Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival.

Deregulated oncogenes such as MYC and RAS are typically insufficient to transform cells on their own due to the activation of pathways that restrain proliferation. Previous studies have shown that oncogenic H-Ras can induce proliferative arrest or senescence, depending on the cellular context. Here, we show that deregulated H-Ras activity can also lead to caspase-independent cell death with features of autophagy. Ras-induced autophagy was associated with upregulation of the BH3-only protein Noxa as well as the autophagy regulator Beclin-1. Silencing of Noxa or Beclin-1 expression reduced Ras-induced autophagy and increased clonogenic survival. Ras-induced cell death was also inhibited by coexpression of Bcl-2 family members that inhibit Beclin-1 function. Ras-induced autophagy was associated with Noxa-mediated displacement of the Bcl-2 family member, Mcl-1, from Beclin-1. Thus, Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death, which represents a mechanism to limit the oncogenic potential of deregulated Ras signals.

Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases.

Interleukin-33 (IL-33) is a member of the IL-1 family and is involved in polarization of T cells toward a T helper 2 (Th2) cell phenotype. IL-33 is thought to be activated via caspase-1-dependent proteolysis, similar to the proinflammatory cytokines IL-1 beta and IL-18, but this remains unproven. Here we showed that IL-33 was processed by caspases activated during apoptosis (caspase-3 and -7) but was not a physiological substrate for caspases associated with inflammation (caspase-1, -4, and -5). Furthermore, caspase-dependent processing of IL-33 was not required for ST2 receptor binding or ST2-dependent activation of the NF-kappaB transcription factor. Indeed, caspase-dependent proteolysis of IL-33 dramatically attenuated IL-33 bioactivity in vitro and in vivo. These data suggest that IL-33 does not require proteolysis for activation, but rather, that IL-33 bioactivity is diminished through caspase-dependent proteolysis within apoptotic cells. Thus, caspase-mediated proteolysis acts as a switch to dampen the proinflammatory properties of IL-33.

Bax- or Bak-induced mitochondrial fission can be uncoupled from cytochrome C release.

Bax and Bak promote apoptosis by perturbing the permeability of the mitochondrial outer membrane and facilitating the release of cytochrome c by a mechanism that is still poorly defined. During apoptosis, Bax and Bak also promote fragmentation of the mitochondrial network, possibly by activating the mitochondrial fission machinery. It has been proposed that Bax/Bak-induced mitochondrial fission may be required for release of cytochrome c from the mitochondrial intermembrane space, although this has been a subject of debate. Here we show that Bcl-xL, as well as other members of the apoptosis-inhibitory subset of the Bcl-2 family, antagonized Bax and/or Bak-induced cytochrome c release but failed to block mitochondrial fragmentation associated with Bax/Bak activation. These data suggest that Bax/Bak-initiated remodeling of mitochondrial networks and cytochrome c release are separable events and that Bcl-2 family proteins can influence mitochondrial fission-fusion dynamics independent of apoptosis.

Inhibitor of apoptosis proteins (IAPs) and their antagonists regulate spontaneous and tumor necrosis factor (TNF)-induced proinflammatory cytokine and chemokine production.

Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings.

Palmitoyl transferase ZDHHC20 promotes pancreatic cancer metastasis.

Metastasis is one of the defining features of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor prognosis. In this study, the palmitoyl transferase ZDHHC20 was identified in an in vivo short hairpin RNA (shRNA) screen as critical for metastatic outgrowth, with no effect on proliferation and migration in vitro or primary PDAC growth in mice. This phenotype is abrogated in immunocompromised animals and animals with depleted natural killer (NK) cells, indicating that ZDHHC20 affects the interaction of tumor cells and the innate immune system. Using a chemical genetics platform for ZDHHC20-specific substrate profiling, a number of substrates of this enzyme were identified. These results describe a role for palmitoylation in enabling distant metastasis that could not have been detected using in vitro screening approaches and identify potential effectors through which ZDHHC20 promotes metastasis of PDAC.

Executioner caspase-3 and caspase-7 are functionally distinct proteases.

