Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.

The iron chelator deferriferrichrysin induces paraptosis via extracellular signal-related kinase activation in cancer cells.

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.

Epigenetic Priming with Decitabine Augments the Therapeutic Effect of Cisplatin on Triple-Negative Breast Cancer Cells through Induction of Proapoptotic Factor NOXA.

Epigenetic alterations caused by aberrant DNA methylation have a crucial role in cancer development, and the DNA-demethylating agent decitabine, is used to treat hematopoietic malignancy. Triple-negative breast cancers (TNBCs) have shown sensitivity to decitabine; however, the underlying mechanism of its anticancer effect and its effectiveness in treating TNBCs are not fully understood. We analyzed the effects of decitabine on nine TNBC cell lines and examined genes associated with its cytotoxic effects. According to the effect of decitabine, we classified the cell lines into cell death (D)-type, growth inhibition (G)-type, and resistant (R)-type. In D-type cells, decitabine induced the expression of apoptotic regulators and, among them, NOXA was functionally involved in decitabine-induced apoptosis. In G-type cells, induction of the cyclin-dependent kinase inhibitor, p21, and cell cycle arrest were observed. Furthermore, decitabine enhanced the cytotoxic effect of cisplatin mediated by NOXA in D-type and G-type cells. In contrast, the sensitivity to cisplatin was high in R-type cells, and no enhancing effect by decitabine was observed. These results indicate that decitabine enhances the proapoptotic effect of cisplatin on TNBC cell lines that are less sensitive to cisplatin, indicating the potential for combination therapy in TNBC.

Regenerative Polarity of the Fin Ray in Zebrafish Caudal Fin and Related Tissue Formation on the Cut Surface.

Zebrafish caudal fin rays are used as a model system for regeneration because of their high regenerative ability, but studies on the regeneration polarity of the fin ray are limited. To investigate this regeneration polarity, we made a hole to excise part of the fin ray and analyzed the regeneration process. We confirmed that the fin rays always regenerated from the proximal margin toward the distal margin, as previously reported; however, regeneration-related genes were expressed at both the proximal and distal edges of the hole in the early stage of regeneration, suggesting that the regenerative response also occurs at the distal edge. One difference between the proximal and distal margins is a sheet-like tissue that is formed on the apical side of the regenerated tissue at the proximal margin. This sheet-like tissue was not observed at the distal edge. To investigate whether the distal margin was also capable of forming this sheet-like tissue and subsequent regeneration, we kept the distal margin separated from the proximal margin by manipulation. Consequently, the sheet-like tissue was formed at the distal margin and regeneration of the fin ray was also induced. The regenerated fin rays from the distal margin protruded laterally from the caudal fin and then bent distally, and their ends showed the same characteristics as those of the normal fin rays. These results suggest that fin rays have an ability to regenerate in both directions; however, under normal conditions, regeneration is restricted to the proximal margin because the sheet-like tissue is preferentially formed on the apical side of the regenerating tissue from the proximal margin.

Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis.

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

Krüppel-Like Factor 4 and Its Activator APTO-253 Induce NOXA-Mediated, p53-Independent Apoptosis in Triple-Negative Breast Cancer Cells.

Inducing apoptosis is an effective treatment for cancer. Conventional cytotoxic anticancer agents induce apoptosis primarily through activation of tumor suppressor p53 by causing DNA damage and the resulting regulation of B-cell leukemia/lymphoma-2 (BCL-2) family proteins. Therefore, the effects of these agents are limited in cancers where p53 loss-of-function mutations are common, such as triple-negative breast cancer (TNBC). Here, we demonstrate that ultraviolet (UV) light-induced p53-independent transcriptional activation of NOXA, a proapoptotic factor in the BCL-2 family, results in apoptosis induction. This UV light-induced NOXA expression was triggered by extracellular signal-regulated kinase (ERK) activity. Moreover, we identified the specific UV light-inducible DNA element of the NOXA promoter and found that this sequence is responsible for transcription factor Krüppel-like factor 4 (KLF4)-mediated induction. In p53-mutated TNBC cells, inhibition of KLF4 by RNA interference reduced NOXA expression. Furthermore, treatment of TNBC cells with a KLF4-inducing small compound, APTO-253, resulted in the induction of NOXA expression and NOXA-mediated apoptosis. Therefore, our results help to clarify the molecular mechanism of DNA damage-induced apoptosis and provide support for a possible treatment method for p53-mutated cancers.

