Jin Sook Jeong

An RNA Aptamer That Binds Carcinoembryonic Antigen Inhibits Hepatic Metastasis of Colon Cancer Cells in Mice

BACKGROUND & AIMS Carcinoembryonic antigen (CEA) is expressed by many types of cancer cells; its overexpression induces cell adhesion, increases resistance to anoikis, and promotes hepatic metastasis of colon cancer cells. The amino acid sequence PELPK in its hinge region, between the N and A1 domains, is required for migration of cancer cells to the liver. We sought to identify ligands of this domain for use in diagnosis and therapy. METHODS We screened for RNA aptamers against the domain of CEA required for metastasis using systematic evolution of ligands by exponential enrichment. The specificity and affinity of the aptamer for CEA protein were characterized by mobility shift, uptake, and surface plasmon resonance assays. We analyzed the effects of the aptamer on metastatic properties of cells, as well as metastasis of colon cancer cells in mice. RESULTS Using systematic evolution of ligands by exponential enrichment, we identified an RNA aptamer that bound to the PELPK sequence in CEA with high affinity and specificity. The isolated aptamer bound specifically to CEA-positive cells and inhibited interactions between CEA and heterogeneous nuclear ribonucleoprotein M4. The aptamer inhibited homotypic aggregation, migration, and invasion by CEA-positive cancer cells, but did not affect adhesion of endothelial cells. The aptamer induced colon cancer cell anoikis by interrupting the interaction between death receptor 5 and CEA. The aptamer prevented metastasis of human colon cancer cells to the livers of mice. CONCLUSIONS An RNA aptamer that binds to the PELPK sequence in CEA inhibits its interactions with heterogeneous nuclear ribonucleoprotein M4 and death receptor 5, migration and invasion by colon cancer cells, and hepatic metastasis of colon cancer cells in mice. It promoted cancer cell anoikis and might be used to identify CEA-positive tumors in patients or be developed as an anti-cancer reagent.

Acceleration of Gastric Tumorigenesis Through MKRN1-Mediated Posttranslational Regulation of p14ARF

Background We investigated whether Makorin ring finger protein 1 (MKRN1), an E3 ligase, affects p14ARF-associated cellular senescence and tumorigenesis by posttranslational modification in gastric tumorigenesis. Methods A link between MKRN1 and ARF was examined in MKRN1 null mouse embryonic fibroblasts (MEFs) and in human fibroblasts and gastric cancer cells by silencing MKRN1 using small interfering RNA (siRNA) and short hairpin RNA (shRNA). Ubiquitination and proteasomal degradation assays were used to assess p14ARF degradation associated with MKRN1. MKRN1 and p14ARF expression levels were analyzed with immunohistochemistry in malignant and normal tissues from gastric cancer patients and with χ2 tests. The tumor growth of gastric cancer cells stably expressing MKRN1 shRNA, p14ARF shRNA, or both was examined in mouse xenograft models (n = 4–6) and analyzed with unpaired t tests. All statistical tests were two-sided. Results MKRN1 knockout MEFs exhibited premature senescence and growth retardation with increased p19ARF protein expression. Similar results were obtained for human fibroblasts or gastric cancer cell lines by MKRN1 knockdown. Biochemical analyses confirmed that MKRN1 targets p14ARF for ubiquitination and subsequent proteasome-dependent degradation. A statistically significant association was shown between MKRN1 overexpression and p14ARF underexpression (P = .016). Xenograft analyses using p53-functional AGS or -dysfunctional SNU601 cells displayed statistically significant tumor growth retardation by silencing MKRN1, which was reversed under depletion of p14ARF (AGS cells, MKRN1 knockdown tumors vs MKRN1 and p14ARF knockdown tumors: 164.6 vs 464.8mm3, difference = 300.2mm3, 95% CI = 189.1 to 411.3mm3, P < .001). Conclusions We demonstrated that MKRN1 functions as a novel E3 ligase of p14ARF and that it potentially regulates cellular senescence and tumorigenesis in gastric cancer.

eIF3m expression influences the regulation of tumorigenesis-related genes in human colon cancer

