Linda Connelly

Epithelial NF-κB activation promotes urethane-induced lung carcinogenesis

Chronic inflammation is linked to carcinogenesis in several organ systems. In the lungs, NF-κB, a central effector of inflammatory responses, is frequently activated in non-small-cell lung cancer, but its role in tumor promotion has not been studied. Several lines of evidence indicate that ethyl carbamate (urethane)-induced lung tumor formation, a prototypical mouse model of multistage lung carcinogenesis, is potentiated by inflammation. We found that mouse strains susceptible to lung tumor formation (FVB, BALB/c) exhibited early NF-κB activation and inflammation in the lungs after urethane treatment. However, a resistant strain (C57B6) failed to activate NF-κB or induce lung inflammation. In FVB mice, we identified urethane-induced NF-κB activation in airway epithelium, as well as type II alveolar epithelial cells and macrophages. Using an inducible transgenic mouse model (FVB strain) to express a dominant inhibitor of NF-κB specifically in airway epithelial cells, we found that urethane-induced lung inflammation was blocked and tumor formation was reduced by >50%. Selective NF-κB inhibition resulted in increased apoptosis of airway epithelial cells at 2 weeks after urethane treatment in association with a marked reduction of Bcl-2 expression. These studies indicate that NF-κB signaling in airway epithelium is integral to tumorigenesis in the urethane model and identify the NF-κB pathway as a potential target for chemoprevention of lung cancer.

Resistance to Endotoxic Shock in Endothelial Nitric-oxide Synthase (eNOS) Knock-out Mice A PRO-INFLAMMATORY ROLE FOR eNOS-DERIVED NO IN VIVO

The expression of inducible nitric-oxide synthase (iNOS) and subsequent “high-output” nitric oxide (NO) production underlies the systemic hypotension, inadequate tissue perfusion, and organ failure associated with septic shock. Therefore, modulators of iNOS expression and activity, both endogenous and exogenous, are important in determining the magnitude and time course of this condition. We have shown previously that NO from the constitutive endothelial NOS (eNOS) is necessary to obtain maximal iNOS expression and activity following exposure of murine macrophages to lipopolysaccharide (LPS). Thus, eNOS represents an important regulator of iNOS expression in vitro. Herein, we validate this hypothesis in vivo using a murine model of sepsis. A temporal reduction in iNOS expression and activity was observed in LPS-treated eNOS knock-out (KO) mice as compared with wild-type animals; this was reflected in a more stable hemodynamic profile in eNOS KO mice during endotoxaemia. Furthermore, in human umbilical vein endothelial cells, LPS leads to the activation of eNOS through phosphoinositide 3-kinase- and Akt/protein kinase B-dependent enzyme phosphorylation. These data indicate that the pathogenesis of sepsis is characterized by an initial eNOS activation, with the resultant NO acting as a co-stimulus for the expression of iNOS, and therefore highlight a novel pro-inflammatory role for eNOS.

Inhibition of NF-kappa B activity in mammary epithelium increases tumor latency and decreases tumor burden.

The transcription factor nuclear factor kappa B (NF-κB) is activated in human breast cancer tissues and cell lines. However, it is unclear whether NF-κB activation is a consequence of tumor formation or a contributor to tumor development. We developed a doxycycline (dox)-inducible mouse model, termed DNMP, to inhibit NF-κB activity specifically within the mammary epithelium during tumor development in the polyoma middle T oncogene (PyVT) mouse mammary tumor model. DNMP females and PyVT littermate controls were treated with dox from 4 to 12 weeks of age. We observed an increase in tumor latency and a decrease in final tumor burden in DNMP mice compared with PyVT controls. A similar effect with treatment from 8 to 12 weeks indicates that outcome is independent of effects on postnatal virgin ductal development. In both cases, DNMP mice were less likely to develop lung metastases than controls. Treatment from 8 to 9 weeks was sufficient to impact primary tumor formation. Inhibition of NF-κB increases apoptosis in hyperplastic stages of tumor development and decreases proliferation at least in part by reducing Cyclin D1 expression. To test the therapeutic potential of NF-κB inhibition, we generated palpable tumors by orthotopic injection of PyVT cells and then treated systemically with the NF-κB inhibitor thymoquinone (TQ). TQ treatment resulted in a reduction in tumor volume and weight as compared with vehicle-treated control. These data indicate that epithelial NF-κB is an active contributor to tumor progression and demonstrate that inhibition of NF-κB could have a significant therapeutic impact even at later stages of mammary tumor progression.

