NF-κB Activation In Cancer Cells Research Articles

MALT1 is required for EGFR-induced NF-κB activation and contributes to EGFR-driven lung cancer progression.

The transcription factor NF-κB has been implicated in playing a crucial role in the tumorigenesis of many types of human cancers. Although Epidermal Growth Factor Receptor (EGFR) can directly activate NF-κB, the mechanism by which EGFR induces NF-κB activation and the role of NF-κB in EGFR-associated tumor progression is still not fully defined. Herein, we found that Mucosa-Associated Lymphoid Tissue 1 (MALT1) is involved in EGFR-induced NF-κB activation in cancer cells, and MALT1 deficiency impaired EGFR-induced NF-κB activation. MALT1 mainly functions as a scaffold protein by recruiting E3 ligase TRAF6 to IKK complex to activate NF-κB in response to EGF stimulation. Functionally, MALT1 inhibition shows significant defects in EGFR-associated tumor malignancy, including cell migration, metastasis and anchorage independent growth. To further access a physiological role of MALT1-dependent NF-κB activation in EGFR-driven tumor progression, we generated triple transgenic mouse model (tetO-EGFRL858R; CCSP-rtTA; Malt1−/−), in which mutant EGFR-driven lung cancer was developed in the absence of MALT1 expression. MALT1-deficient mice show significantly less lung tumor burden when compared to its heterozygous controls, suggesting that MALT1 is required for the progression of EGFR-induced lung cancer. Mechanistically, MALT1 deficiency abolished both NF-κB and STAT3 activation in vivo, which is a result of a defect of IL-6 production. In comparison, MALT1 deficiency does not affect tumor progression in a mouse model (LSL-K-rasG12D; CCSP-Cre; Malt1−/−) in which lung cancer is induced by expressing a K-ras mutant. Thus, our study has provided the cellular and genetic evidence that suggests MALT1-dependent NF-κB activation is important in EGFR-associated solid tumor progression.

Analysis of epidermal growth factor-induced NF-κB signaling.

The nuclear factor kappaB (NF-κB) is a family of transcription factors that control cell survival, cell proliferation, cell differentiation, inflammatory responses, and innate and adaptive immune responses. Its activation is tightly regulated, and incorrect regulation of NF-κB has been linked to a variety of pathological diseases, including cancer initiation and progression. NF-κB is often constitutively activated in cancer cells to promote cell survival, proliferation, migration, and/or epithelial-to-mesenchymal transition (EMT). Although the mechanism of constitutive NF-κB activation in cancer cells is not fully understood, it has been shown that mutation or aberrant expression of epidermal growth factor receptor (EGFR) contributes to this, and the NF-κB activation, in turn, contributes to cell proliferation, survival, metastasis, and drug resistance in various cancers. Recent study from our lab indicates that CARMA3, similar to the function of CARMA1 in mediating antigen receptor-mediated NF-κB activation, plays an essential role in mediating EGFR-induced NF-κB activation. However, the mechanism on how EGFR induces NF-κB activation is not clearly understood. In this chapter, we describe the methods required to test and characterize the role of a potential signaling component in EGFR-induced NF-κB activation.

PHLPP2 suppresses the NF-κB pathway by inactivating IKKβ kinase

The NF-κB growth pathway is constitutively activated in many cancers but its activation mechanism is unclear in most cases. We show that PHLPP2 interacts with IKKβ kinase, decreases its phosphorylation and the subsequent NF-κB activation in cancer cells. PHLPP2 is progressively lost in glioma and colorectal cancer and acts as a bona fide tumor suppressor, depending on IKKβ expression in cells. Physiologically, IKKβ activation by growth factors requires the formation of the Bcl10-MALT1 ubiquitin-ligase complex leading to NEMO/IKKγ non-degradative ubiquitination and IKKβ phosphorylation. PHLPP2 opposes the formation of this complex through interaction with Bcl10 and competitive displacement of MALT1 from Bcl10. Conversely, PHLPP2 loss enhances Bcl10-MALT1 complex formation, NEMO ubiquitination and subsequent IKKβ phosphorylation, resulting in increased NF-κB-dependent transcription of multiple target genes. Our results reveal PHLPP2 as a new biomarker of cancer progression, and implicate it as major negative regulator of NF-κB signaling.

PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65

Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 (PHF20) maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that PHF20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In PHF20 overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between PHF20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that PHF20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies PHF20 as a novel regulator of NF-κB activation and suggests that elevated expression of PHF20 may drive constitutive NF-κB activation in some cancers.

The HSP70 and Autophagy Inhibitor Pifithrin-μ Enhances the Antitumor Effects of TRAIL on Human Pancreatic Cancer

TRAIL and agonistic death receptor-specific antibodies can induce apoptosis in cancer cells with little cytotoxicity to normal cells. To improve TRAIL-induced antitumor effects, we tested its effectiveness in combination with pifithrin (PFT)-μ, which has the potential to inhibit HSP70 function and autophagy, both of which participate in TRAIL resistance in cancer cells. Among the four human pancreatic cancer cell lines tested, MiaPaca-2, Panc-1, and BxPC-3 cells showed varying sensitivities to TRAIL. In MiaPaca-2 and Panc-1 cells, knockdown of HSP70 or beclin-1, the latter an autophagy-related molecule, by RNA interference augmented TRAIL-induced antitumor effects, decreasing cell viability, and increasing apoptosis. On the basis of these findings, we next determined whether the TRAIL-induced antitumor effects could be augmented by its combination with PFT-μ. The combination of TRAIL plus PFT-μ significantly decreased the viability and colony-forming ability of MiaPaca-2 and Panc-1 cells compared with cells treated with either agent alone. When applied alone, PFT-μ increased Annexin V(+) cells in both caspase-dependent and -independent manners. It also promoted TRAIL-induced apoptosis and arrested cancer cell growth. Furthermore, PFT-μ antagonized TRAIL-associated NF-κB activation in cancer cells. In a xenograft mouse model, combination therapy significantly inhibited MiaPaca-2 tumor growth compared with treatment with either agent alone. The results of this study suggest protective roles for HSP70 and autophagy in TRAIL resistance in pancreatic cancer cells and suggest that PFT-μ is a promising agent for use in therapies intended to enhance the antitumor effects of TRAIL.

TAK1 ubiquitination regulates doxorubicin-induced NF-κB activation

Chemotherapeutic agents- and radiation therapy-induced NF-κB activation in cancer cells contributes to aggressive tumor growth and resistance to chemotherapy and ionizing radiation during cancer treatment. TAK1 has been shown to be required for genotoxic stress-induced NF-κB activation. However, whether TAK1 ubiquitination is involved in genotoxic stress-induced NF-κB activation remains unknown. Herein, we demonstrate that TAK1 ubiquitination plays an important role in the positive and negative regulation of doxorubicin (Dox)-induced NF-κB activation. We found that TAK1 was required for Dox-induced NF-κB activation. At the early stage of Dox treatment, Dox induced Lys63-linked TAK1 polyubiquitination at lysine 158 residue. USP4 inhibited Dox-induced TAK1 Lys63-linked polyubiquitination and knockdown of USP4 enhanced Dox-induced NF-κB activation. At the late stage of Dox treatment, Dox induced Lys48-linked TAK1 polyubiquitination to promote TAK1 degradation. ITCH inhibited Dox-induced NF-κB activation by promoting Lys48-linked TAK1 polyubiquitination and its subsequent degradation. Our study indicates that TAK1 ubiquitination plays critical roles in the regulation of Dox-induced NF-κB activation. Thus, intervention of TAK1 kinase activity or TAK1 Lys63-linked polyubiquitination pathways might greatly enhance the therapeutic efficacy of Dox.

