Overexpression Of DEC1 Research Articles

Basic helix-loop-helix transcription factor DEC1 regulates the cisplatin-induced apoptotic pathway of human esophageal cancer cells.

DEC1 [basic helix-loop-helix (BHLH) E40/Stra13/Sharp2] and DEC2 (BHLHE41/Sharp1) are BHLH transcription factors that are associated with the regulation of apoptosis, cell proliferation, and circadian rhythms, as well as malignancy in various cancers. However, the roles of DEC1 and DEC2 expression in esophageal cancer are poorly understood. In this study, we examined the roles of DEC1 and DEC2 in human esophageal cancer TE 5 and TE 10 cells that had been treated with cis-diamminedichloroplatinum (II) (cisplatin: CDDP). Expression of DEC1 and DEC2 was decreased with CDDP treatment in TE 5 cells; however, knockdown or overexpression of DEC1/DEC2 had little effects on CDDP-induced apoptosis in TE 5 cells. DEC1 expression was up-regulated in CDDP-treated TE 10 cells, whereas DEC2 expression was unchanged. DEC1 knockdown by siRNA in TE 10 decreased the amount of cleaved poly (ADP-ribose) polymerase (PARP) after treatment with CDDP, whereas DEC2 knockdown had no effects on the amount of cleaved PARP in both the presence and absence of CDDP. We also demonstrated that DEC1 overexpression promoted cleaved PARP expression, whereas DEC2 overexpression had no effects on the amount of cleaved PARP in TE 10 cells. These results suggested that DEC1 has pro-apoptotic effects on human esophageal cancer TE 10 cells of well-differentiated type.

Involvement of c-Myc in the proliferation of MCF-7 human breast cancer cells induced by bHLH transcription factor DEC2.

Differentiated embryonic chondrocyte expressed gene 1 (DEC1; BHLHE40/Stra13/Sharp2) and differentiated embryonic chondrocyte expressed gene 2 (DEC2; BHLHE41/Sharp1) are basic helix-loop-helix (bHLH) transcriptional factors that are involved in the regulation of cell differentiation, circadian rhythms, response to hypoxia and carcinogenesis. Previous studies have demonstrated that the expression of DECs is induced under hypoxic conditions in various normal and cancer cell lines. In the present study, using RT-qPCR and western blot analysis, we demonstrated that hypoxia induced the expression of DEC1 and DEC2 in MCF-7 human breast cancer cells; their expression levels reached a peak at different time points. In particular, we found that the expression pattern of the hypoxia-inducible factor (HIF)-1α protein was similar to DEC1, and that of the HIF-2α protein was identical to that of DEC2. The knockdown of HIF-2α using siRNA suppressed the phosphorylation of Akt, as well as the expression of DEC2 and c-Myc. Hypoxia failed to affect the expression of DEC2 and c-Myc when the PI3K/Akt signaling pathway was blocked. In addition, the overexpression of DEC1 and DEC2 was induced by transfecting the cells with a pcDNA vector. The overexpression of DEC2, but not that of DEC1, increased the proliferation of the MCF-7 cells under both normoxic and hypoxic conditions. Concomitantly, the expression of c-Myc was upregulated by exposure to hypoxia and by the overexpression of DEC2. In conclusion, DEC2 participates in hypoxia-induced cell proliferation by functioning as a target gene of the PI3K/Akt signaling pathway and regulating the expression of c-Myc.

