Overexpression Of DEC1 Research Articles

DEC1 deficiency promotes osteoclastic activity by augmenting NFATc1 signaling via transactivation and the Ca2+/calcineurin pathway.

We have previously demonstrated that DEC1 promotes osteoblast differentiation. This study aims to evaluate the impact of DEC1 knockout on osteopenic activities, such as osteoclast differentiation and the expression of bone-degrading genes. To gain mechanistic insights, we employed both in vivo and in vitro experiments, utilizing cellular and molecular approaches, including osteoclast differentiation assays and RNA-seq in combination with ChIP-seq. Our results showed that NFATc1, a master regulator of osteoclast differentiation, and PPP3CB, a member of the calcineurin family, were significantly upregulated in DEC1-/- mice. In vitro experiments revealed that osteoclast differentiation significantly increased both the number and size of osteoclasts in DEC1-/- bone marrow macrophages (BMMs) compared to DEC1+/+ BMMs. Additionally, NFATc1 expression was notably higher in DEC1-/- BMMs than in DEC1+/+ BMMs. Overexpression of DEC1 reduced NFATc1 promoter activity, while knockout increased it. Furthermore, intracellular free Ca2+ levels and calcineurin activity were elevated (∼150 %) in DEC1-/- BMMs compared to DEC1+/+ BMMs. Importantly, the use of calcineurin inhibitors and calcium channel blockers effectively abolished the increased osteoclast differentiation observed in DEC1-/- BMMs. In summary, DEC1 deficiency promotes osteoclast differentiation by enhancing NFATc1 signaling through transcriptional regulation and the Ca2+/calcineurin pathway. Clinically, the mRNA levels of DEC1 were reduced by up to 75 % in patients with osteoporosis. The findings of this study establish that inducing DEC1 expression, alongside attenuators of the Ca2+/calcineurin pathway, offers a molecular basis for preventing and treating osteoporosis associated with DEC1 deficiency.

Retracted] Hypoxia‑induced overexpression of DEC1 is regulated by HIF‑1α in hepatocellular carcinoma.

Further to the publication of the above paper, a concerned reader drew to our attention that, in Figs. 1A and 4A, the same loading controls for the RT‑PCR experiments portrayed had apparently been incorporated into these figures, even though the experimental results (i.e., for the DEC1 bands) were otherwise different. In addition, the control GAPDH bands featured in the western blots in Figs. 3A and 3B for the SMMC‑7721 and BRL‑7402 cell lines respectively were strikingly similar, albeit the bands appeared to have been vertically flipped in Fig. 3A relative to Fig. 3B, and possibly stretched. The Editorial Office subsequently investigated this matter, and were able to confirm the concerns of the reader. After having considered the various issues that have been brought to light with this paper, in spite of a request from the authors that a Corrigendum be published, the Editor of Oncology Reports has determined that the article should be retracted on account of a lack of overall confidence in the presented data. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 30: 2957‑2962, 2013; DOI: 10.3892/or.2013.2774].

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.

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.

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.

DEC1 directly interacts with estrogen receptor (ER) α to suppress proliferation of ER-positive breast cancer cells

Aberrant ERα signaling and altered circadian rhythms are both features of ER-positive breast cancer, however, the molecular interaction between them is still not fully understood. Herein, we analyzed the interplay between the circadian rhythm molecule DEC1 and ERα and its effect on the proliferation of ER-positive breast cancer cells, providing a new clue for clarifying the pathogenesis of breast cancer. In this study, we revealed that DEC1 negatively regulates the proliferation of ER-positive breast cancer MCF-7 cells through interaction with ERα protein. DEC1 co-localized with ERα in the nucleus of MCF7 cells, stabilized ERα protein independently of its transcriptional activity and without affecting by estrogen stimulation and inhibited the degradation of ERα mediated by CHX in a time-dependent manner. Moreover, results from luciferase reporter assay showed that overexpression of DEC1 significantly inhibits ERα-mediated transcriptional activity in a dose-dependent manner. These results together suggested that DEC1 may serve as a co-repressor of ERα in ER-positive breast cancer. Although DEC1 improved the stability of ERα and alleviated protein degradation, DEC1 inhibited the proliferation of MCF7 cells by decreasing ERα-mediated signal transduction.