Forced Expression of Heat Shock Protein 27 (Hsp27) Reverses P-Glycoprotein (ABCB1)-mediated Drug Efflux and MDR1 Gene Expression in Adriamycin-resistant Human Breast Cancer Cells

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G(2)/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

Nuclear Factor-κB modulates cellular glutathione and prevents oxidative stress in cancer cells

The NF-κB is best known for its role in inflammation. Here we show that constitutive NF-κB activity in cancer cells promotes the biosynthesis of redox scavenger glutathione (GSH), which in turn confers resistance to oxidative stress. Inhibition of NF-κB significantly decreases GSH in several lines of human leukemia and prostate cancer cells possessing high or moderate NF-κB activities. Concomitantly, NF-κB inhibition by pharmacological and molecular means sensitizes “NF-κB positive” cancer cells to chemically-induced oxidative stress and death. We propose that inhibition of NF-κB can reduce intracellular GSH in “NF-κB-positive” cancers thereby improving the efficacy of oxidative stress-based anti-cancer therapy.

Abstract 1794: TXNL2 is a key regulator of reactive oxygen species levels and NF-κB activity in cancer cells

Abstract Cancer cells usually generate and maintain higher levels of reactive oxygen species (ROS) compared to normal cells, in part because of their higher metabolic rate and defects in the electron transport chain. Because most tumor cells possess stronger antioxidative defense mechanisms to counterbalance excessive ROS, redox proteins such as thioredoxins (TRXs) and glutaredoxins (GRXs) are potential targets of anticancer therapy. We recently found that a novel redox protein called thioredoxin-like 2 (TXNL2) is required for embryonic stem cell functions and is consistently overexpressed in many different cancers. It has a TRX-homology domain and two GRX-homology domains, suggesting that it may have redox function. Using immunohistochemical and cDNA microarray analyses, we found that levels of TXNL2 were significantly higher (P = 0.00036) in human breast cancers than in normal breast tissues; levels also were higher in human breast cancer cell lines than in normal mammary epithelial cell lines. Knockdown of TXNL2 by shRNA in breast cancer cell lines induced a 5-fold increase of ROS levels and inhibited cell proliferation, survival, invasion, and the expression of genes critically involved in these cellular processes. Breast cancer stem-like cell populations and stem-like properties such as mammosphere formation were reduced by TXNL2 silencing and induced by TXNL2 overexpression. Surprisingly, TXNL2 depletion in normal mammary epithelial cells did not significantly affect ROS levels and cell survival. Xenograft models of orthotopic injection indicated that TXNL2 knockdown inhibited mammary tumor growth and metastasis to the lung. Cell signaling studies showed that TXNL2 knockdown inhibited GSH synthase (GSS) expression, and thereby decreased GSH levels and increased ROS levels; N-acetylcysteine (NAC), a GSH precursor, partially reversed these effects. In addition, TXNL2 knockdown suppressed NF-κB activity by inactivating IKK and p65 via glutathionylation, whereas TXNL2 overexpression markedly increased NF-κB activity. Blockade of NF-κB using its small-molecule inhibitors and IκB overexpression mimicked the effect of TXNL2 depletion. Our results suggest that TXNL2 is a potentially promising target for ROS-based anticancer therapies and is a key regulator of the NF-κB pathway. Because high cellular antioxidant capacity is well-known to impair cellular responses to chemotherapy and radiation therapy, our study may also open up a new avenue of maximize the efficacy of conventional anticancer therapies by targeting TXNL2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1794.

Lipopolysaccharide (LPS) increases the invasive ability of pancreatic cancer cells through the TLR4/MyD88 signaling pathway

Inflammation plays a multifaceted role in cancer progression, and NF-kappaB is one of the key factors connecting inflammation with cancer progression. We have shown that lipopolysaccharide (LPS) promotes NF-kappaB activation in colon cancer cells and pancreatic cancer cells. However, it is unclear why inflammatory stimuli can induce NF-kappaB activation in cancer cells. We used two human pancreatic cancer cells, Panc-1 and AsPC-1, as target cells. LPS was used as an inflammatory stimulus. To confirm the participation of TLR4/NF-kappaB signaling pathway, we used three different NF-kappaB inhibitors (PDTC, IkappaBalpha mutant, and NF-kappaB decoy ODN) and siRNAs (against TLR4, MyD88, and MMP-9). Effect of LPS on pancreatic cancer cell invasive ability was determined by Matrigel invasion assay. LPS increased the invasive ability of pancreatic cancer cells, while blockade of NF-kappaB pathway decreased the LPS-dependent increased invasive ability. Blockade of TLR4 or MyD88 by siRNA also decreased the LPS-dependent increased invasive ability. These results suggest that TLR/MyD88/NF-kappaB signaling pathway plays a significant role in connecting inflammation and cancer invasion and progression.

