Levels Of Notch1 Intracellular Domain Research Articles

Autophagy-mediated degradation of NOTCH1 intracellular domain controls the epithelial to mesenchymal transition and cancer metastasis

BackgoundAutophagy controls levels of cellular components during normal and stress conditions; thus, it is a pivotal process for the maintenance of cell homeostasis. In cancer, autophagy protects cells from cancerous transformations that can result from genomic instability induced by reactive oxygen species or other damaged components, but it can also promote cancer survival by providing essential nutrients during the metabolic stress condition of cancer progression. However, the molecular mechanism underlying autophagy-dependent regulation of the epithelial to mesenchymal transition (EMT) and metastasis is still elusive.MethodsThe intracellular level of NOTCH1 intracellular domain (NICD) in several cancer cells was studied under starvation, treatment with chloroquine or ATG7-knockdown. The autophagy activity in these cells was assessed by immunocytochemistry and molecular analyses. Cancer cell migration and invasion under modulation of autophagy were determined by in vitro scratch and Matrigel assays.ResultsIn the study, autophagy activation stimulated degradation of NICD, a key transcriptional regulator of the EMT and cancer metastasis. We also found that NICD binds directly to LC3 and that the NICD/LC3 complex associates with SNAI1 and sequestosome 1 (SQSTM1)/p62 proteins. Furthermore, the ATG7 knockdown significantly inhibited degradation of NICD under starvation independent of SQSTM1-associated proteasomal degradation. In addition, NICD degradation by autophagy associated with the cellular level of SNAI1. Indeed, autophagy inhibited nuclear translocation of NICD protein and consequently decreased the transcriptional activity of its target genes. Autophagy activation substantially suppressed in vitro cancer cell migration and invasion. We also observed that NICD and SNAI1 levels in tissues from human cervical and lung cancer patients correlated inversely with expression of autophagy-related proteins.ConclusionsThese findings suggest that the cellular level of NICD is regulated by autophagy during cancer progression and that targeting autophagy-dependent NICD/SNAI1 degradation could be a strategy for the development of cancer therapeutics.

Loss of ARNT in skeletal muscle limits muscle regeneration in aging.

The ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old (23‐25 months old) mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2‐3 months old) mice. Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle‐specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), which simulates the actions of ARNT, rescued skeletal muscle regeneration in both old and ARNT‐deleted mice. These results suggest that the loss of ARNT in skeletal muscle is partially responsible for diminished myogenic potential in aging and activation of hypoxia signaling holds promise for rescuing regenerative activity in old muscle.

Distinct roles for Notch1 and Notch3 in human adipose-derived stem/stromal cell adipogenesis.

The role of the Notch signaling pathway in adipogenesis has long been controversial as the action of individual Notch receptors appears to vary with experimental conditions. In this study, we offer some explanation for the observed contradictions by comparing the role of both Notch1 and Notch3 in regulating the expression of key adipogenic regulator, PPARγ, in human adipose-derived stem/stromal cells (hADSCs) during in vitro adipogenesis. Utilizing qRT-PCR, western blot, and immunofluorescence staining, we demonstrated that Notch3 was expressed prior to the formation of lipid vesicles, while Notch1 only appeared after vesicle formation. In addition, following the induction of adipogenesis, the levels of Notch1 intracellular domain in the nucleus were significantly reduced, while the siRNA-mediated loss of Notch1 reduced transcript but not protein levels of PPARγ. The knockdown of Notch3 led to increased expression of PPARγ during early adipogenesis that was not paralleled by a decreased expression of Hes1 and Hey1, but was accompanied by a marked decrease in the protein level of β-catenin, the key functional component of the canonical Wnt/β-catenin signaling pathway. This study deepens the understanding of the Notch pathway by clarifying the distinct roles of Notch1 and Notch3 during adipogenesis. We showed that Notch3 is involved in early adipogenic differentiation, while Notch1 functions later in the process. In addition, we begin to uncover the interaction between the Notch and Wnt signaling pathways that may offer novel therapeutic targets aimed at obesity and diabetes.

Genistein Protects Against Burn-Induced Myocardial Injury via Notch1-Mediated Suppression of Oxidative/Nitrative Stress.

