomas via tumor-derived interleukin-1 receptor antagonist

Abstract Many tumor types, including pancreatic adenocarcinomas, have dysregulated circadian rhythms. Previous work in our lab has shown that disseminated dormant tumor cells have increased expression of Dec2, a circadian rhythm gene, and knocking out Dec2 leads to increased overall survival in our murine resectable PDAC model. To see whether the effect of Dec2 is due to a circadian effect, we deleted the master regulator circadian gene, BMAL1. Using CRISPR-generated knockout (KO) cell lines, we saw that loss of Bmal1 completely abrogated metastasis in immunocompetent mice, but not in immunocompromised mice indicating an immune mediated mechanism. Similarly in our murine resectable PDAC model, loss of BMAL1 led to an increase in overall survival in immunocompetent mice only. Looking at the tumor microenvironment, we saw an increase in CD4+ and CD8+ T cell infiltration in the tumors derived from the BMAL KO cells. We then evaluated the BMAL KO cells for their cytokine secretion and saw that loss of BMAL1 led to decreased interleukin-1 receptor antagonist both in vitro and in vivo. IL-Ra has been shown in literature to suppress the immunogenic anti-tumor response by impairing the activities of APCs, CD4+ and CD8+ T cells and disruption of IL-1Ra can reverse the TME towards more immunogenic leading to an antitumor effect. Taken together these studies implicate Bmal1 in regulating anti-tumor immunity in PDAC through its regulation of IL-1Ra secretion. This is a novel function for a circadian rhythm gene in PDAC biology. Citation Format: Orjola Prela, Lan Wang, Chris Harris, Darren Carpizo. The circadian master regulator BMAL1 blocks immune cell recognition of pancreatic ductal adenocarcinomas via tumor-derived interleukin-1 receptor antagonist [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B057.

Exposure to BaA inhibits trophoblast cell invasion and induces miscarriage by regulating the DEC1/ARHGAP5 axis and promoting ubiquitination-mediated degradation of MMP2

Benz[a]anthracene (BaA), a hazardous polycyclic aromatic hydrocarbon classified by the EPA, is a probable reproductive toxicant. Epidemiological studies suggest that BaA exposure may be a risk factor for recurrent miscarriage (RM). However, the underlying mechanisms are not well understood. This study identified DEC1 as a key gene through RNA-seq and single-cell RNA sequencing analysis. DEC1 expression was found to be downregulated in villous tissues from women with RM and in primary extravillous trophoblasts (EVTs) exposed to BaA. BaA suppressed DEC1 expression by promoting abnormal methylation patterns. Further analysis revealed that ARHGAP5 is a direct target of DEC1 in EVTs, where DEC1 inhibits trophoblast invasion by directly regulating ARHGAP5 transcription. Additionally, BaA destabilized matrix metalloproteinase 2 (MMP2) by activating the aryl hydrocarbon receptor (AhR) and promoting E3 ubiquitin ligase MID1-mediated degradation. In a mouse model, BaA induced miscarriage by modulating the DEC1/ARHGAP5 and MID1/MMP2 axes. Notably, BaA-induced miscarriage in mice was prevented by DEC1 overexpression or MID1 knockdown. These findings indicate that BaA exposure leads to miscarriage by suppressing the DEC1/ARHGAP5 pathway and enhancing the MID1/MMP2 pathway in human EVTs.

Novel role of NCoR1 in impairing spatial memory through the mediation of a novel interacting protein DEC2

Long-term memory formation requires de novo RNA and protein synthesis. Using differential display PCR, we found that the NCoR1 cDNA fragment is differentially expressed between fast learners and slow learners, with fast learners showing a lower expression level than slow learners in the water maze learning task. Fast learners also show lower NCoR1 mRNA and protein expression levels. In addition, spatial training decreases both NCoR1 mRNA and protein expression, whereas NCoR1 conditional knockout (cKO) mice show enhanced spatial memory. In studying the molecular mechanism, we found that spatial training decreases the association between NCoR1 and DEC2. Both NCoR1 and DEC2 suppress the expression of BDNF, integrin α3 and SGK1 through C/EBPα binding to their DNA promoters, but overexpression of DEC2 in NCoR1 cKO mice rescues the decreased expression of these proteins compared with NCoR1 loxP mice overexpressing DEC2. Further, spatial training decreases DEC2 expression. Spatial training also enhances C/EBPα binding to Bdnf, Itga3 and Sgk1 promoters, an effect also observed in fast learners, and both NCoR1 and DEC2 control C/EBPα activity. Whereas knockdown of BDNF, integrin α3 or SGK1 expression impairs spatial learning and memory, it does not affect Y-maze performance, suggesting that BDNF, integrin α3 and SGK1 are involved in long-term memory formation, but not short-term memory formation. Moreover, NCoR1 expression is regulated by the JNK/c-Jun signaling pathway. Collectively, our findings identify DEC2 as a novel interacting protein of NCoR1 and elucidate the novel roles and mechanisms of NCoR1 and DEC2 in negative regulation of spatial memory formation.

