Hyun Min Jeon

Wnt/Snail Signaling Regulates Cytochrome c Oxidase and Glucose Metabolism

Wnt signaling plays a critical role in embryonic development, and its deregulation is closely linked to the occurrence of a number of malignant tumors, including breast and colon cancer. The pathway also induces Snail-dependent epithelial-to-mesenchymal transition (EMT), which is responsible for tumor invasion and metastasis. In this study, we show that Wnt suppresses mitochondrial respiration and cytochrome C oxidase (COX) activity by inhibiting the expression of 3 COX subunits, namely, COXVIc, COXVIIa, and COXVIIc. We found that Wnt induced a glycolytic switch via increased glucose consumption and lactate production, with induction of pyruvate carboxylase (PC), a key enzyme of anaplerosis. In addition, Wnt-induced mitochondrial repression and glycolytic switching occurred through the canonical β-catenin/T-cell factor 4/Snail pathway. Short hairpin RNA-mediated knockdown of E-cadherin, a regulator of EMT, repressed mitochondrial respiration and induced a glycolytic switch via Snail activation, indicating that EMT may contribute to Wnt/Snail regulation of mitochondrial respiration and glucose metabolism. Together, our findings provide a new function for Wnt/Snail signaling in the regulation of mitochondrial respiration (via COX gene expression) and glucose metabolism (via PC gene expression) in tumor growth and progression.

Induction of metastasis, cancer stem cell phenotype, and oncogenic metabolism in cancer cells by ionizing radiation

Radiation therapy is one of the major tools of cancer treatment, and is widely used for a variety of malignant tumours. Radiotherapy causes DNA damage directly by ionization or indirectly via the generation of reactive oxygen species (ROS), thereby destroying cancer cells. However, ionizing radiation (IR) paradoxically promotes metastasis and invasion of cancer cells by inducing the epithelial-mesenchymal transition (EMT). Metastasis is a major obstacle to successful cancer therapy, and is closely linked to the rates of morbidity and mortality of many cancers. ROS have been shown to play important roles in mediating the biological effects of IR. ROS have been implicated in IR-induced EMT, via activation of several EMT transcription factors—including Snail, HIF-1, ZEB1, and STAT3—that are activated by signalling pathways, including those of TGF-β, Wnt, Hedgehog, Notch, G-CSF, EGFR/PI3K/Akt, and MAPK. Cancer cells that undergo EMT have been shown to acquire stemness and undergo metabolic changes, although these points are debated. IR is known to induce cancer stem cell (CSC) properties, including dedifferentiation and self-renewal, and to promote oncogenic metabolism by activating these EMT-inducing pathways. Much accumulated evidence has shown that metabolic alterations in cancer cells are closely associated with the EMT and CSC phenotypes; specifically, the IR-induced oncogenic metabolism seems to be required for acquisition of the EMT and CSC phenotypes. IR can also elicit various changes in the tumour microenvironment (TME) that may affect invasion and metastasis. EMT, CSC, and oncogenic metabolism are involved in radioresistance; targeting them may improve the efficacy of radiotherapy, preventing tumour recurrence and metastasis. This study focuses on the molecular mechanisms of IR-induced EMT, CSCs, oncogenic metabolism, and alterations in the TME. We discuss how IR-induced EMT/CSC/oncogenic metabolism may promote resistance to radiotherapy; we also review efforts to develop therapeutic approaches to eliminate these IR-induced adverse effects.

Homeobox gene Dlx-2 is implicated in metabolic stress-induced necrosis

BackgroundIn contrast to tumor-suppressive apoptosis and autophagic cell death, necrosis promotes tumor progression by releasing the pro-inflammatory and tumor-promoting cytokine high mobility group box 1 (HMGB1), and its presence in tumor patients is associated with poor prognosis. Thus, necrosis has important clinical implications in tumor development; however, its molecular mechanism remains poorly understood.ResultsIn the present study, we show that Distal-less 2 (Dlx-2), a homeobox gene of the Dlx family that is involved in embryonic development, is induced in cancer cell lines dependently of reactive oxygen species (ROS) in response to glucose deprivation (GD), one of the metabolic stresses occurring in solid tumors. Increased Dlx-2 expression was also detected in the inner regions, which experience metabolic stress, of human tumors and of a multicellular tumor spheroid, an in vitro model of solid tumors. Dlx-2 short hairpin RNA (shRNA) inhibited metabolic stress-induced increase in propidium iodide-positive cell population and HMGB1 and lactate dehydrogenase (LDH) release, indicating the important role(s) of Dlx-2 in metabolic stress-induced necrosis. Dlx-2 shRNA appeared to exert its anti-necrotic effects by preventing metabolic stress-induced increases in mitochondrial ROS, which are responsible for triggering necrosis.ConclusionsThese results suggest that Dlx-2 may be involved in tumor progression via the regulation of metabolic stress-induced necrosis.

