[Show abstract][Hide abstract] ABSTRACT: NF-κB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IκB kinase (IKK) β results in altered cytokine signaling and marked neutrophilia. To investigate the role of IKKβ in regulation of hematopoiesis, we employed Mx1-Cre mediated IKKβ conditional knockout mice. As previously reported, deletion of IKKβ in hematopoietic cells results in neutrophilia, and we now also noted decreased monocytes and modest anemia. Granulocyte-macrophage progenitors (GMPs) accumulated markedly in bone marrow of IKKβ deleted mice whereas the proportion and number of megakaryocyte-erythrocyte progenitors (MEP) decreased. Accordingly, we found a significantly reduced frequency of proerythroblasts and basophilic and polychromatic erythroblasts, and IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. These changes are associated with elevated expression of C/EBPα, Gfi1, and PU.1 and diminished Gata1, Klf1, and SCL/Tal1 in IKKβ deficient Lineage-Sca1+c-Kit+ (LSK) cells. In contrast, no effect on erythropoiesis or expression of lineage-related transcription factors was found in marrow lacking NF-κB p65. Bone marrow from IKKβ knockout mice has elevated numbers of phenotypic long and short term hematopoietic stem cells (HSC). A similar increase was observed when IKKβ was deleted after marrow transplantation into a wild type host, indicating cell autonomous expansion. Myeloid progenitors from IKKβ- but not p65-deleted mice demonstrate increased serial replating in colony-forming assays, indicating increased cell autonomous self-renewal capacity. In addition, in a competitive repopulation assay deletion of IKKβ resulted in a stable advantage of bone marrow derived from IKKβ knockout mice. In summary, loss of IKKβ resulted in significant effects on hematopoiesis not seen upon NF-κB p65 deletion. These include increased myeloid and reduced erythroid transcription factors, skewing differentiation towards myeloid over erythroid differentiation, increased progenitor self-renewal, and increased number of functional long term HSCs. These data inform ongoing efforts to develop IKK inhibitors for clinical use.
[Show abstract][Hide abstract] ABSTRACT: RNA interference-based gene silencing has become a widely used technology to evaluate how inhibition of expression of individual proteins affects biological readout. Through the use of this technology, a lot has been learned about how different proteins function in a wide variety of biological contexts, including cancer. In this context, RNA interference-mediated gene silencing has contributed to further our understanding of how different proteins in the NF-κB signaling pathway (including the NF-κB members themselves) contribute to cancer. Here, we describe two RNA interference-based protocols in lung cancer cells targeting upstream activators of NF-κB transcription factor: the catalytic subunits of the IKK complex. The first protocol is designed to evaluate the impact of IKKα or IKKβ inhibition on NF-κB transcriptional activity, whereas the second protocol is designed to evaluate how siRNA-mediated IKK inhibition affects lung cancer cell proliferation.
No preview · Article · Mar 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different
downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1
activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity.
We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an
important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of
mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to
phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that
IKKα is an effector of Akt in promoting mTORC1 activity.
[Show abstract][Hide abstract] ABSTRACT: The IKK/nuclear factor-kappaB pathway (NF-κB) is critical in proper immune function, cell survival, apoptosis, cellular proliferation, synaptic plasticity, and even memory. While NF-κB is crucial for both innate and adaptive immunity, defective regulation of this master transcriptional regulator is seen in a variety of diseases including autoimmune disease, neurodegenerative disease, and, important to this review, cancer. While NF-κB functions in cancer to promote a number of critical oncogenic functions, here we discuss the importance of the NF-κB signaling pathway in contributing to cancer through promotion of the tumor microenvironment and through maintenance/expansion of tumor-initiating cells, processes that appear to be functionally interrelated.
