IKK Phosphorylation Research Articles

CCL18/PITPNM3 enhances migration, invasion, and EMT through the NF-κB signaling pathway in hepatocellular carcinoma.

Chemokine ligand 18 (CCL18) has been associated with hepatocellular carcinoma (HCC) metastasis. Here, we demonstrated a novel mechanism through which CCL18 enhances cell migration, invasion, and epithelial-mesenchymal transition (EMT) in HCC. (1) Using immunohistochemistry, we analyzed the expression of PITPNM3, a molecule that correlated with CCL18 signaling, in 149 HCC tissue specimens. The results showed that PITPNM3 expression is highly associated with tumor metastasis and differentiation; (2) in vitro experiments showed that CCL18 enhances cell migration, invasion, and EMT in PITPNM3((+)) HCC cells but not in PITPNM3((-)) cells. Silencing of PITPNM3 by short interfering RNA (siRNA) inhibited the induction of cell migration, invasion, and EMT by CCL18; (3) Cell migration, invasion, and EMT induced by CCL18 accompanied with the phosphorylation of IKK and IKBα as well as p65 nuclear translocation in PITPNM3((+)) HCC cells, but not in the cells that PITPNM3 is silenced with siRNA, implying that the activation of NF-κB signaling is involved in the action of CCL18/PITPNM3. These results suggest that CCL18 enhances HCC cell migration, invasion, and EMT through the expression of PITPNM3 and the activation of the NF-κB signaling pathway.

IFIT5 positively regulates NF-κB signaling through synergizing the recruitment of IκB kinase (IKK) to TGF-β-activated kinase 1 (TAK1)

Precise regulation of NF-κB signaling pathways is essential to effective host immune response. However, the specific molecular mechanism underlying NF-κB activation by different stimuli is not fully understood. Here we demonstrate that IFIT5, one of the interferon induced tetratricopeptide repeat family members, enhances IKK phosphorylation and NF-κB activation through interacting with TAK1 and IKK. Following TNF-α treatment, IFIT5 interacted with TAK1 or IKK complex and synergized the recruitment of IKK to TAK1 in a dose dependent manner. Consistent with these observations, knockdown of IFIT5 decreased the recruitment of IKK to TAK1 and markedly weakened IKK phosphorylation, further reducing the production of NF-κB target genes IL-8 and ICAM-1. Moreover, we found that IFIT5 also promoted SeV-induced IKK phosphorylation and NF-κB activation by regulating the recruitment of IKK to TAK1. Our findings identify a previously unrecognized role of IFIT5 as a positive regulator in IKK phosphorylation and NF-κB activation, highlighting that IFIT5 serves as an important mediator in innate immunity.

HTLV-1 tax hijacks cellular ubiquitination machinery to assemble K63-linked polyubiquitin for canonical NF-κB activation

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of basic cellular processes, including I-κB kinase (IKK) signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in leukemogenesis, but the underlying mechanisms remain unknown. IKK and its upstream kinase, transforming growth factor β activated kinase-1 (TAK1), contain ubiquitin-binding subunits, NF-κB essential modulator (NEMO) and TAK1 binding protein 2 (TAB2) respectively, which interact with K63-linked polyubiquitin. On this signaling platform, auto-phosphorylation and activation of TAK1 occurs, followed by TAK1-catalyzed IKK phosphorylation and activation. Here we demonstrate in vitro and in vivo that Tax stimulates ubiquitin E2 conjugating enzyme Ubc13:Uev1A (or Ubc13:Uev2) and ubiquitin E3 ligase ring finger protein 8 (RNF8) to assemble long and unanchored K63-linked polyubiquitin for TAK1 and IKK activation. The TAK1 so activated by Tax also promotes JNK phosphorylation. The inappropriate activation of RNF8 — an E3 ligase involved in DNA damage repair, cytokinesis, and centrosome function — by Tax can explain the pleiotropic effects of Tax on signaling pathways.

HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation.

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.