Smad3 Suppresses Epithelial Cell Migration and Proliferation via the Clock Gene Dec1, Which Negatively Regulates the Expression of Clock Genes Dec2 and Per1

Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression.

Transcription factor DEC1 is required for maximal experimentally induced periodontal inflammation.

Disruption of transcriptional regulation is a confounding factor associated with a wide range of human inflammatory diseases. To investigate mechanistic links between transcription factor DEC1 and pathways underlying inflammation, wild-type and DEC1 knockout (KO) C57BL/6 mice were treated with Porphyromonas gingivalis (or carboxymethyl cellulose as a control) to induce periodontal inflammation. It provoked an inflammatory response within the oral environment, which showed robust variation in alveolar bone resorption and expression of inflammatory cytokines. Male DEC1KO mice and their wild-type littermates were used for the experimental periodontitis model. Measurement of alveolar bone resorption, micro-computed tomography, isolation of gingival mononuclear cells (GMCs), flow cytometry and immunohistochemical analysis were used in this study. Human gingival fibroblast cells (HGF-1) were used for DEC1 over-expression and short interference RNA (siRNA) studies and quantitative real-time polymerase chain reaction and western blot analysis were performed. Micro-computed tomography analysis demonstrated that P.gingivalis caused a decrease in bone area of wild-type mice compared with DEC1KO mice. Expression of inflammatory and immune markers in GMCs was significantly decreased in DEC1KO mice after treatment with P.gingivalis. Conversely, interleukin (IL)-4 and IL-10 mRNAs were significantly increased in GMCs isolated from DEC1KO mice. The results show that treatment of DEC1KO mice with P.gingivalis decreased the numbers of CD11b+ F4/80+ and CD4+ RANKL+ T cells. Moreover, expression of CD4, F4/80, RANKL and cathepsin K in inflammatory cell infiltrates was significantly reduced in DEC1KO mice treated with P.gingivalis compared with controls. Furthermore, over-expression of DEC1 in HGF-1 cells increased the expression of IL-1β and tumor necrosis factor-α mRNAs and their expression levels reached a maximum in response to treatment with lipopolysaccharide. Inhibition of DEC1 by short interference RNA interference suppressed the P.gingivalis-derived lipopolysaccharide-induced expression of IL-1β, tumor necrosis factor-α and toll-like receptor4. These results suggest that transcription factor DEC1 can modulate P.gingivalis-induced periodontitis in the oral mucosa.

Abstract 456: Functional analysis of DEC1 and DEC2 in cancer metabolism

Abstract Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) and DEC2 (bHLHE41/Sharp1) are clock genes that show a circadian rhythm in various tissues. However, the function of DEC1 and DEC2 in metabolism has not been poorly understood. AMP-activated protein kinase (AMPK) activity plays important roles in the metabolic process and circadian rhythm. However, how circadian rhythm of phosphorylation of AMPK (pAMPK) is regulated has been yet understood. The aim of this study is to investigate whether there is a direct correlation between DEC1 and DEC2 expression and AMPK activity in normal and cancer cells. There was an inverse relationship between DEC1 expression and AMPK activity. DEC1 protein and pAMPK showed a circadian rhythm in the mouse liver with different peak levels. Knocking down DEC1 expression increased pAMPK, whereas overexpression of DEC1 decreased it. DEC1 bound to the E-box of the LKB1 promoter, decreased LKB1 activity and total protein levels. In addition, pAMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. Interestingly, medium change induced the circadian rhythm of DEC2, but not DEC1 in cancer cells. We conclude that DEC1 and DEC2 negatively regulates AMPK activity via LKB1, and play important roles in cancer cell metabolism. Citation Format: Fuyuki Sato, Yasueru Muragaki. Functional analysis of DEC1 and DEC2 in cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 456. doi:10.1158/1538-7445.AM2017-456