No preview · Article · Jun 2014 · Advances in Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Acute graft-versus-host disease (aGvHD) is a major limitation to the use of allogeneic stem cell transplantation for the treatment of patients with relapsed malignant disease. Previous work using animals lacking secondary lymphoid tissue (SLT) suggested that activation of donor T cells in SLT is critically important for the pathogenesis of aGvHD. However, these studies did not determine if impaired migration into, and more importantly, out of SLT, would ameliorate aGvHD. Here, we show that T cells from mice lacking Coronin 1A (Coro 1A−/−), an actin associated protein shown to be important for thymocyte egress, do not mediate acute GvHD. The attenuation of aGvHD was associated with decreased expression of the critical trafficking proteins CCR7 and sphingosine 1 phosphate (S1P) receptor on donor T cells. This was mediated in part by impaired activation of the canonical NF-κB pathway in the absence of Coro 1A. As a result of these alterations, donor T cells from Coro 1A−/− mice were not able to initially traffic to SLT or exit SLT after bone marrow transplantation. However, this alteration did not abrogate the GvL response. Our data suggest that blocking T-cell migration into and out of SLT is a valid approach to prevent aGvHD.This article is protected by copyright. All rights reserved
No preview · Article · Jun 2014 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Activated B-Cell (ABC) Diffuse Large B-Cell Lymphoma (DLBCL) is a common, aggressive and poorly chemoresponsive subtype of DLBCL, characterized by constitutive canonical NF-κB signaling. Inhibition of NF-κB signaling leads to apoptosis of ABC-DLBCL cell lines, suggesting targeted disruption of this pathway may have therapeutic relevance. The selective IKK inhibitor, NEMO Binding Domain (NBD) peptide effectively blocks constitutive NF-κB activity and induces apoptosis in ABC-DLBCL cells in vitro. Here we used a comparative approach to determine the safety and efficacy of systemic NBD peptide to inhibit constitutive NF-κB signaling in privately owned dogs with spontaneous newly diagnosed or relapsed ABC-like DLBCL. Malignant lymph nodes biopsies were taken before and twenty-four hours after peptide administration to determine biological effects. Intravenous administration of <2 mg/kg NBD peptide was safe and inhibited constitutive canonical NF-κB activity in 6/10 dogs. Reductions in mitotic index and Cyclin D expression also occurred in a subset of dogs 24 hours post peptide and in 3 dogs marked, therapeutically beneficial histopathological changes were identified. Mild, grade 1 toxicities were noted in 3 dogs at the time of peptide administration and one dog developed transient subclinical hepatopathy. Long term toxicities were not identified. Pharmacokinetic data suggested rapid uptake of peptide into tissues. No significant hematological or biochemical toxicities were identified. Overall the results from this phase I study indicate that systemic administration of NBD peptide is safe and effectively blocks constitutive NF-κB signaling and reduces malignant B cell proliferation in a subset of dogs with ABC-like DLBCL. These results have potential translational relevance for human ABC-DLBCL.
[Show abstract][Hide abstract] ABSTRACT: Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. These results argue that targeting downstream effectors of RAS will be an alternative route for blocking RAS-driven oncogenic pathways. We and others have shown that oncogenic RAS activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic deletion of IKKβ or the RELA/p65 subunit of NF-κB. Here, genetic and pharmacological approaches were utilized to inactivate IKK in human primary lung epithelial cells transformed by KRAS, as well as KRAS mutant lung cancer cell lines. Administration of the highly specific IKKβ inhibitor Compound A (CmpdA) led to NF-κB inhibition in different KRAS mutant lung cells and siRNA-mediated knockdown of IKKα or IKKβ reduced activity of the NF-κB canonical pathway. Next, we determined that both IKKα and IKKβ contribute to oncogenic properties of KRAS mutant lung cells, particularly when p53 activity is disrupted. Based on these results, CmpdA was tested for potential therapeutic intervention in the Kras-induced lung cancer mouse model (LSL-Kras (G12D)) combined with loss of p53 (LSL-Kras (G12D)/p53 (fl/fl)). CmpdA treatment was well tolerated and mice treated with this IKKβ inhibitor presented smaller and lower grade tumors than mice treated with placebo. Additionally, IKKβ inhibition reduced inflammation and angiogenesis. These results support the concept of targeting IKK as a therapeutic approach for oncogenic RAS-driven tumors with altered p53 activity.