TLR4-MyD88-TRAF6-TAK1 Complex-Mediated NF-κB Activation Contribute to the Anti-Inflammatory Effect of V8 in LPS-Induced Human Cervical Cancer SiHa Cells.

The synthetic compound 7-4-[Bis-(2-hydroxyethyl)-amino]-butoxy-5-hydroxy-8-methoxy-2-phenylchromen-4-one (V8) is a novel flavonoid-derived compound. In this study, we investigated the effects of V8 on Toll-like receptor 4 (TLR4)-mediated inflammatory reaction in human cervical cancer SiHa cells and lipopolysaccharide (LPS)-induced TLR4 activity in cervical cancer SiHa (HPV16+) cells, but not in HeLa (HPV18+) and C33A (HPV-) cells. In addition, V8 inhibited LPS-induced expression of TLR4, MyD88, TRAF6 and phosphorylation of TAK1, and their interaction with TLR4 in SiHa cells, resulting in an inhibition of TLR4-MyD88-TRAF6-TAK1 complex. Moreover, V8 blocked LPS-induced phosphorylation of IκB and IKK, resulting in inhibition of the nuclear translocation of P65-NF-κB in SiHa cells. We also found that V8 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, IL-6, IL-8, CCL-2, and TNF-α in LPS-stimulated SiHa cells. These results suggested that V8 exerted an anti-inflammatory effect on SiHa cells by inhibiting the TLR4-MyD88-TRAF6-TAK1 complex-mediated NF-κB activation.

USP18 negatively regulates NF-κB signaling by targeting TAK1 and NEMO for deubiquitination through distinct mechanisms.

Nuclear factor κB (NF-κB) is a key transcription factor in inflammatory immune responses and cell survival. Multiple types of ubiquitination play critical roles in the activation of NF-κB signaling, yet the molecular mechanisms responsible for their reversible deubiquitination are still poorly understood. In this study, we identified a member of the deubiquitinases family, ubiquitin-specific protease 18 (USP18), as a novel negative regulator in Toll-like receptor (TLR)-mediated NF-κB activation in human macrophages. USP18 is an interferon inducible gene, which is also upregulated by various TLR ligands in human monocytes and macrophages. Knockdown of USP18 enhanced the phosphorylation of IKK, the degradation of IκB, and augmented the expression of pro-inflammatory cytokines. Furthermore, USP18 interacted with TAK1-TAB1 complex and IKKα/β-NEMO complex, respectively. USP18 cleaved the K63-linked polyubiquitin chains attached to TAK1 in a protease-dependent manner. Moreover, USP18 targeted the IKK complex through the regulatory subunit NEMO of IKK, and specifically inhibited K63-linked ubiquitination of NEMO. Mutation analysis revealed direct binding of USP18 to the UBAN motif of NEMO. Our study has identified a previously unrecognized role for USP18 in the negative regulation of NF-κB activation by inhibiting K63-linked ubiquitination of TAK1 and NEMO through distinct mechanisms.

Synergistic action of 5Z-7-oxozeaenol and bortezomib in inducing apoptosis of Burkitt lymphoma cell line Daudi

Treatment failure in cancer chemotherapy is largely due to the toxic effects of chemotherapeutic agents on normal cells/tissues. The proteasome inhibitor bortezomib has been successfully applied to treat multiple myeloma (MM), but there are some common adverse reactions in the clinic including peripheral neuropathy (PN). The TAK1 selective inhibitor 5Z-7-oxozeaenol has been widely studied in cancer therapy. Here, we investigated the potential synergy of bortezomib and 5Z-7-oxozeaenol in Burkitt's lymphoma (BL) cell lines. Cell viability assay showed that co-treatment of bortezomib at 8nM, representing a one-eighth concentration for growth arrest, and 5Z-7-oxozeaenol at 2μM, a dose that exhibited insignificant cytotoxic effects, synergistically induced apoptosis in the cell line Daudi. In parallel with the increasing dose of the bortezomib, and 5Z-7-oxozeaenol at 0.5μM, lower colony formation efficiencies were seen in the cell line Daudi. Western blotting analysis verified that TAK1 inhibition by 5Z-7-oxozeaenol completely blocked JNK, p38, Erk, IKK, and IκB phosphorylation, which was almost instantly activated by TAK1 both directly or indirectly. Both agents synergistically prevented nuclear translocation of NF-κB, a characteristic of NF-κB inactivation. Moreover, a synergistic effect of bortezomib and 5Z-7-oxozeaenol on Western blotting analysis and flow cytometry was disclosed. Collectively, our results indicated that the proteasome inhibitor bortezomib and the TAK1 inhibitor 5Z-7-oxozeaenol displayed synergy on inhibiting BL cell apoptosis by inhibiting NF-κB activity.

