Differential association of STK11 and TP53 with KRAS mutation-associated gene expression, proliferation and immune surveillance in lung adenocarcinoma.

While mutations in the KRAS oncogene are among the most prevalent in human cancer, there are few successful treatments to target these tumors. It is also likely that heterogeneity in KRAS-mutant tumor biology significantly contributes to the response to therapy. We hypothesized that the presence of commonly co-occurring mutations in STK11 and TP53 tumor suppressors may represent a significant source of heterogeneity in KRAS-mutant tumors. To address this, we utilized a large cohort of resected tumors from 442 lung adenocarcinoma patients with data including annotation of prevalent driver mutations (KRAS and EGFR) and tumor suppressor mutations (STK11 and TP53), microarray-based gene expression and clinical covariates, including overall survival (OS). Specifically, we determined impact of STK11 and TP53 mutations on a new KRAS mutation-associated gene expression signature as well as previously defined signatures of tumor cell proliferation and immune surveillance responses. Interestingly, STK11, but not TP53 mutations, were associated with highly elevated expression of KRAS mutation-associated genes. Mutations in TP53 and STK11 also impacted tumor biology regardless of KRAS status, with TP53 strongly associated with enhanced proliferation and STK11 with suppression of immune surveillance. These findings illustrate the remarkably distinct ways through which tumor suppressor mutations may contribute to heterogeneity in KRAS-mutant tumor biology. In addition, these studies point to novel associations between gene mutations and immune surveillance that could impact the response to immunotherapy.

IKBKE is induced by STAT3 and tobacco carcinogen and determines chemosensitivity in non-small cell lung cancer.

Serine/threonine kinase IKBKE is a newly identified oncogene; however, its regulation remains elusive. Here, we provide evidence that IKBKE is a downstream target of signal transducer and activator of transcription 3 (STAT3) and that tobacco components induce IKBKE expression through STAT3. Ectopic expression of constitutively active STAT3 increased IKBKE mRNA and protein levels, whereas inhibition of STAT3 reduced IKBKE expression. Furthermore, expression levels of IKBKE are significantly associated with STAT3 activation and tobacco use history in non-small cell lung cancer (NSCLC) patients examined. In addition, we show induction of IKBKE by two components of cigarette smoke, nicotine and nicotine-derived nitrosamine ketone (NNK). Upon exposure to nicotine or NNK, cells express high levels of IKBKE protein and mRNA, which are largely abrogated by inhibition of STAT3. Characterization of the IKBKE promoter revealed two STAT3-response elements. The IKBKE promoter directly bound to STAT3 and responded to nicotine and NNK stimulation. Notably, enforcing expression of IKBKE induces chemoresistance, whereas knockdown of IKBKE not only sensitizes NSCLC cells to chemotherapy but also abrogates STAT3- and nicotine-induced cell survival. These data indicate for the first time that IKBKE is a direct target of STAT3 and is induced by tobacco carcinogens through STAT3 pathway. In addition, our study also suggests that IKBKE is an important therapeutic target and could have a pivotal role in tobacco-associated lung carcinogenesis.

Targeted mutation of TNF receptor I rescues the RelA-deficient mouse and reveals a critical role for NF-kappa B in leukocyte recruitment.

NF-kappaB binding sites are present in the promoter regions of many acute phase and inflammatory response genes, suggesting that NF-kappaB plays an important role in the initiation of innate immune responses. However, targeted mutations of the various NF-kappaB family members have yet to identify members responsible for this critical role. RelA-deficient mice die on embryonic day 15 from TNF-alpha-induced liver degeneration. To investigate the importance of RelA in innate immunity, we genetically suppressed this embryonic lethality by breeding the RelA deficiency onto a TNFR type 1 (TNFR1)-deficient background. TNFR1/RelA-deficient mice were born healthy, but were susceptible to bacterial infections and bacteremia and died within a few weeks after birth. Hemopoiesis was intact in TNFR1/RelA-deficient newborns, but neutrophil emigration to alveoli during LPS-induced pneumonia was severely reduced relative to that in wild-type or TNFR1-deficient mice. In contrast, radiation chimeras reconstituted with RelA or TNFR1/RelA-deficient hemopoietic cells were healthy and demonstrated no defect in neutrophil emigration during LPS-induced pneumonia. Analysis of RNA harvested from the lungs of mice 4 h after LPS insufflation revealed that the induction of several genes important for neutrophil recruitment to the lung was significantly reduced in TNFR1/RelA-deficient mice relative to that in wild-type or TNFR1-deficient mice. These results suggest that TNFR1-independent activation of RelA is essential in cells of nonhemopoietic origin during the initiation of an innate immune response.

