Hae Yun Nam

GD2 expression is closely associated with neuronal differentiation of human umbilical cord blood-derived mesenchymal stem cells

GD2 ganglioside has been identified as a key determinant of bone marrow-derived mesenchymal stem cells (BM-MSCs). Here, we characterized GD2 ganglioside expression and its implications in umbilical cord blood-derived MSCs (UCB-MSCs). Using immune-selection analysis, we showed that both GD2-positive and GD2-negative UCB-MSCs expressed general stem cell markers and possessed mesodermal lineage differentiation potential. Although the fraction of GD2-expressing cells was lower in UCB-MSC than in BM-MSC populations, inhibition of GD2 synthesis in UCB-MSCs suppressed neuronal differentiation and down-regulated basic helix-loop-helix (bHLH) transcription factors, which are involved in early stage neuronal differentiation. In addition, the levels of bHLH factors in neuronally induced UCB-MSCs were significantly higher in GD2-positive than GD2-negative cells. Our data demonstrate that GD2 ganglioside expression is associated with regulation of bHLH factors and identify neurogenic-capable UCB-MSCs, providing new insights into the potential clinical applications of MSC-based therapy.

Knockdown of β-catenin controls both apoptotic and autophagic cell death through LKB1/AMPK signaling in head and neck squamous cell carcinoma cell lines.

The Wnt/β-catenin pathway regulates the viability and radiosensitivity of head and neck squamous cancer cells (HNSCC). Increased β-catenin predisposes HNSCC patients to poor prognosis and survival. This study was conducted to determine the mechanism by which β-catenin regulates the viability of HNSCC. AMC-HN-3, -HN-8, UM-SCC-38, and -SCC-47 cells, which were established from human head and neck cancer specimens, and underwent cell death following β-catenin silencing. β-Catenin silencing significantly induced G1 arrest and increased the expression of Bax and active caspase-3, which demonstrates the sequential activation of apoptotic cascades following treatment of HNSCC with targeted siRNA. Intriguingly, β-catenin silencing also induced autophagy. Here, we confirm that the number of autophagic vacuoles and the expression of type II light chain 3 were increased in cells that were treated with β-catenin siRNA. These cell death modes are most likely due to the activation of LKB1-dependent AMPK following β-catenin silencing. The activated LKB1/AMPK pathway in AMC-HN-3 cells caused G1 arrest by phosphorylating p53 and suppressing mTOR signaling. In addition, treating AMC-HN-3 cells with LKB1 siRNA preserved cell viability against β-catenin silencing-induced cytotoxicity. Taken together, these results imply that following β-catenin silencing, HNSCC undergo both apoptotic and autophagic cell death that are under the control of LKB1/AMPK. To the best of our knowledge, these results suggest for the first time that novel crosstalk between β-catenin and the LKB1/AMPK pathway regulates the viability of HNSCC. This study thus presents new insights into our understanding of the cellular and molecular mechanisms involved in β-catenin silencing-induced cell death.

Radioresistant Cancer Cells Can Be Conditioned to Enter Senescence by mTOR Inhibition

Autophagy is frequently activated in radioresistant cancer cells where it provides a cell survival strategy. The mTOR inhibitor rapamycin activates autophagy but paradoxically it also enhances radiosensitivity. In this study, we investigated the mechanisms of these opposing actions in radiation-resistant glioma or parotid carcinoma cells. Radiation treatment transiently enhanced autophagic flux for a period of 72 hours in these cells and treatment with rapamycin or the mTOR inhibitor PP242 potentiated this effect. However, these treatments also increased heterochromatin formation, irreversible growth arrest, and premature senescence, as defined by expression of senescence-associated β-galactosidase activity. This augmentation in radiosensitivity seemed to result from a restoration in the activity of the tumor suppressor RB and a suppression of RB-mediated E2F target genes. In tumor xenografts, we showed that administering rapamycin delayed tumor regrowth after irradiation and increased senescence-associated β-galactosidase staining in the tumor. Our findings suggest that a potent and persistent activation of autophagy by mTOR inhibitors, even in cancer cells where autophagy is occurring, can trigger premature senescence as a method to restore radiosensitivity.