Pomegranate inhibits neuroinflammation and amyloidogenesis in IL-1β-stimulated SK-N-SH cells.

Pomegranate fruit, Punica granatum L. (Punicaceae), and its constituents have been shown to inhibit inflammation. In this study, we aimed to assess the effects of freeze-dried pomegranate (PWE) on PGE2 production in IL-1β-stimulated SK-N-SH cells. An enzyme immunoassay (EIA) was used to measure prostaglandin E2 (PGE2) production from supernatants of IL-1β-stimulated SK-N-SH cells. Expression of COX-2, phospho-IκB, and phospho-IKK proteins was evaluated, while NF-κB reporter gene assay was carried out in TNFα-stimulated HEK293 cells to determine the effect of PWE on NF-κB transactivation. Levels of BACE-1 and Aβ in SK-N-SH cells stimulated with IL-1β were measured with an in cell ELISA. PWE (25-200μg/ml) dose dependently reduced COX-2-dependent PGE2 production in SK-N-SH cells stimulated with IL-1β. Phosphorylation of IκB and IKK was significantly (p<0.001) inhibited by PWE (50-200μg/ml). Our studies also show that PWE (50-200μg/ml) significantly (p<0.01) inhibited NF-κB transactivation in TNFα-stimulated HEK293 cells. Furthermore, PWE inhibited BACE-1 and Aβ expression in SK-N-SH cells treated with IL-1β. Taken together, our study demonstrates that pomegranate inhibits inflammation, as well as amyloidogenesis in IL-1β-stimulated SK-N-SH cells. We propose that pomegranate is a potential nutritional strategy in slowing the progression of neurodegenerative disorders such as Alzheimer's disease.

Sinomenine inhibits breast cancer cell invasion and migration by suppressing NF-κB activation mediated by IL-4/miR-324-5p/CUEDC2 axis

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a vital transcription factor that regulates multiple important biological processes, including the epithelial–mesenchymal transition (EMT) and metastasis of breast cancer. Sinomenine is an isoquinoline well known for its remarkable curative effect on rheumatic and arthritic diseases and can induce apoptosis of several cancer cell types. Recently, sinomenine was reported as a tumor suppressor via inhibiting cell proliferation and inducing apoptosis. However, the role and mechanism of sinomenine in invasion and metastasis of breast cancer are largely unknown. Here, we report that sinomenine suppressed the invasion and migration of MDA-MB-231 and 4T1 breast cancer cells in a dose-dependent manner. We detected binding of NF-κB to the inhibitor of NF-κB (IκB) after the MDA-MB-231 cells were treated with 0.25, 0.5, and 1 mM sinomenine. Co-IP analysis revealed that sinomenine enhanced the binding of NF-κB and IκB in a dose-dependent manner, suggesting that sinomenine had an effect on inactivation of NF-κB. Western blotting and ELISA approaches indicated that the suppression effect was closely associated with the phosphorylation of IκB kinase (IKK) and its negative regulator CUEDC2. Sinomenine treatment decreased miR-324-5p expression, thus increased the level of its target gene CUEDC2, and then blocked the phosphorylation of IKK through altering the upstream axis. Finally, transfection of a miR-324-5p mimic inhibited the suppression of invasion and metastasis of MDA-MB-231 and 4T1 cell by sinomenine, providing evidence that sinomenine treatment suppressed breast cancer cell invasion and metastasis via regulation of the IL4/miR-324-5p/CUEDC2 axis. Our findings reveal a novel mechanism by which sinomenine suppresses cancer cell invasion and metastasis, i.e., blocking NF-κB activation.

