Sergei A. Nedospasov

B cell–derived lymphotoxin promotes castration-resistant prostate cancer

Prostate cancer (CaP) progresses from prostatic intraepithelial neoplasia through locally invasive adenocarcinoma to castration-resistant metastatic carcinoma. Although radical prostatectomy, radiation and androgen ablation are effective therapies for androgen-dependent CaP, metastatic castration-resistant CaP is a major complication with high mortality. Androgens stimulate growth and survival of prostate epithelium and early CaP. Although most patients initially respond to androgen ablation, many develop castration-resistant CaP within 12–18 months. Despite extensive studies, the mechanisms underlying the emergence of castration-resistant CaP remain poorly understood and their elucidation is critical for developing improved therapies. Curiously, castration-resistant CaP remains androgen-receptor dependent, and potent androgen-receptor antagonists induce tumour regression in castrated mice. The role of inflammation in castration-resistant CaP has not been addressed, although it was reported that intrinsic NF-κB activation supports its growth. Inflammation is a localized protective reaction to injury or infection, but it also has a pathogenic role in many diseases, including cancer. Whereas acute inflammation is critical for host defence, chronic inflammation contributes to tumorigenesis and metastatic progression. The inflammation-responsive IκB kinase (IKK)-β and its target NF-κB have important tumour-promoting functions within malignant cells and inflammatory cells. The latter, including macrophages and lymphocytes, are important elements of the tumour microenvironment, but the mechanisms underlying their recruitment remain obscure, although they are thought to depend on chemokine and cytokine production. We found that CaP progression is associated with inflammatory infiltration and activation of IKK-α, which stimulates metastasis by an NF-κB-independent, cell autonomous mechanism. Here we show that androgen ablation causes infiltration of regressing androgen-dependent tumours with leukocytes, including B cells, in which IKK-β activation results in production of cytokines that activate IKK-α and STAT3 in CaP cells to enhance hormone-free survival.

A Lymphotoxin-Driven Pathway to Hepatocellular Carcinoma

Hepatitis B and C viruses (HBV and HCV) cause chronic hepatitis and hepatocellular carcinoma (HCC) by poorly understood mechanisms. We show that cytokines lymphotoxin (LT) alpha and beta and their receptor (LTbetaR) are upregulated in HBV- or HCV-induced hepatitis and HCC. Liver-specific LTalphabeta expression in mice induces liver inflammation and HCC, causally linking hepatic LT overexpression to hepatitis and HCC. Development of HCC, composed in part of A6(+) oval cells, depends on lymphocytes and IKappa B kinase beta expressed by hepatocytes but is independent of TNFR1. In vivo LTbetaR stimulation implicates hepatocytes as the major LT-responsive liver cells, and LTbetaR inhibition in LTalphabeta-transgenic mice with hepatitis suppresses HCC formation. Thus, sustained LT signaling represents a pathway involved in hepatitis-induced HCC.

Distinct Role of Surface Lymphotoxin Expressed by B Cells in the Organization of Secondary Lymphoid Tissues

In order to definitively ascertain the functional contribution of lymphotoxin (LT) expressed by B cells, we produced mice with the LTbeta gene deleted from B cells (B-LTbeta KO mice). In contrast to systemic LTbeta deletion, in B-LTbeta KO mice only splenic microarchitecture was affected, while lymph nodes and Peyers patches (PP) were normal, except for PPs reduced size. Even though B-LTbeta KO spleens retained a small number of follicular dendritic cells (FDC) which appeared to be dependent on LTbeta produced by T cells, IgG responses to sheep red blood cells were markedly reduced. Thus, the organogenic function of B-LTbeta is almost entirely restricted to spleen, where it supports the correct lymphoid architecture that is critical for an effective humoral immune response.

