Yong-Nyun Kim

Anoikis resistance: an essential prerequisite for tumor metastasis.

Metastasis is a multistep process including dissociation of cancer cells from primary sites, survival in the vascular system, and proliferation in distant target organs. As a barrier to metastasis, cells normally undergo an apoptotic process known as “anoikis,” a form of cell death due to loss of contact with the extracellular matrix or neighboring cells. Cancer cells acquire anoikis resistance to survive after detachment from the primary sites and travel through the circulatory and lymphatic systems to disseminate throughout the body. Because recent technological advances enable us to detect rare circulating tumor cells, which are anoikis resistant, currently, anoikis resistance becomes a hot topic in cancer research. Detailed molecular and functional analyses of anoikis resistant cells may provide insight into the biology of cancer metastasis and identify novel therapeutic targets for prevention of cancer dissemination. This paper comprehensively describes recent investigations of the molecular and cellular mechanisms underlying anoikis and anoikis resistance in relation to intrinsic and extrinsic death signaling, epithelial-mesenchymal transition, growth factor receptors, energy metabolism, reactive oxygen species, membrane microdomains, and lipid rafts.

Exosomes: A new delivery system for tumor antigens in cancer immunotherapy

Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with plasma membrane. Exosomes are secreted by various cell types including hematopoietic cells, normal epithelial cells and even some tumor cells. They are known to carry MHC class I, various costimulatory molecules and some tetraspanins. Recent studies have shown the potential of using native exosomes as immunologic stimulants. Here, we demonstrate a novel means of using exosomes engineered to express a specific tumor antigen to generate an immune response against tumors. We expressed a target tumor antigen, human MUC1 (hMUC1), in 2 MHC type‐distinct mouse cell lines, CT26 and TA3HA. Analysis of exosomes purified from these cells revealed that exosomes contained the target MUC1 antigen on their surfaces as well as other well‐described exosomal proteins, including Hsc70 and MHC class I molecules. In addition, both autologous and allogenic exosomes were able to stimulate the activation of immune cells and suppress hMUC1‐expressing tumor growth in a MUC1‐specific and dose‐related manner. Therefore, these data suggest that exosomes can be engineered from tumor cell lines to deliver a target immunogen capable of inducing an effective immune response and that they may represent a new cell‐free tumor vaccine.

MYC translocation and an increased copy number predict poor prognosis in adult diffuse large B-cell lymphoma (DLBCL), especially in germinal centre-like B cell (GCB) type.

Aims:  Diffuse large B‐cell lymphoma (DLBCL) is a heterogeneous disease with various genetic alterations. The aim was to investigate MYC, Bcl‐2 and Bcl‐6 translocations and copy number changes in adult DLBCLs to evaluate their clinicopathological features and prognostic implications.

Detection of response-predicting mutations in the kinase domain of the epidermal growth factor receptor gene in cholangiocarcinomas

Purpose: Epidermal growth factor receptor (EGFR) signalings have recently been implicated in the genesis and progression of cholangiocarcinomas. Thus, the EGFR kinase inhibitor appears to be promising in the treatment of this cancer. The response-predicting mutations in the tyrosine kinase domain of EGFR gene have recently been detected in non-small cell lung cancers. This study was, therefore, to investigate if these mutations are also found in cholangiocarcinomas. Methods: Twenty-two consecutive cholangiocarcinoma patients who underwent surgical resection were enrolled. Their resected paraffin-embedded cholangiocarcinoma specimens were used for mutation analysis, which was performed by DNA sequencing of exons 18, 19 and 21 in the EGFR gene. Clinical characteristics were compared between each group according to the presence or absence of mutations. Results: Three patients (13.6%) harbored EGFR mutations. All the mutations found were deletions in exon 19. Mutations were more common in intra-hepatic or poorly differentiated tumors. Differences in age, sex, stage at diagnosis and survival were not observed between mutation-positive and -negative patients. Conclusions: This study, for the first time, demonstrates that a subset of cholangiocarcinoma patients has response-predicting EGFR mutations. Therefore, a highly selected application of the EGFR kinase inhibitor would be therapeutically effective in these patients.