[Show abstract][Hide abstract] ABSTRACT: Arp2/3-branched actin is critical for cytoskeletal dynamics and cell migration. However, perturbations and diseases affecting this network have phenotypes that cannot be fully explained by cell-autonomous effects. In this paper, we report nonautonomous effects of Arp2/3 depletion. We show that, upon Arp2/3 depletion, the expression of numerous genes encoding secreted factors, including chemokines, growth factors, and matrix metalloproteases, was increased, a signature resembling the senescence-associated secretory phenotype. These factors affected epidermal growth factor chemotaxis in a nonautonomous way, resolving the recent contradictions about the role of Arp2/3 in chemotaxis. We demonstrate that these genes were activated by nuclear factor κB via a CCM2-MEKK3 pathway that has been implicated in hyperosmotic stress signaling. Consistent with this, Arp2/3-depleted cells showed misregulation of volume control and reduced actin in the submembranous cortex. The defects in osmotic signaling in the Arp2/3-depleted cells can be rescued by hypoosmotic treatment. Thus, perturbations of Arp2/3 have nonautonomous effects that should be considered when evaluating experimental manipulations and diseases affecting the Arp2/3-actin cytoskeleton.
Full-text · Article · Dec 2013 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Background
The extraordinary invasiveness of human glioblastoma multiforme (GBM) contributes to treatment failure and the grim prognosis of patients diagnosed with this tumor. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in vitro and in vivo in human melanoma models. To determine whether MDA-9/syntenin is a relevant target in GBM, we investigated its expression in tumor samples and involvement in GBM invasion and angiogenesis.MaterialsWe assessed MDA-9/syntenin levels in available databases, patient tumor samples, and human-derived cell lines. Through gain-of-function and loss-of-function studies, we analyzed changes in invasion, angiogenesis, and signaling in vitro. We used orthotopic xenografts with GBM6 cells to demonstrate the role of MDA-9/syntenin in GBM pathogenesis in vivo.ResultsMDA-9/syntenin expression in high-grade astrocytomas is significantly higher than normal tissue counterparts. Forced overexpression of MDA-9/syntenin enhanced Matrigel invasion, while knockdown inhibited invasion, migration, and anchorage-independent growth in soft agar. Moreover, overexpression of MDA-9/syntenin increased activation of c-Src, p38 mitogen-activated protein kinase, and nuclear factor kappa-B, leading to elevated expression of matrix metalloproteinase 2 and secretion of interleukin-8 with corresponding changes observed upon knockdown. GBM6 cells that stably express small hairpin RNA for MDA-9/syntenin formed smaller tumors and had a less invasive phenotype in vivo.Conclusions
Our findings indicate that MDA-9/syntenin is a novel and important mediator of invasion in GBM and a key regulator of pathogenesis, and we identify it as a potential target for anti-invasive treatment in human astrocytoma.
[Show abstract][Hide abstract] ABSTRACT: Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.
[Show abstract][Hide abstract] ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a 5-year survival rate of only 6%. Although the cytosine analog gemcitabine is the drug commonly used to treat PDAC, chemoresistance unfortunately renders the drug ineffective. Thus, strategies that can decrease this resistance will be essential for improving the dismal outcome of patients suffering from this disease. We previously observed that oncogenic Pim-1 kinase was aberrantly expressed in PDAC tissues and cell lines and was responsible for radioresistance. Furthermore, members of the Pim family have been shown to reduce the efficacy of chemotherapeutic drugs in cancer. Therefore, we attempted to evaluate the role of Pim-3 in chemoresistance of PDAC cells. We were able to confirm upregulation of the Pim-3 oncogene in PDAC tissues and cell lines versus normal samples. Biological consequences of inhibiting Pim-3 expression with shRNA-mediated suppression included decreases in anchorage-dependent growth, invasion through Matrigel and chemoresistance to gemcitabine as measured by caspase-3 activity. Additionally, we were able to demonstrate that Pim-1 and Pim-3 play overlapping but non-identical roles as it relates to gemcitabine sensitivity of pancreatic cancer cells. To further support the role of Pim-3 suppression in sensitizing PDAC cells to gemcitabine, we used the pharmacological Pim kinase inhibitor SGI-1776. Treatment of PDAC cells with SGI-1776 resulted in decreased phosphorylation of the proapoptotic protein Bad and cell cycle changes. When SGI-1776 was combined with gemcitabine, there was a greater decrease in cell viability in the PDAC cells versus cells treated with either of the drugs separately. These results suggest combining drug therapies that inhibit Pim kinases, such as Pim-3, with chemotherapeutic agents, to aid in decreasing chemoresistance in pancreatic cancer.