HCINAP negatively regulates NF-κB signaling by recruiting the phosphatase PP1 to deactivate IKK complex

Tight regulation of nuclear factor-κB (NF-κB) signaling is essential to maintain homeostasis in immune system in response to various stimuli, which has been studied extensively and deeply. However, the molecular mechanisms responsible for its negative regulation are not completely understood. Here we demonstrate that human coilin-interacting nuclear ATPase protein (hCINAP) is a novel negative regulator in NF-κB signaling by deactivating IκB kinase (IKK) complex. In response to TNF stimulation, hCINAP dynamically associates with IKKα and IKKβ and inhibits IKK phosphorylation. Notably, hCINAP directly interacts with the catalytic subunits of protein phosphatase 1 (PP1) and mediates the formation of IKK-hCINAP-PP1 complex, serving as an adaptor protein that recruits PP1 to dephosphorylate IKK. Furthermore, decreased levels of hCINAP are observed in several inflammatory diseases with NF-κB hyperactivity. Our study suggests a novel mechanism underlying deactivation of IKK and provides new insight into the negative regulation of NF-κB signaling.

Inhibition of serum- and glucocorticoid-inducible kinase 1 enhances TLR-mediated inflammation and promotes endotoxin-driven organ failure.

Serum- and glucocorticoid-regulated kinase (SGK)1 is associated with several important pathologic conditions and plays a modulatory role in adaptive immune responses. However, the involvement and functional role of SGK1 in innate immune responses remain entirely unknown. In this study, we establish that SGK1 is a novel and potent negative regulator of TLR-induced inflammation. Pharmacologic inhibition of SGK1 or suppression by small interfering RNA enhances proinflammatory cytokine (TNF, IL-12, and IL-6) production in TLR-engaged monocytes, a result confirmed in Cre-loxP-mediated SGK1-deficient cells. SGK1 inhibition or gene deficiency results in increased phosphorylation of IKK, IκBα, and NF-κB p65 in LPS-stimulated cells. Enhanced NF-κB p65 DNA binding also occurs upon SGK1 inhibition. The subsequent enhancement of proinflammatory cytokines is dependent on the phosphorylation of TGF-β-activated kinase 1 (TAK1), as confirmed by TAK1 gene silencing. In vivo relevance was established in a murine endotoxin model, in which we found that SGK1 inhibition aggravates the severity of multiple organ damage and enhances the inflammatory response by heightening both proinflammatory cytokine levels and neutrophil infiltration. These findings have identified an anti-inflammatory function of SGK1, elucidated the underlying intracellular mechanisms, and establish, for the first time, that SGK1 holds potential as a novel target for intervention in the control of inflammatory diseases.

Abstract 349: Sgk1 Is a Pivotal Player in Vascular Inflammation and Atherogenesis

