Hong-Beum Kim

Oncogenic H-Ras Up-Regulates Expression of ERCC1 to Protect Cells from Platinum-Based Anticancer Agents

Tumors frequently contain mutations in the ras genes, resulting in the constitutive activation of the Ras-activated signaling pathway. The activation of Ras is involved not only in tumor progression but also in the development of resistance of the tumor cells to platinum-based chemotherapeutic agents. To investigate the potential mechanisms underlying this resistance, we analyzed the effect of activated H-Ras on the expression of the nucleotide excision repair genes. Here we identified ERCC1, which is one of the key enzymes involved in nucleotide excision repair, as being markedly up-regulated by the activated H-Ras. From promoter analysis of ERCC1, an increase in the Ap1 transcriptional activity as a result of the expression of the oncogenic H-Ras was found to be crucial for this induction. In addition, ERCC1 small interfering RNA expression was shown to reduce the oncogenic H-Ras-mediated increase in the DNA repair activity as well as to suppress the oncogenic H-Ras-mediated resistance of the cells to platinum-containing chemotherapeutic agents. These results suggest that the oncogenic H-Ras-induced ERCC1, which activates the DNA repair capacity, may be involved in the protection of the cells against platinum-based anticancer agents.

Bcl-2 expression suppresses mismatch repair activity through inhibition of E2F transcriptional activity.

Bcl-2 stimulates mutagenesis after the exposure of cells to DNA-damaging agents. However, the biological mechanisms of Bcl-2-mediated mutagenesis have remained largely obscure. Here we demonstrate that the Bcl-2-mediated suppression of hMSH2 expression results in a reduced cellular capacity to repair mismatches. The pathway linking Bcl-2 expression to the suppression of mismatch repair (MMR) activity involves the hypophosphorylation of pRb, and then the enhancement of the E2F–pRb complex. This is followed by a decrease in hMSH2 expression. MMR has a key role in protection against deleterious mutation accumulation and in maintaining genomic stability. Therefore, the decreased MMR activity by Bcl-2 may be an underlying mechanism for Bcl-2-promoted oncogenesis.

Colon cancer progression is driven by APEX1-mediated upregulation of Jagged

Aberrant expression of apurinic-apyrimidinic endonuclease-1 (APEX1) has been reported in numerous human solid tumors and is positively correlated with cancer progression; however, the role of APEX1 in tumor progression is poorly defined. Here, we show that APEX1 contributes to aggressive colon cancer behavior and functions as an upstream activator in the Jagged1/Notch signaling pathway. APEX1 overexpression or knockdown in human colon cancer cell lines induced profound changes in malignant properties such as cell proliferation, anchorage-independent growth, migration, invasion, and angiogenesis in vitro and in tumor formation and metastasis in mouse xenograft models. These oncogenic effects of APEX1 were mediated by the upregulation of Jagged1, a major Notch ligand. Furthermore, APEX1 expression was associated with Jagged1 in various colon cancer cell lines and in tissues from colon cancer patients. This finding identifies APEX1 as a positive regulator of Jagged1/Notch activity and suggests that it is a potential therapeutic target in colon cancers that exhibit high levels of Jagged1/Notch signaling.

Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor α1 Expression

ABSTRACT Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor α1 (GFRα1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRα1 transcription through enhanced binding of NF-κB complexes to the GFRα1 promoter. GFRα1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRα1 expression and invasion in response to GNDF, while overexpression of GFRα1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from β-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRα1 expression may play important roles in pancreatic cancer progression and neuronal cell survival.

Oncogenic H-Ras Up-regulates Expression of Ku80 to Protect Cells from γ-Ray Irradiation in NIH3T3 Cells

The Ras activation contributes to radioresistance, but the mechanism is unclear. This article shows that the expression of the dominant-positive H-Ras increased the Ku80 level, which is one of the key enzymes involved in repairing dsDNA breaks (DSB). After exposing the cells to ionizing radiation and analyzing them using an electrophoretic mobility shift assay and pulsed-field gel electrophoresis, it was found that activated H-Ras expression in NIH3T3 cells increases the DNA-binding activity of Ku80 and increases the DSB repair activity. Ku80 small interfering RNA expression was shown to reduce the oncogenic H-Ras-mediated increase in the DSBs and suppress the oncogenic H-Ras-mediated resistance of the cells to gamma-ray irradiation, whereas Ku80 overexpression in the NIH3T3 cells significantly increased the radioresistance. These results suggest that the Ku80 expression induced by oncogenic H-Ras seems to play an important role in protecting cells against gamma-ray irradiation.