Abstract C033: The circadian rhythm gene Dec2 promotes pancreatic cancer dormancy by facilitating immune evasion

Abstract Metastatic recurrence following curative-intent pancreatic cancer surgery is a major clinical problem as nearly all die of this at both early and latent periods following surgery. Latent recurrences are due to reactivation of dormant tumor cells that disseminate before the primary tumor has been removed. The mechanisms that drive cancer dormancy are poorly understood in part because of the lack of animal models that recapitulate the biology of the resected patient. Here we introduce a novel mouse model of pancreatic cancer dormancy in which we establish distal pancreatic tumors by orthotopic injection, resect them four weeks later and then follow the mice for recurrence. We observed recurrence patterns and survival outcomes that mimic human patients undergoing surgery for pancreatic cancer where two thirds of the mice succumb to early metastatic recurrence (median survival 26 days) and one third of the mice (called dormant mice) live substantially long (median survival 554 days) without clinical evidence of disease yet harbor disseminated tumor cells in most organs of the body. Disseminated tumor cells isolated from the livers of dormant mice were quiescent, exhibited stem cell properties and upregulated the expression of Dec2, a transcriptional repressor previously best known for its importance in maintaining circadian rhythms. Dec2 enabled the dormant tumor cells to evade the immune system by reducing the amount of MHC1 on their surfaces, and thereby lowered the number of CD8+ T cells within the tumor microenvironment. Knocking out Dec2 significantly improved the overall survival of immunocompetent mice and increased CD8+ T cell tumor infiltration. The decrease in cell surface MHCI was likely due to impaired surface localization of the protein. RNA-seq analysis demonstrated a loss of antigen presentation pathway genes in Dec2 WT cells, particularly in genes encoding subunits of the proteasome. Proper localization of MHC1 is known to depend upon proteasome-generated peptides. By cut-and-run analysis, Dec2 bound directly to the promoters of a number of proteasomal genes, lowered expression of these genes, and decreased proteasomal activity. These studies describe a new mechanism for Dec2 in regulating cancer dormancy through its downregulation of proteasomal activity which in turn converts a hot to a cold TME by decreasing cell surface MHC1. Citation Format: Lan Wang, Orjola Prela, Ching-Hua Shih, Juliana Cazarin de Menezes, Brian Altman, Paula Vertino, Chris Harris, Darren Carpizo. The circadian rhythm gene Dec2 promotes pancreatic cancer dormancy by facilitating immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C033.

THU498 Establishing The Link Between Glucocorticoid Signaling And The Circadian Regulator DEC1 In Breast Cancer

Abstract Disclosure: M.I. Castro: None. W.S. Ybañez: None. P.D. Bagamasbad: None. Epidemiological studies have linked altered circadian rhythms due to irregular shift work, chronic jet lag, and heightened night-time light exposure to increased breast cancer (BCa) pathogenesis. Dysregulated glucocorticoid (GC; CORT) signaling in BCa may further promote chronodisruption by directly regulating the cyclic expression of clock genes found in the breast. The differentiated embryo-chondrocyte expressed gene 1 (DEC1; also known as STRA13/SHARP2/ BHLHB2) is a transcription factor involved in the regulation of circadian rhythmicity and tumor progression of several cancer types including the breast and associated with hormone receptor signaling in BCa. In this study, we explored the involvement of DEC1 in the crosstalk between GC signaling and the circadian axis in BCa. To establish DEC1 expression pattern in BCa tumor samples, we assessed publicly available RNA-seq datasets from the Genotype-Tissue Expression Project and found no significant difference in DEC1 expression in tumors when compared to normal tissue controls. However, when tumor samples were stratified into molecular subtypes based on transcriptomic data from the Cancer Genome Atlas, DEC1 expression was significantly downregulated in triple-negative BCa (TNBC) in comparison to luminal subtypes. This pattern of expression was reflected in the BCa cell lines where we observed higher DEC1 expression in the estrogen receptor-positive MCF7 cells relative to nontumorigenic triple-negative MCF10A cells and the highly aggressive TNBC MDA-MB-231 cell line. To determine if DEC1 is regulated by GCs in the breast, we treated non-tumorigenic breast epithelial MCF10A cells and the TNBC MDA-MB-231 cells with increasing concentrations of CORT and observed that DEC1 mRNA expression decreases in response to increasing concentrations of CORT. Treatment of MCF10A and MDA-MB-231 cells with GC receptor (GR)- specific agonist RU486 abolished the GC-dependent repression of DEC1 expression and this pattern of DEC1 expression persisted even in the presence of the protein synthesis inhibitor cycloheximide, suggesting that DEC1 is a direct transcriptional target of GR in mammary epithelial cells. Through in silico analysis of publicly available ChIP-sequencing datasets from BCa cell lines, we identified multiple CORT-responsive cis-regulatory elements in the DEC1 locus that could mediate the CORT-dependent repression of DEC1 expression. Lentiviral knockdown of DEC1 in three breast epithelial cell lines was generated and will be used in cancer hallmark assays on proliferation, apoptosis, survival, and migration to assess the functional role of DEC1 in BCa. Taken together, our data will contribute to understanding the combined effect of GC signaling and circadian gene regulation towards BCa progression. Presentation: Thursday, June 15, 2023