An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells.

Tissue damage induced by infection or injury can result in necrosis, a mode of cell death characterized by induction of an inflammatory response. In contrast, cells dying by apoptosis do not induce inflammation. However, the reasons for underlying differences between these two modes of cell death in inducing inflammation are not known. Here we show that necrotic cells, but not apoptotic cells, activate NF-kappaB and induce expression of genes involved in inflammatory and tissue-repair responses, including neutrophil-specific chemokine genes KC and macrophage-inflammatory protein-2, in viable fibroblasts and macrophages. Intriguingly, NF-kappaB activation by necrotic cells was dependent on Toll-like receptor 2, a signaling pathway that induces inflammation in response to microbial agents. These results have identified a novel mechanism by which cell necrosis, but not apoptosis, can induce expression of genes involved in inflammation and tissue-repair responses. Furthermore, these results also demonstrate that the NF-kappaB/Toll-like receptor 2 pathway can be activated both by exogenous microbial agents and endogenous inflammatory stimuli.

NF-kappa B RelA (p65) is essential for TNF-alpha-induced fas expression but dispensable for both TCR-induced expression and activation-induced cell death.

The Fas death receptor plays a key role in the killing of target cells by NK cells and CTLs and in activation-induced cell death of mature T lymphocytes. These cytotoxic pathways are dependent on induction of Fas expression by cytokines such as TNF-alpha and IFN-gamma or by signals generated after TCR engagement. Although much of our knowledge of the Fas death pathway has been generated from murine studies, little is known about regulatory mechanisms important for murine Fas expression. To this end, we have molecularly cloned a region of the murine Fas promoter that is responsible for mediating TNF-alpha and PMA/PHA-induced expression. We demonstrate here that induction of Fas expression by both stimuli is critically dependent on two sites that associate with RelA-containing NF-kappaB complexes. To determine whether RelA and/or other NF-kappaB subunits are also important for regulating Fas expression in primary T cells, we used CD4 T cells from RelA(-/-), c-Rel(-/-), and p50(-/-) mice. Although proliferative responses were significantly impaired, expression of Fas and activation-induced cell death was unaffected in T cells obtained from these different mice. Importantly, we show that unlike fibroblasts, which consist primarily of RelA-containing NF-kappaB complexes, T cells have high levels of both RelA and c-Rel complexes, suggesting that Fas expression in T cells may be dependent on redundant functions of these NF-kappaB subunits.

The Rela(p65) subunit of NF-kappaB is essential for inhibiting double-stranded RNA-induced cytotoxicity.

Double-stranded RNA (dsRNA) molecules generated during virus infection can initiate a host antiviral response to limit further infection. Such a response involves induction of antiviral gene expression by the dsRNA-activated protein kinase (PKR) and the NF-kappaB transcription factor. In addition, dsRNA can also induce apoptosis by an incompletely understood mechanism that may serve to further limit viral replication. Here we demonstrate a novel role for the RelA subunit of NF-kappaB in inhibiting dsRNA-induced cell death. dsRNA treatment resulted in caspase 3 activation and apoptotic morphological transformations in mouse embryonic fibroblasts (MEFs) derived from RelA-/- mice but not from RelA+/+ mice. Such dsRNA-induced killing could be inhibited by expression of either a dominant-negative mutant of PKR or wild-type RelA. Interestingly, caspase 3 activated following dsRNA treatment of RelA-/- MEFs was essential for apoptotic nuclear changes but dispensable for cytotoxicity. A broader specificity caspase inhibitor was also unable to inhibit dsRNA-induced cytotoxicity, suggesting that caspase activation is not essential for the induction of cell death by dsRNA in MEFs. However, combined inhibition of caspase 3 and reactive oxygen species production resulted in complete inhibition of dsRNA-induced cytotoxicity. These results demonstrate an essential role for NF-kappaB in protecting cells from dsRNA-induced apoptosis and suggest that NF-kappaB may inhibit both caspase-dependent and reactive oxygen species-dependent cytotoxic pathways.

Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon.