Icariin protects against glucocorticoid induced osteoporosis, increases the expression of the bone enhancer DEC1 and modulates the PI3K/Akt/GSK3β/β-catenin integrated signaling pathway

Osteoporosis is a serious public health concern worldwide. Herba epimedii has been used for centuries and even thousands of years to treat osteoporotic conditions. Icariin, a flavonol glycoside, is one of the major active ingredients. In this study, we have shown that icariin protected against glucocorticoid-induced osteoporotic changes in SaoS-2 cells and mice. We have also shown that dexamethasone (a glucocorticoid) suppressed and icariin induced DEC1, a structurally distinct helix-loop-helix protein. DEC1 overexpression promoted whereas DEC1 knockdown decreased osteogenic activity. Likewise, DEC1 overexpression and knockdown inversely regulated the expression of β-catenin and PIK3CA, an essential player in the Wnt/β-catenin and PI3K/Akt signaling pathways, respectively. Interestingly, DKK1, an inhibitor of Wnt/β-catenin signaling inhibitor, and LY294002, an inhibitor of PI3K/Akt signaling, abolished the induction of DEC1 by icariin. It is established that these two pathways are interconnected by the phosphorylation status of GSK3β. Dexamethasone decreased but icariin increased GSK3β phosphorylation. Finally, DEC1 deficient mice developed osteoporotic phenotypes. Taken together, it is concluded that DEC1 likely supports the action of icariin against glucocorticoid induced osteoporosis with an involvement of the PI3K/Akt/GSK3β/β-catenin integrated signaling pathway.

DEC2 expression antagonizes cisplatin‑induced apoptosis in human esophageal squamous cell carcinoma.

Differentiated embryonic chondrocyte expressed gene 1 (DEC1) and differentiated embryonic chondrocyte expressed gene 2 (DEC2) belong to the Hairy/Enhancer of Split subfamily of basic helix-loop-helix factors. Previous studies have demonstrated that DEC proteins are involved in the regulation of circadian rhythms, response to hypoxia, and tumorigenesis. However, the roles of DEC1 and DEC2 in apoptosis of esophageal carcinoma remain unclear. In the present study, alterations in expression of apoptosis-related markers in human esophageal squamous cell carcinoma TE-11 cells treated with cisplatin were examined by western blot, while overall cell viability and apoptosis were analyzed by MTS assay and hematoxylin and eosin staining, respectively. Following cisplatin treatment, expression of DEC2 was downregulated, whereas expression of DEC1 was upregulated. DEC2 overexpression during cisplatin treatment markedly inhibited expression of the pro-apoptotic factor Bim and slightly increased the anti-apoptotic factor Bcl-xL. However, overexpression of DEC1 during cisplatin treatment failed to affect expression of these markers. Additionally, overexpression of DEC2 improved cell viability and decreased cell apoptosis induced by cisplatin. These results suggested that DEC2 exhibits anti-apoptotic effects in TE-11 esophageal squamous cell carcinoma cells. Inhibiting DEC2 may therefore have therapeutic potential for the treatment of esophageal cancer, in combination with cisplatin.

DEC1 promotes hypoxia-induced epithelial-mesenchymal transition (EMT) in human hepatocellular carcinoma cells.

Differentiated embryonic chondrocyte (DEC) 1 has been reported to be involved in cell differentiation, hypoxia response, and cancer progression. Recent studies have demonstrated that hypoxia-inducible factor (HIF)-1α induces epithelial-mesenchymal transition (EMT) in carcinoma cells to facilitate cell invasiveness and metastasis. However, it remains unclear whether DEC1 participates in hypoxia-mediated EMT processes. In the present study, we reported that hypoxia induced DEC1 expression in hepatocellular carcinoma (HCC) HepG2 cells, and DEC1 negatively regulated expression of HIF-1α and E-cadherin in transcriptional/translational levels. Cell morphological changes were evaluated with hematoxylin and eosin (H-E) staining. Exposure to hypoxia caused spindle-like shape in some of the HepG2 cells, and DEC1 overexpression furthered these changes. In conclusions, DEC1 is involved in hypoxia-induced EMT processes via negatively regulating E-cadherin expression in HepG2 cells.