A New Regulatory Mechanism of NF-κB Activation by I-κBβ in Cancer Cells

Transglutaminase 2 (TGase 2) catalyzes covalent isopeptide bond formation between glutamine and lysine residues. Recently, we reported that TGase 2 activates nuclear factor-kappa B (NF-κB) by depleting inhibitor of NF-κBα (I-κBα) levels via polymer formation. Furthermore, TGase 2 expression synergistically increases NF-κB activity with canonical pathway. The major I-κB proteins such as I-κBα and I-κBβ resemble each other in both primary sequence and tertiary structure. However, I-κBβ does not degrade fully, while I-κBα degrades immediately in response to most stimuli. We found that I-κBβ does not contain any of the previously identified TGase 2 target sites. In this study, both an in vitro cross-linking assay and a TGase 2 transfection assay revealed that I-κBβ is independent from TGase 2-mediated polymerization. Furthermore, increased I-κBβ expression reversed NF-κB activation in cancer cells, compensating for the loss of I-κBα via TGase 2 polymerization.

Protein kinase C–associated kinase is required for NF-κB signaling and survival in diffuse large B-cell lymphoma cells

AbstractDiffuse large B-cell lymphoma (DLBCL) is an aggressive and the most common type of non-Hodgkin lymphoma. Despite recent advances in treatment, less than 50% of the patients are cured with current multiagent chemotherapy. Abnormal NF-κB activity not only contributes to tumor development but also renders cancer cells resistant to chemotherapeutic agents. Identifying and targeting signaling molecules that control NF-κB activation in cancer cells may thus yield more effective therapy for DLBCL. Here, we show that while overexpression of protein kinase C–associated kinase (PKK) activates NF-κB signaling in DLBCL cells, suppression of PKK expression inhibits NF-κB activity in these cells. In addition, we show that NF-κB activation induced by B cell–activating factor of tumor necrosis factor family (BAFF) in DLBCL cells requires PKK. Importantly, we show that knockdown of PKK impairs the survival of DLBCL cells in vitro and inhibits tumor growth of xenografted DLBCL cells in mice. Suppression of PKK expression also sensitizes DLBCL cells to treatment with chemotherapeutic agents. Together, these results indicate that PKK plays a pivotal role in the survival of human DLBCL cells and represents a potential target for DLBCL therapy.

Cepharanthin-enhanced radiosensitivity through the inhibition of radiation-induced nuclear factor-κB activity in human oral squamous cell carcinoma cells

We have already demonstrated that human head and neck cancer cells have significantly enhanced levels of transcription factor nuclear factor (NF)-kappaB activity compared to their normal counterparts, suggesting that NF-kappaB plays an important role in the development of head and neck cancer. However, it has been reported that chemotherapeutic agents and radiation activate NF-kappaB activity in cancer cells, thus making the cells radioresistant and chemoresistant. In addition, we have shown that the suppression of NF-kappaB activity enhanced apoptosis in oral squamous cell carcinoma cells. In this study, we examined whether cepharanthin-induced inhibition of NF-kappaB activity enhances radiosensitivity in human oral carcinoma cells. Cepharanthin is a biscoclaurine alkaloid extracted from the roots of Stephania cepharantha hayata, and is widely used in Japan for the treatment of patients with leucopenia, nasal allergy, and venomous snakebites. gamma-irradiation (IR) induces NF-kappaB activity in oral carcinoma cells through the activation of upstream molecules, including Akt and IkappaB kinase. However, a luciferase assay revealed that cepharanthin suppresses IR-induced NF-kappaB activity in oral squamous cell carcinoma cells, thereby enhancing the radio-sensitivity. Western blot analysis showed an enhanced cleavage of poly-(ADP-ribose) polymerase protein in carcinoma cells by both cepharanthin treatment and IR exposure compared to IR or cepharanthin alone. In an in vivo study, B88 cells were s.c. inoculated into the backs of nude mice. Tumor-bearing nude mice received either cepharanthin, IR alone, or a combination of cepharanthin and IR. The combined treatment suppressed tumor growth significantly more than either cepharanthin or IR alone. Cepharanthin inhibited the production of IR-induced IL-6 and IL-8, which are downstream targets of NF-kappaB. In quantitative real-time RT-PCR, IR also induced the expression of anti-apoptotic proteins [cellular inhibitor of apoptosis protein (cIAP)-1 and -2] in carcinoma cells. Treatment of cancer cells with cepharanthin combined with exposure to IR decreased cIAP-1 and -2 mRNA expression. These findings suggested that the combination of radiotherapy and cepharanthin could enhance radiosensitivity in the treatment of human oral cancer.

