Chang-Hwan Yoon

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

Claudin-1 induces epithelial-mesenchymal transition through activation of the c-Abl-ERK signaling pathway in human liver cells

Claudins (CLDNs) are a family of integral membrane proteins central to the formation of tight junctions, structures that are involved in paracellular transport and cellular growth and differentiation, and are critical for the maintenance of cellular polarity. Recent studies have provided evidence that CLDNs are aberrantly expressed in diverse types of human cancers, including hepatocellular carcinomas (HCCs). However, little is known about how CLDN expression is involved in cancer progression. In this study, we show that CLDN1 has a causal role in the epithelial–mesenchymal transition (EMT) in human liver cells, and that the c-Abl-Ras-Raf-1-ERK1/2 signaling axis is critical for the induction of malignant progression by CLDN1. Overexpression of CLDN1 induced expression of the EMT-regulating transcription factors Slug and Zeb1, and thereby led to repression of E-cadherin, β-catenin expression, enhanced expression of N-cadherin and Vimentin, a loss of cell adhesion, and increased cell motility in normal liver cells and HCC cells. In line with these findings, inhibition of either c-Abl or ERK clearly attenuated CLDN1-induced EMT, as evidenced by a reversal of N-cadherin and E-cadherin expression patterns, and restored normal motility. Collectively, these results indicate that CLDN1 is necessary for the induction of EMT in human liver cells, and that activation of the c-Abl-Ras-Raf-1-ERK1/2 signaling pathway is required for CLDN1-induced acquisition of the malignant phenotype. The present observations suggest that CLDN1 could be exploited as a biomarker for liver cancer metastasis and might provide a pivotal point for therapeutic intervention in HCC.

The Rac1/MKK7/JNK pathway signals upregulation of Atg5 and subsequent autophagic cell death in response to oncogenic Ras

To prevent the development of malignancies, mammalian cells activate disposal programs, such as programmed cell death, in response to deregulated oncogene expression. However, the molecular basis for regulation of cellular disposal machinery in response to activated oncogenes is unclear at present. In this study, we show that upregulation of the autophagy-related protein, Atg5, is critically required for the oncogenic H-ras-induced autophagic cell death and that Rac1/mitogen-activated kinase kinase (MKK) 7/c-Jun N-terminal kinase (JNK) signals upregulation of Atg5. Overexpression of H-ras(V12) induced marked autophagic vacuole formation and cell death in normal fibroblasts, which remained unaffected by a caspase inhibitor. Pretreatment with Bafilomycin A1, an autophagy inhibitor, completely attenuated H-ras(V12)-induced cell death as well as autophagic vacuole formation. Selective production of Atg5 was observed in cells overexpressing H-ras(V12), and small interfering RNA (siRNA) targeting of Atg5 clearly inhibited autophagic cell death. Interestingly, inhibition of JNK or c-Jun by specific siRNA suppressed Atg5 upregulation and autophagic cell death. Moreover, inhibition of MKK7, but not MKK4, effectively attenuated H-ras(V12)-induced JNK activation. In addition, ectopic expression of RacN17 or Rac1-siRNA effectively inhibited MKK7-JNK activation, Atg5 upregulation and autophagic cell death. These data support the notion that upregulation of Atg5 is required for the oncogenic H-ras-induced autophagic cell death in normal fibroblasts and that activation of Rac1/MKK7/JNK-signaling pathway leads to upregulation of Atg5 in response to oncogenic H-ras. Our findings suggest that in cells acquiring deregulated oncogene expression, oncogenic stress triggers autophagic cell death, which protects cells against malignant progression.

Claudin-1 Acts through c-Abl-Protein Kinase Cδ (PKCδ) Signaling and Has a Causal Role in the Acquisition of Invasive Capacity in Human Liver Cells

Claudins are identified as members of the tetraspanin family of proteins, which are integral to the structure and function of tight junction. Recent studies showed an increase in expression of claudins during tumorigenesis, which is associated with loss of cell-cell contact, dedifferentiation, and invasiveness. However, the molecular basis for the causal relationship between claudin expression and cancer progression is not fully understood yet. In this study, we show that claudin-1 plays a causal role in the acquisition of invasive capacity in human liver cells and that c-Abl-protein kinase Cδ (PKCδ) signaling is critical for the malignant progression induced by claudin-1. Overexpression of claudin-1 clearly induced expression of matrix metalloproteinase-2 (MMP-2) and cell invasion and migration in normal liver cells as well as in non-invasive human hepatocellular carcinoma (HCC) cells. Conversely, small interfering RNA targeting of claudin-1 in invasive HCC cells completely inhibited cell invasion. Both c-Abl and PKCδ are found to be activated in normal liver cell line clones that stably overexpress claudin-1. Inhibition of either c-Abl or PKCδ alone clearly attenuated MMP-2 activation and impeded cell invasion and migration in both human HCC and normal liver cells expressing claudin-1. These results indicate that claudin-1 is both necessary and sufficient to induce invasive behavior in human liver cells and that activation of c-Abl-PKCδ signaling pathway is critically required for the claudin-1-induced acquisition of the malignant phenotype. The present observations raise the possibility of exploiting claudin-1 as a potential biomarker for the spread of liver cancer and might provide pivotal points for therapeutic intervention in HCC.

