자궁경부암세포에서 SMAD4 와 그 표적 유전자의 발현을 조절하는 GNAI2 에 대한 기능 연구

학위논문 (석사)-- 서울대학교 대학원 : 약학과, 2012. 8. 신영기.GNAI2 (Guanine nucleotide binding protein (G-protein), alpha inhibiting activity polypeptide 2) belongs to a family of Gi alpha proteins that includes three polypeptides: Gi alpha 1 (GNAI1), Gi alpha 2 (GNAI2), and Gi alpha 3 (GNAI3). GNAI2 is a well known proto-oncogene that is involved in onset and propagation of many different types of cancers. Smad4 (Mother Against Decapentaplegic Homologue 4, DPC4) is a well-known tumor suppressor and key elements in TGF-β (Transforming Growth factor- β) signaling pathway. In order to demonstrate the role of GNAI2 in carcinogenesis and to find out its potential interacting partners, we had initially identified Smad4 as one of its interacting partners through a baculovirus protoarray system. In this study we firstly demonstrated the novel interaction between GNAI2 and Smad4 using bimolecular fluorescence complementation (BiFC) assay in cervical cancer (HeLa) cell. We also confirmed the interaction between GNAI2 and Smad4 by endogenous and exogenous immunoprecipitaion (IP) assay. Upon ectopic transfection of GNAI2, the western blot analysis resulted in decrements of the level of Smad4, p15INK4b, p21 protein and an increase the protein expression of C-Myc. In contrast, transient knockdown of GNAI2 resulted in substantial increase in Smad4, p15INK4b, p21 protein and a decrease in the protein expression of C-Myc. However, our qRT-PCR data showed no change at mRNA level of Smad4 but a substantial decrease and increase at mRNA level of p15INK4, p21 after transient overexpression and knockdown of GNAI2 in HeLa cell respectively. Moreover, 26S proteosomal degradation inhibitor MG132 treatment in GNAI2 overexpressed HeLa cell resulted in reduced degradation of Smad4 protein.Through luciferase reporter assay we have found that the overexpression of GNAI2 resulted in reduced p15INK4b promoter activity. In addition, the si-RNA-based knockdown of GNAI2 significantly reduced the proliferation of cervical cancer (HeLa) cells. Taken together our study demonstrated that GNAI2 interacts with Smad4 and negatively regulates the expression of Smad4 and its targeted gene in cervical cancer (HeLa) cells.Elucidate the role GNAI2 in the regulation of Smad4 function and its consequences in cervical carcinogenesis.Maste

Stage dependent expression and tumor suppressive function of FAM134B (JK1) in colon cancer

The aims of the present study are to investigate sub-cellular location, differential expression in different cancer stages and functional role of FAM134B in colon cancer development. FAM134B expression was studied and quantified at protein and mRNA levels in cell lines using immunocytochemistry, Western blot and real-time PCR. In vitro functional assays and an in vivo xenotransplantation mouse models were used to investigate the molecular role of FAM134B in cancer cell biology in response to FAM134B silencing with shRNA lentiviral particles. FAM134B protein was noted in both cytoplasm and nuclei of cancer cells. In cancer cells derived from stage IV colon cancer, FAM134B expression was remarkably reduced when compared to non-cancer colon cells and cancer cells derived from stage II colon cancer. FAM134B knockdown significantly (P < 0.05) increased the proliferation of colon cancer cells following lentiviral transfection. Furthermore, FAM134B suppression significantly increased (34-52%; P < 0.05) the clonogenic capacity, wound healing potential of and increases the proportion of cells performing DNA synthesis (P < 0.01). Xenotransplantation model showed that larger and higher-grade tumors were formed in mice receiving FAM134B knockdown cells. To conclude, expression analysis, in vitro and in vivo indicated that FAM134B acts as a cancer suppressor gene in colon cancer

Cellular expression, in-vitro and in-vivo confirmation of GAEC1 oncogenic properties in colon cancer

GAEC1 (Gene amplified in esophageal cancer 1) alterations have oncogenic properties in oesophageal squamous cell carcinomas and frequent amplifications of the gene were noted in colorectal adenocarcinomas. However, the subcellular localization and expression of GAEC1 at the protein level have never been reported in human cancer cells. The present study aimed to investigate whether GAEC1 is differentially expressed in different stages of colon cancer and to elucidate its underlying cellular and molecular mechanism in colon cancer progression. We found differential expression of GAEC1 protein and mRNA in different pathological stages of colon cancer cells (SW480-Stage II, SW48-Stage III and HCT116-Stage IV) when compared to non-neoplastic colon cells (FHC cells) by immunocytochemistry, immunofluorescence, western blot analysis and real-time polymerase chain reaction. GAEC1 protein was predominantly expressed in the cytoplasm of colon cancer cells (SW480, SW48, and HCT116) and in the nucleus of non-neoplastic colon epithelial cells (FHC cells). The transient knockdown of GAEC1 using siRNA induced apoptosis in SW480 and SW48 cells, which was associated with G2/M phase arrest and decreased expression of bcl-2 and K-ras proteins and increased expression of p53. In addition, down-regulation of GAEC1 significantly inhibited (p<0.05) cell proliferation, reduced migration capacity and decreased clonogenic potentiality of colon cancer cells (SW480 and SW48 cells). Furthermore, a xenotransplantation model showed that stable knockdown of GAEC1 using shRNA constructs in colon cancer cells fully suppressed xenograft tumour growth in mice. Collectively, the expression analysis, in vitro and in vivo data indicated that GAEC1 is differentially expressed in cancer cells and act as an oncogene in colon cancer progression