Basic helix-loop-helix transcription factor DEC2 functions as an anti-apoptotic factor during paclitaxel-induced apoptosis in human prostate cancer cells.

The functions of basic helix-loop-helix (bHLH) transcription factor-differentiated embryonic chondrocyte (DEC)1 (BHLHE40) and 2 (BHLHE41) are involved in various fields such as circadian rhythms, immune responses, cell proliferation, hypoxia reaction as well as malignant tumors. Previous findings showed that DEC served as apoptosis regulators of various cancer cell lines. However, little is known regarding the expression of DEC1 and DEC2 in prostate cancer cells. The present study aimed to examine the roles of DEC1 and DEC2 in human prostate cancer DU145 and PC-3 cells that were treated with paclitaxel. The expression of DEC1 and DEC2 was decreased in DU145 cells but was increased in PC-3 cells when treated with paclitaxel. DU145 cells were more sensitive to paclitaxel than PC-3 cells since the amount of cleaved poly(ADP-ribose) polymerase (PARP) reached its peak at 50 μM of paclitaxel in DU145 cells but at 100 μM in PC-3 cells. In addition, the amount of cleaved PARP was decreased by DEC1 siRNA, while it was increased by DEC2 siRNA in the presence of paclitaxel. Although DEC2 overexpression slightly inhibited cleaved PARP in the two cell lines, the effects of DEC1 overexpression on apoptosis remain to be determined. In conclusion, DEC1, at least partly, exerted a pro-apoptotic effect, whereas DEC2 exerted an anti-apoptotic effect in paclitaxel-induced apoptosis of human prostate cancer cells.

Dec1 expression predicts prognosis and the response to temozolomide chemotherapy in patients with glioma.

Differentiated embryo chondrocyte expressed gene1 (Dec1), a crucial cell differentiation mediator and apoptosis inhibitor, is abundantly expressed in various types of human cancer and is associated with malignant tumor progression. As poor differentiation and low apoptosis are closely associated with poor survival rates and a poor response to radio/chemotherapy in patients with cancer, the prognostic value of Dec1 expression was examined in the present study and its correlation with response to temozolomide (TMZ) chemotherapy was analyzed in patients with glioma. Dec1 expression was analyzed by immunohistochemistry in 157 samples of newly diagnosed glioma and 63 recurrent glioblastoma cases that relapsed during TMZ chemotherapy. Correlations with clinical variables, prognosis and the response to TMZ chemotherapy were analyzed in the newly diagnosed gliomas. Dec1 expression was also compared with the apoptosis index determined by TdT‑mediated dUTP nick ending‑labeling assay in recurrent glioblastomas. The antiglioma effect of TMZ in nude mice xenografts with Dec1 expression was examined invivo. High expression of Dec1, which was significantly associated with high pathological tumor grade and poor response to TMZ chemotherapy, was demonstrated to be an unfavorable independent prognostic factor and predicted poor survival in patients with newly diagnosed glioma. In patients with recurrent glioblastoma, there was a negative correlation between Dec1 expression and the apoptotic index. In nude mice treated with TMZ, Dec1 overexpression potentiated proliferation, but attenuated TMZ‑induced apoptosis. In conclusion, Dec1 is a prognostic factor for the clinical outcome and a predictive factor for the response to TMZ chemotherapy in patients with glioma.