The alpha/beta interferon (IFN-alpha/beta) system represents one of the first lines of defense against virus infections. As a result, most viruses encode IFN antagonistic factors which enhance viral replication in their hosts. We have previously shown that a recombinant influenza A virus lacking the NS1 gene (delNS1) only replicates efficiently in IFN-alpha/beta-deficient systems. Consistent with this observation, we found that infection of tissue culture cells with delNS1 virus, but not with wild-type influenza A virus, induced high levels of mRNA synthesis from IFN-alpha/beta genes, including IFN-beta. It is known that transactivation of the IFN-beta promoter depends on NF-kappaB and several other transcription factors. Interestingly, cells infected with delNS1 virus showed high levels of NF-kappaB activation compared with those infected with wild-type virus. Expression of dominant-negative inhibitors of the NF-kappaB pathway during delNS1 virus infection prevented the transactivation of the IFN-beta promoter, demonstrating a functional link between NF-kappaB activation and IFN-alpha/beta synthesis in delNS1 virus-infected cells. Moreover, expression of the NS1 protein prevented virus- and/or double-stranded RNA (dsRNA)-mediated activation of the NF-kappaB pathway and of IFN-beta synthesis. This inhibitory property of the NS1 protein of influenza A virus was dependent on its ability to bind dsRNA, supporting a model in which binding of NS1 to dsRNA generated during influenza virus infection prevents the activation of the IFN system. NS1-mediated inhibition of the NF-kappaB pathway may thus play a key role in the pathogenesis of influenza A virus.

Induction of necrotic-like cell death by tumor necrosis factor alpha and caspase inhibitors: novel mechanism for killing virus-infected cells.

Induction of apoptotic cell death generally requires the participation of cysteine proteases belonging to the caspase family. However, and similar to most cell types, mouse fibroblasts are normally resistant to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. Surprisingly, TNF-alpha treatment of vaccinia virus-infected mouse fibroblasts resulted in necrotic-like cell death, which was significantly reduced in cells infected with a vaccinia virus mutant lacking the caspase inhibitor B13R. Furthermore, TNF-alpha also induced necrotic-like cell death of fibroblasts in the presence of peptidyl caspase inhibitors. In both cases, necrosis was accompanied by generation of superoxide species. Caspase inhibitors also sensitized fibroblasts to killing by double-stranded RNA and gamma interferon. In all cases, cell death was efficiently blocked by antioxidants or mitochondrial respiratory chain inhibitors. These results define a new mitochondrion-dependent mechanism which may be important in the killing of cells infected with viruses encoding caspase inhibitors.

A mechanism of suppression of TGF-beta/SMAD signaling by NF-kappa B/RelA.

A number of pathogenic and proinflammatory stimuli, and the transforming growth factor-beta (TGF-beta) exert opposing activities in cellular and immune responses. Here we show that the RelA subunit of nuclear factor kappaB (NF-kappaB/RelA) is necessary for the inhibition of TGF-beta-induced phosphorylation, nuclear translocation, and DNA binding of SMAD signaling complexes by tumor necrosis factor-alpha (TNF-alpha). The antagonism is mediated through up-regulation of Smad7 synthesis and induction of stable associations between ligand-activated TGF-beta receptors and inhibitory Smad7. Down-regulation of endogenous Smad7 by expression of antisense mRNA releases TGF-beta/SMAD-induced transcriptional responses from suppression by cytokine-activated NF-kappaB/RelA. Following stimulation with bacterial lipopolysaccharide (LPS), or the proinflammatory cytokines TNF-alpha and interleukin-1beta (IL-1beta, NF-kappaB/RelA induces Smad7 synthesis through activation of Smad7 gene transcription. These results suggest a mechanism of suppression of TGF-beta/SMAD signaling by opposing stimuli mediated through the activation of inhibitory Smad7 by NF-kappaB/RelA.

The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor.

Ionotropic GABA receptors generally require the products of three subunit genes. By contrast, the GABA receptor needed for locomotion in Caenorhabditis elegans requires only the unc-49 gene. We cloned unc-49 and demonstrated that it possesses an unusual overlapping gene structure. unc-49 contains a single copy of a GABA receptor N terminus, followed by three tandem copies of a GABA receptor C terminus. Using a single promoter, unc-49 generates three distinct GABAA receptor-like subunits by splicing the N terminus to each of the three C-terminal repeats. This organization suggests that the three UNC-49 subunits (UNC-49A, UNC-49B, and UNC-49C) are coordinately rescued and therefore might coassemble to form a heteromultimeric GABA receptor. Surprisingly, only UNC-49B and UNC-49C are expressed at high levels, whereas UNC-49A expression is barely detectable. Green fluorescent protein-tagged UNC-49B and UNC-49C subunits are coexpressed in muscle cells and are colocalized to synaptic regions. UNC-49B and UNC-49C also coassemble efficiently in Xenopus oocytes and HEK-293 cells to form a heteromeric GABA receptor. Together these data argue that UNC-49B and UNC-49C coassemble at the C. elegans neuromuscular junction. Thus, C. elegans is able to encode a heteromeric GABA receptor with a single locus.