A Transgenic Model Reveals Important Roles for the NF-κB Alternative Pathway (p100/p52) in Mammary Development and Links to Tumorigenesis

A regulated pattern of nuclear factor κB (NF-κB) activation is essential for normal development of the mammary gland. An increase in NF-κB activity has been implicated in breast cancer. We have generated a novel transgenic mouse model to investigate the role of the alternative NF-κB pathway in ductal development and identify possible mediators of tumorigenesis downstream of p100/p52. By overexpressing the NF-κB p100/p52 subunit in mammary epithelium using the β-lactoglobulin milk protein promoter, we found that transgene expression resulted in increased overall NF-κB activity during late pregnancy. During pregnancy, p100/p52 expression resulted in delayed ductal development with impaired secondary branching and increased levels of Cyclin D1, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and cyclo-oxygenase-2 (COX-2) in the mammary gland. After multiple pregnancies the p100 transgenics exhibited a ductal thickening accompanied by small hyperplastic foci. In tumors from mice expressing the polyoma middle T oncoprotein (PyVT) in the mammary gland, increased levels of p100/p52 were present at the time of tumor development. These results show that increased p100/p52 disrupts normal ductal development and provides insight into the mechanism by which this may contribute to human breast cancer.

Silent information regulator (Sir)T1 inhibits NF-κB signaling to maintain normal skeletal remodeling.

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF‐κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF‐κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast‐ or osteoblast‐specific SirT1 deletion using the Sirtflox/flox mice crossed to lysozyme M‐cre and the 2.3 kb col1a1‐cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF‐κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF‐κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF‐κB. In further support of the critical role of NF‐κB signaling in bone remodeling, elevated NF‐κB activity in IκBα+/− mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell‐autonomous manner in both osteoblasts and osteoclasts, through control of NF‐κB and bone cell differentiation.

Activation of nuclear factor-kappa B in mammary epithelium promotes milk loss during mammary development and infection

We investigated whether nuclear factor kappa B (NF‐κB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and re‐modeling. We generated a doxycycline inducible transgenic mouse model to activate NF‐κB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF‐κB signaling cascade. We found that activation of NF‐κB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase‐3, an indicator of apoptosis. We also found that activation of NF‐κB during lactation with no additional involution signals had a similar effect. The observation that NF‐κB is a key regulator of milk production led us to investigate the role of NF‐κB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF‐κB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF‐κB in mammary epithelium. Together, our data reveal that activation of NF‐κB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF‐κB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact. J. Cell. Physiol. 222:73–81, 2010.

NF-kappaB activation within macrophages leads to an anti-tumor phenotype in a mammary tumor lung metastasis model.

IntroductionMetastasis from primary tumor to the lungs is a major cause of the mortality associated with breast cancer. Both immune and inflammatory responses impact whether circulating mammary tumor cells successfully colonize the lungs leading to established metastases. Nuclear factor -kappaB (NF-κB) transcription factors regulate both immune and inflammatory responses mediated in part by the activities of macrophages. Therefore, NF-κB activity specifically within macrophages may be a critical determinant of whether circulating tumor cells successfully colonize the lungs.MethodsTo investigate NF-κB signaling within macrophages during metastasis, we developed novel inducible transgenic models which target expression of the reverse tetracycline transactivator (rtTA) to macrophages using the cfms promoter in combination with inducible transgenics that express either an activator (cIKK2) or an inhibitor (IκBα-DN). Doxycyline treatment led to activation or inhibition of NF-κB within macrophages. We used a tail vein metastasis model with mammary tumor cell lines established from MMTV-Polyoma Middle T-Antigen-derived tumors to investigate the effects of modulating NF-κB in macrophages during different temporal windows of the metastatic process.ResultsWe found that activation of NF-κB in macrophages during seeding leads to a reduction in lung metastases. The mechanism involved expression of inflammatory cytokines and reactive oxygen species, leading to apoptosis of tumor cells and preventing seeding in the lung. Activation of NF-κB within macrophages after the seeding phase has no significant impact on establishment of metastases.ConclusionsOur results have identified a brief, defined window in which activation of NF-κB has significant anti-metastatic effects and inhibition of NF-κB results in a worse outcome.