Hepatic Differentiated Embryo-Chondrocyte-expressed Gene 1 (Dec1) Inhibits Sterol Regulatory Element-binding Protein-1c (Srebp-1c) Expression and Alleviates Fatty Liver Phenotype

Hepatic steatosis, characterized by ectopic hepatic triglyceride accumulation, is considered as the early manifestation of non-alcoholic fatty liver diseases (NAFLD). Increased SREBP-1c level and activity contribute to excessive hepatic triglyceride accumulation in NAFLD patients; however, negative regulators of Srebp-1c are not well defined. In this study, we show that Dec1, a critical regulator of circadian rhythm, negatively regulates hepatic Srebp-1c expression. Hepatic Dec1 expression levels are markedly decreased in NAFLD mouse models. Restored Dec1 gene expression levels in NAFLD mouse livers decreased the expression of Srebp-1c and lipogenic genes, subsequently ameliorating the fatty liver phenotype. Conversely, knockdown of Dec1 expression by an adenovirus expressing Dec1-specific shRNA led to an increase in hepatic TG content in normal mouse livers. Correspondingly, expression levels of lipogenic genes, including Srebp-1c, Fas, and Acc, were increased in livers of mice with Dec1 knockdown. Moreover, a functional lipogenesis assay suggested that Dec1 overexpression repressed lipid synthesis in primary hepatocytes. Finally, a luciferase reporter gene assay indicates that DEC1 inhibits Srebp-1c gene transcription via the E-box mapped to the promoter region. Chromatin immunoprecipitation confirmed that DEC1 proteins bound to the identified E-box element. Our studies indicate that DEC1 is an important regulator of Srebp-1c expression and links circadian rhythm to hepatic lipogenesis. Activation of Dec1 can alleviate the nonalcoholic fatty liver phenotype.

The transcription factor DEC1 (BHLHE40/STRA13/SHARP-2) is negatively associated with TNM stage in non-small-cell lung cancer and inhibits the proliferation through cyclin D1 in A549 and BE1 cells

The objective of the current study was to investigate the expression pattern and clinicopathological significance of differentiated embryo-chondrocyte-expressed gene 1 (DEC1) in patients with non-small-cell lung cancer (NSCLC). In 118 archived NSCLC tissues, the positive rate of DEC1 was reduced in human lung cancer samples (36/118, 30.5 %) compared with adjacent normal lung tissues (106/118, 89.8 %), as measured by immunohistochemical staining. Loss of DEC1 was correlated with poor differentiation (p=0.005) and high p-TNM stage (p=0.002). Consistently, downregulation of DEC1 by siRNA knockdown promoted the growth and colony formation in the A549 lung cancer cell line, and overexpression of DEC1 inhibited the growth and colony formation in the BE1 lung cancer cell line. In addition, a significant negative correlation was found between DEC1 and cyclin D1 (p=0.014) in 118 cases of NSCLC. Knockdown of DEC1 resulted in the upregulation of cyclin D1, and overexpression of DEC1 led to the downregulation of cyclin D1. Together with the observation that DEC1 bound directly to the promoter region of cyclin D1 in A549 cells, these results indicate that loss of DEC1 may promote tumor progression in NSCLC through upregulation of cyclin D1, and DEC1 might serve as a novel therapeutic target of NSCLC.

Overexpression of the DEC1 Protein Induces Senescence In Vitro and Is Related to Better Survival in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related death in China and has limited effective therapeutic options except for early surgery, since the underlying molecular mechanism driving its precursor lesions towards invasive ESCC is not fully understood. Cellular senescence is the state of the permanent growth arrest of a cell, and is considered as the initial barrier of tumor development. Human differentiated embryo chondrocyte expressed gene 1 (Dec1) is an important transcription factor that related to senescence. In this study, DEC1 immunohistochemical analysis was performed on tissue microarray blocks constructed from ESCC combined with adjacent precursor tissues of 241 patients. Compared with normal epithelia, DEC1 expression was significantly increased in intraepithelial neoplasia and DEC1 expression was significantly decreased in ESCC in comparison with intraepithelial neoplasia. In vitro, DEC1 overexpression induced cellular senescence, and it inhibited cell growth and colony formation in ESCC cell line EC9706. Fresh esophagectomy tissue sections from five ESCC patients were detected by immunohistochemistry of DEC1 and senescence-associated β-galactosidase (SA-β-Gal) activity, and strongly positive expression of DEC1 was correlated to more senescent cells in these fresh tissue sections. Kaplan – Meier method analysis of the 241 patients revealed that DEC1 expression levels were significantly correlated with the survival of ESCC patients after surgery. The expression levels of DEC1 were also correlated with age, tumor embolus, depth of invasion of ESCC, lymph metastasis status and pTNMs. These results suggest that DEC1 overexpression in precursor lesions of ESCC is a protective mechanism by inducing cellular senescence in ESCC initiation, and DEC1 may be a potential prognostic marker of ESCC.