Objective: Atherosclerosis, an inflammatory disease of arterial vessel wall, requires migration and MMP-9-dependent invasion of macrophages into the vascular wall. MMP-9 expression is stimulated by transcription factor NF-κB, which is regulated by IκB and thus IκB-kinase IKK. Regulators of NF-κB include SGK1. The present study explored involvement of SGK1 in vascular inflammation and atherogenesis. Approach and Results: Gene-targeted ApoE-deficient mice without (apoe-/-sgk1+/+) or with (apoe-/-sgk1-/-) additional SGK1 knockout received 16-weeks cholesterol-rich diet. Atherosclerotic lesions in aorta and carotid artery, vascular CD45+ leukocyte infiltration, Mac-3+ macrophage infiltration, VSMC content, MMP-2 and MMP-9 positive areas in atherosclerotic tissue were significantly less in apoe-/-sgk1-/-mice. Migration of SGK1-deficient CD11b+F4/80+ macrophages was significantly diminished in vitro and in vivo. Zymographic MMP-2 and MMP-9 production, MMP-9 activity and invasion through matrigel in vitro were significantly less in sgk1-/- than in sgk1+/+ macrophages and in control plasmid- or inactive K127NSGK1-transfected than in constitutively active S422DSGK1-transfected THP-1 cells. Plaques of apoe-/-sgk1-/- mice displayed reduced macrophage number and macrophage MMP-9 content. In THP-1 cells MMP-inhibitor GM6001 abrogated S422DSGK1-induced MMP-9 production and invasion. MMP-9 transcript levels were significantly reduced in sgk1-/- macrophages and strongly upregulated in S422DSGK1-transfected THP-1 cells compared to control plasmid- or K127NSGK1-transfected THP-1 cells. Phosphorylation of IKK and IκB and nuclear translocation of p50 were significantly lower in sgk1-/- macrophages than in sgk1+/+ macrophages and significantly higher in S422DSGK1-transfected THP-1 cells than in control plasmid- or K127NSGK1-transfected THP-1 cells. Treatment of S422DSGK1-transfected THP-1 cells with IKK-inhibitor BMS-345541 abolished S422DSGK1-induced increase of MMP-9 transcription and gelatinase activity. Conclusions: SGK1 plays a pivotal role in vascular inflammation during atherogenesis. SGK1 participates in the regulation of macrophage migration and MMP-9 transcription via regulation of NF-κB.

Abstract 487: Saturated FFA Induced NF-κB Activation Is Independent of IKK/IκBα in Aortic Endothelial Cells

Accumulating evidence has suggested a linkage between free fatty acids (FFAs) and chronic inflammation. It has been shown that saturated FFAs inhibits insulin signal in adipocytes though the activation of IKK. In this study, we examined the effect of FFAs on NF-κB activation in bovine aortic endothelial cells. We found that the saturated FFAs, but not the unsaturated FFAs, caused the activation of NF-κB at a low but sustained level. As a result, ICAM-1 expression was also increased. Interestingly, inconsistent with the previous reports on insulin signal, no changes in IKK phosphorylation and IκBα protein level were observed. IKK16, an IKK inhibitor, failed to block saturated FFA induced NF-κB nuclear translocation. These results imply that saturated FFA induced NF-κB activation is independent of IKK activation and IκBα degradation in aortic endothelial cells, suggesting a novel mechanism for NF-κB activation.

Cyclo(L-phe-pro), Vibrio vulnificus produced metabolite, suppresses innate immune response (INM3P.418)

Abstract Cyclo(Phe-Pro)(cFP), known as a secondary metabolite of some bacteria and fungi, is also produced by Vibrio vulnificus. It has been suggested that cFP has a role in bacterial cell-to-cell communication. However, the role of cFP in host immune response has not been reported yet. In this study, we investigated whether cFP modulates host innate immune responses. cFP suppressed production of pro-inflammatory cytokines, TNFa and IL6, nitric oxide, and reactive oxygen species in LPS-induced macrophages and bone marrow-derived macrophages. cFP also inhibited IKK phosphorylation, IkBa degradation, and NF-kB translocation to the nucleus, indicating that cFP affects the NF-kB pathway. These results provide evidences that cFP has immune suppressive effects that enhance the pathogen’s ability to infect and survive in host environment. Moreover, it may contribute to reveal the role of cFP in host-pathogen interaction and establish potential therapeutic strategies for the diseases related to Vibrio vulnificus.

Abstract P4-05-03: Cytoplasmic PELP1 promotes breast cancer initiation via NF-κB signaling

