David F. Kashatus

Tumour maintenance is mediated by eNOS

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K–AKT–eNOS–(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.

Erk2 Phosphorylation of Drp1 Promotes Mitochondrial Fission and MAPK-Driven Tumor Growth

Ras is mutated in up to 30% of cancers, including 90% of pancreatic ductal adenocarcinomas, causing it to be constitutively GTP-bound, and leading to activation of downstream effectors that promote a tumorigenic phenotype. As targeting Ras directly is difficult, there is a significant effort to understand the downstream biological processes that underlie its protumorigenic activity. Here, we show that expression of oncogenic Ras or direct activation of the MAPK pathway leads to increased mitochondrial fragmentation and that blocking this phenotype, through knockdown of the mitochondrial fission-mediating GTPase Drp1, inhibits tumor growth. This fission is driven by Erk2-mediated phosphorylation of Drp1 on Serine 616, and both this phosphorylation and mitochondrial fragmentation are increased in human pancreatic cancer. Finally, this phosphorylation is required for Ras-associated mitochondrial fission, and its inhibition is sufficient to block xenograft growth. Collectively, these data suggest mitochondrial fission may be a target for treating MAPK-driven malignancies.

NF-κB and IκBα Are Found in the Mitochondria EVIDENCE FOR REGULATION OF MITOCHONDRIAL GENE EXPRESSION BY NF-κB

The transcription factor NF-κB has been shown to be predominantly cytoplasmically localized in the absence of an inductive signal. Stimulation of cells with inflammatory cytokines such as tumor necrosis factor α or interleukin-1 induces the degradation of IκB, the inhibitor of NF-κB, allowing nuclear accumulation of NF-κB and regulation of specific gene expression. The degradation of IκB is controlled initially by phosphorylation induced by the IκB kinase, which leads to ubiquitination and subsequent proteolysis of the inhibitor by the proteasome. We report here that NF-κB and IκBα (but not IκBβ) are also localized in the mitochondria. Stimulation of cells with tumor necrosis factor α leads to the phosphorylation of mitochondrial IκBα and its subsequent degradation by a nonproteasome-dependent pathway. Interestingly, expression of the mitochondrially encoded cytochromec oxidase III and cytochrome bmRNAs were reduced by cytokine treatment of cells. Inhibition of activation of mitochondrial NF-κB by expression of the superrepressor form of IκBα inhibited the loss of expression of both cytochromec oxidase III and cytochrome b mRNA. These data indicate that the NF-κB regulatory pathway exists in mitochondria and that NF-κB can negatively regulate mitochondrial mRNA expression.

Mitochondrial Control by DRP1 in Brain Tumor Initiating Cells

Brain tumor initiating cells (BTICs) co-opt the neuronal high affinity glucose transporter, GLUT3, to withstand metabolic stress. We investigated another mechanism critical to brain metabolism, mitochondrial morphology, in BTICs. BTIC mitochondria were fragmented relative to non-BTIC tumor cell mitochondria, suggesting that BTICs increase mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), showed activating phosphorylation in BTICs and inhibitory phosphorylation in non-BTIC tumor cells. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca2+-calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTIC tumor cells, suggesting that tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

The p65/RelA subunit of NF-κB suppresses the sustained, antiapoptotic activity of Jun kinase induced by tumor necrosis factor

ABSTRACT Tumor necrosis factor (TNF) signaling through the TNF receptors involves the recruitment of key signaling factors, leading to the activation of both the transcription factor NF-κB and the stress-activated Jun kinase (JNK). In most cells, TNF signaling leads to a rapid and transient increase in JNK activity. However, we show that TNF treatment leads to the sustained activation of JNK in cells that are null for the p65/RelA subunit of NF-κB as well as in cells expressing the super-repressor form of IκB. In addition, the data indicate that the ability of p65/RelA to regulate gene expression is required to suppress the persistent activation of JNK. Interestingly, this suppression occurs upstream of JNK, within the signal transduction cascade leading to JNK activation, without affecting the stress-activated kinase p38. Since NF-κB has previously been shown to be involved in the suppression of TNF-induced apoptosis, we were interested in determining the role of deregulated JNK activity, induced by the loss of NF-κB, in controlling the cell death response. Through the use of different approaches for inhibition of JNK, we show that the suppression of JNK activity in cells that lack active NF-κB enhances the apoptotic response to TNF. These data suggest that the activity of JNK in cells blocked for NF-κB function provides an antiapoptotic signal and explains, at least partly, why a significant number of NF-κB null cells remain viable following TNF treatment.