EPITHELIAL NUCLEAR FACTOR-κB SIGNALING PROMOTES LUNG CARCINOGENESIS VIA RECRUITMENT OF REGULATORY T LYMPHOCYTES

The mechanisms by which chronic inflammatory lung diseases, particularly chronic obstructive pulmonary disease, confer enhanced risk for lung cancer are not well-defined. To investigate whether nuclear factor (NF)-κB, a key mediator of immune and inflammatory responses, provides an interface between persistent lung inflammation and carcinogenesis, we utilized tetracycline-inducible transgenic mice expressing constitutively active IκB kinase β in airway epithelium (IKTA (IKKβ trans-activated) mice). Intraperitoneal injection of ethyl carbamate (urethane), or 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) was used to induce lung tumorigenesis. Doxycycline-treated IKTA mice developed chronic airway inflammation and markedly increased numbers of lung tumors in response to urethane, even when transgene expression (and therefore epithelial NF-κB activation) was begun after exposure to carcinogen. Studies using a separate tumor initiator/promoter model (MCA+BHT) indicated that NF-κB functions as an independent tumor promoter. Enhanced tumor formation in IKTA mice was preceded by increased proliferation and reduced apoptosis of alveolar epithelium, resulting in increased formation of premalignant lesions. Investigation of inflammatory cells in lungs of IKTA mice revealed a substantial increase in macrophages and lymphocytes, including functional CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Importantly, Treg depletion using repetitive injections of anti-CD25 antibodies limited excessive tumor formation in IKTA mice. At 6 weeks following urethane injection, antibody-mediated Treg depletion in IKTA mice reduced the number of premalignant lesions in the lungs in association with an increase in CD8 lymphocytes. Thus, persistent NF-κB signaling in airway epithelium facilitates carcinogenesis by sculpting the immune/inflammatory environment in the lungs.

Osteoprotegerin in breast cancer: beyond bone remodeling

Osteoprotegerin (OPG) is a secreted protein and member of the Tumor Necrosis Factor (TNF) Receptor superfamily. OPG has been well characterized as a regulator of bone metabolism which acts by blocking osteoclast maturation and preventing bone breakdown. Given this role, early studies on OPG in breast cancer focused on the administration of OPG in order to prevent the osteolysis observed with bone metastases. However OPG is also produced by the breast tumor cells themselves. Research focusing on OPG produced by breast tumor cells has revealed actions of OPG which promote tumor progression. In vitro studies into the role of OPG produced by breast tumor cells have demonstrated that OPG can block TNF-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Furthermore, in vivo studies show that OPG expression by breast tumors can promote tumor growth and metastasis. In addition it has been shown that OPG stimulates endothelial cell survival and tube formation thus it may indirectly promote breast tumor progression through impacting angiogenesis. This article will present a summary of the data concerning the tumor-promoting effects of OPG in breast cancer.

Interleukin-5 Facilitates Lung Metastasis by Modulating the Immune Microenvironment

Although the lung is the most common metastatic site for cancer cells, biologic mechanisms regulating lung metastasis are not fully understood. Using heterotopic and intravenous injection models of lung metastasis in mice, we found that IL5, a cytokine involved in allergic and infectious diseases, facilitates metastatic colonization through recruitment of sentinel eosinophils and regulation of other inflammatory/immune cells in the microenvironment of the distal lung. Genetic IL5 deficiency offered marked protection of the lungs from metastasis of different types of tumor cells, including lung cancer, melanoma, and colon cancer. IL5 neutralization protected subjects from metastasis, whereas IL5 reconstitution or adoptive transfer of eosinophils into IL5-deficient mice exerted prometastatic effects. However, IL5 deficiency did not affect the growth of the primary tumor or the size of metastatic lesions. Mechanistic investigations revealed that eosinophils produce CCL22, which recruits regulatory T cells to the lungs. During early stages of metastasis, Treg created a protumorigenic microenvironment, potentially by suppressing IFNγ-producing natural killer cells and M1-polarized macrophages. Together, our results establish a network of allergic inflammatory circuitry that can be co-opted by metastatic cancer cells to facilitate lung colonization, suggesting interventions to target this pathway may offer therapeutic benefits to prevent or treat lung metastasis.