Abstract Progress in breast cancer prevention is limited by an inability to reliably predict which women will develop breast cancer and which high-risk women will respond to chemoprevention therapies. Thus, there is a critical need to determine the molecular mechanisms that promote breast cancer initiation in order to 1) identify biological markers of disease susceptibility and 2) to develop novel targeted chemoprevention agents. A promising target for preventing breast cancer initiation is proline, glutamic acid, and leucine rich protein 1 (PELP1). PELP1 was first identified as an estrogen receptor (ER) co-activator in ER+ breast cancer cell lines. Subsequent studies found that PELP1 functions in ER+ and ER- breast cancer cell lines and is overexpressed in 80% of invasive breast cancers. Interestingly, while PELP1 is primarily localized to the nucleus in the normal breast epithelial cells, in about 40% of invasive breast tumors a significant amount of PELP1 is localized in the cytoplasm. Interestingly, in a mammary gland specific transgenic mouse model, PELP1-cyto expression induced mammary gland hyperplasia. We therefore analyzed breast needle aspirates from asymptomatic high-risk women, and found cytoplasmic PELP1 in 4/11 (36%) samples. These findings suggest that altered localization of PELP1 may be an early event in breast cancer initiation. The objective of this research project is to identify the molecular mechanisms associated with PELP1-induced breast cancer initiation. We performed global gene expression analyses of our in vitro human mammary epithelial cell (HMEC) models to identify potential downstream pathways regulated by PELP1-cyto. Interestingly, genes regulated by PELP1-cyto differ greatly from those regulated by nuclear PELP1. Our results showed that PELP1-cyto induces expression of cell survival genes, and inflammatory cytokines and chemokines, and activates the NF-κB signaling pathway. Thus, we hypothesize that cytoplasmic PELP1 induces activation of inflammatory cytokines and chemokines via NF-κB, which promotes tumor initiation and a pro-tumorigenic microenvironment. In support of our hypothesis we have found that PELP1-cyto expression promotes phosphorylation of the non-canonical IKK, TBK1, and the NF-κB subunit p65. Additionally, we found that conditioned medium from PELP1-cyto cells, induced expression of IL-1β, TNFα, and IL-8 in THP-1 cells that had been differentiated into macrophages, suggesting that PELP1-cyto HMECs release cytokines that promote the activation of macrophages. The goal of future studies is to validate these findings in vitro and in vivo and identify the cytokines and/or chemokines induced by PELP1-cyto that promote breast cancer initiation and activation of tumor associated macrophages. Citation Format: Julie H Ostrander, Brian J Girard, Todd Knutson, Bethanie Kuker, Victoria Seewaldt. Cytoplasmic PELP1 promotes breast cancer initiation via NF-κB signaling [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-03.

Abstract P5-11-04: Inhibition of alternative NF-κB activity prevents the expansion of genetically unstable progenitor cells in BRCA1-deficient mammary glands

Abstract Understanding the biological mechanisms underlying the initiation and progression of breast cancer is an important step for prevention and treatment. Individuals with mutations in the breast cancer-associated gene 1 (BRCA1) have a lifetime increased risk of up to 85% of developing breast cancer. BRCA1 participates in DNA damage repair and cell cycle checkpoint control, serving as a tumor suppressor gene to maintain global genomic stability. However, BRCA1 has also been shown to play a key role in the expansion of mammary stem/progenitor cells, which are the targets for carcinogenesis in individuals who have undergone loss of heterozygosity (LOH) for BRCA1. We have used in vitro and in vivo models to demonstrate that BRCA1 loss or mutation induces alternative NF-κB pathway activation and leads to the expansion of a genetically unstable progenitor cell population in the mammary gland. Our data showed that BRCA1 loss or mutation is responsible for activation of the alternative NF-κB pathway evidenced by IκB kinase-α (IKKα) phosphorylation, processing of p100 to p52 and p52/RelB nuclear localization. Remarkably, we found that RelB and p100/p52 were highly expressed in 20-50% of the lobular structures in histologically normal breast tissue obtained from human BRCA1 mutation carriers. After DNA damage, ATM directs the nuclear export of NEMO (NF-κB essential modifier) that in turn activates the alternative NF-κB pathway. We found high levels of phospho-ATM in breast tissue from human BRCA1-mutation carriers and in progenitor cells from BRCA1-knockout mouse mammary glands. Moreover, co-immunoprecipitation studies showed increased ATM/NEMO-containing complexes in BRCA1-deficient cells. Progesterone injections into MMTV-cre;BRCA1f/f mice induced an increase in Ki-67 and phospho-H2AX levels, demonstrating a role for progesterone in DNA damage amplification in mouse mammary glands. Mammary epithelial progenitor cells obtained from BRCA1-mutated carriers as well as BRCA1-/- mouse glands can form acini in Matrigel in a progesterone-independent manner. We found that in vivo inhibition of IKKα/β using the IKK inhibitor dimethylaminoparthenolide (DMAPT) completely blocked mammary acini formation in Matrigel, requiring several estrus cycles post-injection to recover. Importantly, knockdown studies showed that p100/p52 was necessary for progenitor cell proliferation in Matrigel. Consistent with the continued dependence on alternative NF-κB activity in tumors derived from BRCA1-deficient progenitor cells, human breast cancer xenografts derived from BRCA1-mutation carriers infected with lenti-shp100/p52 showed a significant growth inhibition. Overall, these results suggest an exciting new approach for the prevention of breast cancer in patients wherein intermittent or cyclic therapy using DMAPT (generic name LC-1) has the potential to mitigate breast cancer risk in BRCA1 mutation carriers through acute reduction of aberrant progenitor activity. As such, NF-κB-directed chemopreventive therapies may provide promising alternatives to prophylactic mastectomy in this high risk patient population. Citation Format: Andrea Sau, Rosanna Lau, Emma Nolan, Peter A Crooks, Jane E Visvader, Christine MA Pratt. Inhibition of alternative NF-κB activity prevents the expansion of genetically unstable progenitor cells in BRCA1-deficient mammary glands [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-11-04.