C-Jun N-terminal kinase has a pivotal role in the maintenance of self-renewal and tumorigenicity in glioma stem-like cells

Uncovering the mechanisms that govern the maintenance of stem-like cancer cells is critical for developing therapeutic strategies for targeting these cells. Constitutive activation of c-Jun N-terminal kinase (JNK) has been reported in gliomas and correlates with histological grade. Here, we found that JNK signaling is crucial for the maintenance of ‘stemness’ in glioma cells. Sphere-cultured glioma cells showed more phosphorylation of JNK compared with serum-containing monolayer cultures. Importantly, blockade of JNK signaling with SP600125 or small interfering RNAs targeting JNK1 or JNK2 significantly reduced the CD133+/Nestin+ population and suppressed sphere formation, colony formation in soft agar, and expression of stem cell markers in sphere-cultured glioma cells. Intriguingly, sphere-cultured glioma cells exhibited enhanced expression of Notch-2, but not Notch-1, -3 or -4, and JNK inhibition almost completely abrogated this increase. Blocking the phosphoinoside 3-kinase (PI3K)/Akt pathway with LY294002 or si-Akt also suppressed the self-renewal of sphere-cultured glioma cells. PI3K, but not Akt, had a role as an upstream kinase in JNK1/2 activation. In addition, treatment with si-JNK greatly increased etoposide- and ionizing radiation (IR)-induced cell death in glioma spheres. Consistent with glioma cell lines, glioma stem-like cells isolated from primary patient glioma cells also had a higher activity of JNK and Notch-2 expression. Importantly, inhibition of JNK2 led to a decrease of Notch-2 expression and suppressed the CD133+/Nestin+ cell population in patient-derived primary glioma cells. Finally, downregulation of JNK2 almost completely suppressed intracranial tumor formation by glioma cells in nude mice. Taken together, these data demonstrate that JNK signaling is crucial for the maintenance of self-renewal and tumorigenicity of glioma stem-like cells and drug/IR resistance, and can be considered a promising target for eliminating stem-like cancer cells in gliomas.

Reactive Oxygen Species-Dependent Activation of Bax and Poly(ADP-ribose) Polymerase-1 Is Required for Mitochondrial Cell Death Induced by Triterpenoid Pristimerin in Human Cervical Cancer Cells

Naturally occurring triterpenoid compounds have long been used as anti-inflammatory, antimalarial, and insecticidal agents. It has become evident that some of the natural or synthetic triterpenoids have promising clinical potential as both a therapeutic and chemopreventive agent for cancer. However, the molecular basis for the antitumor activity of triterpenoid has yet to be defined. In this study, we show that pristimerin, a natural triterpenoid, induces mitochondrial cell death in human cervical cancer cells and that reactive oxygen species (ROS)-dependent activation of both Bax and poly(ADP-ribose) polymerase-1 (PARP-1) is critically required for the mitochondrial dysfunction. We also showed that c-Jun N-terminal kinase (JNK) is involved in ROS-dependent Bax activation. Treatment of pristimerin induced an increase in intracellular ROS, JNK activation, conformational change, and mitochondrial redistribution of Bax, mitochondrial membrane potential loss, and cell death. The PARP-1 was also found to be activated by pristimerin treatment. An antioxidant, N-acetyl-l-cysteine (NAC), inhibited pristimerin-induced JNK activation, Bax relocalization, and PARP-1 activation, as well as mitochondrial cell death. Moreover, inhibition of JNK clearly suppressed conformational change and mitochondrial translocation of Bax and subsequent mitochondrial cell death but did not affect PARP-1 activation. Inhibition of PARP-1 with 1,5-dihydroxyisoquinoline (DIQ) or with small interfering RNA of PARP-1 significantly attenuated pristimerin-induced mitochondrial membrane potential loss and cell death but did not affect JNK activation and Bax relocalization. These results indicate that the natural triterpenoid pristimerin induces mitochondrial cell death through ROS-dependent activation of both Bax and PARP-1 in human cervical cancer cells and that JNK is involved in ROS-dependent Bax activation.