Expression of circadian regulatory genes is dysregulated by increased cytokine production in mice subjected to concomitant intestinal injury and parenteral nutrition.

We have developed a mouse model of Parenteral Nutrition Associated Cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of bile acid and sterol signaling and transport. In the liver, the master circadian gene regulators Bmal/Arntl and Clock drive circadian modulation of hepatic functions, including bile acid synthesis. Once activated, Bmal and Clock are downregulated by several transcription factors including Reverbα (Nr1d1), Dbp (Dbp), Dec1/2 (Bhlhe40/41), Cry1/2 (Cry1/2) and Per1/2 (Per1/2). The aim of this study was to examine the effects of PN on expression of hepatic circadian rhythm (CR) regulatory genes in mice. WT, IL1KO or TNFRKO mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN) and the expression of key CR regulatory transcription factors evaluated. Animals were NPO on a 14 hr light-dark cycle and were administered PN continuously over 24 hrs. Mice were sacrificed, and hepatic tissue obtained at 9-10AM (Zeitgeber Z+3/Z+4 hrs). PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and total bilirubin and the effect of i.p. injection of recombinant IL-1β (200ng/mouse) or TNFα (200ng/mouse) on CR expression was examined after 4 hrs. In the PNAC model, DSS-PN increased serum biomarkers of hepatic injury (ALT, AST, serum bile acids) which was suppressed in both DSS-PN IL1KO and DSS-PN TNFRKO mice. In WT DSS-PN, mRNA expression of Arntl and Dec1 was suppressed corresponding to increased Nr1d1, Per2, Dbp and Dec2. These effects were ameliorated in both DSS-PN IL1KO and DSS-PN TNFRKO groups. Western analysis of the circadian transcription factor network revealed in WT mice DSS-PN significantly suppressed Reverbα, Bmal, Dbp, Per2 and Mtnr1b. With the exception of Dbp, DSS-PN mediated suppression was ameliorated by both IL1KO and TNFRKO. Intraperitoneal injection of IL-1β or TNFα into WT mice increased serum AST and ALT and suppressed mRNA expression of Nr1d1, Arntl and Clock and increased Dbp and Per2. Altered expression of CR-dependent regulatory genes during PNAC accompanies cholestasis and is, in part, due to increased cytokine (IL-1β and TNFα) production. Evaluation of the effects of modulating CR in PNAC thus deserves further investigation.

Knockdown of DEC2 expression inhibits the proliferation of mesangial cells through suppressed glycolysis and p38 MAPK/Erk pathway in lupus nephritis

BackgroundTo elucidate the mechanism by which DEC2 modulates the proliferation of mesangial cells (MCs) in lupus nephritis (LN).MethodsThe 32-week-old female Fcgr2b−/− mice and their serum-treated MCs were used as in vivo and in vitro LN model. MCs knocked down of DEC2 and overexpressed with DEC2 were also established. The expression of DEC2 was measured in the kidneys of Fcgr2b−/− mice and LN serum-treated MCs using RT-qPCR and Western blot. MCs proliferation was detected by 5-ethynyl-2′-deoxyuridine (EdU) labeling assay and PCNA expression using immunofluorescence. The glucose metabolism was evaluated in LN serum-treated MCs, and the levels of lactate production, glucose consumption, ATP production and mitochondrial membrane potential were assayed. The glycolysis and mitochondrial respiration function of the MCs were measured using the Extracellular Flux Analyzer. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were dynamically monitored and multiple important bioenergetic parameters can be calculated. The expression of Toll like receptor 4 (TLR4) and glucose transporter 1 (GLUT1) were detected in the MCs. Multiple signaling proteins were screened.ResultsDEC2 was found overexpressed in the kidney of Fcgr2b−/− LN mice. Knockdown of DEC2 inhibited LN serum-induced MCs proliferation. DEC2 was associated with the glucose metabolism in LN serum-treated MCs. DEC2 regulated glycolysis in LN serum-treated MCs. DEC2 was associated with mitochondrial fitness in LN serum treated MCs. DEC2 activated MCs glycolysis through TLR4 and glucose transporter 1 (GLUT1) regulation. DEC2 regulated MCs proliferation through two signaling pathways including dependent and independent of glycolysis, which located in the downstream of TLR4 signaling.ConclusionKnockdown of DEC2 expression inhibits the proliferation of MCs through suppressed glycolysis and p38 MAPK/ERK pathway in LN.