Inositol Hexaphosphate and Paclitaxel: Symbiotic Treatment of Oral Cavity Squamous Cell Carcinoma

Nuclear factor (NF)-kappaB is an early response gene that has been associated with head and neck squamous cell cancer (HNSCC) progression. NF-kappaB activation is induced by some chemotherapy agents, including paclitaxel. The activation of this gene can be correlated with apoptosis resistance. Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate. NF-kappaB levels were evaluated in oral cavity HNSCC lines after treatment with paclitaxel and IP6, alone and in combination. Resulting levels of cell death and apoptosis were assessed, and conclusions are drawn regarding a possible synergistic relationship between paclitaxel and IP6. NF-kappaB activation in cancer cells treated with paclitaxel and IP6, alone and in combination, was measured by transient transfection, and results were interpreted by luminometry. Cell proliferation of treated cells was measured by MTT assay. Cell viability and apoptosis of cancer cells treated with paclitaxel and IP6 combinations were quantitated by trypan blue staining and Caspase-Glo 3/7 assay, respectively. IP6 was observed to significantly downregulate NF-kappaB activation in both NA and CA-9-22 oral cavity HNSCC cell lines. Paclitaxel treatments caused increased NF-kappaB activation in the same cell lines. IP6 was observed to mitigate paclitaxel-induced NF-kappaB activation in the CA-9-22 cell line. IP6, when combined with paclitaxel, reduces CA-9-22 cell proliferation, increases cell death, and increases apoptosis, when compared with treatment with paclitaxel alone. IP6 reduces paclitaxel induced NF-kappaB activation and increases paclitaxel-mediated cell killing and apoptosis. As a well-tolerated and safe supplement, IP6 deserves further study in the treatment of oral cavity squamous cell carcinoma.

Enhanced IκB kinase activity is responsible for the augmented activity of NF-κB in human head and neck carcinoma cells

The nuclear transcription factor κB (NF-κB) plays an important role in the development and progression of cancers. However, the mechanism by which cancer cells in the head and neck region acquire high NF-κB activity has not yet been clarified. In this study, we examined the NF-κB binding activity and the expression of the signal-transduction-related proteins of NF-κB in head and neck carcinoma cell lines. These cancer cells showed significantly higher NF-κB binding activity than normal oral epithelial and salivary gland cells. We also demonstrated the increased phosphorylation and degradation of IκB-α protein in cancer cells. Thus, enhanced NF-κB activity in cancer cells is attributable to the rapid phosphorylation and degradation of IκB-α protein. To further elucidate the mechanism involved in this phenomenon, we analyzed both the expression levels of upstream kinases (IκB kinase- (IKK-) α, IKK-β, IKK-γ, and NF-κB-inducing kinase (NIK)) and the IKK activity in cells. Although there was no significant difference in the expression levels of NIK, IKK-β, or IKK-γ in cancer cell lines compared to those in normal cells, increased expression of IKK-α protein was observed in cancer cells. In addition, IKK activity was significantly augmented in cancer cells as compared to normal cells. Thus, our results suggest that enhanced NF-κB activity in head and neck cancer cells may be due to the augmentation of IKK activity.