Abstract 732: Overexpression of the basic helix-loop-helix protein DEC1 induces cellular senescence and is related to better survival in esophageal squamous cell carcinoma

Abstract Objective: Human differentiated embryo chondrocyte expressed gene 1 (Dec1), also known as Bhlhb2/Stra13/Sharp-2, is a basic helix-loop-helix transcription factor that involves in cell proliferation, differentiation, apoptosis, and cellular senescence. In this study, the expression, function, diagnosis and prognostic impact of DEC1 in normal esophageal epithelia, esophageal squamous cell carcinoma (ESCC) and its precursor lesions (intraepithelial neoplasia) were investigated. Methods: ESCC combined with adjacent precursor tissues from 241 patients were collected to construct tissue microarray (TMA) blocks. DEC1 expression among normal epithelia, introepithelial neoplasia and ESCC was analyzed by immunohistochemistry (IHC), and the correlations between Dec1 and clinicopathological parameters were also analyzed statistically. In vitro study, DEC1 was transient transfected into ESCC cell line EC9706, and then cellular senescence was analyzed by senescence associated β-galactosidase (SA-α-Gal) activity. Fresh esophagectomy tissues from ESCC patients were embedded and fixed, and tissue sections were detected by immunohistochemistry of DEC1 and SA-α-Gal activity. Results: Compared with normal epithelia, DEC1 expression was significantly increased in intraepithelial neoplasia and DEC1 expression was significantly decreased in ESCC in comparison with intraepithelial neoplasia. DEC1 overexpression both induced cellular senescence in EC9706 cell line and correlated to more senescent cells in fresh tissue sections. Kaplan - Meier method analysis revealed that DEC1 expression levels were significantly correlated with the survival of ESCC patients after surgery. The expression levels of DEC1 were also correlated with the tumor embolus, depth of invasion of ESCC, lymph metastasis status and pTNMs. Conclusions: These results suggest that DEC1 overexpression is a protective mechanism by inducing cellular senescence in ESCC progression, and DEC1 is a potential prognostic marker of ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 732. doi:1538-7445.AM2012-732

BHLH transcription factor DEC2 regulates pro-apoptotic factor Bim in human oral cancer HSC-3 cells

DEC1 (BHLHE40/Stra13/Sharp2) and DEC2 (BHLHE41/Sharp1) are basic helix-loop-helix (bHLH) transcription factors that are involved in the regulation of apoptosis, cell proliferation, circadian rhythms and the response to hypoxia. We previously showed the functional effects of DEC1 and DEC2 on apoptosis in human breast cancer MCF-7 cells. However, the roles of DEC1 and DEC2 in oral cancer are poorly understood. We examined whether DEC1 and DEC2 are involved in the regulation of apoptosis in human oral cancer HSC-3 and CA9-22 cells. The expression of DEC2 was upregulated by cis-diamminedichloroplatinum (II) (cisplatin: CDDP) treatment in HSC-3 cells, whereas CDDP treatment had little effects on the expression of DEC2 in CA9-22 cells. We showed that DEC2 overexpression inhibits pro-apoptotic factor Bim and inhibits apoptosis induced by CDDP in HSC-3 cells, whereas it had little effects on apoptosis in CA9-22 cells. DEC1 overexpression had little effects on apoptosis induced by CDDP in these cells. We also found that CDDP upregulated the amounts of DEC2 in the nucleus in HSC-3 cells. These results suggest that DEC2 has anti-apoptotic effects on apoptosis induced by CDDP in HSC-3 cells.