p53 is a suppressor of inflammatory response in mice

Chronic inflammation is known to promote cancer, suggesting that negative regulation of inflammation is likely to be tumor suppressive. We found that p53 is a general inhibitor of inflammation that acts as an antagonist of nuclear factor κB (NFκB). We first observed striking similarities in global gene expression profiles in human prostate cancer cells LNCaP transduced with p53 inhibitory genetic element or treated with TNF, suggesting that p53 inhibits transcription of TNF‐inducible genes that are largely regulated by NFκB. Consistently, ectopically expressed p53 acts as an inhibitor of transcription of NFκB‐dependent promoters. Furthermore, suppression of inflammatory response by p53 was observed in vivo in mice by comparing wild‐type and p53 null animals at molecular (inhibition of transcription of genes encoding cytokines and chemokines, reducing accumulation of reactive oxygen species and protein oxidation products), cellular (activation of macrophages and neutrophil clearance) and organismal (high levels of metabolic markers of inflammation in tissues of p53‐deficient mice and their hypersensitivity to LPS) levels. These observations indicate that p53, acting through suppression of NFκB, plays the role of a general “buffer” of innate immune response in vivo that is well consistent with its tumor suppressor function and frequent constitutive activation of NFκB in tumors.

Tumor Necrosis Factor Alpha Transcription in Macrophages Is Attenuated by an Autocrine Factor That Preferentially Induces NF-κB p50

ABSTRACT Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-α. This activity, termed TNF-α-inhibiting factor (TIF), suppressed the induction of TNF-α expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1β [IL-1β], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-α expression by macrophage conditioned medium was associated with selective induction of the NF-κB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-α promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-α gene. Repression of the TNF-α promoter by TIF required a distal region that includes three NF-κB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-α promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-α expression in activated macrophages. TIF is distinct from the known TNF-α-inhibiting factors IL-4, IL-10, and transforming growth factor β and may represent a novel cytokine.

Non-random cleavage of SV40 DNA in the compact minichromosome and free in solution by micrococcal nuclease

Abstract The distribution of the primary cleavage sites produced by micrococcal nuclease on SV40 DNA in the compact minichromosome was analysed. Minichromosomes purified by improved method were digested in mild conditions in order to minimize possible changes in nucleosomal and supranucleosomal arrangement. The primary cuts were found to be non-randomly and unevenly distributed through the whole SV40 genome and those located in the “early” half of SV40 DNA were mapped. Some of them were also preferentially attacked on the naked DNA treated under the same conditions. Possible relation of the results to the nucleosomal organization of the compact minichromosome is discussed.

B regulatory cells and the tumor-promoting actions of TNF-α during squamous carcinogenesis

The inflammatory cytokine TNF-α has been recognized as a critical tumor promoter, but the effector cells that mediate its action have not been fully characterized. Because B cells regulate squamous and prostate carcinogenesis, and Tnf−/− mice harbor B-cell defects, we investigated the hypothesis that B cells are important effector cells for TNF-α–mediated promotion of cancer development. Using an adoptive transfer strategy and the 7,12-dimethylbenz[α]anthracene/terephthalic acid (DMBA/TPA) two-stage model of skin carcinogenesis, we found that both B cells and TNF-α are critical for the development of DMBA/TPA-induced papilloma. Transfer of B cells from DMBA/TPA-treated wild-type mice to Tnf−/− mice rescued papilloma development to a wild-type level, a result not observed when B cells from Tnf−/− mice were transferred to Rag2−/− mice or when TNF-α was eliminated selectively in B cells. Resistance to papilloma development in Tnf−/− mice was associated with increased IFN-γ and CD8+ T cells in skin and a significant reduction in IL-10–producing B regulatory cells alongside an increase in IFN-γ–producing CD8+ T cells in the spleen. These data indicate that during DMBA/TPA-induced squamous carcinogenesis TNF-α mediates tumor-promoting activity via regulatory B cells that repress antitumor immunity.