STAT3 inhibits the degradation of HIF-1α by pVHL-mediated ubiquitination

Hypoxia-inducible factor 1α (HIF-1α) is rapidly degraded by the ubiquitin-proteasome pathway under normoxic conditions. Ubiquitination of HIF-1α is mediated by interaction with von Hippel-Lindau tumor suppressor protein (pVHL). In our previous report, we found that hypoxia-induced active signal transducer and activator of transcription3 (STAT3) accelerated the accumulation of HIF-1α protein and prolonged its half-life in solid tumor cells. However, its specific mechanisms are not fully understood. Thus, we examined the role of STAT3 in the mechanism of pVHL-mediated HIF-1α stability. We found that STAT3 interacts with C-terminal domain of HIF-1α and stabilizes HIF-1α by inhibition of pVHL binding to HIF-1α. The binding between HIF-1α and pVHL, negative regulator of HIF-1α stability, was interfered dose-dependently by overexpressed constitutive active STAT3. Moreover, we found that the enhanced HIF-1α protein levels by active STAT3 are due to decrease of poly-ubiquitination of HIF-1α protein via inhibition of interaction between pVHL and HIF-1α. Taken together, our results suggest that STAT3 decreases the pVHL-mediated ubiquitination of HIF-1α through competition with pVHL for binding to HIF-1α, and then stabilizes HIF-1α protein levels.

Cholesterol depletion induces anoikis-like apoptosis via FAK down-regulation and caveolae internalization

Caveolae (lipid rafts), microdomains of the plasma membrane, are known to contain various signalling molecules and consequently are involved in the regulation of many biological functions. To investigate the role of the caveolae in cell survival and adhesion, we disrupted the caveolae by depletion of cholesterol, a major lipid component of the caveolae, with methyl‐β cyclodextrin (MβCD) treatment of A431 cells. We found that cholesterol depletion induced an anoikis‐like cell death involving actin reorganization, resulting in a decrease in cell spreading and an increase in cell detachment, which was reversed by cholesterol addition. Disruption of caveolae led to the down‐regulation of FAK, Src activation, tyrosine phosphorylation of caveolin‐1 and mobilization of caveolae markers, GM1 and caveolin‐1, from the cell surface to the cytoplasm, which were also recovered by cholesterol addition. The expression of dominant‐active FAK was able to delay caveolae internalization and apoptosis and attenuated Akt inactivation by MβCD, whereas dominant‐negative FAK expression resulted in enhanced apoptosis. Moreover, FAK down‐regulation by si‐RNA resulted in Akt inactivation and thus increased cell death by MβCD treatment. Our results suggest that the cholesterol content and/or surface levels of the caveolae affect the activity of FAK, which in turn regulates caveolae internalization and cell survival. Copyright

Oxidative stress attenuates Fas-mediated apoptosis in Jurkat T cell line through Bfl-1 induction

Many types of mammalian cells produce ROS in response to many different stimuli to modulate a number of cellular functions, including apoptosis. However, the correlation between ROS and apoptosis remains controversial, and the mechanisms whereby ROS-induced signals are propagated to critical downstream targets remain largely undefined. Here, we demonstrate that hydrogen peroxide (H2O2) upregulates the expression of Bfl-1, an antiapoptotic member of the Bcl-2 family, and that this is responsible for the antiapoptotic activity of ROS. When Jurkat, human leukemic T cells, were pretreated with 100 μM H2O2 and then treated with anti-Fas antibody, apoptosis was impaired without change of cell surface Fas expression. An investigation of the expression patterns of Bcl-2 family genes revealed that H2O2 treatment induced Bfl-1 gene expression, but left other genes unchanged, and this Bfl-1 expression and H2O2-induced antiapoptotic effect was inhibited by antioxidants or NF-κB inhibitor. In addition, an electromobility shift assay revealed that the p65/p50 subunits of NF-κB activated by H2O2 bound to a bfl-1 promoter. Neither the induction of Bfl-1 nor the antiapoptotic effect of H2O2 was detected in Bfl-1-knockdown Jurkat cell line containing Bfl-1 antisense (Bfl-1AS). These data indicate that oxidative stress induces the expression of Bfl-1 via NF-κB activation, and this early-response gene protects cells from Fas-mediated apoptosis. This may be a cellular survival mechanism of cells exposed to phagocytes-derived ROS.