HIF-1α induction suppresses excessive lipid accumulation in alcoholic fatty liver in mice

Chronic alcohol intake stimulates hepatic oxygen consumption and subsequently causes liver hypoxia, leading to activation of hypoxia inducible factor-1 (HIF-1). Although HIF-1 plays a crucial role in the metabolic switch from aerobic to anaerobic metabolism in response to hypoxia, its roles in the regulation of lipid metabolism in alcoholic fatty liver remain unknown. Wild-type and hepatocyte-specific HIF-1α-null mice were subjected to a 6% ethanol-containing liquid diet for 4 weeks, and functional effects of loss of the HIF-1α gene on lipid metabolism were examined in the liver. Hepatocyte-specific HIF-1α-null mice developed severe hypertriglyceridemia with enhanced accumulation of lipids in the liver of mice exposed to a 6% ethanol-containing liquid diet for 4 weeks. Sterol regulatory element-binding protein 1c (SREBP-1c) and its downstream target acetyl-CoA carboxylase were greatly activated as the hepatic steatosis progressed, and these alterations were inversely correlated with the expression of the HIF-1-regulated gene DEC1. Overexpression of DEC1 in the mutant liver abrogated the detrimental effects of loss of HIF-1α gene on ethanol-induced fatty liver with reduced SREBP-1c expression. Conversely, co-administration of the HIF hydroxylase inhibitor dimethyloxalylglycine for the last 2 weeks improved markedly the ethanol-induced fatty liver in mice. The current results provide direct evidence for protective roles of HIF-1 induction in the development of ethanol-induced fatty liver via activation of the HIF-1-regulated transcriptional repressor DEC1.

Basic helix‐loop‐helix transcription factor DEC1 negatively regulates cyclin D1

DEC1 (also known as Stra13/Bhlhb2/Sharp2) and DEC2 (also known as Bhlhb3/Sharp1) are two paralogous basic helix-loop-helix (bHLH) transcriptional regulators which exhibit a robust circadian gene expression pattern in the suprachiasmatic nucleus (SCN) and in peripheral organs. DEC1 has been suggested to play key roles in mammalian cell differentiation, the cell cycle and circadian regulation, hypoxia response, and carcinogenesis. Here we show that DEC1 overexpression exhibits delayed wound healing and reduces cell proliferation, migration, and invasion. DEC1 strongly repressed the promoter activity of cyclin D1. We further identify a possible DEC-response element in the cyclin D1 promoter region, and confirmed the direct binding of DEC1 to that element. Forced expression of DEC1 efficiently repressed the cyclin D1 promoter and expression. Our clinical data provide the first evidence that there is a strong inverse correlation between DEC1 and cyclin D1 expression in oral cancer, and DEC1 expression significantly correlated with clinicopathological parameters. We suggest that radiation-induced DEC1 overexpression and Akt phosphorylation in cancer cells are mediated via PI-3K signalling. Overexpression of DEC1 activates the PI-3K/Akt signalling pathway through reactive oxygen species (ROS).

Anti‐apoptotic effect of the basic helix‐loop‐helix (bHLH) transcription factor DEC2 in human breast cancer cells

DEC1 (BHLHB2/Stra13/Sharp2) and DEC2 (BHLHB3/Sharp1) are basic helix-loop-helix (bHLH) transcription factors that are involved in circadian rhythms, differentiation and the responses to hypoxia. We examined whether DEC1 and DEC2 are involved in apoptosis regulation, in human breast cancer MCF-7 cells. We found that siRNA-mediated knockdown of DEC2 resulted in marked enhancement of apoptosis compared with that in control cells transfected with nonspecific siRNA. However, knockdown of DEC1 by siRNA did not affect cell survival. Knockdown of DEC2 affected the expression of mRNA or proteins related to apoptosis, such as Fas, c-Myc, caspase-8, poly (ADP-ribose) polymerase (PARP) and Bax. We also showed that tumor necrosis factor-alpha (TNF-alpha) up-regulates the expression of DEC1 and DEC2. DEC2 over-expression caused by the transfection of an expression vector reduced the amounts of cleaved PARP and caspase-8 induced by TNF-alpha treatment, whereas DEC1 over-expression increased it. Finally, we revealed that treatment with double knockdown against both DEC1 and DEC2 decreased the amounts of cleaved PARP and caspase-8 induced by DEC2 siRNA with or without TNF-alpha. These data indicate that DEC2 has an anti-apoptotic effect, whereas DEC1 has a pro-apoptotic effect, which are involved in the balance of survival of human breast cancer MCF-7 cells.