Genistein (Gen) exhibits strong anti-oxidative/antinitrative activity and cardioprotective effects in several models; however, its role in burn-induced myocardial injury is unknown. This study investigated the protective effect of Gen on burn-induced myocardial injury and aimed to elucidate the mechanism of protection. Mice were injected with Gen, intraperitoneally, at different dose immediately after burn injury. The expression levels of Notch-1 intracellular domain (NICD1) and hairy and enhancer of split (Hes-1) were determined by immunoblotting. Conditional Notch-RBP-J knockout mice were used to investigate the mechanisms of Gen-induced cardioprotection. Gen alleviated burn-induced myocardial injury, as shown by improved left ventricle ejection fraction, decreased serum lactate dehydrogenase and creatine kinase levels, and apoptosis. Moreover, Gen decreased expressions of inducible nitric oxide (NO) synthase and gp, reduced NO and superoxide anions production, and ameliorated their cytotoxic reaction product, peroxynitrite. More importantly, Gen significantly up-regulated the expression of NICD1 and Hes1 after burn injury. In addition, genetic knockout of Notch1 not only blocked the cardioprotection of Gen but also markedly attenuated Gen-induced anti-oxidative/antinitrative effect. These results demonstrate, for the first time, that Gen treatment attenuates burn-induced myocardial injury via the Notch1 mediated suppression of oxidative/nitrative stress.

Deubiquitylation and stabilization of Notch1 intracellular domain by ubiquitin-specific protease 8 enhance tumorigenesis in breast cancer.

Notch, an essential factor in tissue development and homoeostasis, has been reported to play an oncogenic function in a variety of cancers. Here, we report ubiquitin-specific protease 8 (USP8) as a novel deubiquitylase of Notch1 intracellular domain (NICD). USP8 specifically stabilizes and deubiquitylates NICD through a direct interaction. The inhibition of USP8 downregulated the Notch signalling pathway via NICD destabilization, resulting in the retardation of cellular growth, wound closure, and colony forming ability of breast cancer cell lines. These phenomena were restored by the reconstitution of NICD or USP8, supporting the direct interaction between these two proteins. The expression levels of NICD and USP8 proteins were positively correlated in patients with advanced breast cancer. Taken together, our results suggest that USP8 functions as a positive regulator of Notch signalling, offering a therapeutic target for breast cancer.

MiR-34a affects hepatocyte proliferation during hepatocyte regeneration through regulating Notch/HIF-1α signaling pathway.

To explore the influences of micro ribonucleic acid (miR)-34a on liver function and hepatocyte proliferation during hepatocyte regeneration in rats and its mechanism. A total of 80 Sprague-Dawley rats were randomly divided into 4 groups: Sham-2 d group (2 days after hepatectomy), Sham-10 d group (10 days after hepatectomy), miR-34a siRNA-2d group (miR-34a knockdown + 2 days after hepatectomy) and miR-34a siRNA-10 d group (miR-34a knockdown + 10 days after hepatectomy), with 20 rats in each group. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were detected at 2 d and 10 d after the operation. The rat liver was harvested for calculating the liver/body weight ratio. In addition, the deoxyribonucleic acid (DNA) content in rat hepatocytes was detected via Feulgen staining. The pathological changes in rat liver were detected via hematoxylin-eosin (H&E) staining. Moreover, the hepatocyte apoptosis in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Expression levels of proliferating cell nuclear antigen (PCNA), Notch1 intracellular domain (NICD), and hypoxia-inducible factor-1α (HIF-1α) in liver tissues of each group were detected via immunohistochemistry and Western blotting. No significant differences in the liver/body weight ratio, serum levels of ALT, AST, LDH, pathological structure of the liver, hepatocyte apoptosis level, and PCNA expression in hepatocytes were found between miR-34a siRNA-2 d group and Sham-2 d group. However, the expression levels of NICD and HIF-1α in the liver significantly increased in miR-34a siRNA-2 d group compared with those in Sham-2 d group (p<0.05). On the contrary, compared with those in Sham-10 d group, the liver function and hepatocyte regeneration level significantly increased in miR-34a siRNA-10 d group. Increased liver/body weight ratio, remarkable decline in serum levels of ALT, AST, and LDH, significant alleviation of pathological injury of liver tissues, decreased the apoptosis level and upregulated PCNA protein were observed in miR-34a siRNA-10 d group than those of Sham-10 d group. The Notch/HIF-1α signaling pathway was also significantly activated. MiR-34a knockdown can significantly enhance the liver function and hepatocyte regeneration ability in rats at 10 d after hepatectomy through activating the Notch/HIF-1α signaling pathway.

FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

AbstractNOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

Doxorubicin combined with Notch1-targeting siRNA for the treatment of gastric cancer.

Notch1, a transmembrane receptor that has a notable role in gastric cancer (GC) as an oncogene, has been reported to be involved in doxorubicin resistance. Thus, Notch1 is a potential therapeutic target for GC. In the present study, the protein levels of Notch1 intracellular domain (NICD; a marker of Notch1 activation) in human GC cell lines and tumor tissues was measured by western blotting. Next, the effects of Notch1 depletion in SGC7901 cells were evaluated. Finally, the efficacy of Notch1 small interfering RNA (siRNA) combined with doxorubicin therapy for GC was examined in vitro and in vivo. The results revealed that NICD levels were high in GC cells, and that the inhibition of NICD by transfection with Notch1 siRNA induced apoptosis and inhibited proliferation. Ectopic downregulation of Notch1 expression enhanced the sensitivity of GC tumors to doxorubicin, which suppressed the development of GC. These data demonstrated that Notch1 was a significant regulator of cell proliferation and apoptosis in GC. Thus, the combination of doxorubicin with Notch1 siRNA is a potential strategy for the treatment of GC.

The function of Notch1 intracellular domain in the differentiation of gastric cancer.

Due to the complex function of the Notch signal pathway in gastric cancer (GC), the association between Notch homolog 1 (Notch1) intracellular domain (NICD) and differentiation of GC remains unknown. The present study aimed to investigate the potential association between NICD and GC differentiation, and demonstrated that poorly differentiated GC expressed increased NICD levels compared with well differentiated GC. A γ-secretase inhibitor inhibited the growth of AGS cells through downregulating NICD level. Additional data suggested that a COX-2 inhibitor caused a marked reduction of NICD level in comparison with a control group treated with dimethyl sulfoxide. Combined administration of γ-secretase and COX-2 inhibitor produced a marked inhibition of growth in AGS cells, which suggests that patients with poorly differentiated GC may benefit from the blockage of NICD, which potentially serves a role in GC differentiation.

MED12 mutations and NOTCH signalling in chronic lymphocytic leukaemia.

Mutations in the N-terminus of MED12 protein occur at high frequency in uterine leiomyomas and breast fibroepithelial tumours, and are frequently found in chronic lymphocytic leukaemia (CLL). MED12 mutations have been previously linked to aberrant Cyclin C-CDK8 kinase activity, but the exact oncogenic function in CLL is unknown. Here, we characterized MED12 mutations in CLL and identified recurrent mutations in 13 out of 188 CLL patients (6·9%), which clustered in the N-terminus. MED12 mutations were associated with unmutated IGHV (P=0·024). Protein analysis of NOTCH1 in primary CLL samples revealed increased levels of NOTCH1 intracellular domain (NICD), the active form of NOTCH1, in the context of MED12 mutations. We found evidence that NICD is the target of Cyclin C-CDK8 kinase using a specific CDK8 inhibitor. In line with these findings, MED12 mutations were mutually exclusive to mutations in NOTCH1 in CLL, based on a meta-analysis of 1429 CLL patients (P=0·011). Our results suggest that MED12 mutations may contribute to CLL pathogenesis by activating NOTCH signalling.

Regulation of the epithelial to mesenchymal transition and metastasis by Raf kinase inhibitory protein-dependent Notch1 activity.