Oncogenic Ras-mediated downregulation of Clast1/LR8 is involved in Ras-mediated neoplastic transformation and tumorigenesis in NIH3T3 cells.

Oncogenic Ras proteins transform cells by way of multiple downstream signaling pathways that promote the genesis of human cancers. However, the exact cellular mechanisms by which downstream targets are regulated are not fully understood. Here, we show that oncogenic Ras reduced Clast1/LR8 transcript levels in mouse NIH3T3 fibroblasts and human WI38 fibroblasts. Clast1/LR8 transcript was undetectable in H460, A549, and H1299 cells showing high Ras activity, but was relatively abundant in DMS53 cells displaying low Ras activity. We also showed that K‐Ras siRNA restored Clast1/LR8 expression in H460 and A549 cells, and that inhibitors of DNA methylation and histone deacetylation reversed oncogenic H‐Ras‐mediated suppression of Clast1/LR8 transcription. Additionally, ectopic expression of Clast1/LR8 inhibited serum‐stimulated phosphorylation of ERK1/2 and Akt in H‐RasV12‐transformed NIH3T3 cells. We further showed that the expression of Clast1/LR8 interfered with oncogenic Ras‐induced NIH3T3 cell transformation and invasion. Finally, our results showed that Clast1/LR8 inhibited Ras‐induced proliferation of, and tumor formation by, oncogenic H‐RasV12‐transformed NIH3T3 cells in vivo. This study identifies the downregulation of Clast1/LR8 as a potentially important mechanism by which oncogenic Ras‐mediated neoplastic transformation occurs. (Cancer Sci 2010)

Evaluation of the prevalence and clinical impact of toxocariasis in patients with eosinophilia of unknown origin.

Background/Aims Eosinophilia has numerous diverse causes, and in many patients, it is not possible to establish the cause of eosinophilia. Recently, toxocariasis was introduced as one cause of eosinophilia. The aims of this study were to evaluate the prevalence of toxocariasis and the clinical impact of albendazole treatment for toxocariasis in patients suspected of eosinophilia of unknown origin. Methods We performed a retrospective chart review. After evaluation of cause of eosinophilia, the patients suspected of eosinophilia of unknown origin performed immunoglobulin G antibody specific assay for the Toxocara canis larval antigen by enzyme-linked immunosorbent assay. Results This study evaluated 113 patients, 69 patients (61%) were suspected of eosinophilia of unknown origin. Among these 69 patients, the frequency of T. canis infection was very high (45 patients, 65.2%), and albendazole treatment for 45 eosinophilia with toxocariasis was highly effective for a cure of eosinophilia than no albendazole group regardless of steroid (82.3%, p = 0.007). Furthermore, among the nonsteroid treated small group (19 patients), albendazole treatment for eosinophilia were more effective than no albendazole group, too (83.3% vs. 28.6 %, p = 0.045). Conclusions The prevalence of toxocariasis was high among patients suspected of eosinophilia of unknown origin; therefore, evaluation for T. canis infection is recommended for patients with eosinophilia of unknown origin. Furthermore, for patients suspected of eosinophilia of unknown origin who have positive results for T. canis, albendazole treatment may be considered a valuable treatment option.