DEC1, a Basic Helix-Loop-Helix Transcription Factor and a Novel Target Gene of the p53 Family, Mediates p53-dependent Premature Senescence

Cellular senescence plays an important role in tumor suppression. p53 tumor suppressor has been reported to be crucial in cellular senescence. However, the underlying mechanism is poorly understood. In this regard, a cDNA microarray assay was performed to identify p53 targets involved in senescence. Among the many candidates is DEC1, a basic helix-loop-helix transcription factor that has been recently shown to be up-regulated in K-ras-induced premature senescence. However, it is not clear whether DEC1 is capable of inducing senescence. Here, we found that DEC1 is a novel target gene of the p53 family and mediates p53-dependent premature senescence. Specifically, we showed that DEC1 is induced by the p53 family and DNA damage in a p53-dependent manner. We also found that the p53 family proteins bind to, and activate, the promoter of the DEC1 gene. In addition, we showed that overexpression of DEC1 induces G(1) arrest and promotes senescence. Moreover, we found that targeting endogenous DEC1 attenuates p53-mediated premature senescence in response to DNA damage. Furthermore, overexpression of DEC1 induces cellular senescence in p53-knockdown cells, albeit to a lesser extent. Finally, we showed that DEC1-induced senescence is p21-independent. Taken together, our data provided strong evidence that DEC1 is one of the effectors downstream of p53 to promote premature senescence.

Transforming Growth Factor-β Promotes Survival of Mammary Carcinoma Cells through Induction of Antiapoptotic Transcription Factor DEC1

Transforming growth factor-beta (TGF-beta) signaling facilitates tumor growth and metastasis in advanced cancer. In the present study, we identified differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stra13) as a downstream target of TGF-beta signaling, which promotes the survival of breast cancer cells. In the mouse mammary carcinoma cell lines JygMC(A) and 4T1, the TGF-beta type I receptor kinase inhibitors A-44-03 and SB431542 induced apoptosis of cells under serum-free conditions. Oligonucleotide microarray and real-time reverse transcription-PCR analyses revealed that TGF-beta induced DEC1 in these cells, and the increase of DEC1 was suppressed by the TGF-beta type I receptor kinase inhibitors as well as by expression of dominant-negative TGF-beta type II receptor. Overexpression of DEC1 prevented the apoptosis of JygMC(A) cells induced by A-44-03, and knockdown of endogenous DEC1 abrogated TGF-beta-promoted cell survival. Moreover, a dominant-negative mutant of DEC1 prevented lung and liver metastasis of JygMC(A) cells in vivo. Our observations thus provide new insights into the molecular mechanisms governing TGF-beta-mediated cell survival and metastasis of cancer.