Ribosomal Stress Couples with the Hypoxia Response in Dec1-Dependent Orthodontic Tooth Movement.

This study characterized the effects of a deficiency of the hypoxia-responsive gene, differentiated embryonic chondrocyte gene 1 (Dec1), in attenuating the biological function of orthodontic tooth movement (OTM) and examined the roles of ribosomal proteins in the hypoxic environment during OTM. HIF-1α transgenic mice and control mice were used for hypoxic regulation of periodontal ligament (PDL) fibroblasts. Dec1 knockout (Dec1KO) and wild-type (WT) littermate C57BL/6 mice were used as in vivo models of OTM. The unstimulated contralateral side served as a control. In vitro, human PDL fibroblasts were exposed to compression forces for 2, 4, 6, 24, and 48 h. HIF-1α transgenic mice had high expression levels of Dec1, HSP105, and ribosomal proteins compared to control mice. The WT OTM mice displayed increased Dec1 expression in the PDL fibroblasts. Micro-CT analysis showed slower OTM in Dec1KO mice compared to WT mice. Increased immunostaining of ribosomal proteins was observed in WT OTM mice compared to Dec1KO OTM mice. Under hypoxia, Dec1 knockdown caused a significant suppression of ribosomal protein expression in PDL fibroblasts. These results reveal that the hypoxic environment in OTM could have implications for the functions of Dec1 and ribosomal proteins to rejuvenate periodontal tissue homeostasis.

HIF-1α contributes to metastasis in choriocarcinoma by regulating DEC1 expression.

To elucidate the underlying mechanism of HIF-1α in migration and invasion of choriocarcinoma. Cell proliferation was determined by CCK-8 assay when cell invasion was detected by transwell assay. The protein expression was detected by western blotting, immunohistochemistry, and qPCR assay. HIF-1α was shown to be strongly expressed in both clinical tumour tissues and cell lines in choriocarcinoma. When HIF-1α was efficiently knocked down in JEG3 cells, the proliferation rate was reduced by approximately 50% and the number of cells that migrated through the transwell insert was greatly decreased. The cell invasion rate was also significantly reduced. Moreover, typical markers of epithelial-mesenchymal transition such as E-cadherin, were increased, while vimentin and α-SMA were decreased after HIF-1α knockdown. In contrast, overexpression of DEC1 reversed the effects of HIF-1α knockdown. Cell proliferation, migration, and invasion were partially recovered. The level of E-cadherin was decreased, while the level of vimentin and α-SMA was increased. In addition, the level of β-catenin and LEF1 was downregulated after HIF-1α knockdown. The expression of MMP2 and MMP9 also declined. However, overexpression of DEC1 after HIF-1α knockdown partially reversed the expression pattern of these molecules. HIF-1α contributed to EMT and metastasis through activation of canonical β-catenin signalling in choriocarcinoma and this process was dependent on DEC1. This study provides a new mechanism of HIF-1α in choriocarcinoma and suggests that intervention with DEC1 might be a promising therapeutic choice for choriocarcinoma.

Obstructive sleep apnea-increased DEC1 regulates systemic inflammation and oxidative stress that promotes development of pulmonary arterial hypertension.

Obstructive sleep apnea (OSA), characterized by chronic intermittent hypoxia (CIH), is a common risk factor for pulmonary arterial hypertension (PAH). As a hypoxia-induced transcription factor, differentially expressed in chondrocytes (DEC1) negatively regulates the transcription of peroxisome proliferative activated receptor-γ (PPARγ), a recognized protective factor of PAH. However, whether and how DEC1 is associated with PAH pathogenesis remains unclear. In the present study, we found that DEC1 was increased in lungs and pulmonary arterial smooth muscle cells (PASMCs) of rat models of OSA-associated PAH. Oxidative indicators and inflammatory cytokines were also elevated in the blood of the rats. Similarly, hypoxia-treated PASMCs displayed enhanced DEC1 expression and reduced PPARγ expression in vitro. Functionally, DEC1 overexpression exacerbated reactive oxygen species (ROS) production and the expression of pro-inflammatory cytokines (such as TNFα, IL-1β, IL-6, and MCP-1) in PASMCs. Conversely, shRNA knockdown of Dec1 increased PPARγ expression but attenuated hypoxia-induced oxidative stress and inflammatory responses in PASMCs. Additionally, DEC1 overexpression promoted PASMC proliferation, which was drastically attenuated by a PPARγ agonist rosiglitazone. Collectively, these results suggest that hypoxia-induced DEC1 inhibits PPARγ, and that this is a predominant mechanism underpinning oxidative stress and inflammatory responses in PASMCs during PAH. DEC1 could be used as a potential target to treat PAH.