No preview · Article · Jun 2013 · Cancer biology & therapy
[Show abstract][Hide abstract] ABSTRACT: Hematopoiesis is a tightly regulated process resulting in the production of blood cells. Self-renewal and differentiation of hematopoietic stem cells (HSCs) are key processes in hematopoietic development. Disruption of these steps can lead to altered cell distribution and disease. To investigate the role of the NF-κB subunit RelA/p65 in the regulation of HSCs in vivo, we generated mice lacking RelA/p65 in the hematopoietic compartment. Using this model system, we show that loss of p65 severely impairs HSC function, and occurs in conjunction with increased HSPC cycling, extramedullary hematopoiesis, and differentiation defects. Gene array studies of phenotypic HSCs indicate the up-regulation of genes normally expressed in lineage restricted cells, as well as the down-regulation of genes involved in HSC maintenance and homeostasis. We hypothesize that changes in gene expression in p65-definicent cells lead to decreased self-renewal and differentiation efficiency of hematopoietic stem and progenitor cells. These studies demonstrate that p65 is an important regulator of hematopoiesis through the transcription of genes required for HSC fate.
[Show abstract][Hide abstract] ABSTRACT: Mutations in KRAS drive the oncogenic phenotype in a variety of tumors of epithelial origin. The NF-κB transcription factor pathway is important for oncogenic RAS to transform cells and to drive tumorigenesis in animal models. Recently TAK1, an upstream regulator of IKK, which controls canonical NF-κB, was shown to be important for chemoresistance in pancreatic cancer and for regulating KRAS+ colorectal cancer cell growth and survival. Here we show that KRAS+ upregulates GSK-3α leading to its interaction with TAK1 to stabilize the TAK1/TAB complex to promote IKK activity. Additionally, GSK-3α is required for promoting critical non-canonical NF-κB signaling in pancreatic cancer cells. Pharmacologic inhibition of GSK-3 suppresses growth of human pancreatic tumor explants, consistent with the loss of expression of oncogenic genes such as c-myc and TERT. These data identify GSK-3α as a key downstream effector of oncogenic KRAS via its ability to coordinately regulate distinct NF-κB signaling pathways.
No preview · Article · Apr 2013 · Cancer Discovery
[Show abstract][Hide abstract] ABSTRACT: Tumor-initiating cells (TICs) are a sub-population of cells that exhibit a robust ability to self-renew and contribute to the formation of primary tumors, the relapse of previously treated tumors and the development of metastases. TICs have been identified in various tumors including those of the breast, and are particularly enriched in the basal-like and claudin-low subtypes of breast cancer. The signaling pathways that contribute to the function and maintenance of TICs are under intense study. We explored the potential involvement of the nuclear factor-κB (NF-κB) family of transcription factors in TICs in cell lines that are representative of basal-like and claudin-low breast cancer. NF-κB was found to be activated in breast cancer cells that form tumorspheres efficiently. Moreover, both canonical and non-canonical NF-κB signaling is required for these cells to self-renew in vitro and to form xenograft tumors efficiently in vivo using limiting dilutions of cells. Consistent with this fact, canonical and non-canonical NF-κB signaling is activated in TICs isolated from breast cancer cell lines. Experimental results indicate that NF-κB promotes the function of TICs by stimulating epithelial-to-mesenchymal transition and by upregulating the expression of the inflammatory cytokines interleukin-1β and interleukin-6. The results suggest the use of NF-κB inhibitors for clinical therapy of certain breast cancers.Oncogene advance online publication, 11 March 2013; doi:10.1038/onc.2013.64.