Senescence-Associated MCP-1 Secretion Is Dependent on a Decline in BMI1 in Human Mesenchymal Stromal Cells

AIMS Cellular senescence and its secretory phenotype (senescence-associated secretory phenotype [SASP]) develop after long-term expansion of mesenchymal stromal cells (MSCs). Further investigation of this phenotype is required to improve the therapeutic efficacy of MSC-based cell therapies. In this study, we show that positive feedback between SASP and inherent senescence processes plays a crucial role in the senescence of umbilical cord blood-derived MSCs (UCB-MSCs). RESULTS We found that monocyte chemoattractant protein-1 (MCP-1) was secreted as a dominant component of the SASP during expansion of UCB-MSCs and reinforced senescence via its cognate receptor chemokine (c-c motif) receptor 2 (CCR2) by activating the ROS-p38-MAPK-p53/p21 signaling cascade in both an autocrine and paracrine manner. The activated p53 in turn increased MCP-1 secretion, completing a feed-forward loop that triggered the senescence program in UCB-MSCs. Accordingly, knockdown of CCR2 in UCB-MSCs significantly improved their therapeutic ability to alleviate airway inflammation in an experimental allergic asthma model. Moreover, BMI1, a polycomb protein, repressed the expression of MCP-1 by binding to its regulatory elements. The reduction in BMI1 levels during UCB-MSC senescence altered the epigenetic status of MCP-1, including the loss of H2AK119Ub, and resulted in derepression of MCP-1. INNOVATION Our results provide the first evidence supporting the existence of the SASP as a causative contributor to UCB-MSC senescence and reveal a so far unappreciated link between epigenetic regulation and SASP for maintaining a stable senescent phenotype. CONCLUSION Senescence of UCB-MSCs is orchestrated by MCP-1, which is secreted as a major component of the SASP and is epigenetically regulated by BMI1.

Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription

For therapeutic applications of siRNA, there are technical challenges with respect to targeted and systemic delivery. We here report a new siRNA carrier, RNAtr NPs, in a way that multiple tandem copies of RNA hairpins as a result of rolling circle transcription (RCT) can be readily adapted in tumour-targeted and systemic siRNA delivery. RNAtr NPs provide a means of condensing large amounts of multimeric RNA transcripts into the compact nanoparticles, especially without the aid of polycationic agents, and thus reduce the risk of immunogenicity and cytotoxicity by avoiding the use of synthetic polycationic reagents. This strategy allows the design of a platform technology for systemic delivery of siRNA to tumour sites, because RCT reaction, which enzymatically generates RNA polymers in multiple copy numbers at low cost, can lead to directly accessible routes to targeted and systemic delivery. Therefore, RNAtr NPs suggest great potentials as the siRNA therapeutics for cancer treatment.

A monitoring method for Atg4 activation in living cells using peptide-conjugated polymeric nanoparticles

To date, several principal methods are presently used to monitor the autophagic process, but they have some potential experimental pitfalls or limitations that make them not applicable to living cells. In order to improve on the currently developed detection methods for autophagy, we report here fluorescent peptide-conjugated polymeric nanoparticles loaded with a lysosome staining dye in their core. The fluorescent peptide is designed to be specifically cleaved by the Atg4 cysteine protease, which plays a crucial role in autophagy activation. In this study, we demonstrate that peptide-conjugated polymeric nanoparticles can be used to visualize Atg4 activity in both cell-free and cell culture systems. The fluorescence imaging of cells incubated with nanoparticles demonstrates that Atg4 activity is activated in the autophagy-induced conditions, but suppressed in the autophagy-inhibited conditions. These results indicate that Atg4 activity is correlated with autophagic flux through its own regulatory pathway. Therefore, our strategy provides an alternative detection method that can clearly distinguish between an “autophagy active” and “autophagy inactive” state in cultured cells. As our nanoparticles are highly cell-permeable and biocompatible, this detection system has general applicability to living cells and can be extended to cell-based screening to evaluate newly developed compounds.