Nonredundant Function of Soluble LTα3 Produced by Innate Lymphoid Cells in Intestinal Homeostasis

Command and Control Innate lymphoid cells are vital for the development of gut-associated lymphoid tissues, maintenance of the epithelial barrier, and protection against intestinal microbes; their dysfunction can promote immune pathology. Immunoglobulin A (IgA) production is important for maintenance of the gut epithelial barrier and the composition of the gut microbiota. Through the generation of knockout mouse models, Kruglov et al. (p. 1243) were able to distinguish how soluble and membrane-bound lymphotoxins expressed by innate lymphoid cells in the gut specifically regulate IgA production and thereby control gut microbiota composition. Soluble lymphotoxin plays a paracrine role in controlling immunoglobulin A responses and regulating gut microbiota. Immunoglobulin A (IgA) production at mucosal surfaces contributes to protection against pathogens and controls intestinal microbiota composition. However, mechanisms regulating IgA induction are not completely defined. We show that soluble lymphotoxin α (sLTα3) produced by RORγt+ innate lymphoid cells (ILCs) controls T cell–dependent IgA induction in the lamina propria via regulation of T cell homing to the gut. By contrast, membrane-bound lymphotoxin β (LTα1β2) produced by RORγt+ ILCs is critical for T cell–independent IgA induction in the lamina propria via control of dendritic cell functions. Ablation of LTα in RORγt+ cells abrogated IgA production in the gut and altered microbiota composition. Thus, soluble and membrane-bound lymphotoxins produced by ILCs distinctly organize adaptive immune responses in the gut and control commensal microbiota composition.

Complex NF-kappaB interactions at the distal tumor necrosis factor promoter region in human monocytes

We describe a dense cluster of DNA-protein interactions located 600 nucleotides upstream of the transcriptional start site of the human tumor necrosis factor (TNF) gene. This area was identified as being of potential importance for lipopolysaccharide-inducible TNF expression in the human monocyte cell line Mono Mac 6, based on reporter gene analysis of point mutations at a number of nuclear factor κB (NF-κB)-like motifs within the human TNF promoter region. The area contains two NF-κB sites, which are here shown by DNase I and methylation interference footprinting to flank a novel binding site. UV cross-linking studies reveal that the novel site can also bind NF-κB as well as an unknown protein(s) of approximately 40 kDa. We show that these three adjacent κB-binding sites differ markedly in their relative affinities for p50/p50, p65/p65, and p65/p50, yet this 39-nucleotide segment of DNA appears capable of binding up to three NF-κB heterodimers simultaneously. Reporter gene studies indicate that each element of the cluster contributes to lipopolysaccharide-induced transcriptional activation in Mono Mac 6 cells. These findings suggest that NF-κB acts in a complex manner to activate TNF transcription in human monocytes.

Crosstalk between the canonical NF-κB and Notch signaling pathways inhibits Pparγ expression and promotes pancreatic cancer progression in mice

The majority of human pancreatic cancers have activating mutations in the KRAS proto-oncogene. These mutations result in increased activity of the NF-κB pathway and the subsequent constitutive production of proinflammatory cytokines. Here, we show that inhibitor of κB kinase 2 (Ikk2), a component of the canonical NF-κB signaling pathway, synergizes with basal Notch signaling to upregulate transcription of primary Notch target genes, resulting in suppression of antiinflammatory protein expression and promotion of pancreatic carcinogenesis in mice. We found that in the Kras(G12D)Pdx1-cre mouse model of pancreatic cancer, genetic deletion of Ikk2 in initiated pre-malignant epithelial cells substantially delayed pancreatic oncogenesis and resulted in downregulation of the classical Notch target genes Hes1 and Hey1. Tnf-α stimulated canonical NF-κB signaling and, in collaboration with basal Notch signals, induced optimal expression of Notch targets. Mechanistically, Tnf-α stimulation resulted in phosphorylation of histone H3 at the Hes1 promoter, and this signal was lost with Ikk2 deletion. Hes1 suppresses expression of Pparg, which encodes the antiinflammatory nuclear receptor Pparγ. Thus, crosstalk between Tnf-α/Ikk2 and Notch sustains the intrinsic inflammatory profile of transformed cells. These findings reveal what we believe to be a novel interaction between oncogenic inflammation and a major cell fate pathway and show how these pathways can cooperate to promote cancer progression.