Abnormal regulation of gene expression is essential for tumorigenesis. Recent studies indicate that regulation of oncogene expression and neoplastic transformation are controlled by subunits of eukaryotic translation initiation factors (eIFs). Here we focused on eIF3 performing a pivotal role in protein synthesis and the differential expression of its subunits in cancer. The most uncharacterized non-core subunit eIF3m was confirmed to be highly expressed in human cancer cell lines and colon cancer patient tissues. By expression silencing with eIF3m-specific small interfering RNA (siRNA), we confirmed that eIF3m influences cell proliferation, cell cycle progression and cell death in human colon cancer cell line HCT-116. Using a ribonomics approach, we identified a subset of elF3m-influenced genes and showed that the expression of two highly represented tumorigenesis-related genes, MIF and MT2, were affected by eIF3m at the mRNA level. We also confirmed eIF3m-dependent regulation of MT2A downstream molecule CDC25A, which is necessary for cell cycle progression in HCT-116 cells. These results suggest that eIF3m mediates regulation of tumorigenesis-related genes in human colon cancer. Further investigations on tumorigenesis-related genes and their regulation by eIFs will provide clues for designing targeted therapy for cancer.

Activation of nuclear PTEN by inhibition of Notch signaling induces G2/M cell cycle arrest in gastric cancer

Mutation in PTEN has not yet been detected, but its function as a tumor suppressor is inactivated in many cancers. In this study we determined that, activated Notch signaling disables PTEN by phosphorylation and thereby contributes to gastric tumorigenesis. Notch inhibition by small interfering RNA or γ-secretase inhibitor (GSI) induced mitotic arrest and apoptosis in gastric cancer cells. Notch inhibition induced dephosphorylation in the C-terminal domain of PTEN, which led to PTEN nuclear localization. Overexpression of activated Notch1-induced phosphorylation of PTEN and reversed GSI-induced mitotic arrest. Dephosphorylated nuclear PTEN caused prometaphase arrest by interaction with the cyclin B1-CDK1 complex, resulting in their accumulation in the nucleus and subsequent apoptosis. We found a correlation between high expression levels of Notch1 and low survival rates and, similarly, between reduced nuclear PTEN expression and increasing the TNM classification of malignant tumours stages in malignant tissues from gastric cancer patients. The growth of Notch1-depleted gastric tumors was significantly retarded in xenografted mice, and in addition, PTEN deletion restored growth similar to control tumors. We also demonstrated that combination treatment with GSI and chemotherapeutic agents significantly reduced the orthotopically transplanted gastric tumors in mice without noticeable toxicity. Overall, our findings suggest that inhibition of Notch signaling can be employed as a PTEN activator, making it a potential target for gastric cancer therapy.

Efficacy of hepatic arterial infusion chemotherapy in advanced hepatocellular carcinoma

AIM To investigate the efficacy of hepatic arterial infusion chemotherapy (HAIC) using floxuridine (FUDR) in patients with advanced hepatocellular carcinoma (HCC) confined to the liver. METHODS Thirty-four patients who had advanced HCC with unresectability or unsuccessful previous therapy in the absence of extrahepatic metastasis were treated with intra-arterial FUDR chemotherapy at our hospital between March 2005 and May 2008. Among the 34 patients, 9 patients were classified as Child class C, and 18 patients had portal vein tumor thrombus (PVTT). One course of chemotherapy consisted of continuous infusion of FUDR (0.3 mg/kg during day 1-14) and dexamethasone (10 mg on day 1, 4, 7 and 11), and this treatment was repeated every 28 d. RESULTS Two patients (5.9%) displayed a complete response, and 12 patients (35.3%) had a partial response. The tumor control rate was 61.8%. The median overall survival times were 15.3 mo, 12.4 mo and 4.3 mo for the patients who were classified as Child class A, Child class B and Child class C, respectively (P = 0.0392). The progression-free survival was 12.9 mo, 7.7 mo and 2.6 mo for the patients who were classified as Child class A, Child class B and Child class C, respectively (P = 0.0443). The cumulative survival differed significantly according to the Child-Pugh classification and the presence of PVTT. In addition to hepatic reserve capacity and PVTT, the extent of HCC was an independent factor in determining a poor prognosis. The most common adverse reactions to HAIC were mucositis, diarrhea and peptic ulcer disease, but most of these complications were improved by medical treatment and/or a delay of HAIC. CONCLUSION The present study demonstrates that intra-arterial FUDR chemotherapy is a safe and effective treatment for advanced HCC that is recalcitrant to other therapeutic modalities, even in patients with advanced cirrhosis.

Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma.

Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy.

Focal thickening at the fundus of the gallbladder: computed tomography differentiation of fundal type adenomyomatosis and localized chronic cholecystitis.

