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 E₂ 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. [Cancer Res 2009;69(12):5039–48]

Gastric cancer is the second leading cause of cancer-related death worldwide. Herein, we investigated the role of transcription factor Yin Yang 1 (YY1), a multi-functional protein, in tumorigenesis of gastric cancer cells. Results showed that YY1 contributed to gastric carcinogenesis of SC-M1 cells including growth, viability, and abilities of colony formation, migration, invasion, and tumorsphere formation. Levels of pluripotency genes CD44, Oct4, SOX-2, and Nanog were also up-regulated by YY1 in SC-M1 cells. Additionally, the 3′-untranslated region (3′-UTR) of YY1 mRNA was the target of microRNA-34 (miR-34) family consisting of miR-34a, miR-34b, and miR-34c. Overexpression of miR-34 family suppressed carcinogenesis through down-regulation of YY1 in NUGC-3 gastric cancer cells scarcely expressing miR-34 family. Alternatively, knockdown of miR-34 family promoted tumorigenesis via up-regulation of YY1 in SC-M1 and AZ521 gastric cancer cells with higher levels of miR-34 family. The miR-34 family also affected tumorsphere ultra-structure and inhibited the xenografted tumor growth as well as lung metastasis of SC-M1 cells through YY1. Expressions of miR-34a and miR-34c in gastric cancer tissues of patients were lower than those in normal tissues. Taken together, these results suggest that miR-34 family-YY1 axis plays an important role in the control of gastric carcinogenesis.

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 alpha-enolase and c-myc promoter binding protein 1 (MBP-1). Both alpha-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 alpha-enolase or MBP-1. Furthermore, N1IC, YY1, and alpha-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 alpha-enolase and MBP-1. In addition, both alpha-enolase and MBP-1 suppressed the N1IC-enhanced colony-forming ability through c-myc. These results indicate that the activated Notch1 receptor and alpha-enolase or MBP-1 cooperate in controlling c-myc expression through binding the YY1 response element of the c-myc promoter to regulate tumorigenesis.

Transcription factor Ying Yang 1 (YY1) indirectly regulates the C promoter-binding factor 1 (CBF1)-dependent Notch1 signaling via direct interaction with the Notch1 receptor intracellular domain (N1IC) on CBF1-response elements. To evaluate the possibility that the N1IC might modulate the gene expression of YY1 target genes through associating with YY1 on the YY1-response elements, we herein investigated the effect of Notch1 signaling on the expression of YY1 target genes. We found that the N1IC bound to the double-stranded oligonucleotides of YY1-response element to activate luciferase activity of the reporter gene with YY1-response elements through a CBF1-independent manner. Furthermore, the N1IC also bound to the promoter of human c-myc oncogene, a YY1 target gene, to elevate c-myc expression via a CBF1-independent pathway. The activation of reporter genes with YY1-response elements or human c-myc promoter by N1IC depended on the formation of N1IC–YY1-associated complex. To delineate the role of the Notch signal pathway in tumorigenesis, K562 cell lines expressing the N1IC were established. Compared with control cells, the proliferation and the tumor growth of N1IC-expressing K562 cells were suppressed. Taken together, these results suggest that the N1IC enhances the human c-myc promoter activity that is partially modulated by YY1 through a CBF1-independent pathway. However, the enhancement of c-myc expression by N1IC is insufficient to promote the tumor growth of K562 cells.

The 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) is a potential regulatory node in the mevalonate pathway that is frequently dysregulated in tumors. This study found that HMGCS1 expression is upregulated in stomach adenocarcinoma samples of patients and tumorspheres of gastric cancer cells. HMGCS1 elevates the expression levels of the pluripotency genes Oct4 and SOX-2 and contributes to tumorsphere formation ability in gastric cancer cells. HMGCS1 also promotes in vitro cell growth and progression and the in vivo tumor growth and lung metastasis of gastric cancer cells. After blocking the mevalonate pathway by statin and dipyridamole, HMGCS1 exerts nonmetabolic functions in enhancing gastric cancer progression. Furthermore, the level and nuclear translocation of HMGCS1 in gastric cancer cells are induced by serum deprivation. HMGCS1 binds to and activates Oct4 and SOX-2 promoters. HMGCS1 also enhances the integrated stress response (ISR) and interacts with the endoplasmic reticulum (ER) stress transducer protein kinase RNA-like endoplasmic reticulum kinase (PERK). Our results reveal that HMGCS1 contributes to gastric cancer progression in both metabolic and nonmetabolic manners.