Tien-Shun Yeh

The Activated Notch1 Signal Pathway Is Associated with Gastric Cancer Progression through Cyclooxygenase-2

Gastric carcinoma is one of the most common cancers and lethal malignancies worldwide. Thus far, the regulatory mechanisms of its aggressiveness are still poorly understood. To understand the pathogenesis and to develop new therapeutic strategies, it is essential to dissect the molecular mechanisms that regulate progression of gastric cancer. Herein, we sought to address whether Notch1 signal pathway is involved in the control of progression in gastric cancer. We found that expression of Notch ligand Jagged1 was correlated with aggressiveness of human gastric cancer. Patients with Jagged1 expression in gastric cancer tissues had a poor survival rate compared with those without Jagged1 expression. The Notch1 receptor intracellular domain (N1IC), the activated form of Notch1 receptor, promoted the colony-forming ability and xenografted tumor growth of human stomach adenocarcinoma SC-M1 cells. Migration and invasion abilities of SC-M1 cells were enhanced by N1IC. Furthermore, N1IC and C promoter-binding factor 1 (CBF1) bound to cyclooxygenase-2 (COX-2) promoter and elevated COX-2 expression in SC-M1 cells through a CBF1-dependent manner. The colony-forming, migration, and invasion abilities enhanced by N1IC were suppressed in SC-M1 cells after treatment with the COX-2 inhibitor NS-398 or knockdown of COX-2. These cellular processes inhibited by Notch1 knockdown were restored by prostaglandin E(2) or exogenous COX-2. Taken together, these results suggest that activation of Notch1 signal pathway promotes progression of gastric cancer, at least in part through COX-2.

Maintenance of mitochondrial DNA copy number and expression are essential for preservation of mitochondrial function and cell growth

To examine whether a reduction in the mtDNA level will compromise mitochondrial biogenesis and mitochondrial function, we created a cell model with depleted mtDNA. Stable transfection of small interfering (si)RNA of mitochondrial transcription factor A (Tfam) was used to interfere with Tfam gene expression. Selected stable clones showed 60–95% reduction in Tfam gene expression and 50–90% reduction in cytochrome b (Cyt b) gene expression. Tfam gene knockdown clones also showed decreased mtDNA‐encoded cytochrome c oxidase subunit I (COX I) protein expression. However, no significant differences in protein expression were observed in nuclear DNA (nDNA)‐encoded mitochondrial respiratory enzyme subunits. The cell morphology changed from a rhombus‐like to a spindle‐like form as determined in clones with decreased expressions of Tfam, mtRNA, and mitochondrial proteins. The mitochondrial respiratory enzyme activities and ATP production in such clones were significantly lower. The proportions of mtDNA mutations including 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), a 4,977‐bp deletion, and a 3,243‐point mutation were also examined in these clones. No obvious increase in mtDNA mutations was observed in mitochondrial dysfunctional cell clones. The mitochondrial respiratory activity and ATP production ability recovered in cells with increased mtDNA levels after removal of the specific siRNA treatment. These experimental results provide direct evidence to substantiate that downregulation of mtDNA copy number and expression may compromise mitochondrial function and subsequent cell growth and morphology. J. Cell. Biochem. 103: 347–357, 2008.

The autonomous notch signal pathway is activated by baicalin and baicalein but is suppressed by niclosamide in K562 cells

The Notch signaling pathway plays important roles in a variety of cellular processes. Aberrant transduction of Notch signaling contributes to many diseases and cancers in humans. The Notch receptor intracellular domain, the activated form of Notch receptor, is extremely difficult to detect in normal cells. However, it can activate signaling at very low protein concentration to elicit its biological effects. In the present study, a cell based luciferase reporter gene assay was established in K562 cells to screen drugs which could modulate the endogenous CBF1‐dependent Notch signal pathway. Using this system, we found that the luciferase activity of CBF1‐dependent reporter gene was activated by baicalin and baicalein but suppressed by niclosamide in both dose‐ and time‐dependent manners. Treatment with these drugs modulated endogenous Notch signaling and affected mRNA expression levels of Notch1 receptor and Notch target genes in K562 cells. Additionally, erythroid differentiation of K562 cells was suppressed by baicalin and baicalein yet was promoted by niclosamide. Colony‐forming ability in soft agar was decreased after treatment with baicalin and baicalein, but was not affected in the presence of niclosamide. Thus, modulation of Notch signaling after treatment with any of these three drugs may affect tumorigenesis of K562 cells suggesting that these drugs may have therapeutic potential for those tumors associated with Notch signaling. Taken together, this system could be beneficial for screening of drugs with potential to treat Notch signal pathway‐associated diseases. J. Cell. Biochem. 106: 682–692, 2009.