Arterioportal shunt incidental to treatment with oxaliplatin that mimics recurrent gastric cancer

Arterioportal shunt (APS) is an organic communication between the hepatic arterial system and the portal venous system. The APS is one of the major causes of transient hepatic attenuation differences on dynamic computed tomography (CT) or magnetic resonance imaging (MRI). This condition is usually associated with trauma, liver cirrhosis, and malignancies of the liver. However, there has been no report about oxaliplatin-induced APS. A 41-year-old male was diagnosed with Stage IIIB gastric cancer. The patient initially underwent neoadjuvant chemotherapy with capecitabine and oxaliplatin After 3 cycles of therapy, the mass had markedly decreased, and a total gastrectomy with splenectomy was performed. Since the malignancy was locally invasive, the patient was continued on the same regimen of the adjuvant chemotherapy. After 3 more cycles, a computed tomography revealed a 1 cm sized arterial-enhancing nodule in the right lobe of the liver. An MRI revealed an arterial enhancing lesion, and a positron emission tomography CT scan showed a hypermetabolic lesion in the same portion of the liver. We tried to perform a liver biopsy; however, an ultrasonography could not detect any mass. A presumptive diagnosis of an APS due to a recurred cancer was made. We found a similar but slightly different case report of an oxaliplatin-induced liver injury, mimicking a metastatic tumor on an MRI. Based on a prior report, the patient was continued on treatment with adjuvant chemotherapy following discontinuation of oxaliplatin. After 2 cycles, the arterial enhancing liver mass resolved, supporting the final diagnosis of an APS, related to oxaliplatin-induced sinusoidal injury. The patient has not experienced any a relapse after two years of additional follow up recurrent gastric cancer upon interpretation of multiple imaging modalities.

Clinical implications of APEX1 and Jagged1 as chemoresistance factors in biliary tract cancer

Purpose Biliary cancer is a highly malignant neoplasm with poor prognosis and most patients need to undergo palliative chemotherapy, however major clinical problem associated with the use of chemotherapy is chemoresistance. So far, we aimed at investigating clinical implications of apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and Jagged1 as chemoresistance factors in biliary tract cancer. Methods We used 5 human biliary tract cancer cell lines (SNU-245, SNU-308, SNU-478, SNU-1079, and SNU-1196), and investigated the chemosensitivity of APEX1 and Jagged1 through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and Western blot. Alternately, the 10 patients of advanced biliary cancer consist of 2 group according to the chemotherapy response examined by immunohistochemistry using APEX1 and Jagged1 antibody, and protein expression level was scored for staining intensity and percent positive cell. Results The result of MTT assay after APEX1 knockdown showed that strong coexpression of APEX1 and Jagged1 cell line (SNU-245, SNU-1079, and SNU-1196) showed a greater decrease in IC50 of chemotherapeutic agent (5-fluorouracil, gemcitabine and cisplatin). The Western blot analysis of APEX1 and Jagged1 expression in biliary cancer cell lines after APEX1 knockdown definitively demonstrated decreased Jagged1 expression. The APEX1 and Jagged1expression level of immunohistochemistry represented that chemorefractory patients had higher than chemoresponsive patients. Conclusion These results demonstrate that simultaneous high expression of APEX1 and Jagged1 is associated with chemoresistance in biliary cancer and suggest that is a potential therapeutic target for chemoresistance in advanced biliary cancer.

APEX-1 Regulates Cell Proliferation through GDNF/ GFRα1 Signaling

Human apurinic/apyrimidinic endonuclease (APEX-1) is a multifunctional protein that is capable of repairing abasic sites and single-strand breaks in damaged DNA. In addition, it serves as a redox-modifying factor for a number of transcription factors. Identifying the transcriptional targets of APEX-1 is essential for understanding how it affects various cellular outcomes. Expression array analysis was used to identify glial cell-derived neurotropic factor receptor α1 (GFRα1), which is an encoding receptor for the glial cell-derived neurotropic factor (GDNF) family, the expression of which is induced by APEX-1. A target of GDNF/GFRα signaling, c-Src (Tyr418) was strongly phosphorylated by GNDF in the APEX-1 expressing cells. Moreover, GDNF initiated cell proliferation, measured by counting the number of cells, in the APEX-1 expressing cells. Importantly, the down-regulation of APEX-1 by siRNA caused a marked reduction in the GFRα1 expression level, and it reduced the ability of GDNF to phosphorylate c-Src (Tyr418) and stimulate cell proliferation. These results demonstrate an association between APEX-1 and GDNF/GFRα signaling and suggest a potential molecular mechanism for the involvement of APEX-1 in cell survival and proliferation.