Members of the caspase family of cysteine proteases play central roles in coordinating the stereotypical events that occur during apoptosis. Because the major executioner caspases, caspase-3 and caspase-7, exhibit almost indistinguishable activity toward certain synthetic peptide substrates, this has led to the widespread view that these proteases occupy functionally redundant roles within the cell death machinery. However, the distinct phenotypes of mice deficient in either of these caspases, as well as mice deficient in both, is at odds with this view. These distinct phenotypes could be related to differences in the relative expression levels of caspase-3 and caspase-7 in vivo, or due to more fundamental differences between these proteases in terms of their ability to cleave natural substrates. Here we show that caspase-3 and caspase-7 exhibit differential activity toward multiple substrate proteins, including Bid, XIAP, gelsolin, caspase-6, and cochaperone p23. Caspase-3 was found to be generally more promiscuous than caspase-7 and appears to be the major executioner caspase during the demolition phase of apoptosis. Our observations provide a molecular basis for the different phenotypes seen in mice lacking either caspase and indicate that these proteases occupy nonredundant roles within the cell death machinery.

Value of cerebral microhemorrhages detected with susceptibility-weighted MR Imaging for prediction of long-term outcome in children with nonaccidental trauma.

PURPOSE: To determine the prevalence of parenchymal brain microhemorrhages (MHs) in infants with nonaccidental trauma (NAT) by using susceptibility-weighted (SW) magnetic resonance (MR) imaging and to assess whether the presence of MH results in improved prediction of the long-term neurologic outcome. MATERIALS AND METHODS: A retrospective case-control analysis of the data for 101 children aged 1-32 months with forensic pediatric specialist-confirmed NAT was performed with institutional review board approval. Sixty-two patients were boys (mean age, 8.4 months +/- 7.4 [standard deviation]), and 39 were girls (mean age, 7.4 months +/- 7.8). The imaging findings and clinical data of the children who were examined with SW imaging were collected. Exclusion criteria included pre-existing cognitive delays, central nervous system malformations, previous brain injuries, and/or birth before 30 weeks gestation. Dichotomized long-term neurologic outcomes (good [normal, mild disability, or moderate disability] versus poor [severe disability, vegetative state, or death]) at greater than or equal to 6 months (mean, 33 months; range 6-95 months) were available for 53 patients (36 boys [mean age, 7.3 months +/- 5.9]; 17 girls [mean age, 7.4 months +/- 7.9]; overall range, 2-32 months). Logistic regression was used to determine whether the presence of SW imaging-depicted MH, as compared with other radiologic findings, resulted in improved prediction of long-term neurologic outcome. RESULTS: Imaging findings showed that of the 101 patients, 29 (29%) had MH at SW imaging, 66 (65%) had extraaxial hemorrhages, 52 (51%) had retinal hemorrhages, and 35 (35%) had evidence of acute ischemic injury. A significantly larger number of children with poor outcomes than children with good outcomes had brain MH (nine of 14 vs seven of 39; P = .001) and ischemic injury (13 of 14 vs 17 of 39; P = .006). Logistic regression analysis revealed presence of MH at SW imaging-followed by acute ischemic injury, initial Glasgow Coma Scale score, and age-to be the most significant single variable in the final model, with an overall predictive accuracy of 92.5%. CONCLUSION: Presence of intraparenchymal brain MH in children with NAT, as detected on SW images, correlates with significantly poor long-term neurologic outcome, improves outcome prediction compared with the predictions made by using other tested clinical and imaging findings, and is most predictive when combined with presence of ischemic injury.

Overview of KRAS-Driven Genetically Engineered Mouse Models of Non-Small Cell Lung Cancer.

KRAS, the most frequently mutated oncogene in non-small cell lung cancer, has been utilized extensively to model human lung adenocarcinomas. The results from such studies have enhanced considerably an understanding of the relationship between KRAS and the development of lung cancer. Detailed in this overview are the features of various KRAS-driven genetically engineered mouse models (GEMMs) of non-small cell lung cancer, their utilization, and the potential of these models for the study of lung cancer biology.

Inhibiting the RAS-PI3K pathway in cancer therapy.

The PI3K pathway is over-activated in the majority of human cancers. This may occur through oncogenic activation of upstream RAS isoforms and tyrosine kinase receptors, or by mutational activation of components of the PI3K pathway themselves. Stimulation of the PI3K pathway enhances growth, survival, and metabolism of cancer cells. Migration, invasion, and angiogenesis are also supported by PI3K signaling. Thus, the PI3K pathway is an attractive candidate for the therapeutic targeting of tumors. Multiple kinases within the PI3Ks, AKT, and mTOR pathway have been selected for inhibition, and dual inhibitors have also been produced. Recently, the development of kinase inhibitors with enhanced specificity and improved pharmacokinetics has facilitated the investigation of PI3K pathway inhibition in clinical trials. Initial reports are encouraging, with tolerable toxicity profiles reported. PI3K inhibitors have provided some benefit as single-agent treatments of advanced solid tumors and the possibilities for enhanced effect with combination treatments look promising. In this chapter, we describe the PI3K inhibitors currently under investigation for the treatment of cancer and discuss the opportunities and obstacles that have been revealed by the latest preclinical and clinical studies.