Galectin-1 promotes lung cancer progression and chemoresistance by upregulating p38 MAPK, ERK and cyclooxygenase-2

Purpose: This study is aimed at investigating the role and novel molecular mechanisms of galectin-1 in lung cancer progression. Experimental Design: The role of galectin-1 in lung cancer progression was evaluated both in vitro and in vivo by short hairpin RNA (shRNA)-mediated knockdown of galectin-1 in lung adenocarcinoma cell lines. To explore novel molecular mechanisms underlying galectin-1–mediated tumor progression, we analyzed gene expression profiles and signaling pathways using reverse transcription PCR and Western blotting. A tissue microarray containing samples from patients with lung cancer was used to examine the expression of galectin-1 in lung cancer. Results: We found overexpression of galectin-1 in non–small cell lung cancer (NSCLC) cell lines. Suppression of endogenous galectin-1 in lung adenocarcinoma resulted in reduction of the cell migration, invasion, and anchorage-independent growth in vitro and tumor growth in mice. In particular, COX-2 was downregulated in galectin-1–knockdown cells. The decreased tumor invasion and anchorage-independent growth abilities were rescued after reexpression of COX-2 in galectin-1–knockdown cells. Furthermore, we found that TGF-β1 promoted COX-2 expression through galectin-1 interaction with Ras and subsequent activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal–regulated kinase (ERK), and NF-κB pathway. Galectin-1 knockdown sensitized lung cancer cells to platinum-based chemotherapy (cisplatin). In addition, galectin-1 and COX-2 expression was correlated with the progression of lung adenocarcinoma, and high clinical relevance of both proteins was evidenced (n = 47). Conclusions: p38 MAPK, ERK, and COX-2 activation are novel mediators for the galectin-1–promoted tumor progression and chemoresistance in lung cancer. Galectin-1 may be an innovative target for combined modality therapy for lung cancer. Clin Cancer Res; 18(15); 4037–47. ©2012 AACR.

Activation of the Notch1/STAT3/Twist signaling axis promotes gastric cancer progression

Gastric carcinoma is one of the most common malignancies and a lethal cancer in the world. Notch signaling and transcription factors STAT3 (signal transducer and activator of transcription 3) and Twist regulate tumor development and are critical regulators of gastric cancer progression. Herein, the relationship among Notch, STAT3 and Twist pathways in the control of gastric cancer progression was studied. We found that Twist and phosphorylated STAT3 levels were promoted by the activated Notch1 receptor in human stomach adenocarcinoma SC-M1, embryonic kidney HEK293 and erythroleukemia K562 cells. Notch1 signaling dramatically induced Twist promoter activity through a C promoter binding factor-1-independent manner and STAT3 phosphorylation. Overexpression of Notch1 receptor intracellular domain (N1IC) enhanced the interaction between nuclear STAT3 and Twist promoter in cells. Gastric cancer progression of SC-M1 cells was promoted by N1IC through STAT3 phosphorylation and Twist expression including colony formation, migration and invasion. STAT3 regulated gastric cancer progression of SC-M1 cells via Twist. N1IC also elevated the progression of other gastric cancer cells such as AGS and KATO III cells through STAT3 and Twist. The N1IC-promoted tumor growth and lung metastasis of SC-M1 cells in mice were suppressed by the STAT3 inhibitor JSI-124 and Twist knockdown. Furthermore, Notch1 and Notch ligand Jagged1 expressions were significantly associated with phosphorylated STAT3 and Twist levels in gastric cancer tissues of patients. Taken together, these results suggest that Notch1/STAT3/Twist signaling axis is involved in progression of human gastric cancer and modulation of this cascade has potential for the targeted combination therapy.

Abnormal mitochondrial structure in human unfertilized oocytes and arrested embryos

Abstract: To clarify the relationship between mitochondria and embryo development, we collected human unfertilized oocytes, early embryos, and arrested embryos. Unfertilized oocytes and poor‐quality embryos were collected, and the ultrastructure of mitochondria was determined by transmission electron micrography. Four criteria for determining the mitochondrial state were mitochondrial morphology, cristae shape, location, and number of mitochondria. In mature oocytes, mitochondria were rounded with arched cristae and a dense matrix and were distributed evenly in the ooplasm. In pronuclear zygotes, the size and shape of mitochondria were similar to those in mature oocytes; however, mitochondria appeared to migrate and concentrate around pronuclei. In this study, 67% of examined unfertilized oocytes had fewer mitochondria in the cytoplasm. A decreased number of mitochondria located near the nucleus was also demonstrated in 60% of arrested embryos. Fewer differentiated cristae were determined in all three arrested blastocyst stages of embryos. The relative expressions of oxidative phosphorylation genes in oocytes and embryos were also determined. These data imply that inadequate redistribution of mitochondria, unsuccessful mitochondrial differentiation, or decreased mitochondrial transcription may result in poor oocyte fertilization and compromised embryo development.