IKKβ Regulates VEGF Expression and Is a Potential Therapeutic Target for Ovarian Cancer as an Antiangiogenic Treatment.

The prolongation of progression-free survival (PFS) in patients with advanced ovarian cancer by antiangiogenic therapy has been shown in several clinical trials. However, although an anti-VEGF antibody (bevacizumab) is the only option currently available, its efficacy is limited and it is not cost effective for use in all patients. Therefore, the development of a novel antiangiogenic drug, especially composed of small-molecule compounds, could be a powerful armament for ovarian cancer treatment. As NF-κB signaling has the potential to regulate VEGF expression, we determined to identify whether VEGF expression is associated with NF-κB activation and to investigate the possibility of a novel IKKβ inhibitor, IMD-0354 (IMMD Inc.), as an antiangiogenic drug. Tissue microarrays from 94 ovarian cancer tissues were constructed and immunohistochemical analyses performed. We revealed that IKK phosphorylation is an independent prognostic factor (PFS: 26.1 vs. 49.8 months, P = 0.011), and is positively correlated with high VEGF expression. In in vitro analyses, IMD-0354 robustly inhibited adhesive and invasive activities of ovarian cancer cells without impairing cell viabilities. IMD-0354 significantly suppressed VEGF production from cancer cells, which led to the inhibition of angiogenesis. In a xenograft model, the treatment of IMD-0354 significantly inhibited peritoneal dissemination with a marked reduction of intratumoral blood vessel formation followed by the inhibition of VEGF expression from cancer cells. IMD-0354 is a stable small-molecule drug and has already been administered safely to humans in other trials. Antiangiogenic therapy targeting IKKβ is a potential future option to treat ovarian cancer.

Involvement of DNA-PKcs in the type I IFN response to CpG-ODNs in conventional dendritic cells in TLR9-dependent or -independent manners.

CpG-ODNs activate dendritic cells (DCs) to produce interferon alpha (IFNα) and beta (IFNβ). Previous studies demonstrated that Toll-like receptor 9 (TLR9) deficient DCs exhibited a residual IFNα response to CpG-A, indicating that yet-unidentified molecules are also involved in induction of IFNα by CpG-A. Here, we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) but not Ku70 deficient BMDCs showed defective IFNα and IFNβ responses to CpG-A or CpG-B. Loss of both DNA-PKcs and TLR9 further reduced the IFNα response to CpG-A. These DNA-PKcs and TLR9 effects were mediated by their downstream Akt/mTORC1 pathway and downstream events IRAK1 and IKKα. Loss of DNA-PKcs, TLR9, MyD88 or IRAK4 impaired phosphorylation of Akt(S473), S6K, S6, IRAK1, or IKKα in BMDCs in response to CpG-ODNs. The residual IFNα and IFNβ in DNA-PKcs-deficient BMDCs were partially responsible for the induction of IL-6 and IL-12 by CpG-ODNs and their stimulatory effect was blocked by IFNAR1 neutralizing antibodies. Further analysis indicated that CpG-ODN associated with DNA-PKcs and Ku70, and induced DNA-PKcs’s interaction with TRAF3. Intriguingly, DNA-PKcs but not Ku70 expression level was reduced in TLR9-deficient BMDCs. Taken together, our data suggest that DNA-PKcs is an important mediator in the type I IFN response to CpG-ODNs in TLR9-dependent or -independent fashions.

