Sung Ouk Nam

Molecular Hierarchy of Heparin-Binding EGF-like Growth Factor–Regulated Angiogenesis in Triple-Negative Breast Cancer

Heparin-binding EGF-like growth factor (HB-EGF) is one of several proangiogenic factors and represents a possible therapeutic target for patients with triple-negative breast cancer (TNBC). However, the role of HB-EGF in promoting tumor aggressiveness in TNBC remains unclear. To investigate specific genes and pathways involved in TNBC tumorigenesis, we profiled gene expression changes in two TNBC cell lines under two-dimensional culture (2DC) and three-dimensional culture (3DC) and in a tumor xenograft model. We identified simultaneous upregulation of HB-EGF, VEGFA, and angiopoietin-like 4 (ANGPTL4) in 3DC and tumor xenografts, compared with 2DC. We show that HB-EGF regulates the expression of VEGFA or ANGPTL4 via transcriptional regulation of hypoxia-inducible factor-1α and NF-κB. Furthermore, suppression of VEGFA or ANGPTL4 expression enhanced HB-EGF expression, highlighting a unique regulatory loop underlying this angiogenesis network. Targeted knockdown of HB-EGF significantly suppressed tumor formation in a TNBC xenograft model, compared with individual knockdown of either VEGFA or ANGPTL4, by reducing the expression of both VEGFA and ANGPTL4. In patients with TNBC, VEGFA or ANGPTL4 expression was also significantly correlated with HB-EGF expression. Low concentrations of exogenously added HB-EGF strongly activated the proliferation of endothelial cells, tube formation, and vascular permeability in blood vessels, in a similar fashion to high doses of VEGFA and ANGPTL4. Taken together, these results suggest that HB-EGF plays a pivotal role in the acquisition of tumor aggressiveness in TNBC by orchestrating a molecular hierarchy regulating tumor angiogenesis. Mol Cancer Res; 11(5); 506–17. ©2013 AACR.

Warburg effect regulated by amphiregulin in the development of colorectal cancer.

Colorectal cancer (CRC) is one of the most frequently occurring cancers with high morbidity and mortality worldwide. Amphiregulin (AREG), a member of the epidermal growth factor family and a rational target for CRC therapy, is essential for the three‐dimensional structure of tumor formation. To clone the genes associated with increased AREG expression, we performed a cDNA microarray analysis in two CRC cell lines undergoing two‐dimensional (2DC) and three‐dimensional culture (3DC). Upregulated (>2.0‐fold) and downregulated (<0.5‐fold) genes in 3DC compared with 2DC were selected. Pathway analysis using DAVID based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases revealed a number of genes involved in glycolysis. In CRC cells, glucose elevated the expression of GLUT1 and AREG as well as the activity of the hypoxia‐inducible factor 1 (HIF‐1) luciferase reporter promoter. The suppression of AREG expression reduced the uptake of glucose and production of lactate. Luciferase assay identified a critical regulatory region for AREG expression between −130 and −180 bp upstream of the start site, which contained a carbohydrate response element (ChoRE). Max‐like protein X (MLX) bound to ChoRE and enhanced the expression of AREG. Together these data suggest that AREG plays a pivotal role in the development of CRC through activation of the Warburg effect.

Contribution of transcription factor, SP1, to the promotion of HB‐EGF expression in defense mechanism against the treatment of irinotecan in ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is a worst histological subtype than other ovarian malignant tumor. Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is a promising target for ovarian cancer therapy. The aims of this study were to validate the efficacy of HB‐EGF–targeted therapy for OCCC and to identify the transcription factor that contributed to the induction of HB‐EGF by SN38 treatment in OCCC cells. HB‐EGF was highly expressed in OCCC cells, and an increase of HB‐EGF was induced by SN38 which had only antitumor effect among conventional anticancer agents on OCCC. A specific inhibitor of HB‐EGF, a cross‐reacting material 197 (CRM197), led to a synergistic increase in the number of apoptotic OCCC cells with the treatment of SN38. The luciferase assay with 5′‐deletion promoter constructs identified a GC‐rich element between −125 and −178 (the distal transcription start site was denoted +1) as a cis‐regulatory region, and the treatment of SN38 induced luciferase activity in this region. An in silico and chromatin immunoprecipitation analysis estimated that SP1 bound to the cis‐regulatory region of HB‐EGF in OCCC cells. Real‐time PCR and cell viability assays showed that the transfection of a small interfering RNA targeting SP1 suppressed the expression of HB‐EGF induced by SN38, resulting in the enhanced sensitivity of SN38. Taken together, these results indicate that induction of HB‐EGF expression contributed to defense mechanism against treatment of SN38 through the transcriptional activity of SP1 in OCCC cells.

MicroRNA-135a-3p as a promising biomarker and nucleic acid therapeutic agent for ovarian cancer

Ovarian cancer is the most lethal gynecologic malignancy. Recently, several molecularly targeted anticancer agents have been developed for ovarian cancer; however, its prognosis remains extremely poor. The development of molecularly targeted therapy, as well as companion diagnostics, is required to improve outcomes for patients with ovarian cancer. In this study, to identify microRNAs (miRNAs) involved in the progression of ovarian cancer we analyzed serum miRNAs in patients with ovarian cancer using miRNA array and quantitative RT‐PCR and examined the anticancer properties of miRNA expression in ovarian cancer cells. In patients with ovarian cancer, high amount of miR‐135a‐3p in serum samples was significantly associated with favorable clinical prognosis. The amount of miR‐135a‐3p was significantly decreased in patients with ovarian cancer compared with patients with ovarian cysts or normal ovaries. In SKOV‐3 and ES‐2 human ovarian cancer cells, enhanced expression of miR‐135a‐3p induced drug sensitivity to cisplatin and paclitaxel and suppressed cell proliferation and xenograft tumor growth. These findings suggest that miR‐135a‐3p may be considered as a biomarker and a therapeutic agent in ovarian cancer.