Raf kinase inhibitory protein (RKIP), an endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway, has been implicated as a suppressor of metastasis and a prognostic marker in cancers. However, how RKIP acts as a suppressor during metastasis is not fully understood. Here, we show that RKIP activity in cervical and stomach cancer is inversely correlated with endogenous levels of the Notch1 intracellular domain (NICD), which stimulates the epithelial to mesenchymal transition (EMT) and metastasis. The levels of RKIP were significantly decreased in tumor tissues compared to normal tissues, whereas NICD levels were increased. Overexpression of RKIP in several cell lines resulted in a dramatic decrease of NICD and subsequent inhibition of several mesenchymal markers, such as vimentin, N-cadherin, and Snail. In contrast, knockdown of RKIP exhibited opposite results both in vitro and in vivo using mouse models. Nevertheless, knockdown of Notch1 in cancer cells had no effect on the expression of RKIP, suggesting that RKIP is likely an upstream regulator of the Notch1 pathway. We also found that RKIP directly interacts with Notch1 but has no influence on the intracellular level of the γ-secretase complex that is necessary for Notch1 activation. These data suggest that RKIP plays a distinct role in activation of Notch1 during EMT and metastasis, providing a new target for cancer treatment.

Inhibition of perillyl alcohol on cell invasion and migration depends on the Notch signaling pathway in hepatoma cells.

Cell metastasis, especially the process of invasion and migration, is considered as the main cause for the high mortality rate of hepatocellular carcinoma (HCC), which has become the sixth most common cancer worldwide and the third leading cause of cancer death. In this present study, we aimed to exploit the effects of perillyl alcohol on cell invasion and migration and the underlying molecular mechanisms in HCC. According to the transwell assays, cell invasiveness and migratory capacity were markedly higher in hepatoma cells (HepG2, SMMC-7721 and MHCC97H) than those in normal liver cells (HL-7702), and then significantly suppressed by perillyl alcohol treatment (P<0.05). Meanwhile, the mRNA levels of Notch signaling pathway downstream target genes, HES1, HES5, and HEY1, were notably higher in hepatoma cells detected with real-time reverse transcription polymerase chain reaction (RT-PCR) (P<0.05). After treated with perillyl alcohol, these mRNA levels were significantly decreased in hepatoma cells (P<0.05). In addition, compared with the normal liver cells, the protein expression levels of Notch1 intracellular domain (N1ICD) and Snail were significantly increased, while E-cadherin protein expression was significantly decreased in hepatoma cells (P<0.05). However, perillyl alcohol treatment significantly decreased N1ICD and Snail protein expressions and increased E-cadherin protein expression in hepatoma cells (P<0.05). In conclusion, perillyl alcohol might play an important role in the process of hepatoma cell invasion and migration via decreasing the activity of Notch signaling pathway and increasing E-cadherin expression regulated by Snail.

Bevacizumab modulates retinal pigment epithelial-to-mesenchymal transition via regulating Notch signaling.

To investigate the effect of bevacizumab treatment on Notch signaling and the induction of epithelial-of-mesenchymal transition (EMT) in human retinal pigment epithelial cells (ARPE-19) in vitro. In vitro cultivated ARPE-19 cells were treated with 0.25 mg/mL bevacizumab for 12, 24, and 48h. Cell morphology changes were observed under an inverted microscope. The expression of zonula occludens-1 (ZO-1), vimentin and Notch-1 intracellular domain (NICD) was examined by immunofluorescence. The mRNA levels of ZO-1, α-SMA, Notch-1, Notch-2, Notch-4, Dll4, Jagged-1, RBP-Jk and Hes-1 expression were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of α-SMA, NICD, Hes-1 and Dll-4 expression were examined with Western blot. Bevacizumab stimulation increased the expression of α-SMA and vimentin in ARPE-19 cells which changed into spindle-shaped fibroblast-like cells. Meanwhile, the mRNA expression of Hes-1 increased and the protein expression of Hes-1 and NICD also increased, which Notch signaling was activated. The mRNA expression of Notch-1, Jagged-1 and RBP-Jk increased at 48h, and while Dll4 mRNA and protein expression did not change after bevacizumab treatment. Jagged-1/Notch-1 signaling may play a critical role in bevacizumab-induced EMT in ARPE-19 cells, which provides a novel insight into the pathogenesis of intravitreal bevacizumab-associated complication.

