Yeon Sung Son

Heat Shock 70‐kDa Protein 8 Isoform 1 Is Expressed on the Surface of Human Embryonic Stem Cells and Downregulated upon Differentiation

The cell‐surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC‐specific cell‐surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz‐hES1, and selected 26 MAbs that were able to bind to Miz‐hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz‐hES1 cells, either. Of these, MAb 20‐202S (IgG1, κ) immunoprecipitated a cell‐surface protein of 72‐kDa from the lysate of biotin‐labeled Miz‐hES1 cells, which was identified to be heat shock 70‐kDa protein 8 isoform 1 (HSPA8) by quadrupole time‐of‐flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz‐hES4, Miz‐hES6, and HSF6. Two‐color flow cytometric analysis of Miz‐hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage‐specific embryonic antigen 3 (SSEA3), SSEA4, TRA‐1‐60, and TRA‐1‐81. Flow cytometric and Western blot analyses using various cells showed that MAb 20‐202S specifically bound to the HSPA8 protein on the surface of Miz‐hES1, contrary to other anti‐HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz‐hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20‐202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell‐surface marker for undifferentiated hESCs.

L1 Cell Adhesion Molecule Is a Novel Therapeutic Target in Intrahepatic Cholangiocarcinoma

Purpose: Intrahepatic cholangiocarcinoma (ICC), a highly malignant hepatobiliary cancer, has a poor prognosis and is refractory to conventional therapies. The aim of this study is to discover a novel molecular target for the treatment of ICC. Experimental Design: To discover novel cancer-associated membrane antigens expressed in ICC cells, we generated monoclonal antibodies (mAb) by immunizing mice with intact ICC cell lines and screened for those that bind to the plasma membrane of ICC cells but not to normal cells. The mAb A10-A3 was selected and its target antigen was identified as the L1 cell adhesion molecule. Expression of L1 in ICC was evaluated by immunohistochemical analysis of tumor samples from 42 ICC patients. The functional significance of L1 expression in the tumor progression of ICC was investigated by L1 suppression, L1 overexpression, and antibody treatment. Results: L1 was not expressed in normal hepatocytes and intrahepatic bile duct epithelium but highly expressed in 40.5% of ICC patients, remarkably at the invasive front of the tumors. Suppression of L1 with short hairpin RNA significantly decreased proliferation, migration, and invasion of ICC cells in vitro. Consistently, L1 overexpression in ICC cells enhanced proliferation, migration, invasion, and apoptosis resistance. In addition, L1 short hairpin RNA or anti-L1 mAb significantly reduced the tumor growth in nude mice bearing ICC xenograft. Conclusions: We identified that L1 is expressed in ICC. L1 plays an important role in the tumor progression of ICC by enhancing cell proliferation, migration, invasion, and survival. L1 may represent a novel therapeutic target for ICC. Clin Cancer Res; 16(14); 3571–80. ©2010 AACR.

Stanniocalcin-2 (STC2): A potential lung cancer biomarker promotes lung cancer metastasis and progression ☆

The homodimeric glycoprotein, stanniocalcin 2 (STC2) is previously known to be involved in the regulation of calcium and phosphate transport in the kidney and also reported to play multiple roles in several cancers. However, its function and clinical significance in lung cancer have never been reported and still remain uncertain. Here, we investigated the possibility of STC2 as a lung cancer biomarker and identified its potential role in lung cancer cell growth, metastasis and progression. Proteomic analysis of secretome of primary cultured lung cancer cells revealed higher expression of STC2 in cancers compared to that of adjacent normal cells. RT-PCR and Western blot analyses showed higher mRNA and protein expressions of STC2 in lung cancer tissues compared to the adjacent normal tissues. Knockdown of STC2 in H460 lung cancer cells slowed down cell growth progression and colony formation. Further analysis revealed suppression of migration, invasion and delayed G0/G1 cell cycle progression in the STC2 knockdown cells. STC2 knockdown also attenuated the H202-induced oxidative stress on H460 cell viability with a subsequent increase in intracellular ROS levels, which suggest a protective role of STC2 in redox regulatory system of lung cancer. These findings suggest that STC2 can be a potential lung cancer biomarker and plays a positive role in lung cancer metastasis and progression. This article is part of a Special Issue entitled: Medical Proteomics.

Brief report: L1 cell adhesion molecule, a novel surface molecule of human embryonic stem cells, is essential for self-renewal and pluripotency.

Despite the recent identification of surface markers of undifferentiated human embryonic stem cells (hESCs), the crucial cell‐surface molecules that regulate the self‐renewal capacity of hESCs remain largely undefined. Here, we generated monoclonal antibodies (MAbs) that specifically bind to undifferentiated hESCs but not to mouse embryonic stem cells. Among these antibodies, we selected a novel MAb, 4‐63, and identified its target antigen as the L1 cell adhesion molecule (L1CAM) isoform 2. Notably, L1CAM expressed in hESCs lacked the neuron‐specific YEGHH and RSLE peptides encoded by exons 2 and 27, respectively. L1CAM colocalized with hESC‐specific cell‐surface markers, and its expression was markedly downregulated on differentiation. Stable L1CAM depletion markedly decreased hESC proliferation, whereas L1CAM overexpression increased proliferation. In addition, the expression of octamer‐binding transcription factor 4, Nanog, sex‐determining region Y–box 2, and stage‐specific embryonic antigen (SSEA)‐3 was markedly downregulated, whereas lineage‐specific markers and SSEA‐1 were upregulated in L1CAM‐depleted hESCs. Interestingly, the actions of L1CAM in regulating the proliferation and differentiation of hESCs were exerted predominantly through the fibroblast growth factor receptor 1 signaling pathway. Taken together, our results suggest that L1CAM is a novel cell‐surface molecule that plays an important role in the maintenance of self‐renewal and pluripotency in hESCs. Stem Cells 2011;29:2094–2099.