Abstract 2026: DEC1 negatively regulates AMPK activity via LKB1

Abstract Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) is one of the clock genes that show a circadian rhythm in various tissues. AMP-activated protein kinase (AMPK) activity plays important roles in metabolism and in cell death induced by glucose depletion. Recent reports have shown that AMPK activity exhibited a circadian rhythm. However, how the circadian rhythm of AMPK activity is regulated is not known. The aim of this study is the direct correlation between DEC1 expression and AMPK activity. DEC1 protein and AMPK activity showed a circadian rhythm in the mouse liver with different peak levels. A medium change and serum shock led to rhythmic patterns of DEC1 protein and AMPK activity levels in WI-38 cells, and their peak levels occurred at different times. However, changing the medium did not induce circadian rhythms of DEC1 protein or AMPK activity levels in MCF-7 and U2OS cells, in which the intensities of these levels had inversely correlated patterns. Knocking down DEC1 expression increased AMPK activity, whereas DEC1 overexpression decreased it. DEC1 bound to the E-box of the LKB1 promoter, decreasing the LKB1 activity and total protein levels. In addition, knocking down DEC1 expression inhibited cell death under the condition of glucose depletion, increasing the activity of both AMPK and LKB1. Taken together, our results showed that the levels of DEC1 protein and AMPK activity were affected even long after the medium change and that the rhythmic patterns of these levels were inversely correlated. We concluded that DEC1 negatively regulated AMPK activity via LKB1. Citation Format: Fuyuki Sato, Yasuteru Muragaki. DEC1 negatively regulates AMPK activity via LKB1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2026.

Podoplanin-mediated TGF-β-induced epithelial-mesenchymal transition and its correlation with bHLH transcription factor DEC in TE-11 cells.

Podoplanin is reported involved in the collective cell invasion, another tumor invasion style which is distinct from the single cell invasion, so-called epithelial-mesenchymal transition (EMT). In this study, we investigated the correlation between podoplanin and EMT-related markers in esophageal squamous cell carcinoma (ESCC), and evaluated its linkage with the basic helix-loop-helix (bHLH) transcription factor differentiated embryonic chondrocyte (DEC) 1 and DEC2. Three ESCC cell lines and human squamous cell carcinoma A431 cells were subjected to western blot analyses for podoplanin and EMT markers, as well as the expression of DEC1 and DEC2. By RT-qPCR and western blotting, we found that TGF-β increased the expression of podoplanin and mensenchymal markers (e.g., N-cadherin and vimentin), while decreased the expression of epithelial markers (e.g., Claudin-4 and E-cadherin), accompanied by Smad2 phosphorylation and slug activation. Moreover, TGF-β has different effects on the expression of DEC1 and DEC2, that is, it upregulates DEC1, but downregulates DEC2. Capability of cell proliferation, invasion and migration were further analyzed using CCK-8 assay, Matrigel-invasion assay, and the wound-healing assay, respectively. The proliferation, invasion and migration ability were significantly lost in podoplanin-knockdown cells when compared with the scrambled siRNA group. In addition to these changes, the expression of Claudin-4, but not that of Claudin-1 or E-cadherin, was induced by the siRNA against podoplanin. On the contrary, overexpression of DEC1 and DEC2 exhibits opposite effects on podoplanin, but only slight effect on Claudin-4 was detected. These data indicated that podoplanin is significantly associated with EMT of TE-11 cells, and may be directly or indirectly regulated by bHLH transcription factors DEC1 and DEC2.

The anti-metastatic effect of 8-MOP on hepatocellular carcinoma is potentiated by the down-regulation of bHLH transcription factor DEC1

