Histone deacetylase inhibitors repress macrophage migration inhibitory factor (MIF) expression by targeting MIF gene transcription through a local chromatin deacetylation.

The cytokine macrophage migration inhibitory factor plays a central role in inflammation, cell proliferation and tumorigenesis. Moreover, macrophage migration inhibitory factor levels correlate with tumor aggressiveness and metastatic potential. Histone deacetylase inhibitors are potent antitumor agents recently introduced in the clinic. Therefore, we hypothesized that macrophage migration inhibitory factor would represent a target of histone deacetylase inhibitors. Confirming our hypothesis, we report that histone deacetylase inhibitors of various chemical classes strongly inhibited macrophage migration inhibitory factor expression in a broad range of cell lines, in primary cells and in vivo. Nuclear run on, transient transfection with macrophage migration inhibitory factor promoter reporter constructs and transduction with macrophage migration inhibitory factor expressing adenovirus demonstrated that trichostatin A (a prototypical histone deacetylase inhibitor) inhibited endogenous, but not episomal, MIF gene transcription. Interestingly, trichostatin A induced a local and specific deacetylation of macrophage migration inhibitory factor promoter-associated H3 and H4 histones which did not affect chromatin accessibility but was associated with an impaired recruitment of RNA polymerase II and Sp1 and CREB transcription factors required for basal MIF gene transcription. Altogether, this study describes a new molecular mechanism by which histone deacetylase inhibitors inhibit MIF gene expression, and suggests that macrophage migration inhibitory factor inhibition by histone deacetylase inhibitors may contribute to the antitumorigenic effects of histone deacetylase inhibitors.

The GraftConnector experience. Long-term patency and histological work up in an animal model.

BACKGROUND: A device to perform sutureless end-to-side coronary artery anastomosis has been developed by means of stent technology (GraftConnector). The present study assesses the long-term quality of the GraftConnector anastomosis in a sheep model. METHODS: In 8 adult sheep, 40-55 kg in weight, through left anterior thoracotomy, the right internal mammary artery (RIMA) was prepared and connected to the left anterior descending artery (LAD) by means of GraftConnector, on beating heart, without using any stabilizer. Ticlopidine 250 mg/day for anticoagulation for 4 weeks and Aspirin 100 mg/day for 6 months were given. The animals were sacrificed after 6 months and histological examination of anastomoses was carried out after slicing with the connector in situ for morphological analysis. RESULTS: All animals survived at 6 months. All anastomoses were patent and mean luminal width at histology was 1.8 +/- 0.2 mm; mean myotomia hyperplasia thickness was 0.21 +/- 0.1 mm. CONCLUSIONS: Long-term results demonstrate that OPCABGs performed with GraftConnector had 100% patency rate. The mean anastomotic luminal width corresponds to mean LAD's adult sheep diameter. We may speculate that myotomia hyperplasia occurred as a result of local device oversizing.

Absence of ectopic epithelial inclusions in 3,904 axillary lymph nodes examined in sentinel technique.

Intraoperative examination of sentinel axillary lymph nodes can be done by imprint cytology, frozen section, or, most recently, by PCR-based amplification of a cytokeratin signal. Using this technique, benign epithelial inclusions, representing mammary tissue displaced along the milk line, will likely generate a positive PCR signal and lead to a false-positive diagnosis of metastatic disease. To better appreciate the incidence of ectopic epithelial inclusions in axillary lymph nodes, we have performed an autopsy study, examining on 100 μm step sections 3,904 lymph nodes obtained from 160 axillary dissections in 80 patients. The median number of lymph nodes per axilla was 23 (15, 6, and 1 in levels 1, 2, and 3, respectively). A total of 30,450 hematoxylin-eosin stained slides were examined, as well as 8,825 slides immunostained with pan-cytokeratin antibodies. Despite this meticulous work-up, not a single epithelial inclusion was found in this study, suggesting that the incidence of such inclusions is much lower than the assumed 5% reported in the literature.

Viral superantigen drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Mouse mammary tumor virus (MMTV[SW]) encodes a superantigen expressed by infected B cells. It evokes an antibody response specific for viral envelope protein, indicating selective activation of antigen-specific B cells. The response to MMTV(SW) in draining lymph nodes was compared with the response to haptenated chicken gamma globulin (NP-CGG) using flow cytometry and immunohistology. T cell priming occurs in both responses, with T cells proliferating in association with interdigitating dendritic cells in the T zone. T cell proliferation continues in the presence of B cells in the outer T zone, and B blasts then undergo exponential growth and differentiation into plasma cells in the medullary cords. Germinal centers develop in both responses, but those induced by MMTV(SW) appear later and are smaller. Most T cells activated in the T zone and germinal centers in the MMTV(SW) response are superantigen specific and these persist for weeks in lymph nodes draining the site MMTV(SW) injection: this contrasts with the selective loss of superantigen-specific T cells from other secondary lymphoid tissues. The results indicate that this viral superantigen, when expressed by professional antigen-presenting cells, drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Non steroidal anti-inflammatory drugs and COX-2 inhibitors as anti-cancer therapeutics: hypes, hopes and reality.