Background/Aims The objective of our study was to identify useful computed tomography (CT) findings for differentiating fundal type adenomyomatosis from localized chronic cholecystitis involving the fundus of the gallbladder. Methods We retrospectively identified cases of 41 patients with pathologically proven adenomyomatosis (n=21) or chronic cholecystitis (n=20) who had fundal thickening of the gallbladder on preoperative abdominal CT. Analysis of the CT findings included evaluation of the thickness, contour, border, intralesional cystic area, adjacent gallbladder wall thickening, presence of inner layer enhancement, enhancement grade, enhancement pattern, and presence of stones. Statistical analyses were performed using the Mann-Whitney U test and Fisher exact test. Results Oval contour, inner layer enhancement and intralesional cystic area were more frequently noted in adenomyomatosis than in chronic cholecystitis (p<0.05 for each finding). Flat contour and adjacent gallbladder wall thickening were more frequently observed in chronic cholecystitis than in adenomyomatosis. No differences between adenomyomatosis and chronic cholecystitis in terms of the thickness, enhancement grade, enhancement pattern and presence of stones were apparent. Conclusions CT may help to differentiate fundal type adenomyomatosis from localized chronic cholecystitis involving the fundus of the gallbladder.

Intracellular efficacy of tumor-targeting group I intron-based trans-splicing ribozyme

Group I intron‐based trans‐splicing ribozyme, which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA, could be a useful tool for tumor‐targeted gene therapy. In the present study, the therapeutic feasibility of this ribozyme was investigated by analyzing trans‐splicing efficacy in vivo as well as in cells.

Sodium Iodide Symporter and Phosphatase and Tensin Homolog Deleted on Chromosome Ten Expression in Cholangiocarcinoma Analysis with Clinicopathological Parameters

Background/Aims This study was performed to investigate the correlation of sodium iodide symporter (NIS) expression with the functionality and loss of phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression in human cholangiocarcinoma (CCA). Methods Immunohistochemistry for the expression of NIS and PTEN was performed in 60 biopsy specimens of CCA. The clinicopathological parameters were retrospectively identified from medical records. The expression pattern of NIS and loss of PTEN expression were analyzed in association with the clinicopathological characteristics, including survival. Results Normal biliary trees displayed NIS expression, but hepatocytes did not. NIS expression was divided into two patterns: cytoplasmic and membranous. Fifty-nine cases, all except for one case, displayed NIS expression in tumor cells. Twenty-two cases (33.3%) were mixed pattern, and 39 cases (65.05%) were cytoplasmic pattern; the pure membranous pattern was not noted. There was no association between the NIS expression pattern and clinicopathological parameters, including age, sex, differentiation grade, T stage and tumor, node, metastasis stage (p>0.05). The survival rates were similar among various NIS expression patterns. Normal hepatocytes and biliary trees exhibited PTEN expression in the nucleus and cytoplasm. CCA cells displayed nuclear staining. Thirty-six (60.0%) of 60 cases displayed a loss of PTEN expression. The loss of PTEN expression was observed in the advanced T-stage group (p=0.0036), but there was no association between the loss of PTEN expression and other clinicopathological parameters (p>0.05). No association between the loss of PTEN expression and survival was noted. Conclusions NIS is expressed in most types of human CCA. The expression pattern suggests a role in cancer development. PTEN loss expression is common in the context of human CCA, especially in the advanced T stage.

A Case of Hepatic Angiomyolipoma Which Was Misdiagnosed as Hepatocellular Carcinoma in a Hepatitis B Carrier

We report a rare case of resected hepatic AML, which was misdiagnosed as hepatocellular carcinoma in a chronic hepatitis B carrier. A 45-year-old woman who was a carrier of hepatitis B virus infection presented with a hepatic tumor. Her serum alpha-fetoprotein level was normal. Ultrasonography revealed a round and well-circumscribed echogenic hepatic tumor measuring 2.5 cm in the segment VI. On contrast-enhanced computed tomography, a hypervascular tumor was observed in the arterial phase and washing-out of the contrast medium in the portal phase and delayed phase. On MR T1-weighted in-phase images, the mass showed low signal intensity, and on out-of-phase images, the mass showed signal drop and dark signal intensity. On MR T2-weighted images, the mass showed high signal intensity. The mass demonstrated high signal intensity on arterial phase after contrast injection, suggestive of hepatocellular carcinoma. The patient underwent hepatic wedge resection and histopathological diagnosis was a hepatic angiomyolipoma.