Crosstalk between microRNA-21–5p and the transcription factor Dec1 maintains osteoblast function

MicroRNAs are associated with pivotal post-transcriptional gene regulation in bone formation. Human differentiated embryonic chondrocyte expressed gene 1 (Dec1) is also involved in regulating osteoblastogenesis. In the present study, we aimed to investigate the distinctive role of miR-21–5p and Dec1 in osteoblast function and to determine their biological functions. MC3T3-E1 pre-osteoblastic cells were used for in vitro analyses. miR-21–5p knockout (KO) mice, Dec1KO mice and age-matched wild-type (WT) mice were used to characterize the influence of miR-21–5p and Dec1 deficiencies on bone formation. Morphological analyses [micro-computed tomography (micro-CT)] were performed, and measurements were collected to validate miR-21-5pKO mice. Histopathological changes in mouse femur tissues were assessed by H-E staining, Azan staining, Masson's Trichrome staining, and Toluidine Blue staining. Quantitative real-time RT-PCR, western blotting and immunohistochemical staining were used to characterize the expression levels of Alkaline Phosphatase, Runx2, Osterix, Osteopontin, Dec1 and miR-21–5p. Bioinformatics analyses and dual-luciferase reporter assays were performed to confirm Dec1 as a target of miR-21–5p. Dec1 expression was gradually increased from day 7 of osteoblast induction, while miR-21–5p showed a peak at day 21. In non-induced osteoblasts, a mechanistically gain-of-function transfection study with a miR-21–5p mimic enhanced Runx2 and Osterix expression but suppressed Dec1. miR-21-5pKO mice had reduced bone growth. Dec1-deficient mice showed advanced bone formation at the age of 12 weeks compared to WT mice. The Dec1 deficiency upregulated Runx2 and Osterix expression in Dec1KO mouse femurs. Those changes, however, were reversed in miR-21-5pKO mouse femurs compared to WT mouse femurs. Dual-luciferase reporter assays showed that Dec1 is a possible downstream target of miR-21–5p. These findings showed that the reduced osteogenic potential due to a miR-21–5p deficiency is achieved by enhanced Dec1 expression and that the miR-21–5p/Dec1 axis is involved in regulating osteoblast function.

P53 status modifies cytotoxic activity of lactoferrin under hypoxic conditions.

Lactoferrin (LF) is an iron binding glycoprotein of the transferrin family with a wide spectrum of biological effects, including anti-cancer activity. However, the detailed molecular mechanisms of anti-cancer activity of LF have not been fully determined. In this study, we tried to clarify cytotoxic functions of LF on various cell lines under hypoxic conditions and elucidate those molecular mechanisms. Cytotoxic activity of LF on cell lines was found to have a range of sensitivities. Hypoxia decreased sensitivity to LF in KD (lip fibroblast) but increased that in HSC2 (oral squamous cell carcinoma). Expression analyses further revealed that LF treatments increased hypoxic HIF-1α, -2α and p53 proteins in KD but attenuated them in HSC2 cells, and decreased HIF-1 target gene, DEC2, in KD but increased it in HSC2, suggesting a possible relationship between LF-modified DEC2 expression and HIF-α protein. MTT assay strikingly demonstrated that cells expressing mutant-type p53 (MT5) were more sensitive to LF than control HepG2 (hepatoma), suggesting an important role of the p53 signal. Knock-down of TP53 (p53 gene) interestingly reduced sensitivity to LF in HepG2, suggesting that p53 may be a target of LF cytotoxic activity. Further analyses with a ferroptosis promoter or inhibitor demonstrated that LF increased ACSL4 in hypoxic MT5, suggesting LF-induced ferroptosis in cells expressing mutant-type p53. In conclusion, hypoxia was found to regulate cytotoxic activities of LF differently among various cell lines, possibly through the p53 signaling pathway. LF further appeared to regulate ferroptosis through a modification of ACSL4 expression.

Circadian clock disruptions link oxidative stress and systemic inflammation to metabolic syndrome in obstructive sleep apnea patients.