Aurora-A Phosphorylates, Activates, and Relocalizes the Small GTPase RalA

ABSTRACT The small GTPase Ras, which transmits extracellular signals to the cell, and the kinase Aurora-A, which promotes proper mitosis, can both be inappropriately activated in human tumors. Here, we show that Aurora-A in conjunction with oncogenic Ras enhances transformed cell growth. Furthermore, such transformation and in some cases also tumorigenesis depend upon S194 of RalA, a known Aurora-A phosphorylation site. Aurora-A promotes not only RalA activation but also translocation from the plasma membrane and activation of the effector protein RalBP1. Taken together, these data suggest that Aurora-A may converge upon oncogenic Ras signaling through RalA.

The Nuclear Factor κB Subunits RelA/p65 and c-Rel Potentiate but Are Not Required for Ras-Induced Cellular Transformation

Extensive data indicate that oncoproteins, such as oncogenic H-Ras, initiate signal transduction cascades that ultimately lead to the activation of specific transcription factors. We and others have previously demonstrated that Ras activates the inherent transcriptional activation function of the transcription factor nuclear factor κB (NF-κB). Supportive of the importance of NF-κB in transformation, Ras-induced cellular transformation can be suppressed by expression of IκBα, an inhibitor of NF-κB, or by dominant-negative forms of the upstream activator IκB kinase (IKK). However, conclusive evidence for a requirement for NF-κB subunits in oncogenic transformation has not been reported. Furthermore, there is little understanding of the gene targets controlled by NF-κB that might support oncogenic conversion. The data presented here demonstrate that, although both p65 and c-Rel enhance the frequency of Ras-induced cellular transformation, these NF-κB subunits are not essential for Ras to transform spontaneously immortalized murine fibroblasts. Microarray analysis identified a set of genes induced by Ras that is dependent on NF-κB for their expression and that likely play contributory roles in promoting Ras-induced oncogenic transformation.

Ral GTPases in tumorigenesis: Emerging from the shadows

Oncogenic Ras proteins rely on a series of key effector pathways to drive the physiological changes that lead to tumorigenic growth. Of these effector pathways, the RalGEF pathway, which activates the two Ras-related GTPases RalA and RalB, remains the most poorly understood. This review will focus on key developments in our understanding of Ral biology, and will speculate on how aberrant activation of the multiple diverse Ral effector proteins might collectively contribute to oncogenic transformation and other aspects of tumor progression.

cPLA2 Regulates the Expression of Type I Interferons and Intracellular Immunity to Chlamydia trachomatis

Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase RAS and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA2 are activated and are important for the onset of inflammatory responses. In this study we tested if activation of ERK and cPLA2 occurred as a result of RAS signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. We provide genetic and pharmacological evidence that during infection RAS, ERK, and, to a lesser extent, cPLA2 activation are uncoupled, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA2 signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells, inhibition of ERK and cPLA2 played a significant protective role against C. trachomatis. We determined that cPLA2-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of β interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Furthermore, the MAPK p38 was primarily responsible for cPLA2 activation in Chlamydia-infected cells and IRG expression. Overall, these findings define a previously unrecognized role for cPLA2 in the induction of cell autonomous cellular immunity to Chlamydia and highlight the many non-canonical signaling pathways engaged during infection.

Expression of Nuclear Factor-kappaB Family Proteins in Hepatocellular Carcinomas

Purpose: Nuclear factor-ĸB (NF-ĸB) has been shown to be abnormally activated in some human hepatocellular carcinomas (HCCs), but most studies of NF-ĸB in patient samples have focused on the p65 subunit. Recent information has implicated IĸB family members (e.g. Bcl-3) as possible mediators of NF-ĸB activation. Therefore, we examined the expression of all NF-ĸB family members and downstream targets in HCC. Study Design: Archived HCCs from 30 patients were evaluated by immunohistochemistry for NF-ĸB family proteins, Bcl-3 and targets of NF-ĸB/IĸB function. Results were validated by Western blotting in frozen paired HCC and adjacent normal tissue in a subset of cases. Results: NF-ĸB p50 and p52 subunits were frequently localized to tumor cell nuclei (40 and 48%), whereas p65 positivity was infrequent. Bcl-3 was overexpressed in 90% of tumor cell nuclei compared with 26% of adjacent non-neoplastic liver (p < 0.001). Conclusions: Aberrant Bcl-3 nuclear expression occurs in the vast majority of HCCs compared with adjacent normal or cirrhotic liver tissue. Bcl-3 is known to interact with NF-ĸB p50 and p52 homodimers, and our study demonstrates very frequent nuclear colocalization of Bcl-3 and p50/p52, suggesting that the Bcl-3/p50 or Bcl-3/p52 interactions are important in HCC pathogenesis.