PTTG1 Oncogene Promotes Tumor Malignancy via Epithelial to Mesenchymal Transition and Expansion of Cancer Stem Cell Population

Background: PTTG1 is an oncogene with its expression levels correlating with tumor development and metastasis. Results: Modulation of PTTG1 expression levels revealed that PTTG1 promotes invasive and migratory properties and expansion of CD44high CD24low cell population via AKT activation in breast cancer cells. Conclusion: PTTG1 induces EMT and promotes cancer stem cells via activation of AKT. Significance: PTTG1 represents a potential target for therapeutic intervention against the spread of breast cancer. The prognosis of breast cancer patients is related to the degree of metastasis. However, the mechanisms by which epithelial tumor cells escape from the primary tumor and colonize at a distant site are not entirely understood. Here, we analyzed expression levels of pituitary tumor-transforming gene-1 (PTTG1), a relatively uncharacterized oncoprotein, in patient-derived breast cancer tissues with corresponding normal breast tissues. We found that PTTG1 is highly expressed in breast cancer patients, compared with normal tissues. Also, PTTG1 expression levels were correlated with the degree of malignancy in breast cancer cell lines; the more migratory and invasive cancer cell lines MDA-MB-231 and BT549 displayed the higher expression levels of PTTG1 than the less migratory and invasive MCF7 and SK-BR3 and normal MCF10A cell lines. By modulating PTTG1 expression levels, we found that PTTG1 enhances the migratory and invasive properties of breast cancer cells by inducing epithelial to mesenchymal transition, as evidenced by altered morphology and epithelial/mesenchymal cell marker expression patterns and up-regulation of the transcription factor Snail. Notably, down-regulation of PTTG1 also suppressed cancer stem cell population in BT549 cells by decreasing self-renewing ability and tumorigenic capacity, accompanying decreasing CD44high CD24low cells and Sox2 expression. Up-regulation of PTTG1 had the opposite effects, increasing sphere-forming ability and Sox2 expression. Importantly, PTTG1-mediated malignant tumor properties were due, at least in part, to activation of AKT, known to be a key regulator of both EMT and stemness in cancer cells. Collectively, these results suggest that PTTG1 may represent a new therapeutic target for malignant breast cancer.

Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

Background Titanium dioxide (TiO2) has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO2 particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO2 has yet to be defined. Methods and results In this study, we demonstrated that combined treatment with TiO2 nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO2 nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO2P25–70) together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO2-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO2P25–70 and ultraviolet A irradiation. Conclusion These results indicate that sub-100 nm sized TiO2 treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein, Bax. Elucidating the molecular mechanisms by which nanosized particles induce activation of cell death signaling pathways would be critical for the development of prevention strategies to minimize the cytotoxicity of nanomaterials.

The small GTPase Rac1 is involved in the maintenance of stemness and malignancies in glioma stem-like cells

A subpopulation of cancer cells with stem cell properties is responsible for tumor formation, maintenance, and malignant progression; however, the molecular mechanisms underlying the maintenance of cancer stem‐like cell properties have remained unclear. Here, we show that the Rho family GTPase Rac1 is involved in the glioma stem‐like cell (GSLC) maintenance and tumorigenicity in human glioma. The Rac1‐Pak signaling was markedly activated in GSLCs. Knockdown of Rac1 caused reduction of expression of GSLC markers, self‐renewal‐related proteins and neurosphere formation. Moreover, down‐regulation of Rac1 suppressed the migration, invasion, and malignant transformation in GSLCs. Furthermore, inhibition of Rac1 enhanced radiation sensitivity of GSLCs. These results indicate that the small GTPase Rac1 is involved in the maintenance of stemness and malignancies in GSLCs.

Importance of PKCδ signaling in fractionated-radiation-induced expansion of glioma-initiating cells and resistance to cancer treatment.

Brain tumors frequently recur or progress as focal masses after treatment with ionizing radiation. However, the mechanisms underlying the repopulation of tumor cells after radiation have remained unclear. In this study, we show that cellular signaling from Abelson murine leukemia viral oncogene homolog (Abl) to protein kinase Cδ (PKCδ) is crucial for fractionated-radiation-induced expansion of glioma-initiating cell populations and acquisition of resistance to anticancer treatments. Treatment of human glioma cells with fractionated radiation increased Abl and PKCδ activity, expanded the CD133-positive (CD133+) cell population that possesses tumor-initiating potential and induced expression of glioma stem cell markers and self-renewal-related proteins. Moreover, cells treated with fractionated radiation were resistant to anticancer treatments. Small interfering RNA (siRNA)-mediated knockdown of PKCδ expression blocked fractionated-radiation-induced CD133+ cell expansion and suppressed expression of glioma stem cell markers and self-renewal-related proteins. It also suppressed resistance of glioma cells to anticancer treatments. Similarly, knockdown of Abl led to a decrease in CD133+ cell populations and restored chemotherapeutic sensitivity. It also attenuated fractionated-radiation-induced PKCδ activation, suggesting that Abl acts upstream of PKCδ. Collectively, these data indicate that fractionated radiation induces an increase in the glioma-initiating cell population, decreases cellular sensitivity to cancer treatment and implicates activation of Abl–PKCδ signaling in both events. These findings provide insights that might prove pivotal in the context of ionising-radiation-based therapeutic interventions for brain tumors.