Objectives: Obstructive sleep apnea (OSA) is an independent risk factor for metabolic syndrome (MetS). Recent studies have indicated that circadian clock genes were dysregulated in OSA. In addition, it is clear that the impairment of circadian clocks drives the progression of MetS. Therefore, we hypothesized that circadian rhythm disruption links OSA with MetS. Methods: A total of 118 participants, who underwent polysomnography (PSG) and were diagnosed as healthy snorers (control, n = 29) or OSA (n = 89) patients based on the apnea–hypopnea index (AHI), were enrolled in the present study. General information, anthropometric data, blood biochemical indicators, clock gene expressions, and levels of oxidative and inflammatory indicators were collected, determined, and compared in all the participants. Results: We found that Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Differentiated embryo chondrocyte 1 (Dec1) were upregulated, while Period 1 (Per1) was reduced in OSA patients. In addition, these changing trends were closely associated with the hypoxia indicator of AHI and have a significant impact on the presence of MetS components, such as hyperglycemia (Dec1 and Per1, p < 0.05 and 0.001, respectively), hypertension (Bmal1 and Dec1, p < 0.001 and 0.01, respectively), hyperlipidemia (Dec1, p < 0.01), and obesity (Dec1, p < 0.05). Notably, expressions of Dec1 correlated with IR and predicted the presence of MetS in OSA patients. Finally, we also observed that Dec1 expression was interrelated with levels of both oxidative indicators and inflammatory biomarkers (IL-6) in OSA. Conclusion: This study concluded that circadian clock disruptions, especially Dec1, link OSA with MetS in an oxidative and inflammatory-related manner. Circadian clock Dec1 can be used as a specific biomarker (p < 0.001) and therapeutic target in OSA combined with Mets patients.

Dec1 Deficiency Ameliorates Pulmonary Fibrosis Through the PI3K/AKT/GSK-3β/β-Catenin Integrated Signaling Pathway.

Tissue remodeling/fibrosis is a main feature of idiopathic pulmonary fibrosis (IPF), which results in the replacement of normal lung parenchyma with a collagen-rich extracellular matrix produced by fibroblasts and myofibroblasts. Epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells is a key process in IPF, which leads to fibroblasts and myofibroblasts accumulation and excessive collagen deposition. DEC1, a structurally distinct class of basic helix-loop-helix proteins, is associated with EMT in cancer. However, the functional role of DEC1 in pulmonary fibrosis (PF) remains elusive. Herein, we aimed to explore DEC1 expression in IPF and bleomycin (BLM)-induced PF in mice and the mechanisms underlying the fibrogenic effect of DEC1 in PF in vivo and in vitro by Dec1-knockout (Dec1 −/−) mice, knockdown and overexpression of DEC1 in alveolar epithelial cells (A549 cells). We found that the expression of DEC1 was increased in IPF and BLM-injured mice. More importantly, Dec1 −/− mice had reduced PF after BLM challenge. Additionally, DEC1 deficiency relieved EMT development and repressed the PI3K/AKT/GSK-3β/β-catenin integrated signaling pathway in mice and in A549 cells, whereas DEC1 overexpression in vitro had converse effects. Moreover, the PI3K/AKT and Wnt/β-catenin signaling inhibitors, LY294002 and XAV-939, ameliorated BLM-meditated PF in vivo and relieved EMT in vivo and in vitro. These pathways are interconnected by the GSK-3β phosphorylation status. Our findings indicated that during PF progression, DEC1 played a key role in EMT via the PI3K/AKT/GSK-3β/β-catenin integrated signaling pathway. Consequently, targeting DEC1 may be a potential novel therapeutic approach for IPF.

Endothelial Dec1-PPARγ Axis Impairs Proliferation and Apoptosis Homeostasis Under Hypoxia in Pulmonary Arterial Hypertension.

Background: The hypoxia-induced pro-proliferative and anti-apoptotic characteristics of pulmonary arterial endothelial cells (PAECs) play critical roles in pulmonary vascular remodeling and contribute to hypoxic pulmonary arterial hypertension (PAH) pathogenesis. However, the mechanism underlying this hypoxic disease has not been fully elucidated.Methods: Bioinformatics was adopted to screen out the key hypoxia-related genes in PAH. Gain- and loss-function assays were then performed to test the identified hypoxic pathways in vitro. Human PAECs were cultured under hypoxic (3% O2) or normoxic (21% O2) conditions. Hypoxia-induced changes in apoptosis and proliferation were determined by flow cytometry and Ki-67 immunofluorescence staining, respectively. Survival of the hypoxic cells was estimated by cell counting kit-8 assay. Expression alterations of the target hypoxia-related genes, cell cycle regulators, and apoptosis factors were investigated by Western blot.Results: According to the Gene Expression Omnibus dataset (GSE84538), differentiated embryo chondrocyte expressed gene 1-peroxisome proliferative-activated receptor-γ (Dec1-PPARγ) axis was defined as a key hypoxia-related signaling in PAH. A negative correlation was observed between Dec1 and PPARγ expression in patients with hypoxic PAH. In vitro observations revealed an increased proliferation and a decreased apoptosis in PAECs under hypoxia. Furthermore, hypoxic PAECs exhibited remarkable upregulation of Dec1 and downregulation of PPARγ. Dec1 was confirmed to be crucial for the imbalance of proliferation and apoptosis in hypoxic PAECs. Furthermore, the pro-surviving effect of hypoxic Dec1 was mediated through PPARγ inhibition.Conclusion: For the first time, Dec1-PPARγ axis was identified as a key determinant hypoxia-modifying signaling that is necessary for the imbalance between proliferation and apoptosis of PAECs. These novel endothelial signal transduction events may offer new diagnostic and therapeutic options for patients with hypoxic PAH.