NOTCH1 Mutations Are Associated with Low CD20 Expression in Chronic Lymphocytic Leukemia: Evidences for a NOTCH1-Mediated Epigenetic Regulatory Mechanism

Background.NOTCH1 mutations are found in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, and with higher frequencies in chemorefractory CLL, CLL in advanced disease phases and in Richter Syndrome transformations (Rossi et al. Blood, 2013). In CLL, NOTCH1 mutations have been recently associated with clinical resistance to anti-CD20 immunotherapy (Stilgenbauer et al. Blood, 2014, Dal Bo et al. AHO, 2014). In lymphoproliferative disorders, susceptibility to rituximab is determined by CD20 levels (Golay et al. Blood, 2001), which are in turn epigenetically modulated via HDAC (Shimizu et al., Leukemia, 2010).Aim. To investigate whether NOTCH1 mutations could affect CD20 expression in CLL.Methods.NOTCH1 mutations and NOTCH1 mutational burden were evaluated by ARMS PCR, QRT-PCR, conventional and next generation sequencing. NOTCH1 protein levels were evaluated by western blot. CD20 expression was evaluated by flow cytometry. Transcript levels of MS4A1, encoding for CD20 protein, were evaluated by QRT-PCR. CLL cells and CLL-like cell line MEC-1 cells were treated with the HDAC inhibitor valproic acid (VPA) at a concentration of 3mM. Susceptibility to rituximab was evaluated by complement dependent cytotoxicity (CDC) assay. MEC-1 cells were transfected with a vector containing a NOTCH1 intracellular domain (NICD) carrying either the 7544-7545delCT or a stop codon at the beginning of NICD sequence (c.5304G>A), as control.Results.i) In a cohort of 452 CLL, 54 cases carried NOTCH1 mutations. NOTCH1-mutated cases (NOTCH1-mut) with high mutational burden showed 3.0- fold higher transmembrane NOTCH1 and 2.1- fold higher NICD protein expression compared to NOTCH1 wild-type cases (NOTCH1-wt). NOTCH1-mut showed a lower Mean Fluorescent Intensity (MFI) of CD20 expression than NOTCH1-wt both in trisomy 12 (tris12, 18 NOTCH1-mut vs. 44 NOTCH1-wt, p<0.001) and in non-tris12 (36 NOTCH1-mut vs. 354 NOTCH1-wt, p=0.005) cases. MS4A1 transcript levels were lower in NOTCH1-mut than in NOTCH1-wt (tris12 CLL, p=0.024; non-tris12 CLL, p=0.002). By performing cell sorting to isolate CD20low and CD20high subpopulations in 4 cases with subclonal NOTCH1 mutations (range 26%-45%), the CD20low subpopulation always showed an enrichment in NOTCH1 mutational burden when compared to the CD20high counterpart, along with lower MS4A1 expression levels. NICD mutated MEC-1 cells, expressing higher NICD transcript (2.2 over the control) and NICD (1.3 over the control) protein, were characterized by lower CD20 (0.1 over the control) and MS4A1 transcript (0.77 over the control) expression. ii) In keeping with CD20 levels, primary NOTCH1-mut CLL cells showed lower relative lysis induced by rituximab than NOTCH1-wt CLL cells (mean relative lysis: NOTCH1-mut, 5% vs. NOTCH1-wt, 30%, p=0.008). This phenomenon was confirmed by using the MEC-1 cell model (mean relative lysis: NICD mutated, 3% vs. control, 30%, p=0.006). iii) NICD mutated MEC-1 cells expressed higher levels of both HDAC1 (2.0 over the control) and HDAC2 (1.4 over the control) transcripts. Moreover, in co-immunoprecipitation experiments, NICD mutated MEC-1 cells were characterized by lower levels of HDAC1/HDAC2 bound to the transcriptional factor CBF-1, due to a competition for the binding to CBF1 with the high NICD levels present in these cells, and suggesting a nuclear interplay between NOTCH1 and HDAC where CBF-1 is the connecting element. The dependency of low CD20 expression by HDAC activity was demonstrated by the capability of VPA to increase CD20 expression in primary NOTCH1-mut CLL cells (1.3 over the control) and NICD mutated MEC-1 transfectants (1.4 over the control).Conclusions. CLL cases carrying NOTCH1 mutations are characterized by low CD20 expression levels that may confer resistance to anti-CD20 immunotherapy. The low CD20 expression, at least in part due to HDAC-dependent repression mechanism(s), can be reverted by HDAC inhibitor therapy. DisclosuresNo relevant conflicts of interest to declare.