The cell adhesion molecule L1 promotes gallbladder carcinoma progression in vitro and in vivo

Recent studies have demonstrated that the cell adhesion molecule, L1, is expressed in several malignant tumor types and its expression correlates with tumor progression and metastasis. However, the role of L1 in gallbladder carcinoma (GBC) remains unclear. Here, we demonstrate that L1 is expressed in GBC cells and plays an important role in the growth, motility, invasiveness, and adhesiveness of GBC cells. Specific depletion or overexpression of L1 in the GBC cell lines JCRB1033 and SNU-308, respectively, was achieved by lentivirus-mediated transduction and expression of an L1 mRNA-specific short hairpin RNA or full-length human L1. Stable depletion of L1 led to a significant decrease in GBC cell proliferation, migration and invasion, as well as decreased intracellular signaling through AKT and FAK. Overexpression of L1 in GBC cells enhanced these cellular activities. In a GBC xenograft nude mouse model, suppression of L1 markedly reduced tumor growth and increased the survival of tumor-bearing mice whereas L1 overexpression stimulated tumorigenicity. Taken together, these results suggest that L1 plays a crucial role in GBC progression and may be a novel therapeutic target in GBC treatment.

Hedgehog-Interacting Protein (HIP) Regulates Apoptosis Evasion and Angiogenic Function of Late Endothelial Progenitor Cells

Late endothelial progenitor cells (LEPCs) are derived from mononuclear cells (MNCs) and are thought to directly incorporate into blood vessels and differentiate into mature endothelial cells (ECs). Using transcriptome and proteome analysis, we identified distinctive LEPC profiles and found that Hedgehog-interacting protein (HIP) is strongly expressed in LEPCs. Inhibition of HIP by lentiviral knockdown activated canonical hedgehog signaling in LEPCs, while it activated non-canonical hedgehog signaling in ECs. In LEPCs, HIP knockdown induced much enhanced tube formation and resistance to apoptosis under oxidative stress conditions via canonical hedgehog signaling. Although HIP is strongly expressed in proliferating LEPCs, HIP expression is down-regulated during angiogenesis and under oxidative stress condition. Moreover, when LEPCs are treated with angiogenic triggers such as VEGF and FGF2, HIP expression is reduced. Our findings suggest that HIP blocks LEPC angiogenesis and regulate survival when there is no angiogenic stimulation. HIP inhibition in LEPCs enhanced tube formation and reduced apoptosis, resulting in improved angiogenesis.

Paraoxonase-1 (PON1) induces metastatic potential and apoptosis escape via its antioxidative function in lung cancer cells

Paraoxonase-1 (PON1) gene polymorphisms have been closely associated with the development of advanced cancers while PON1 secretion to the serum is linked with inhibition of oxidized high-density lipoprotein by its antioxidative function. Our group previously demonstrated that post-translational modification of serum PON1 in form of fucosylated PON1 is a potential biomarker of small cell lung cancer. Here, we interrogated the role of PON1 in the pathobiology of lung cancer (LC) by addressing cell-autonomous mechanisms using gain-of-function and loss-of-function approaches and protein expression profiling of tissue samples in our clinical biobank. PON1 expression in LC patient tissues varied between overexpression in squamous cell carcinoma and minimal loss in adenocarcinoma sub-types. Simultaneous overexpression of PON1 both at the gene and protein stability levels induced pro-oncogenic characteristics in LC cells and xenografts. PON1 overexpression supported metastatic progression of LC by decreasing G1/S ratio and LC cell senescence involving p21Waf1/Cip1. PON1 suppressed drug- and ligand-induced cell death and protected LC cells from genotoxic damages with maintained ATP levels, requiring p53-directed signals. PON1 promoted ROS deregulation protecting the mitochondria from dysregulation. PON1 knockdown resulted in the blockage of its antioxidant function in LC cells through Akt signaling with reduced invasive signature as a consequence of scant expression. Targeted glycolysis stimulated PON1 antioxidant activity regulating phosphorylation of AMPK-α. The functional data imply that exploitation of the antioxidative function of PON1 is consequential in driving LC pathogenesis at the cell-autonomous mechanistic level with consequences on tumor growth.

IFN‐γ enhances the wound healing effect of late EPCs (LEPCs) via BST2‐mediated adhesion to endothelial cells

Circulating late endothelial progenitor cells (LEPCs) home to injured vessels, initiating blood vessel regeneration. This process requires the initial adhesion of LEPCs to endothelial cells within the wounded site. In this study, treating LEPCs with IFN‐γ enhanced wound healing through BST2‐mediated adhesion to endothelial cells. We found that IFN‐γ significantly upregulated BST2 expression in both LEPCs and ECs and increased tube formation in LEPCs. Upregulated BST2 increased LEPC adhesion to ECs through a tight homophilic interaction of its extracellular domain. Finally, when the IFN‐γ‐treated LEPCs were injected into the wounded mouse tail vein, superior therapeutic effects of wound closure were observed. This study provides a useful application to enhance the adhesion of LEPCs for vessel regeneration and wound closure.