Abstract 121: A genetic signature of cancer dormancy: implications for human disease derived from a novel murine model

Abstract Pancreatic cancer (PC) is a highly lethal malignancy characterized by local tumor aggressiveness and early metastatic dissemination. While surgical resection is used to treat localized cancers, the majority of patients do recur suggesting the presence of disseminated tumor cells (DTC) at the time of surgery. These DTC's represent minimal residual disease. While most patients recur early, a subset recur late due to the reactivation of dormant tumor cells. Currently, the mechanisms of cancer dormancy are poorly understood in part due to a lack of animal models that reflect human disease. Here we describe a murine model of PC dormancy that mimics outcomes in resected human patients. Using single-cell transcriptomics and an assay for chromatin accessibility, we found dormancy is a distinct cellular state from pre or post-dormant cancer cells. Mechanisms of dormancy include increased expression of transcriptional repressor Dec2, which functionally drives quiescence, and monoallelic suppression of mutant KRAS by DNA methylation. Pathway analysis of dormant tumor cell transcriptomics indicated an up-regulation of enzymes involved in linoleic acid metabolism among others. Linoleic acid treatment of tumor cells resulted in inhibited cell proliferation, decreased tumor growth, and an increase in Dec2 expression. We have identified a dormancy gene signature that is distinct from non-dormant PC cells and found that this correlates with human resected patients with long term survivals, and inversely in patients with short term survivals. We demonstrate that dormancy is characterized by large scale transcriptomic changes and global chromatin remodeling. We developed methods for isolating DTCs from the livers of early stage PC patients undergoing resection. Using these methods we have found that the murine dormancy signature correlated with the gene expression of the DTCs and not primary tumor cells indicating this dormancy signature has human relevance with potential novel therapeutic avenues. Citation Format: Anthony S. Casabianca, Crissy Dudgeon, Chris Harris, Subhajyoto De, Mihir Shah, Arthur Roberts, Eric Collisson, Vinod Balachandran, Darren Carpizo. A genetic signature of cancer dormancy: implications for human disease derived from a novel murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 121.

The NutriClock Study Protocol - Assessing the Impact of a Chrononutrition Intervention in Patients With Cardiometabolic Disturbances

Abstract Objectives The NutriClock Study aims to test the effect of a chronotype-based nutritional intervention in circadian homeostasis and in clinical and laboratory biomarkers of dysmetabolism. Methods The NutriClock Study is a randomised, controlled, parallel trial, conducted over 10 weeks (2 weeks of baseline assessments and 8 weeks of intervention), with a 3- and 6-months follow-up. The study includes 2 groups of patients with cardiometabolic disturbances: an experimental group with mealtimes defined according to their chronotype, without caloric restriction; and a control group benefitting only from treatment as usual, with no specific nutritional intervention. The primary endpoint is insulin sensitivity (HOMA Index), and secondary endpoints include cardiometabolic clinical and analytical features (body weight and composition, serum glucose, insulin, glycated haemoglobin, lipid profile, leptin, ghrelin), inflammatory biomarkers (CRP, IL-6, adiponectin) and chronobiological features (salivary clock gene Bmal1, Clock, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, Rev-erbβ, Dec1 expression, melatonin and cortisol). Circadian oscillations of target genes will be measured through bioinformatics and mathematical analysis of rhythmicity, period and amplitude. This method will be used to determine the participants’ chronotype along with the Munich Chronotype Questionnaire and salivary cortisol and melatonin and will assist in optimizing mealtimes based on the circadian clock phenotypes. A sample size of 60 patients (30 per group) is required for a pairwise comparison with overall power of 80% to detect a true between-group difference of 20% in the primary endpoint after 8 weeks of intervention. Results Not applicable. Conclusions Chrononutrition emerges as an innovative intervention to enhance the health of patients with cardiometabolic diseases. This study will characterize circadian rhythms patterns in this population to set up an algorithm that defines pinpointed feeding intervals based on chronotype assessment. Funding Sources M. Lages holds a PhD Scholarship FCT/UIDB/05,704/2020.