Coordinate direct input of both KRAS and IGF1 receptor to activation of PI3 kinase in KRAS-mutant lung cancer.

UNLABELLED: Using a panel of non-small cell lung cancer (NSCLC) lines, we show here that MAP-ERK kinase (MEK) and RAF inhibitors are selectively toxic for the KRAS-mutant genotype, whereas phosphoinositide 3-kinase (PI3K), AKT, and mTOR inhibitors are not. IGF1 receptor (IGF1R) tyrosine kinase inhibitors also show selectivity for KRAS-mutant lung cancer lines. Combinations of IGF1R and MEK inhibitors resulted in strengthened inhibition of KRAS-mutant lines and also showed improved effectiveness in autochthonous mouse models of Kras-induced NSCLC. PI3K pathway activity is dependent on basal IGF1R activity in KRAS-mutant, but not wild-type, lung cancer cell lines. KRAS is needed for both MEK and PI3K pathway activity in KRAS-mutant, but not wild-type, lung cancer cells, whereas acute activation of KRAS causes stimulation of PI3K dependent upon IGF1R kinase activity. Coordinate direct input of both KRAS and IGF1R is thus required to activate PI3K in KRAS-mutant lung cancer cells. SIGNIFICANCE: It has not yet been possible to target RAS proteins directly, so combined targeting of effect or pathways acting downstream of RAS, including RAF/MEK and PI3K/AKT, has been the most favored approach to the treatment of RAS -mutant cancers. This work sheds light on the ability of RASto activate PI3K through direct interaction, indicating that input is also required from a receptor tyrosinekinase, IGF1R in the case of KRAS -mutant lung cancer. This suggests potential novel combination therapeutic strategies for NSCLC.

Requirement for interaction of PI3-kinase p110α with RAS in lung tumor maintenance.

RAS proteins directly activate PI3-kinases. Mice bearing a germline mutation in the RAS binding domain of the p110α subunit of PI3-kinse are resistant to the development of RAS-driven tumors. However, it is unknown whether interaction of RAS with PI3-kinase is required in established tumors. The need for RAS interaction with p110α in the maintenance of mutant Kras-driven lung tumors was explored using an inducible mouse model. In established tumors, removal of the ability of p110α to interact with RAS causes long-term tumor stasis and partial regression. This is a tumor cell-autonomous effect, which is improved significantly by combination with MEK inhibition. Total removal of p110α expression or activity has comparable effects, albeit with greater toxicities.

Mitochondrial fission/fusion dynamics and apoptosis.

Mitochondria play an important role in the progression of apoptosis through the release of pro-apoptotic factors, such as cytochrome c, from the mitochondrial intermembrane space. During this process, mitochondrial networks are dramatically reorganised from long filamentous interconnected tubules into small punctate spheres. Whether remodelling of mitochondrial networks is necessary for apoptosis-associated cytochrome c release, or merely an accompanying process, has been a subject of debate. Here we discuss evidence for and against the role of mitochondrial fragmentation in the progression of apoptosis and highlight recent advances which indicate that mitochondrial fission is not a critical requirement for apoptosis-associated cytochrome c release. We also discuss an emerging role for Bcl-2 family members as regulators of mitochondrial fission and fusion dynamics, independent of the role of this family in the regulation of apoptosis.

Magnetic resonance spectroscopy predicts outcomes for children with nonaccidental trauma.

OBJECTIVE: We evaluated proton magnetic resonance spectroscopic imaging (MRSI) findings for children with traumatic brain injury attributable to nonaccidental trauma (NAT) early after injury, to determine whether brain metabolite changes predicted outcomes. METHODS: Proton MRSI (1.5 T) was performed (mean: 5 days after injury [range: 1-30 days]) through the level of the corpus callosum for 90 children with confirmed NAT. Regional N-acetylaspartate/total creatine, N-acetylaspartate/total choline, and choline/creatine ratios and the presence of lactate were measured. Data on long-term outcomes defined at > or =6 months were collected for 44 of 90 infants. We grouped patients into good (normal, mild disability, or moderate disability; n = 32) and poor (severe disability, vegetative state, or dead; n = 12) outcome groups. RESULTS: We found that N-acetylaspartate/creatine and N-acetylaspartate/choline ratios (mean total, corpus callosum, and frontal white matter) were significantly decreased in patients with poor outcomes (P < .001). A logistic regression model using age, initial Glasgow Coma Scale score, presence of retinal hemorrhage, lactate on MRSI scans, and mean total N-acetylaspartate/creatine ratio predicted outcomes accurately in 100% of cases. CONCLUSIONS: Reduced N-acetylaspartate levels (ie, neuronal loss/dysfunction) and elevated lactate levels (altered energy metabolism) correlated with poor neurologic outcomes for infants with NAT. Elevated lactate levels may reflect primary or secondary hypoxic-ischemic injury, which may occur with NAT. Our data suggest that MRSI performed early after injury can be used for long-term prognosis.