A critical role for the RelA subunit of nuclear factor kappaB in regulation of multiple immune-response genes and in Fas-induced cell death.

Binding sites for the nuclear factor (NF)-kappaB transcription factor have been identified within control regions of many genes involved in inflammatory and immune responses. Such kappaB sites are often found adjacent to those of interferon (IFN)-gamma-inducible transcription factors, suggesting a requirement for multiple signaling pathways for gene regulation. Using fibroblasts from RelA (p65)-deficient mice generated by gene targeting, we have investigated the role of this subunit of NF-kappaB in gene activation by microbial lipopolysaccharide, tumor necrosis factor alpha, and in possible synergism with the IFN-gamma-signaling pathway. Our results indicate not only that RelA is required for activation of key genes involved in adaptive (acquired) immune responses, including major histocompatibility complex class I, CD40, and the Fas death receptor, but also that both NF-kappaB-inducing signals and IFN-gamma are necessary for maximal activation. In contrast, neutrophil-specific chemokine genes KC and MIP-2, which can function as nonspecific mediators in innate immune responses, were strongly induced by RelA in the absence of IFN-gamma. Our results show that RelA plays a critical role in activation of immune system genes in response to nonspecific stimuli and demonstrate a novel proapoptotic function for this protein in Fas-induced cell death.

Oncogenic Ha-Ras-induced signaling activates NF-kappaB transcriptional activity, which is required for cellular transformation.

Ras proteins function in stimulating cell proliferation and differentiation through the activation of Raf-dependent and Raf-independent signal transduction pathways and the subsequent activation of specific transcription factors. The transcription factor NF-kappaB has been widely studied as a regulator of genes involved in immune and inflammatory responses. A variety of stimuli activate NF-kappaB through the induced phosphorylation and degradation of the inhibitor IkappaB followed by nuclear translocation of NF-kappaB. We show here that oncogenic forms of Ha-Ras activate NF-kappaB, not through induced nuclear translocation, but rather through the activation of the transcriptional function of the NF-kappaB RelA/p65 subunit. Importantly, RelA/p65 -/- cells are inefficient in the activation of kappaB-dependent gene expression in response to oncogenic Ras expression. Furthermore, IkappaBalpha expression blocks focus formation in NIH3T3 cells induced by oncogenic Ras. These results demonstrate that NF-kappaB is a critical downstream mediator of Ha-Ras signaling and oncogenic potential.

An essential role for NF-kappaB in preventing TNF-alpha-induced cell death.

Studies on mice deficient in nuclear factor kappa B (NF-kappaB) subunits have shown that this transcription factor is important for lymphocyte responses to antigens and cytokine-inducible gene expression. In particular, the RelA (p65) subunit is required for induction of tumor necrosis factor-alpha (TNF-alpha)-dependent genes. Treatment of RelA-deficient (RelA-/-) mouse fibroblasts and macrophages with TNF-alpha resulted in a significant reduction in viability, whereas RelA+/+ cells were unaffected. Cytotoxicity to both cell types was mediated by TNF receptor 1. Reintroduction of RelA into RelA-/- fibroblasts resulted in enhanced survival, demonstrating that the presence of RelA is required for protection from TNF-alpha. These results have implications for the treatment of inflammatory and proliferative diseases.

Constitutive NF-kappa B activation, enhanced granulopoiesis, and neonatal lethality in I kappa B alpha-deficient mice.