Visualization of extracellular vesicles in the regenerating caudal fin blastema of zebrafish using in vivo electroporation.

Zebrafish have high regenerative ability in several organs including the fin. Although various mechanisms underlying fin regeneration have been revealed, some mechanisms remain to be elucidated. Recently, extracellular vesicles (EVs) have been the focus of research with regard to their role in cell-to-cell communication. It has been suggested that cells in regenerating tissues communicate using EVs. In this study, we examined the involvement of EVs in the caudal fin regeneration of zebrafish using an in vivo electroporation method. The process of regeneration appeared normal after in vivo electroporation, and the transferred plasmid showed mosaic expression in the blastema. We took advantage of this mosaic expression to observe the distribution of exosomal markers in the blastema. We transferred exosomal markers by in vivo electroporation and identified EVs in the regenerating caudal fin. The results suggest that blastemal cells communicate with other cells via EVs during caudal fin regeneration.

Cochlear supporting cells function as macrophage-like cells and protect audiosensory receptor hair cells from pathogens.

To protect the audiosensory organ from tissue damage from the immune system, the inner ear is separated from the circulating immune system by the blood-labyrinth barrier, which was previously considered an immune-privileged site. Recent studies have shown that macrophages are distributed in the cochlea, especially in the spiral ligament, spiral ganglion, and stria vascularis; however, the direct pathogen defence mechanism used by audiosensory receptor hair cells (HCs) has remained obscure. Here, we show that HCs are protected from pathogens by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs). In isolated murine cochlear sensory epithelium, we established Theiler's murine encephalomyelitis virus, which infected the SCs and GERCs, but very few HCs. The virus-infected SCs produced interferon (IFN)-α/ß, and the viruses efficiently infected the HCs in the IFN-α/ß receptor-null sensory epithelium. Interestingly, the virus-infected SCs and GERCs expressed macrophage marker proteins and were eliminated from the cell layer by cell detachment. Moreover, lipopolysaccharide induced phagocytosis of the SCs without cell detachment, and the SCs phagocytosed the bacteria. These results reveal that SCs function as macrophage-like cells, protect adjacent HCs from pathogens, and provide a novel anti-infection inner ear immune system.

DMPK is a New Candidate Mediator of Tumor Suppressor p53-Dependent Cell Death.

Tumor suppressor p53 plays an integral role in DNA-damage induced apoptosis, a biological process that protects against tumor progression. Cell shape dramatically changes when cells undergo apoptosis, which is associated with actomyosin contraction; however, it remains entirely elusive how p53 regulates actomyosin contraction in response to DNA-damaging agents. To identify a novel p53 regulating gene encoding the modulator of myosin, we conducted DNA microarray analysis. We found that, in response to DNA-damaging agent doxorubicin, expression of myotonic dystrophy protein kinase (DMPK), which is known to upregulate actomyosin contraction, was increased in a p53-dependent manner. The promoter region of DMPK gene contained potential p53-binding sequences and its promoter activity was increased by overexpression of the p53 family protein p73, but, unexpectedly, not of p53. Furthermore, we found that doxorubicin treatment induced p73 expression, which was significantly attenuated by downregulation of p53. These data suggest that p53 induces expression of DMPK through upregulating p73 expression. Overexpression of DMPK promotes contraction of the actomyosin cortex, which leads to formation of membrane blebs, loss of cell adhesion, and concomitant caspase activation. Taken together, our results suggest the existence of p53-p73-DMPK axis which mediates DNA-damage induced actomyosin contraction at the cortex and concomitant cell death.

p53-independent Noxa induction by cisplatin is regulated by ATF3/ATF4 in head and neck squamous cell carcinoma cells.