Inhibition of DEC2 is necessary for exiting cell dormancy in salivary adenoid cystic carcinoma

BackgroundPatients were prone to have poor prognosis once dormant tumor cells being reactivated. However, the molecular mechanism of tumor cell dormancy remains poorly understood. This study aimed to investigate the function of DEC2 in the dormancy of salivary adenoid cystic carcinoma (SACC) in vitro and vivo.MethodsThe function of DEC2 in tumor dormancy of SACC was investigated in nude mice by establishing primary and lung metastasis model. Meanwhile, the interaction between hypoxia and SACC dormancy and the role of DEC2 were demonstrated through CoCl2 induced hypoxia–mimicking microenvironments. Furthermore, the expression of DEC2 was detected by immunohistochemical staining in primary SACC samples with and without recurrence.ResultsIn the primary SACC, DEC2 overexpression inhibited cell proliferation, increased cell population arrested in G0/G1 phase, and participated in dormancy regulation, which limited tumor growth. Intriguingly, in the model of lung metastasis, the level of DEC2 was reduced significantly and resulted in dormancy exit and growth resumption of SACC cells. Then, we found that DEC2 may associate with hypoxia in contributing to tumor dormancy, which might provide a possible cue to explain the different roles of DEC2 in primary and metastasis lesions. And overexpression of DEC2 induced dormancy and promoted migration and invasion through activating EMT program. Finally, DEC2 positive expression was shown to be significantly correlated with recurrence and dormancy of SACC patients.ConclusionsThese findings provide a novel insight into the role of DEC2 gene in tumor dormancy and metastasis.

Reciprocal Expression of Differentiated Embryonic Chondrocyte Expressed Genes Result in Functional Antagonism in Gastric Cancer.

Differentiated embryonic chondrocyte expressed genes (DECs) are critical regulators of cellular proliferation and differentiation. However, DEC1 and DEC2 as family member have opposite or identical roles in tumor, acting as an "accelerator" or a "brake" in progression. The possible crosstalk between DEC1 and DEC2 in the gastric cancer (GC). The association of DEC1 and DEC2 expression with prognosis was investigated by immunohistochemistry. The expression pattern of DECs in GC cells was examined using the CCLE database. DECs knockdown or overexpression was conducted via lentiviral transfection. The proliferation of GC cells was evaluated by CCK8, EdU, and Colony forming. ChIP and luciferase reporter assays were used to verify interaction between DEC1 and the DEC2 promoter. The combination downstream with DEC1 and DEC2 was predicted by bioinformation, with Western blot providing further verification. We found that reciprocal expression of DEC1 and DEC2 works together to sustain the progression of GC by promoting cell growth. We confirmed this observation in vivo, showing that inhibition DEC1expression could increase DEC2 expression. DEC1 suppresses DEC2 expression by directly binding to the E-box of the DEC2 promoter in GC cells. Furthermore, this regulation of DEC1 on DEC2 enables the further indirect or cooperative activation of additional downstream target genes, MAPK, and STAT3. Our data demonstrate that DEC1 and DEC2 interact physically and functionally and identify a novel mode of cross-regulatory interaction between DECs that abrogates their functional activity.

Downregulation of DEC1 inhibits proliferation, migration and invasion, and induces apoptosis in ovarian cancer cells via regulation of Wnt/β-catenin signaling pathway.

DEC1 has been reported to regulate the expression of multiple target genes, participate in cell differentiation, apoptosis, aging and the development and progression of numerous tumors, but the detailed effects and possible mechanisms of DEC1 in ovarian cancer (OC) remain unknown. The present study aimed to investigate the expression and mechanism of function of DEC1 in OC. The present results demonstrated that DEC1 was highly expressed in OC tissues and cell lines using reverse transcription-quantitative PCR, western blotting and immunohistochemistry, and high expression of DEC1 was negatively associated with the prognosis of patients with OC. In addition, knockdown of DEC1 significantly inhibited proliferation in SKOV3 and OVCAR3 cells compared with control. DEC1 knockdown also induced apoptosis and increased the expression of apoptosis-related proteins in OC cells. The results suggested that knockdown of DEC1 inhibited OC cell migration and invasion via regulation of epithelial-mesenchymal transition-related protein. It was also found that DEC1 knockdown significantly inhibited the Wnt/β-catenin pathway. Collectively, the current results indicated that knockdown of DEC1 inhibited proliferation, migration and invasion, and induced apoptosis in OC cells via modulating the Wnt/β-catenin signaling pathway. Thus, DEC1 may participate in malignant progression of OC, and may be a target for treatment and diagnosis of OC.