Notch1 Activation Up-Regulates Pancreatic and Duodenal Homeobox-1

Transcription factor pancreatic and duodenal homeobox-1 (PDX-1) plays an essential role in pancreatic development, β-cell differentiation, maintenance of normal β-cell function and tumorigenesis. PDX-1 expression is tightly controlled through a variety of mechanisms under different cellular contexts. We report here that overexpression of Notch1 intracellular domain (NICD), an activated form of Notch1, enhanced PDX-1 expression in both PDX-1 stable HEK293 cells and mouse insulinoma β-TC-6 cells, while NICD shRNA inhibited the enhancing effect. NICD-enhanced PDX-1 expression was accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells, which was reversed by NICD shRNA. Cre activation-induced specific expression of NICD in islet β cells of transgenic βNICD+/+ mice induced increased expression of PDX-1, insulin and proliferating cell nuclear antigen (PCNA) and decreased expression of p27 with accompanied fasting hyperinsulinemia and hypoglycemia and altered responses to intraperitoneal glucose tolerance test. Systemically delivered NICD shRNA suppressed islet expression of PDX-1 and reversed the hypoglycemia and hyperinsulinemia. Moreover, expression levels of NICD were correlated with those of PDX-1 in human pancreatic neuroendocrine tumor. Thus, Notch1 acts as a positive regulator for PDX-1 expression, cooperates with PDX-1 in the development of insulin overexpression and islet cell neoplasia and represents a potential therapeutic target for islet neoplasia.

Abstract 4147: Xanthohumol decreases Notch1 expression and cell growth in human epithelial ovarian cancer cell lines

Abstract Epithelial ovarian cancer is the most lethal of all gynecologic malignancies in the United States with the majority of cases detected with advanced disease. Therefore, it is critical that novel therapies be developed that can effectively treat ovarian cancer. Notch1 is a transmembrane receptor that upon activation is cleaved by gamma-secretase resulting in Notch1 intracellular domain (NICD) translocation to the nucleus where it heterodimerizes with the transcription factor CBF-1 converting CBF-1 from a transcriptional repressor to a transcriptional activator via recruitment of coactivators. Deregulation of the Notch pathway has been observed in cancers such as T cell lymphoblastic leukemia, pancreatic, and breast. In addition, it has been shown that NICD is expressed in ovarian cancer cell lines and ovarian tumors. Xanthohumol, a prenylated chalcone derived from hops used in brewing beer, has been shown to exhibit anti-cancer properties. Since xanthohumol has been shown to inhibit cancer cell growth and invasion, we hypothesized that the Notch1 signaling pathway is suppressed by xanthohumol leading to decreased cell growth. OVCAR3 and SKOV3 are human epithelial ovarian cancer cell lines that express high levels of NICD. These cells were treated with increasing doses of xanthohumol, and cell growth was measured by 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay on days 0, 2, 4, and 6. In addition, protein lysates were isolated on days 2 and 4 of treatment, detected by western blot using a NICD primary antibody, and protein expression was evaluated by densitometry. These treatments resulted in decreased cell growth when measured by MTT assay. In addition, when analyzed by western blot, there was a decrease in NICD protein expression. In conclusion, xanthohumol was a potent inhibitor of ovarian cancer cell growth, and our results suggest that xanthohumol is affecting the Notch pathway. Therefore, it is essential that further investigation into the role that xanthohumol has on the Notch pathway be pursued. These outcomes could prompt the use of xanthohumol as a potential chemotherapeutic agent in ovarian cancer patients who do not respond long term to standard chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4147.

