p52 expression enhances lung cancer progression.

While many studies have demonstrated that canonical NF-κB signaling is a central pathway in lung tumorigenesis, the role of non-canonical NF-κB signaling in lung cancer remains undefined. We observed frequent nuclear accumulation of the non-canonical NF-κB component p100/p52 in human lung adenocarcinoma. To investigate the impact of non-canonical NF-κB signaling on lung carcinogenesis, we employed transgenic mice with doxycycline-inducible expression of p52 in airway epithelial cells. p52 over-expression led to increased tumor number and progression after injection of the carcinogen urethane. Gene expression analysis of lungs from transgenic mice combined with in vitro studies suggested that p52 promotes proliferation of lung epithelial cells through regulation of cell cycle-associated genes. Using gene expression and patient information from The Cancer Genome Atlas (TCGA) database, we found that expression of p52-associated genes was increased in lung adenocarcinomas and correlated with reduced survival, even in early stage disease. Analysis of p52-associated gene expression in additional human lung adenocarcinoma datasets corroborated these findings. Together, these studies implicate the non-canonical NF-κB component p52 in lung carcinogenesis and suggest modulation of p52 activity and/or downstream mediators as new therapeutic targets.

Steroid treatment can inhibit nuclear localization of members of the NF-κB pathway in human disc cells stimulated with TNF-α.

Steroid applications are able to repress inflammatory activity in various conditions, including herniation of the nucleus pulposus (HNP), by inhibiting tumour necrosis factor (TNF)-α, but the effects of long-term use are unknown. Here, we investigated the effect of dexamethasone (DEXA) on TNF-α-stimulated intervertebral disc cells by monitoring the expression and localization of NF-κB in the cytoplasm and nucleus. Cultured human intervertebral disc cells were left untreated or treated with only TNF-α, only DEXA, or with TNF-α and DEXA simultaneously. Cytoplasmic and nuclear proteins were extracted and Western blotted after 10 min, 1 or 2 h, to evaluate the expression of p50, p65, p52, and p100 (components of NF-κB). Immunofluorescence analysis was used to determine the subcellular localization of the proteins at 1 h. DEXA had limited effects on NF-κB expression in TNF-α-stimulated disc cells within the first 10 min. At 1 h, DEXA prevented the TNF-α-stimulated translocation of p50, p52, and p65. After 2 h, DEXA reduced the nuclear expression of p50, p65, and p52. Thus, DEXA resulted in delayed expression of NF-κB components and inhibited the translocation of p50, p52, and p65 to the nucleus, which would prevent expression of the corresponding genes. Therefore, following stimulation with TNF-α, transcriptional regulation of NF-κB in disc cells is mainly mediated via the classical pathway, but also to some extent via the alternative pathway. Hence, blockade of sub-acute inflammatory changes in HNP can be achieved by early injection of steroids, whereas long-term injection of a steroid may initiate NF-κB autophosphorylation.

Immunohistochemical analysis of NF-kappaB signaling proteins IKKepsilon, p50/p105, p52/p100 and RelA in prostate cancers.

Activation of nuclear factor-kappa B (NF-kappaB) signaling is considered an important mechanism in the development of prostate cancers. A recent study revealed that IkappaB kinase epsilon (IKKepsilon), an activator of NF-kappaB, was overexpressed in breast cancers and acted as an oncogene. Expression of NF-kappaB members has been reported in prostate cancer tissues, but expression of IKKepsilon has not yet been studied in prostate cancers. In this study, we attempted to explore as to whether expressions of IKKepsilon and NF-kappaB members p50/105, p52/p100 and RelA are altered in prostate cancers. We analyzed the expression of IKKepsilon, p50/105, p52/p100 and RelA in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. In the TMA, IKKepsilon is expressed in basal cells, but not in alveolar cells in normal prostate glands. IKKepsilon is expressed in 60.0% of prostate intraepithelial neoplasm (PIN) and 70.1% of the prostate cancers in the cytoplasm. Nuclear immunostainings of NF-kappaB members p50/105, p52/p100 and RelA, which are considered activation of NF-kappaB signaling, were observed respectively in 28.0%, 18.7% and 37.4% of the cancers. Nuclear staining was detected neither in normal alveolar cells nor in PIN. However, none of the expression of p50/105 nor p52/p100 nor RelA nor IKKepsilon was associated with pathologic characteristics, including size of the cancers, age, Gleason score and stage. The increased cytoplasmic expression of IKKepsilon as well as the increased nuclear expressions of p50/105, p52/p100 and RelA in the prostate cancers compared to normal alveolar cells suggested that overexpression of these proteins may be related to activation of the NF-kappaB pathway and might play a role in tumorigenesis of prostate cancers.