Transcription factors belonging to the NF-kappa B family are controlled by inhibitory I kappa B proteins, mainly I kappa B alpha and I kappa B beta. Apparently normal at birth, I kappa B alpha-/- mice exhibit severe runting, skin defects, and extensive granulopoiesis postnatally, typically dying by 8 days. Hematopoietic tissues from these mice display elevated levels of both nuclear NF-kappa B and mRNAs of some, but not all, genes thought to be regulated by NF-kappa B. NF-kappa B elevation results in these phenotypic abnormalities because mice lacking both I kappa B alpha and the p50 subunit of NF-kappa B show a dramatically delayed onset of abnormalities. In contrast to hematopoietic cells, I kappa B alpha-/- embryonic fibroblasts show minimal constitutive NF-kappa B, as well as normal signal-dependent NF-kappa B activation that is concomitant with I kappa B beta degradation. Our results indicate that I kappa b beta, but not I kappa B alpha, is required for the signal-dependent activation of NF-kappa B in fibroblasts. However, I kappa B alpha is required for the postinduction repression of NF-kappa B in fibroblasts. These results define distinct roles for the two forms of I kappa B and demonstrate the necessity for stringent control of NF-kappa B.

Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kappa B.

NF-kappa B, which consists of two polypeptides, p50 (M(r) 50K) and p65/RelA (M(r) 65K), is thought to be a key regulator of genes involved in responses to infection, inflammation and stress. Indeed, although developmentally normal, mice deficient in p50 display functional defects in immune responses. Here we describe the generation of mice deficient in the RelA subunit of NF-kappa B. Disruption of the relA locus leads to embryonic lethality at 15-16 days of gestation, concomitant with a massive degeneration of the liver by programmed cell death or apoptosis. Embryonic fibroblasts from RelA-deficient mice are defective in the tumour necrosis factor (TNF)-mediated induction of messenger RNAs for I kappa B alpha and granulocyte/macrophage colony stimulating factor (GM-CSF), although basal levels of these transcripts are unaltered. These results indicate that RelA controls inducible, but not basal, transcription in NF-kappa B-regulated pathways.

Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors.

The ubiquitous transcription factor NF-kappa B is regulated by its cytoplasmic inhibitor I kappa B. A variety of cellular stimuli cause the dissociation of NF-kappa B from I kappa B, allowing NF-kappa B to translocate to the nucleus and regulate gene expression. Although the activation of NF-kappa B in vivo is associated with the phosphorylation and degradation of I kappa B alpha, it has remained unclear how each of these events contributes to this process. Recently, studies utilizing protease inhibitors have suggested that the proteolysis of I kappa B alpha is a necessary event in the activation of NF-kappa B. We demonstrate in this study that these and an additional protease inhibitor also completely repress inducible phosphorylation of I kappa B alpha. This surprising result suggests a more complex role of proteases in NF-kappa B activation. In addition, data presented here indicate that many of these inhibitors also directly modify NF-kappa B and inhibit its DNA binding activity. Due to the pleiotropic effects of these protease inhibitors, it is difficult to conclude from their use how I kappa B alpha phosphorylation and degradation contribute to NF-kappa B activation. In the present study, a more direct approach demonstrates that phosphorylation of I kappa B alpha alone is not sufficient for NF-kappa B activation.

Activation of multiple NF-kappa B/Rel DNA-binding complexes by tumor necrosis factor.

NF-kappa B is an inducible transcription factor that regulates the expression of numerous genes involved in immune and inflammation responses and in cellular growth control. Typically, NF-kappa B is localized in the cytoplasm complexed with members of the I kappa B family. The most well characterized form of NF-kappa B is comprised of a heterodimer of a 50 kD (p50/NFKB1) and a 65 kD (p65/RelA) protein. This heterodimeric protein was thought to be primarily responsible for transcriptional regulation of target genes. However, recent studies have led to the identification of other kappa B binding proteins such as c-Rel, RelB and p52 (NFKB2/lyt-10) although their role in gene regulation has been less clear. Here, using gel mobility shift assays as well as a highly sensitive DNA-protein crosslinking assay, we provide evidence for the existence of multiple tumor necrosis factor (TNF)- inducible kappa B binding complexes containing various members of the NF-kappa B/Rel family, namely p50 and p65 as well as the c-Rel and p52 oncoproteins. Dimeric complexes containing various combinations of these proteins appear rapidly in nuclei of TNF-alpha-stimulated cells and include, along with a p50-p65 heterodimer, p50-c-Rel, p65-c-Rel, p52-c-Rel and p52-p65 complexes. The presence of multiple inducible complexes containing distinct combinations of NF-kappa B/Rel family members indicate that specific kappa B responsive genes may be regulated in an NF-kappa B subunit-dependent manner.