Effects of overexpression of basic helix–loop–helix transcription factor Dec1 on osteogenic and adipogenic differentiation of mesenchymal stem cells

We recently reported that forced expression of basic helix–loop–helix transcription factor Dec1 accelerated chondrogenic differentiation of mesenchymal stem cells (MSC) in pellet cultures (Shen, M., Yoshida, E., Yan, W., Kawamoto, T., Suardita, K., Koyano, Y., Fujimoto, K., Noshiro, M., Kato, Y., 2002. Basic helix–loop–helix protein DEC1 promotes chondrocyte differentiation at the early and terminal stages. J. Biol. Chem. 277, 50112–50120). Since MSC have multilineage differentiation potential, we investigated the roles of Dec1 in osteogenic and adipogenic differentiation of human bone marrow-derived MSC. After osteogenic induction of MSC in medium containing dexamethasone, β-glycerophosphate, and ascorbic acid, Dec1 expression gradually increased from day 5 to day 14, while expression levels of Dec1 mRNA markedly decreased on days 3 and 7 after adipogenic induction. Infection with adenovirus expressing Dec1 raised mRNA levels of several bone characteristic molecules such as osteopontin, PTH receptor, and alkaline phosphatase, even in the absence of the osteogenic induction medium, although it had little effect on Runx2 expression or calcification. In the osteogenic induction medium, Dec1 overexpression enhanced the expression of osteopontin and alkaline phosphatase and induced matrix calcification. Knockdown of Dec1 with siRNA suppressed the expression of osteoblastic phenotype by the induced MSC. Using MSC cultures, we also confirmed that forced expression of Dec1 suppressed adipogenic differentiation. These findings suggest that Dec1 modulates osteogenic differentiation of MSC by inducing the expression of several, but not all, bone-related genes.

A novel autofeedback loop of Dec1 transcription involved in circadian rhythm regulation

An autofeedback loop associated with transcription of clock gene(s), Per(s), is generally accepted as the molecular machinery of circadian rhythm generation, in which CLOCK/BMAL act as positive regulators and PER/CRY as negative ones. We show here an autofeedback loop of Dec1 encoding a basic helix–loop–helix transcription factor: CLOCK/BMAL increased the promoter activity of Dec1, and DEC1 and DEC2 as well as PERs and CRYs suppressed the induced expression. Three CACGTG E-boxes are responsible for both the activation and the suppression of Dec1 transcription. Forced expression of Clock/Bmal increased endogenous Dec1 mRNA level, and overexpression of Dec1 resulted in suppression of Dec2, Per2, and Dbp expression. The level of Dec1 expression in the heart of Clock/Clock mutant mice was continuously low throughout the day. These findings suggest that Dec1 is positively regulated by CLOCK/BMAL and is involved in circadian rhythm regulation by suppressing CLOCK/BMAL-induced gene expression. The autofeedback loop of Dec1 may be interlocked with the core feedback loop of Per in some situations.

Basic Helix-loop-helix Protein DEC1 Promotes Chondrocyte Differentiation at the Early and Terminal Stages

The mRNA level of basic helix-loop-helix transcription factor DEC1 (BHLHB2)/Stra13/Sharp2 was up-regulated during chondrocyte differentiation in cultures of ATDC5 cells and growth plate chondrocytes, and in growth plate cartilage in vivo. Forced expression of DEC1 in ATDC5 cells induced chondrogenic differentiation, and insulin increased this effect of DEC1 overexpression. Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) suppressed DEC1 expression and the differentiation of ATDC5 cells, but DEC1 overexpression antagonized this inhibitory action of PTH/PTHrP. Transforming growth factor-beta or bone morphogenetic protein-2, as well as insulin, induced DEC1 expression in ATDC5 cultures where it induced chondrogenic differentiation. In pellet cultures of bone marrow mesenchymal stem cells exposed to transforming growth factor-beta and insulin, DEC1 was induced at the earliest stage of chondrocyte differentiation and also at the hypertrophic stage. Overexpression of DEC1 in the mesenchymal cells induced the mRNA expressions of type II collagen, Indian hedgehog, and Runx2, as well as cartilage matrix accumulation; overexpression of DEC1 in growth plate chondrocytes at the prehypertrophic stage increased the mRNA levels of Indian hedgehog, Runx2, and type X collagen, and also increased alkaline phosphatase activity and mineralization. To our knowledge, DEC1 is the first transcription factor that can promote both chondrogenic differentiation and terminal differentiation.