Bicaudal is a conserved substrate for Drosophila and mammalian caspases and is essential for cell survival.

Members of the caspase family of cysteine proteases coordinate cell death through restricted proteolysis of diverse protein substrates and play a conserved role in apoptosis from nematodes to man. However, while numerous substrates for the mammalian cell death-associated caspases have now been described, few caspase substrates have been identified in other organisms. Here, we have utilized a proteomics-based approach to identify proteins that are cleaved by caspases during apoptosis in Drosophila D-Mel2 cells, a subline of the Schneider S2 cell line. This approach identified multiple novel substrates for the fly caspases and revealed that bicaudal/betaNAC is a conserved substrate for Drosophila and mammalian caspases. RNAi-mediated silencing of bicaudal expression in Drosophila D-Mel2 cells resulted in a block to proliferation, followed by spontaneous apoptosis. Similarly, silencing of expression of the mammalian bicaudal homologue, betaNAC, in HeLa, HEK293T, MCF-7 and MRC5 cells also resulted in spontaneous apoptosis. These data suggest that bicaudal/betaNAC is essential for cell survival and is a conserved target of caspases from flies to man.

Commitment in apoptosis: slightly dead but mostly alive.

Although the molecular players are well known, the signaling thresholds that shape the decision of a cell to undergo apoptosis remain poorly understood. Using quantitative single-cell analysis approaches, a recent study has generated new insight into the molecular events that influence individual cell-death decisions. Surprisingly, this study demonstrates that cells partly committed to apoptosis can recover and also indicates, although this is as yet unproven, that such cells might harbor DNA damage that could act as a driver of oncogenesis.

Expression and purification of recombinant annexin V for the detection of membrane alterations on apoptotic cells.

Apoptosis is a mode of cell death that is accompanied by specific alterations to the plasma membrane that promote the recognition and engulfment of these cells by phagocytes. Although several such membrane alterations have been defined, redistribution of phosphatidylserine from the inner to the outer plasma membrane leaflet has become one of the most widely used markers for apoptotic cells in mammals. This is largely due to the availability of a sensitive and specific probe for this event in the form of the phosphatidylserine-binding protein, annexin V. Here, we describe methods for the expression and purification of recombinant polyhistidine-tagged annexin V from Escherichia coli. Recombinant annexin V is highly soluble and is thus readily expressed and purified to high yields; typically in the region of 4microg of protein per ml of bacterial culture. We also describe methods for conjugation of this protein to the FITC fluorophore and for its use for the detection of apoptotic cells by flow cytometry or fluorescence microscopy.

Oncogenic B-RafV600E inhibits apoptosis and promotes ERK-dependent inactivation of Bad and Bim.

Recent studies have revealed that B-Raf mutations are very common in malignant melanoma and are required for tumor growth and maintenance. The majority of melanoma-associated B-Raf mutations involve a single point mutation, V600E, which results in greatly elevated B-Raf kinase activity and constitutive activation of MAPK/ERK downstream. Here we show that B-Raf(V600E) increases resistance to apoptosis induced by chemotherapeutic drugs and promotes ERK-dependent phosphorylation of the BH3-only proteins Bim and Bad that are involved in setting thresholds for apoptosis. ERK-dependent phosphorylation of Bim resulted in degradation of this BH3-only protein, whereas phosphorylation of Bad has previously been shown to result in its sequestration by 14-3-3 proteins. Consistent with this, inhibition of ERK activity in a panel of melanoma cell lines resulted in stabilization of Bim and dephosphorylation of Bad. Furthermore, apoptosis induced through overexpression of Bad or Bim was efficiently blocked by coexpression of mutant B-Raf(V600E). However, small interfering RNA-mediated silencing of Bim and Bad expression conferred only modest protection against cytotoxic drugs, whereas oncogenic B-Raf strongly protected against the same stimuli. These observations suggest that B-Raf-initiated inactivation of Bad and Bim only partly contributes to the anti-apoptotic activities of this oncogene and that other points within the cell death machinery are also targeted by deregulated ERK signaling.