Implication of Snail in Metabolic Stress-Induced Necrosis

Background Necrosis, a type of cell death accompanied by the rupture of the plasma membrane, promotes tumor progression and aggressiveness by releasing the pro-inflammatory and angiogenic cytokine high mobility group box 1. It is commonly found in the core region of solid tumors due to hypoxia and glucose depletion (GD) resulting from insufficient vascularization. Thus, metabolic stress-induced necrosis has important clinical implications for tumor development; however, its regulatory mechanisms have been poorly investigated. Methodology/Principal Findings Here, we show that the transcription factor Snail, a key regulator of epithelial-mesenchymal transition, is induced in a reactive oxygen species (ROS)-dependent manner in both two-dimensional culture of cancer cells, including A549, HepG2, and MDA-MB-231, in response to GD and the inner regions of a multicellular tumor spheroid system, an in vitro model of solid tumors and of human tumors. Snail short hairpin (sh) RNA inhibited metabolic stress-induced necrosis in two-dimensional cell culture and in multicellular tumor spheroid system. Snail shRNA-mediated necrosis inhibition appeared to be linked to its ability to suppress metabolic stress-induced mitochondrial ROS production, loss of mitochondrial membrane potential, and mitochondrial permeability transition, which are the primary events that trigger necrosis. Conclusions/Significance Taken together, our findings demonstrate that Snail is implicated in metabolic stress-induced necrosis, providing a new function for Snail in tumor progression.

Early growth response 1 regulates glucose deprivation-induced necrosis

Necrosis is commonly found in the core region of solid tumours due to metabolic stress such as hypoxia and glucose deprivation (GD) resulting from insufficient vascularization. Necrosis promotes tumour growth and development by releasing the tumour-promoting cytokine high mobility group box 1 (HMGB1); however, the molecular mechanism underlying necrotic cell death remains largely unknown. In this study, we show that early growth response 1 (Egr-1) is induced in a reactive oxygen species (ROS)-dependent manner by GD in several cell lines such as A549, MDA-MB-231 and HepG2 cells that exhibit necrosis upon GD. We found that Egr-1 short hairpin RNA (shRNA) prevented GD-induced necrosis and HMGB1 release. Necrosis-inhibiting activity of Egr-1 shRNA was also seen in multicellular tumour spheroids (MTSs), an in vitro tumour model system. In contrast, Egr-1 overexpression appeared to make tumour cells more susceptible to GD-induced necrosis. Finally, Egr-1 shRNA suppressed the growth of MTSs. These findings demonstrate that Egr-1 is implicated in GD-induced necrosis and tumour progression.

Dlx-2 and glutaminase upregulate epithelial-mesenchymal transition and glycolytic switch

Most cancer cells depend on enhanced glucose and glutamine (Gln) metabolism for growth and survival. Oncogenic metabolism provides biosynthetic precursors for nucleotides, lipids, and amino acids; however, its specific roles in tumor progression are largely unknown. We previously showed that distal-less homeobox-2 (Dlx-2), a homeodomain transcription factor involved in embryonic and tumor development, induces glycolytic switch and epithelial-mesenchymal transition (EMT) by inducing Snail expression. Here we show that Dlx-2 also induces the expression of the crucial Gln metabolism enzyme glutaminase (GLS1), which converts Gln to glutamate. TGF-β and Wnt induced GLS1 expression in a Dlx-2-dependent manner. GLS1 shRNA (shGLS1) suppressed in vivo tumor metastasis and growth. Inhibition of Gln metabolism by shGLS1, Gln deprivation, and Gln metabolism inhibitors (DON, 968 and BPTES) prevented Dlx-2-, TGF-β-, Wnt-, and Snail-induced EMT and glycolytic switch. Finally, shDlx-2 and Gln metabolism inhibition decreased Snail mRNA levels through p53-dependent upregulation of Snail-targeting microRNAs. These results demonstrate that the Dlx-2/GLS1/Gln metabolism axis is an important regulator of TGF-β/Wnt-induced, Snail-dependent EMT, metastasis, and glycolytic switch.