Despite progress in diagnostics and treatment of hepatocellular carcinoma (HCC), its prognosis remains poor. 8-Methoxypsoralen (8-MOP), a formerly considered photosensitizing agent, has been reported to induce cell apoptosis in HepG2 cells in a modest way when used alone. In this study, it was demonstrated that 8-MOP inhibited HCC HepG2 cells and SMMC-7721 cells migratory and invasive potentiality, as well as modulated the expression of various EMT-associated genes such as enhancing E-cadherin and reducing N-cadherin, vimentin, α-SMA and MMP9 in a concentration-dependent way. Differentiated embryonic chondrocyte-expressed gene 1, DEC1 (BHLHE40/Stra13/Sharp2), is a basic helix-loop-helix (bHLH) transcription factor that regulates cell growth, differentiation, apoptosis and tumorigenesis. 8-MOP suppressed the expression of DEC1 in a concentration- and time-dependent manner. Overexpression of DEC1 endorsed the HepG2 cells a higher metastatic phenotype, while totally abolished 8-MOP-repressed metastatic capability. In the meanwhile, overexpression of DEC1 promoted EMT process by suppressing expression of epithelial protein and enhancing expression of mesenchymal proteins, while potently antagonized the regulation of EMT-associated genes by 8-MOP. In vivo experiments revealed that the treatment of 8-MOP (5 or 20mg/kg) resulted in a dose-dependent decreases in the lung metastasis of hepatoma H22-transplanted mice without any obvious toxicity to the organs, as well as increased expression of E-cadherin in lung tissues. Consistently, 8-MOP down-regulated the expression of DEC1 in the lungs of tumor-bearing mice, which further confirms that DEC1 was correlated with 8-MOP-induced anti-metastatic effect. The present findings establish a function for DEC1 in HCC metastatic progression and suggest its candidacy as a novel target for the anti-metastasis effect of 8-MOP.

DEC1 negatively regulates AMPK activity via LKB1

Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) is one of the clock genes that show a circadian rhythm in various tissues. AMP-activated protein kinase (AMPK) activity plays important roles in the metabolic process and in cell death induced by glucose depletion. Recent reports have shown that AMPK activity exhibited a circadian rhythm. However, little is known regarding the regulatory mechanisms involved in the circadian rhythm of AMPK activity. The aim of this study is to investigate whether there is a direct correlation between DEC1 expression and AMPK activity. DEC1 protein and AMPK activity showed a circadian rhythm in the mouse liver with different peak levels. Knocking down DEC1 expression increased AMPK activity, whereas overexpression of DEC1 decreased it. Overexpressing the DEC1 basic mutants had little effect on the AMPK activity. DEC1 bound to the E-box of the LKB1 promoter, decreased LKB1 activity and total protein levels. There was an inverse relationship between DEC1 expression and AMPK activity. Our results suggest that DEC1 negatively regulates AMPK activity via LKB1.

DEC1 regulates breast cancer cell proliferation by stabilizing cyclin E protein and delays the progression of cell cycle S phase.

Breast cancer that is accompanied by a high level of cyclin E expression usually exhibits poor prognosis and clinical outcome. Several factors are known to regulate the level of cyclin E during the cell cycle progression. The transcription factor DEC1 (also known as STRA13 and SHARP2) plays an important role in cell proliferation and apoptosis. Nevertheless, the mechanism of its role in cell proliferation is poorly understood. In this study, using the breast cancer cell lines MCF-7 and T47D, we showed that DEC1 could inhibit the cell cycle progression of breast cancer cells independently of its transcriptional activity. The cell cycle-dependent timing of DEC1 overexpression could affect the progression of the cell cycle through regulating the level of cyclin E protein. DEC1 stabilized cyclin E at the protein level by interacting with cyclin E. Overexpression of DEC1 repressed the interaction between cyclin E and its E3 ligase Fbw7α, consequently reducing the level of polyunbiquitinated cyclin E and increased the accumulation of non-ubiquitinated cyclin E. Furthermore, DEC1 also promoted the nuclear accumulation of Cdk2 and the formation of cyclin E/Cdk2 complex, as well as upregulating the activity of the cyclin E/Cdk2 complex, which inhibited the subsequent association of cyclin A with Cdk2. This had the effect of prolonging the S phase and suppressing the growth of breast cancers in a mouse xenograft model. These events probably constitute the essential steps in DEC1-regulated cell proliferation, thus opening up the possibility of a protein-based molecular strategy for eliminating cancer cells that manifest a high-level expression of cyclin E.