TNF-α down-regulates sarcoplasmic reticulum Ca²⁺ ATPase expression and leads to left ventricular diastolic dysfunction through binding of NF-κB to promoter response element.

TNF-alpha (TNF-α) causes left ventricular diastolic dysfunction. Down-regulation of sarcoplasmic reticulum Ca(2+)-ATPase 2a protein (SERCA2a) expression is one of the major mechanisms underlying diastolic dysfunction. We investigated whether TNF-α modulates SERCA2a expression and alters cardiac diastolic function, and its detailed signalling pathway. We used both in vitro cellular cardiomyocyte model and in vivo rat model to address this issue. We found that TNF-α decreased the levels of both SERCA2a mRNA and protein in the cardiomyocytes, with corresponding impairment of diastolic calcium reuptake, a cellular phenotype of cardiac diastolic function. An ∼2 kb promoter of the SERCA2a gene (atp2a2) along with its serial deletions was cloned into the luciferase reporter system. TNF-α significantly decreased the promoter activity, and truncation of the SERCA2a gene promoter with the putative nuclear factor kappa-B (NF-κB) response element abolished TNF-α-induced SERCA2a gene suppression. Chromatin immunoprecipitation and gel retardation also confirmed the binding of NF-κB to this putative-binding site. TNF-α increased the phosphorylation of IKK and the degradation of IκB, resulted in NF-κB nuclear translocation, and decreased SERCA2a gene promoter activity. This process was attenuated by NF-κB blockers and simvastatin. In the in vivo rat model, lipopolysaccharide treatment significantly elevated the serum TNF-α level, as well as phosphorylation of IKK, resulting in a decrease in myocardial SERCA2a expression, diastolic calcium reuptake, and diastolic dysfunction. Oral treatment with simvastatin led to an increase in SERCA2a expression, alleviation, and prevention of the diastolic dysfunction. TNF-α suppresses SERCA2a gene expression via the IKK/IκB/NF-κB pathway and binding of NF-κB to the SERCA2a gene promoter, and its effect is blocked by simvastatin, demonstrating the potential therapeutic effect of statins in treating inflammation-related diastolic dysfunction.

Resveratrol inhibits enterovirus 71 replication and pro-inflammatory cytokine secretion in rhabdosarcoma cells through blocking IKKs/NF-κB signaling pathway.

Polydatin and resveratrol, as major active components in Polygonum cuspidatum, have anti-inflammatory, antioxidant and antitumor functions. However, the effect and mechanism of polydatin and resveratrol on enterovirus 71 (EV71) have not been reported. In this study, resveratrol revealed strong antiviral activity on EV71, while polydatin had weak effect. Neither polydatin nor resveratrol exhibited influence on viral attachment. Resveratrol could effectively inhibit the synthesis of EV71/VP1 and the phosphorylation of IKKα, IKKβ, IKKγ, IKBα, NF-κB p50 and NF-κB p65, respectively. Meanwhile, the remarkably increased secretion of IL-6 and TNF-α in EV71-infected rhabdosarcoma (RD) cells could be blocked by resveratrol. These results demonstrated that resveratrol inhibited EV71 replication and cytokine secretion in EV71-infected RD cells through blocking IKKs/NF-κB signaling pathway. Thus, resveratrol may have potent antiviral effect on EV71 infection.