The platinum-based DNA damaging agent cisplatin is used as a standard therapy for locally advanced head and neck squamous cell carcinoma (HNSCC). However, the mechanisms underpinning the cytotoxic effects of this compound are not entirely elucidated. Cisplatin produces anticancer effects primarily via activation of the DNA damage response, followed by inducing BCL-2 family dependent mitochondrial apoptosis. We have previously demonstrated that cisplatin induces the expression of proapoptotic BCL-2 family protein, Noxa, that can bind to the prosurvival BCL-2 family protein, MCL-1, to inactivate its function and induce cell death. Here, we show that the upregulation of Noxa is critical for cisplatin-induced apoptosis in p53-null HNSCC cells. This induction is regulated at the transcriptional level. With a series of Noxa promoter-luciferase reporter assays, we find that the CRE (cAMP response element) in the promoter is critical for the Noxa induction by cisplatin treatment. Among the CREB/ATF transcription factors, ATF3 and ATF4 are induced by cisplatin, and downregulation of ATF3 or ATF4 reduced cisplatin-induced Noxa. ATF3 and ATF4 bind to and cooperatively activate the Noxa promoter. Furthermore, ERK1 is involved in cisplatin-induced ATF4 and Noxa induction. In conclusion, ATF3 and ATF4 are important regulators that induce Noxa by cisplatin treatment in a p53-independent manner.

Chaperone-mediated autophagy promotes lung cancer cell survival through selective stabilization of the pro-survival protein, MCL1.

Autophagy is a dynamic recycling system using lysosomal proteolysis that produces new proteins and energy for cellular renovation and homeostasis. Although macroautophagy is known to serve as a survival pathway in many cancer cells, the role of chaperone-mediated autophagy (CMA), a selective protein degradation system, in cancer is not fully understood. Here, we demonstrated that lysosomal proteolysis, but not macroautophagy, attenuated apoptosis induced by the tyrosine kinase inhibitor, crizotinib, in the non-small-cell lung cancer (NSCLC) cell line, EBC1. In EBC1 cells, crizotinib induced BIM-dependent apoptosis, which was enhanced by inhibition of lysosomal proteolysis. Moreover, degradation of the pro-survival protein, MCL1, by the ubiquitin-proteasome system was induced by inhibition of lysosomal proteolysis, and by inhibition of the expression of the CMA mediators, HSC70 (heat shock cognate protein 70 kDa) and LAMP2A (lysosome membrane protein type 2A), suggesting the existence of a CMA-mediated MCL1 stabilization system in cancer cells. Indeed, the same MCL1 stabilization system was also observed in several NSCLC cell lines; in these cells, their specific molecular-targeted drug or ABT-263 (Navitoclax), the specific inhibitor of BCL-2 and BCL-XL, but not of MCL1, effectively induced apoptosis in combination with CMA inhibition. Therefore, our results indicate a novel mechanism of MCL1 stabilization in lung cancers by CMA, and a candidate efficient combination chemotherapy method against lung cancers.

DNA damaging agent-induced apoptosis is regulated by MCL-1 phosphorylation and degradation mediated by the Noxa/MCL-1/CDK2 complex.

Noxa, a BH3-only pro-apoptotic BCL-2 family protein, causes apoptosis by specifically interacting with the anti-apoptotic protein MCL-1 to induce its proteasome-mediated degradation. We show here that the DNA damaging agents cisplatin and etoposide upregulate Noxa expression, which is required for the phosphorylation of MCL-1 at Ser64/Thr70 sites, proteasome-dependent degradation, and apoptosis. Noxa-induced MCL-1 phosphorylation at these sites occurs at the mitochondria and is primarily regulated by CDK2. MCL-1 and CDK2 form a stable complex and Noxa binds to this complex to facilitate the phosphorylation of MCL-1. When Ser64 and Thr70 of MCL-1 are substituted with alanine, the mutated MCL-1 is neither phosphorylated nor ubiquitinated, and becomes more stable than the wild-type protein. As a consequence, this mutant can inhibit apoptosis induced by Noxa overexpression or cisplatin treatment. These results indicate that Noxa-mediated MCL-1 phosphorylation followed by MCL-1 degradation is critical for apoptosis induced by DNA damaging agents through regulation of the Noxa/MCL-1/CDK2 complex.

Cisplatin-induced apoptosis in non-small-cell lung cancer cells is dependent on Bax- and Bak-induction pathway and synergistically activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells.