The Activated Notch1 Receptor Cooperates with α-Enolase and MBP-1 in Modulating c-myc Activity

ABSTRACT The Notch signal pathway plays multifaceted roles to promote or suppress tumorigenesis. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, activates the c-myc proto-oncogene. The complex of N1IC and transcription factor YY1 binds to the human c-myc promoter to enhance c-myc expression in a CBF1-independent manner. Here we demonstrated that N1IC interacted with the c-Myc-regulating proteins α-enolase and c-myc promoter binding protein 1 (MBP-1). Both α-enolase and MBP-1 suppressed the N1IC-enhanced activity of the c-myc promoter in a CBF1-independent manner. The YY1 response element in front of the P2 c-myc promoter was essential and sufficient for the modulation of c-myc by N1IC and α-enolase or MBP-1. Furthermore, N1IC, YY1, and α-enolase or MBP-1 but not CBF1 bound to the c-myc promoter through associating with the YY1 response element. Hemin-induced erythroid differentiation was suppressed by N1IC in K562 cells. This suppression was relieved by the expression of α-enolase and MBP-1. In addition, both α-enolase and MBP-1 suppressed the N1IC-enhanced colony-forming ability through c-myc. These results indicate that the activated Notch1 receptor and α-enolase or MBP-1 cooperate in controlling c-myc expression through binding the YY1 response element of the c-myc promoter to regulate tumorigenesis.

YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model to study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation.

Notch2‐induced COX‐2 expression enhancing gastric cancer progression

Gastric carcinoma is one of the most common and mortal types of malignancy worldwide. To date, the mechanisms controlling its aggressiveness are not yet fully understood. Notch signal pathway can function as either an oncogene or a tumor suppressor in tumorigenesis. Four members (Notch1–4) of Notch receptors were found in mammals and each exhibits distinct roles in tumor progression. Previous study showed that the activated Notch1 receptor promoted gastric cancer progression through cyclooxygenase‐2 (COX‐2). This study addressed whether Notch2 signal pathway is also involved in gastric cancer progression. Constitutive expression of Notch2 intracellular domain (N2IC), the activated form of Notch2 receptor, promoted both cell proliferation and xenografted tumor growth of human stomach adenocarcinoma SC‐M1 cells. The colony formation, migration, invasion, and wound‐healing abilities of SC‐M1 cells were enhanced by N2IC expression, whereas these abilities were suppressed by Notch2 knockdown. Similarly, Notch2 knockdown inhibited cancer progressions of AGS and AZ521 gastric cancer cells. Expression of N2IC also caused epithelial–mesenchymal transition in SC‐M1 cells. Furthermore, N2IC bound to COX‐2 promoter and induced COX‐2 expression through a CBF1‐dependent manner in SC‐M1 cells. The ability of N2IC to enhance tumor progression in SC‐M1 cells was suppressed by knockdown of COX‐2 or treatment with NS‐398, a COX‐2 inhibitor. Moreover, the suppression of tumor progression by Notch2 knockdown in SC‐M1 cells was reversed by exogenous COX‐2 or its major enzymatic product PGE2. Taken together, this study is the first to demonstrate that the Notch2‐COX‐2 signaling axis plays an important role in controlling gastric cancer progression.

MBP-1 suppresses growth and metastasis of gastric cancer cells through COX-2.

The c-Myc promoter binding protein 1 (MBP-1) is a transcriptional suppressor of c-myc expression and involved in control of tumorigenesis. Gastric cancer is one of the most frequent neoplasms and lethal malignancies worldwide. So far, the regulatory mechanism of its aggressiveness has not been clearly characterized. Here we studied roles of MBP-1 in gastric cancer progression. We found that cell proliferation was inhibited by MBP-1 overexpression in human stomach adenocarcinoma SC-M1 cells. Colony formation, migration, and invasion abilities of SC-M1 cells were suppressed by MBP-1 overexpression but promoted by MBP-1 knockdown. Furthermore, the xenografted tumor growth of SC-M1 cells was suppressed by MBP-1 overexpression. Metastasis in lungs of mice was inhibited by MBP-1 after tail vein injection with SC-M1 cells. MBP-1 also suppressed epithelial-mesenchymal transition in SC-M1 cells. Additionally, MBP-1 bound on cyclooxygenase 2 (COX-2) promoter and downregulated COX-2 expression. The MBP-1-suppressed tumor progression in SC-M1 cells were through inhibition of COX-2 expression. MBP-1 also exerted a suppressive effect on tumor progression of other gastric cancer cells such as AGS and NUGC-3 cells. Taken together, these results suggest that MBP-1-suppressed COX-2 expression plays an important role in the inhibition of growth and progression of gastric cancer.