The hepatoprotective effects of adenine nucleotide translocator-2 against aging and oxidative stress

Mitochondrial adenine nucleotide translocator (ANT) plays important roles in the regulation of mitochondrial permeability transition and cell bioenergetics. The mouse has three ANT isoforms (1, 2 and 4) showing tissue-specific expression patterns. Although ANT1 is known to have a pro-apoptotic property, the specific functions of ANT2 have not been well determined. In the present study, ANT2 expression was significantly lower in the aged rat liver and in a liver fibrosis model. To explore the protective role of ANT2 in the liver, we established a hepa1c1c7 cell line overexpressing ANT2. Overexpression of ANT2 caused hepa1c1c7 cells to be more resistant to oxidative stress, and mitochondrial membrane potential (MMP, ∆Ψm) was relatively intact in ANT2-overexpressing cells under oxidative stress. In addition, ANT2 was found to increase ATP production by influencing mitochondrial bioenergetics. These results imply that the hepatoprotective effect of ANT2 is due to the stabilization of MMP and enhanced ATP production, and thus, maintaining ANT2 levels in the liver might be important to enhance resistance to aging and oxidative stress.

Snail Switches 5-FU-induced Apoptosis to Necrosis through Akt/PKB Activation and p53 Down-regulation

Snail is a zinc finger transcription factor that induces epithelial-to-mesenchymal transition (EMT), which promotes tumor invasion and metastasis by repressing E-cadherin expression. In addition, Snail restricts the cellular apoptotic response to apoptotic stimuli or survival factor withdrawal; however, its molecular mechanism remains largely unknown. In this study, we have investigated the mechanism underlying Snail-mediated chemoresistance to 5-fluorouracil (5-FU), one of the most widely used anti-cancer drugs. When Snail was overexpressed by doxycycline (DOX) in MCF-7 #5 cells, it inhibited 5-FU-induced apoptotic cell death and switched the cell death mode to necrosis. Snail expression, either by DOX treatment in MCF-7 #5 cells or by the transfection of Snail expression vectors pCR3.1-Snail-Flg, phosphorylation-resistant pCR3.1-S104, and 107A Snail-Flg in MCF-7 cells specifically induced PTEN down-regulation/inactivation and Akt/PKB activation, without affecting ERK1/2 activity. In addition, Snail prominently suppressed 5-FU-induced increases in p53 levels. These findings demonstrate that Snail switches 5-FU-induced apoptosis to necrosis through the activation of Akt/PKB and the down-regulation of p53 levels.

Early Growth Response 1 Induces Epithelial-to-mesenchymal Transition via Snail

The epithelial-to-mesenchymal transition (EMT) plays an essential role in embryogenesis and is involved in tumor metastasis and invasion; it significantly contributes to tumor progression and aggressiveness. The EMT is characterized by a loss of epithelial cell polarity as a result of the reduced expression of epithelial E-cadherin, a hallmark of the EMT, and the acquisition of mesenchymal-like cell morphology. Reactive oxygen species (ROS) such as O₂ -, H₂O₂, and OH- have been demonstrated to induce the EMT; although Snail is involved in ROS-induced EMT by transcriptionally repressing E-cadherin, its mechanism is not fully understood. In this study, we examined the effects of early growth response 1 (Egr-1) overexpression in noninvasive breast tumor cell line MCF-7 cells. Upon Egr-1 overexpression, MCF-7 cells lost epithelial cell polarity and became more spindle-shaped, indicating that Egr-1 may induce EMT. We found that Snail is implicated in Egr-1 induced EMT. We further demonstrate that the Egr-1-Snail axis is activated by ROS and plays a critical role(s) in ROS-induced EMT.

HMGB1 Switches Alkylating DNA Damage-Induced Apoptosis to Necrosis

Necrosis is characterized by the cell membrane rupture and release of the cellular contents, including high-mobility group box 1 protein (HMGB1), into the extracellular microenvironment. HMGB1 acts as a transcriptional regulator in nuclei, but exerts a pro-inflammatory and tumor-promoting cytokine activity when released into the extracellular space. Its overexpression is associated with tumor progression and chemoresistance. Thus, HMGB1 acts as a clinically important molecule in tumor biology. In this study, we examined whether HMGB1 affects cell death induced by anti-cancer drugs. Here we show that HMGB1 prevented cisplatin (alkylating agent)-induced apoptosis and switched the cell fate to necrosis in MCF-7, MDA-MB231, and MDA-MB361 cells. Similar apoptosis-to-necrosis switch effects of HMGB1 were observed in cells treated with 4-HC, another alkylating agent. In contrast, HMGB1 did not exert any significant effects on docetaxel (DOC)-induced apoptosis in MCF-7 cells. We also show that cisplatin-induced apoptosis was switched to necrosis in MCF-7 multicellular tumor spheroids (MTS) that were cultured for 8 days and had necrotic cores, but DOC-induced apoptosis was prevented without the apoptosis-to-necrosis switch. Finally, the levels of RAGE, a receptor of HMGB1, were increased with extended culture of MTS. These findings demonstrate that HMGB1 switches alkylating agent-induced apoptosis to necrosis, suggesting that the strategy to prevent necrosis occurring as an undesirable action of alkylating agent-based chemotherapy should be delineated to improve the efficacy of chemotherapy for cancer.