Cisplatin is a highly effective anticancer drug for treatment of various tumors including non-small-cell lung cancer (NSCLC), and is especially useful in cases nonresponsive to molecular-targeted drugs. Accumulating evidence has shown that cisplatin activates the p53-dependent apoptotic pathway, but it also induces apoptosis in p53-mutated cancer cells. Here we demonstrated that DNA-damage inducible proapoptotic BH3 (Bcl-2 homology region 3)-only Bcl-2 family members, Noxa, Puma, Bim and Bid, are not involved in cisplatin-induced apoptosis in human NSCLC cell lines. In contrast, the expression of proapoptotic multidomain Bcl-2-family members, Bak and Bax, was induced by cisplatin in p53-dependent and -independent manners, respectively. Moreover, in wild-type p53-expressing cells, cisplatin mainly used the Bak-dependent apoptotic pathway, but this apoptotic pathway shifted to the Bax-dependent pathway by loss-of-function of p53. Furthermore, both Bak- and Bax-induced apoptosis was enhanced by the antiapoptotic Bcl-2 family member, Bcl-XL knockdown, but not by Mcl-1 knockdown. From this result, we tested the effect of ABT-263 (Navitoclax), the specific inhibitor of Bcl-2 and Bcl-XL, but not Mcl-1, and found that ABT-263 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells in the presence or absence of p53. These results indicate a novel regulatory system in cisplatin-induced NSCLC cell apoptosis, and a candidate efficient combination chemotherapy method against lung cancers.

Exploitation of the Apoptosis-Primed State of MYCN-Amplified Neuroblastoma to Develop a Potent and Specific Targeted Therapy Combination.

Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.

Combination with vorinostat overcomes ABT-263 (navitoclax) resistance of small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive tumor type with high mortality. One promising approach for SCLC treatment would be to utilize agents targeting molecular abnormalities regulating resistance to apoptosis. BH3 mimetic antagonists, such as ABT-737 and its orally available derivative ABT-263 (navitoclax) have been developed to block the function of pro-survival BCL-2 family members. The sensitivity of SCLC to these drugs varies over a broad range in vitro and in clinical trials. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is a critical determinant of sensitivity to ABT-737. Thus, pharmacological up-regulation of Noxa could enhance cell death induced by the BH3 mimetics. We find that the combination of ABT-263 and a HDAC inhibitor, vorinostat, efficiently induces apoptosis in a variety of SCLC cell lines, including ABT-263 resistant cell lines. Cell death induced by combined treatment is Noxa- and/or BIM-dependent in some cell lines but in others appears to be mediated by down-regulation of BCL-XL and release of BAK from BCL-XL and MCL-1. These results suggest that combination of HDAC inhibitors and BCL-2 inhibitors could be an alternative and effective regimen for SCLC treatment.

Yet another function of p53--the switch that determines whether radiation-induced autophagy will be cytoprotective or nonprotective: implications for autophagy inhibition as a therapeutic strategy.

The influence of autophagy inhibition on radiation sensitivity was studied in human breast, head and neck, and non-small cell lung cancer cell lines, in cell lines that were either wild type or mutant/null in p53, and in cells where p53 was inducible or silenced. Whereas ionizing radiation promoted autophagy in all tumor cell lines studied, pharmacological inhibition of autophagy and/or genetic silencing of autophagy genes failed to influence sensitivity to radiation in p53 mutant Hs578t breast tumor cells, HN6 head and neck tumor cells, and H358 non-small cell lung cancer cells. The requirement for functional p53 in the promotion of cytoprotective autophagy by radiation was confirmed by the observation that radiation-induced autophagy was nonprotective in p53 null H1299 cells but was converted to the cytoprotective form with induction of p53. Conversely, whereas p53 wild-type HN30 head and neck cancer cells did show sensitization to radiation upon autophagy inhibition, HN30 cells in which p53 was knocked down using small hairpin RNA failed to be sensitized by pharmacological autophagy inhibition. Taken together, these findings indicate that radiation-induced autophagy can be either cytoprotective or nonprotective, a functional difference related to the presence or absence of function p53. Alternatively, these findings could be interpreted to suggest that whereas radiation can induce autophagy independent of p53 status, inhibition of autophagy promotes enhanced radiation sensitivity through a mechanism that requires functional p53. These observations are likely to have direct implications with respect to clinical efforts to modulate the response of malignancies to radiation through autophagy inhibition.

p53-mediated activation of the mitochondrial protease HtrA2/Omi prevents cell invasion.