Increased quantity of tumor-infiltrating FOXP3-positive regulatory T cells is an independent predictor for improved clinical outcome in extranodal NK/T-cell lymphoma

BACKGROUND Extranodal natural killer/T-cell lymphoma (NKTCL) is a clinically heterogeneous disease with a poor prognosis, requiring risk-stratified management in affected patients. Recently, tumor microenvironment including regulatory T cells (Tregs) has been implicated as a prognostic marker in certain types of lymphoma. PATIENTS AND METHODS We collected 64 NKTCL cases and numerically quantified the amount of tumor-infiltrating FOXP3-positive Tregs by automated slide scanning and image analysis program after immunohistochemical staining using anti-FOXP3 antibody. RESULTS Patients were able to be classified into two end groups by their level of Tregs. Twenty-eight (44%) patients had Tregs <50/0.40 mm(2), while 36 (56%) had Tregs > or =50/0.40 mm(2) within the tumor. The decreased number of Tregs (<50/0.40 mm(2)) was more common in patients with poor performance status or in those presented in non-upper aerodigestive tract. However, the level of Tregs was not associated with other prognostic factors, including stage, lactate dehydrogenase level, International Prognostic Index, and NKTCL Prognostic Index. Importantly, patients with increased numbers of Tregs (> or =50/0.40 mm(2)) showed prolonged overall and progression-free survival (P = 0.0005 and P = 0.0079, respectively). The number of FOXP3-positive Tregs was an independent prognostic factor (P = 0.001) by multivariate analysis. CONCLUSION Increased quantity of tumor-infiltrating Tregs predicted improved clinical outcome in NKTCL patients.

The heat-shock protein 90 inhibitor, geldanamycin, induces apoptotic cell death in Epstein-Barr virus-positive NK/T-cell lymphoma by Akt down-regulation.

NK/T‐cell lymphoma (NKTL) is strongly associated with latent Epstein–Barr virus (EBV) infection. Recently, latent membrane protein 1 (LMP1), an EBV oncoprotein, was reported to activate the phosphatidylinositol‐3 kinase (PI3K)/Akt pathway for cell survival. Because geldanamycin (GA) and its derivative, 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG), exhibit anti‐tumour activity by degrading HSP90 client proteins, including Akt, we investigated the effect of GA and 17‐AAG on the survival of NKTL cell lines. EBV‐positive NKTL cell lines, Hank‐1 and NK‐YS, and an EBV‐negative NK leukaemia cell line, NK‐L, were treated with PI3K and Akt inhibitors, GA, and 17‐AAG, and were subjected to apoptosis and cell viability assays, and immunoblot analysis. EBV‐positive B‐lymphoblastoid cell lines IM9 and LMP1‐transfected IM9 (IM9‐LMP1) were also included. Hank‐1 and NK‐YS cell viability was compromised and apoptosis was induced by LY294002 (PI3K inhibitor) or Akt inhibitor II. GA or 17‐AAG administration resulted in the apoptosis of NKTL cells, accompanied by Akt and pAkt down‐regulation, caspase 3 activation, and mitochondrial membrane potential disruption. The intrinsic level of pAkt was higher in EBV‐positive NKTL cells than in EBV‐negative NK‐L, and GA or 17‐AAG decreased the viability of NKTL cells more efficiently than NK‐L. Moreover, IM9‐LMP1 was more sensitive to Akt inhibitor II or HSP90 inhibitors than IM9. Importantly, GA showed little effect on the viability of normal peripheral NK cells as non‐neoplastic counterparts for comparison. In conclusion, this study suggests that the PI3K/Akt pathway is frequently activated in EBV‐positive NKTL and that therapeutic modalities based on targeting the PI3K/Akt pathway with HSP90 inhibitors could be useful for achieving NKTL control. Copyright

Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis.

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function.