Prolonged autophagy by MTOR inhibitor leads radioresistant cancer cells into senescence

Radiotherapy is one of the well-established therapeutic modalities for cancer treatment. However, the emergence of cells refractory to radiation is a major obstacle to successful treatment with radiotherapy. Many reports suggest that inhibitors targeting the mechanistic target of rapamycin (MTOR) can sensitize cancer cells to the effect of radiation, although by which mechanism MTOR inhibitors enhance the efficacy of radiation toward cancer cells remains to be elucidated. Our studies indicate that a potent and persistent activation of autophagy via inhibition of the MTOR pathway, even in cancer cells where autophagy is occurring, can trigger premature senescence, cellular proliferation arrest. Combined treatment of MTOR inhibitor and radiation induce heterochromatin formation, an irreversible growth arrest and an increase of senescence-associated GLB1 (β-galactosidase) activity, which appear to result from a constant activation of TP53 and a restoration in the activity of retinoblastoma 1 protein (RB1)-E2F1. Thus, this study provides evidence that promoting cellular senescence via inhibition of the MTOR pathway may serve as an avenue to augment radiosensitivity in cancer cells that initiate an autophagy-survival mode to radiotherapy.

Promoter methylation of WNT inhibitory factor-1 and expression pattern of WNT/β-catenin pathway in human astrocytoma: pathologic and prognostic correlations.

WNT inhibitory factor-1 (WIF1) is an antagonist of the WNT signaling pathway. We investigated the relationship between WIF1 promoter methylation and regulation of the WNT/β-catenin signaling pathway, tumor grade, and survival in patients with astrocytoma. This study included 86 cases of astrocytoma, comprising 20 diffuse astrocytomas and 66 glioblastomas. In addition, 17 temporal lobectomy specimens from patients with epilepsy were included as controls. The ratio of methylated DNA to total methylated and unmethylated DNA (% methylation) was measured by methylation- and unmethylation-specific PCR. Representative tumor tissue was immunostained for WIF1, β-catenin, cyclin D1, c-myc, and isocitrate dehydrogenase 1. Levels of WIF1 promoter methylation, mRNA expression, and protein expression in a glioblastoma cell line were compared before and after demethylation treatment. The mean percent methylation of the WIF1 promoter in astrocytomas was higher than that in control brain tissue. WIF1 protein expression was lower in the tumor group with >5% methylation than in the group with <5% methylation. Cytoplasmic β-catenin staining was more frequently observed in tumors with a low WIF1 protein expression level. Demethylation treatment of a glioblastoma cell line increased WIF1 mRNA and protein expression. Increased WIF1 promoter methylation and decreased WIF1 protein expression were not related to patient survival. In conclusion, WIF1 expression is downregulated by promoter methylation and is an important mechanism of aberrant WNT/β-catenin pathway activation in astrocytoma pathogenesis.

Effect of β‐catenin silencing in overcoming radioresistance of head and neck cancer cells by antagonizing the effects of AMPK on Ku70/Ku80

We attempted to elucidate the mechanism of cell death after radiation by studying how β‐catenin silencing controls the radiation sensitivity of radioresistant head and neck cancer cells.

Activation of p53-p21 is closely associated with the acquisition of resistance to apoptosis caused by β1-integrin silencing in head and neck cancer cells.

The issue of whether aberrant expression of β1-integrin is associated with cancer progression and development of resistance to cytotoxic therapy is of considerable interest. Studies to date have shown that the anchorage-independent survival of cancer is attributed, in part, to epithelial-to-mesenchymal transition (EMT). Here, we have reported a novel alternative mechanism of anchorage-independent survival of cancer cells. Cell lines derived from head and neck cancer patients (AMC-HN-3 and AMC-HN-9) and the well-known EMT cancer cell line, MDA-MB231, were examined. The EMT features of AMC-HN-9 cells were comparable to those of MDA-MB231, whereas AMC-HN-3 cells showed no EMT characteristics. Although the pattern and degree of β1-integrin expression were similar in all three cell lines, sensitivities of the cells to β1-integrin knockdown with small interfering RNA (siRNA) were different. Cancer cells with no EMT features underwent cell death to a more significant extent following β1-integrin silencing than those with EMT. Intriguingly, we observed reactive activation of the p53-p21 pathway after β1-integrin silencing in AMC-HN-9 cells lacking an apparent cell death response. Simultaneous knockdown of wild-type p53 and β1-integrin in this cell line promoted cell death. Our data collectively indicate that β1-integrin-related cell death is closely associated with EMT phenotypes and activation of the p53-p21 pathway is partly involved in the acquisition of resistance to apoptosis induced by β1-integrin silencing. Further clarification of the mechanisms underlying p53 integration with β1-integrin signaling may facilitate the development of novel anti-cancer strategies.