Oncogenic Ras induces cell transformation and promotes an invasive phenotype. The tumor suppressor p53 has a suppressive role in Ras-driven invasion. However, its mechanism remains poorly understood. Here we show that p53 induces activation of the mitochondrial protease high-temperature requirement A2 (HtrA2; also known as Omi) and prevents Ras-driven invasion by modulating the actin cytoskeleton. Oncogenic Ras increases accumulation of p53 in the cytoplasm, which promotes the translocation of p38 mitogen-activated protein kinase (MAPK) into mitochondria and induces phosphorylation of HtrA2/Omi. Concurrently, oncogenic Ras also induces mitochondrial fragmentation, irrespective of p53 expression, causing the release of HtrA2/Omi from mitochondria into the cytosol. Phosphorylated HtrA2/Omi therefore cleaves ß-actin and decreases the amount of filamentous actin (F-actin) in the cytosol. This ultimately down-regulates p130 Crk-associated substrate (p130Cas)-mediated lamellipodia formation, countering the invasive phenotype initiated by oncogenic Ras. Our novel findings provide insights into the mechanism by which p53 prevents the malignant progression of transformed cells.

Paclitaxel-induced apoptosis is BAK-dependent, but BAX and BIM-independent in breast tumor.

Paclitaxel (Taxol)-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death) plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim(-/-) MEFs (mouse embryonic fibroblasts), the bim(-/-) mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak (-/-) MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax(-/-) MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance.

Noxa induces apoptosis in oncogene-expressing cells through catch-and-release mechanism operating between Puma and Mcl-1.

Tumor suppressor p53 induces apoptosis by transcriptional induction of Noxa and Puma, which encode the proapoptotic BH3-only member of the Bcl-2 family proteins. In the p53-mediated tumor surveillance system, p53 induces apoptosis or replicative senescence in oncogene-expressing cells, resulting in elimination of such cells. In this context, we previously found that Noxa and Puma synergistically induce apoptosis. Here, we found the adenovirus oncogene E1A to induce p53-dependently expression of Puma, but not Noxa. The induced Puma associates with antiapoptotic Bcl-2 protein Mcl-1, accompanied by accumulated Mcl-1 protein on mitochondria. Moreover, E1A also reduces expression of the antiapoptotic Bcl-2 protein Bcl-X(L). In contrast, the DNA-damaging agent adriamycin induces Noxa expression in E1A-expressing cells. Interestingly, Mcl-1 knockdown itself induced apoptosis in E1A-expressing MEFs. Furthermore, Noxa displaced Puma's association with Mcl-1, accompanied by Mcl-1 degradation and apoptosis induction by activating mitochondrial apoptotic executers Bax and Bak. These results suggest that p53-induced apoptosis in oncogene-expressing cells is regulated by differential induction and sequential activation of Noxa and Puma. Accumulated Puma by oncogene enhances susceptibility to apoptosis through "catch" in mitochondria by Mcl-1. Subsequently, in response to DNA-damage, Noxa efficiently induces apoptosis by "release" of Puma from Mcl-1.

Interleukin 6 enhances glycolysis through expression of the glycolytic enzymes hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3.

Enhanced glycolysis is important for oncogenesis and for the survival and proliferation of cancer cells in the tumor microenvironment. Recent studies have also shown that proinflammatory cytokine signaling, such as that mediated by nuclear factor kappaB and signal transducer and activator of transcription 3 (STAT3), is important for the generation of inflammation-associated tumors. However, the link between inflammation and enhanced glycolysis has not been identified. In the present study, we found that the proinflammatory cytokine interleukin (IL)-6 enhanced glycolysis in mouse embryonic fibroblasts and human cell lines. Moreover, STAT3 activated by IL-6 enhanced the expression of the glycolytic enzymes hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3). Ectopic expression of PFKFB3 enhanced glycolysis, suggesting that the IL-6-STAT3 pathway enhances glycolysis through the induction of these enzymes. Our findings may provide a novel mechanism for inflammation-associated oncogenesis.