Cancer stem cells in oesophageal squamous cell carcinoma: Identification, prognostic and treatment perspectives

Cancer stem cells (CSCs) are a vital subpopulation of cells to target for the treatment of cancers. In oesophageal squamous cell carcinoma (ESCC), there are several markers such as CD44, ALDH, Pygo2, MAML1, Twist1, Musashi1, Side population (SP), CD271 and CD90 that have been proposed to identify the cancer stem cells in individual cancer masses. It has also been demonstrated that stem cell markers like ALDH1, HIWI, Oct3/4, ABCG2, SOX2, SALL4, BMI-1, NANOG, CD133 and podoplanin are associated with patient's prognosis, pathological stages, cancer recurrence and therapy resistance. Finding new cancer stem cell targets or designing drugs to manipulate the known molecular targets in CSCs could be useful for improvements in clinical outcomes of the disease. To conclude, data suggest that CSCs in oesophageal squamous cell carcinoma are related to resistance to therapy and poor prognosis of patients with ESCC. Therefore, innovative insights into CSC biology and CSC-targeted therapies will help to achieve more effective management of patients with oesophageal squamous cell carcinoma

Deregulation of miR-126 expression in colorectal cancer pathogenesis and its clinical significance

In this study, we investigated the expression profiles and clinicopathological significance of miR-126 in large cohort of patients with colorectal cancers as well the cellular repercussions of miR-126 in colon cancer cells along with its targets in-vitro. Down regulation of miR-126 expression was associated with histological subtypes, peri-neural tumour infiltration, microsatellite instability and pathological staging of colorectal cancers (p<0.05). Low miR-126 expression was also associated with poorer survival in patients with colorectal cancer. Analysis of matched tissues from the same patient revealed that approximately 70% of the tested patients had similar levels of expression of miR-126 in primary cancer and cancer metastases in both lymph node and distant metastases. In addition, induced overexpression of miR-126 showed reduced cell proliferation, increased apoptosis and decreased accumulation of cells in the G0-G1 phase of the colon cancer cells. Furthermore, SW480(+miR-126) cells showed reduced BCL-2 and increased P53 protein expression. To conclude, deregulation of miR-126 in colorectal cancer at the tissue and cellular levels as well as its correlation with various clinicopathological parameters confirm the cancer suppressive role of miR-126 in colorectal cancer

Cancer-on-a-Chip: Models for Studying Metastasis

The microfluidic-based cancer-on-a-chip models work as a powerful tool to study the tumor microenvironment and its role in metastasis. The models recapitulate and systematically simplify the in vitro tumor microenvironment. This enables the study of a metastatic process in unprecedented detail. This review examines the development of cancer-on-a-chip microfluidic platforms at the invasion/intravasation, extravasation, and angiogenesis steps over the last three years. The on-chip modeling of mechanical cues involved in the metastasis cascade are also discussed. Finally, the popular design of microfluidic chip models for each step are discussed along with the challenges and perspectives of cancer-on-a-chip models

GAEC1 mutations and copy number aberration is associated with biological aggressiveness of colorectal cancer

GAEC1 (gene amplified in oesophageal cancer 1) is a transforming oncogene with tumorigenic potential observed in both oesophageal squamous cell carcinoma and colorectal cancer. Nonetheless, there has been a lack of study done on this gene to understand how this gene exert its oncogenic properties in cancer. This study aims to identify novel mutation sites in GAEC1. To do so, seventy-nine matched colorectal cancers were tested for GAEC1 mutation via Sanger sequencing. The mutations noted were investigated for the correlations with the clinicopathological parameters of the patients with the cancer. Additionally, GAEC1 copy number aberration (CNA), mRNA and protein expression were determined with the use of droplet digital (dd) polymerase chain reaction (PCR), real-time PCR and Western blot (confirmed with immunofluorescence analysis). GAEC1 mutation was noted in 8.8% (n = 7/79) of the cancer tissues including one missense mutation, four loss of heterozygosity (LOH) and two substitutions. These mutations were significantly associated with cancer perforation (p = 0.021). GAEC1 mutation is frequently associated with increased GAEC1 protein expression. Nevertheless, GAEC1 mRNA and protein are only weakly associated. Taken together, GAEC1 mutation affects GAEC1 expression and is associated with poorer clinical outcomes. This further strengthens the role of GAEC1 as an oncogene.</p