Rearranged NF-kappa B2 gene in an adult T-cell leukemia cell line.

Adult T-cell leukemia (ATL) is an aggressive type of leukemia, originating from T-cells infected with human T-cell leukemia virus type 1. Accumulating evidence suggests the aberrant activation of NF-kappaB to be a causative factor mediating the abnormal proliferation of leukemic cells, thus resulting in the development of ATL. A rearranged NF-kappa B2/p100 gene was isolated from an ATL-derived cell line, which was generated by a chromosomal translocation. The isolated NF-kappa B2 mutant is fused with the with no (lysine) deficient protein kinase 1 gene, coding for a 58 kDa protein that retains the DNA binding Rel homology domain, but it lacks the entire ankyrin repeat inhibitory domain, thus suggesting its constitutive activation. This rearranged NF-kappa B2 gene product (p58) was localized in the nucleus, and formed a complex with NF-kappaB p65 or RelB. Moreover, a T-cell line expressing p58 increased the amount of an NF-kappa B2-inducible gene, NF-kappa B2/p100 by itself. These results suggest that such NF-kappa B2 gene rearrangement may therefore be a factor in the constitutive activation of NF-kappaB in ATL, and thereby playing a role in the ATL pathogenesis.

Evaluation of data-dependent versus targeted shotgun proteomic approaches for monitoring transcription factor expression in breast cancer.

In breast cancer, there is a significant degree of molecular diversity among tumors. Multiple perturbations in signal transduction pathways impinge on transcriptional networks that in turn dictate malignant transformation and metastatic progression. Detailed knowledge of the sequence-specific transcription factors that become activated or repressed within a tumor and comparison of their relative levels of expression in cancer versus normal tissue should therefore provide insight into disease mechanisms, improving patient stratification and facilitating personalized treatment. While high-throughput tandem mass spectrometry methods for global proteome profiling have been developed, existing approaches have limited sensitivity and are often unable to detect low-abundance transcription factors in a complex biological specimen like a biopsy or tumor cell extract. To this end, we have undertaken a systematic comparative evaluation of three MS/MS methods for the ability to detect reference transcription factors spiked in known amounts into a cell-free breast cancer nuclear extract: Data-Dependent Acquisition (DDA), wherein precursor ion intensity dictates selection for fragmentation; Targeted Peptide Monitoring (TPM), a directed approach using successive isolation and fragmentation of predefined m/ z ratios; and Multiple Reaction Monitoring (MRM), in which specific precursor ion to product ion transitions are selectively monitored. Through a series of controlled, parallel benchmarking experiments, we have determined the relative figures-of-merit of each approach, and have established that prior knowledge of signature proteotypic peptides markedly improves overall detection sensitivity, reliability, and quantification.

Varicella-zoster virus modulates NF-kappaB recruitment on selected cellular promoters.

Intercellular adhesion molecule 1 (ICAM-1) expression is down-regulated in the center of cutaneous varicella lesions despite the expression of proinflammatory cytokines such as gamma interferon and tumor necrosis factor alpha (TNF-alpha). To study the molecular basis of this down-regulation, the ICAM-1 induction of TNF-alpha was analyzed in varicella-zoster virus (VZV)-infected melanoma cells (MeWo), leading to the following observations: (i) VZV inhibits the stimulation of icam-1 mRNA synthesis; (ii) despite VZV-induced nuclear translocation of p65, p52, and c-Rel, p50 does not translocate in response to TNF-alpha; (iii) the nuclear p65 present in VZV-infected cells is no longer associated with p50 and is unable to bind the proximal NF-kappaB site of the icam-1 promoter, despite an increased acetylation and accessibility of the promoter in response to TNF-alpha; and (iv) VZV induces the nuclear accumulation of the NF-kappaB inhibitor p100. VZV also inhibits icam-1 stimulation of TNF-alpha by strongly reducing NF-kappaB nuclear translocation in MRC5 fibroblasts. Taken together, these data show that VZV interferes with several aspects of the immune response by inhibiting NF-kappaB binding and the expression of target genes. Targeting NF-kappaB activation, which plays a central role in innate and adaptive immune responses, leads to obvious advantages for the virus, particularly in melanocytes, which are a site of viral replication in the skin.