Presenilin modulates EGFR signaling and cell transformation by regulating the ubiquitin ligase Fbw7

The epidermal growth factor receptor (EGFR) and Notch signaling pathways have antagonistic roles during epidermal differentiation and carcinogenesis. The molecular mechanisms regulating the crosstalk between EGFR and Notch during epidermal transformation are largely unknown. We found enhanced EGFR-dependent signaling, proliferation and oncogenic transformation caused by loss of presenilins (PS), the catalytic components of gamma-secretase that generates the Notch1 intracellular domain (NICD). The underlying mechanism for abnormal EGFR signaling in PS-deficient cells involves gamma-secretase-independent transcriptional upregulation of the E3 ubiquitin ligase Fbw7. Fbw7alpha, which targets NICD for degradation, regulates positively EGFR by affecting a proteasome-dependent ubiquitination step essential for constitutive degradation and stability of EGFR. To investigate the pathological relevance of this findings in vivo, we generated a novel epidermal conditional PS-deficient (ePS cDKO) mouse by deleting both PS in keratinocytes of the basal layer of the epidermis. The ePS cDKO mice develop epidermal hyperplasia associated with enhanced expression of both EGFR and Fbw7 and reduced NICD levels in keratinocytes. These findings establish a novel role for PS on epidermal growth and transformation by reciprocally regulating the EGFR and Notch signaling pathways through Fbw7.

Hepatitis B Virus X Protein Blunts Senescence-Like Growth Arrest of Human Hepatocellular Carcinoma by Reducing Notch1 Cleavage

One of the serious sequelae of chronic hepatitis B virus (HBV) infection is hepatocellular carcinoma (HCC). Among all the proteins encoded by the HBV genome, hepatitis B virus X protein (HBx) is highly associated with the development of HCC. Although Notch1 signaling has been found to exert a tumor-suppressive function during HCC development, the mechanism of interaction between HBx expression and Notch1 signaling needs to be explored. In this study, we report that HBx expression in hepatic and hepatoma cells resulted in decreased endogenous protein levels of Notch1 intracellular domain (ICN1) and messenger RNA levels of its downstream target genes. These effects were due to a reduction of Notch1 cleavage by HBx through the suppression of presenilin1 (Psen1) transcription rather than inhibition of Notch1 transcription or its ligands' expression. Through transient HBx expression, decreased ICN1 resulted in enhanced cell proliferation, induced G1-S cell cycle progression, and blunted cellular senescence in vitro. Furthermore, the effect of blunted senescence-like growth arrest by stable HBx expression through suppression of ICN1 was shown in a nude mouse xenograft transplantation model. The correlation of inhibited Psen1-dependent Notch1 signaling and blunted senescence-like growth arrest was also observed in HBV-associated HCC patient tumor samples. Our results reveal a novel function of HBx in blunting senescence-like growth arrest by decreasing Notch1 signaling, which could be a putative molecular mechanism mediating HBV-associated hepatocarcinogenesis.

Beta-catenin modulates the level and transcriptional activity of Notch1/NICD through its direct interaction

Wnt and Notch1 signaling pathways play an important role in a variety of biological processes including embryonic induction, the polarity of cell division, cell fate, and cell growth. Although there is evidence that the two main signaling pathways can modulate each other, the precise mechanism is not completely understood. This report shows that β-catenin can regulate the level and transcriptional activity of the Notch1 and Notch1 intracellular domain (NICD). The in vivo and in vitro results demonstrate that β-catenin binds with Notch1 and NICD, for which its Armadillo repeat domain is essential. It was further demonstrated that β-catenin could upregulate the level of Notch1 and NICD, possibly by competing the common ubiquitin-dependent degradation machinery. In addition, β-catenin enhanced the transcriptional activity of NICD on the hairy and enhancer of split 1 (HES1) and CSL through its C-terminal transactivation domain. This effect of cooperative regulation by β-catenin could also be observed in bone morphogenetic protein 2 (BMP2) induced osteogenic differentiation of C2C12 cells. β-catenin coexpression with NICD enhanced the alkaline phosphatase (ALP) activity in C2C12 cells compared with either β-catenin or NICD expression alone. Culturing C2C12 cells on Delta-1 coated dishes together with Wnt3-conditioned media induced noticeable increases in ALP staining, verifying that employed physiological levels of NICD and β-catenin are sufficient to induce ALP activation. Furthermore, effects of β-catenin on Notch1 were dramatically diminished by overexpressed LEF1. Overall, our data suggest that β-catenin can act as a switching molecule between the classical TCF/LEF1 mediated pathway and NICD mediated pathway.