Non-steroidal anti-inflammatory drugs (NSAIDs) and specific inhibitors of cyclooxygenase (COX)-2, are therapeutic groups widely used for the treatment of pain, inflammation and fever. There is growing experimental and clinical evidence indicating NSAIDs and COX-2 inhibitors also have anti-cancer activity. Epidemiological studies have shown that regular use of Aspirin and other NSAIDs reduces the risk of developing cancer, in particular of the colon. Molecular pathology studies have revealed that COX-2 is expressed by cancer cells and cells of the tumor stroma during tumor progression and in response to chemotherapy or radiotherapy. Experimental studies have demonstrated that COX-2 over expression promotes tumorigenesis, and that NSAIDs and COX-2 inhibitors suppress tumorigenesis and tumor progression. Clinical trials have shown that NSAIDs and COX-2 inhibitors suppress colon polyp formation and malignant progression in patients with familial adenomatous polyposis (FAP) syndrome. Recent advances in the understanding of the cellular and molecular mechanisms of the anti-cancer effects of NSAIDs and COX-2 inhibitors have demonstrated that these drugs target both tumor cells and the tumor vasculature. The therapeutic benefits of COX-2 inhibitors in the treatment of human cancer in combination with chemotherapy or radiotherapy are currently being tested in clinical trials. In this article we will review recent advances in the understanding of the anti-tumor mechanisms of these drugs and discuss their potential application in clinical oncology.

FBXW7α attenuates inflammatory signalling by downregulating C/EBPδ and its target gene Tlr4.

Toll-like receptor 4 (Tlr4) has a pivotal role in innate immune responses, and the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ, Cebpd) is a Tlr4-induced gene. Here we identify a positive feedback loop in which C/EBPδ activates Tlr4 gene expression in macrophages and tumour cells. In addition, we discovered a negative feedback loop whereby the tumour suppressor FBXW7α (FBW7, Cdc4), whose gene expression is inhibited by C/EBPδ, targets C/EBPδ for degradation when C/EBPδ is phosphorylated by GSK-3β. Consequently, FBXW7α suppresses Tlr4 expression and responses to the ligand lipopolysaccharide. FBXW7α depletion alone is sufficient to augment pro-inflammatory signalling in vivo. Moreover, as inflammatory pathways are known to modulate tumour biology, Cebpd null mammary tumours, which have reduced metastatic potential, show altered expression of inflammation-associated genes. Together, these findings reveal a role for C/EBPδ upstream of Tlr4 signalling and uncover a function for FBXW7α as an attenuator of inflammatory signalling.

Characterization of the effects of radiation therapy on the tumour microenvironment and their consequences on cancer progression

RESUME La radiothérapie est utilisée avec succès pour le traitement d'un grand nombre de pathologies tumorales (1). Cependant, les récidives post-actiniques sont associées à un risque accru de développer des métastases régionales et à distance (2, 3). La prise en charge de ce type de patients demeure insatisfaisante à l'heure actuelle, principalement parce que les mécanismes physio-pathologiques sous- sous-jacents restent mal compris. Etant donné le rôle primordial du stroma dans la progression tumorale (4) et l'importance des effets de la radiothérapie sur le micro-environnement des tumeurs (5), nous avons émis l'hypothèse que la radiothérapie pouvait engendrer des modifications stromales susceptibles de contribuer à l'émergence d'un phénotype tumoral plus agressif. Nous avons observé que l'exposition préalable d'un environnement tumoral à des radiations ionisantes engendre une inhibition locale et à long terme de l'angiogenèse. Cette inhibition conduit à la création d'un environnement tumoral hypoxique favorisant l'invasion et la métastatisation tumorale. Les mécanismes sous-jacents impliquent l'activation de gènes prométastatiques sous le contrôle du facteur de transcription HIF-1, ainsi que la sélection hypoxique de cellules hautement invasives et métastatiques. Par des analyses de profile d'expression génétique ainsi que par des analyses fonctionnelles, nous avons identifié la protéine matri-cellulaire CYR61 ainsi que ses partenaires d'interaction, les intégrines aVb5/aVb3, comme médiateurs importants de ces effets. De plus, une corrélation significative a également été trouvée entre le niveau d'expression de CYR61 et le taux d'hypoxie dans un grand nombre de carcinomes mammaires chez l'humain. Une association a aussi été observée entre le niveau d'expression de CYR61 et le pronostic de patientes souffrant d'un cancer du sein traité par chimiothérapie adjuvante. Globalement ces résultats identifient l'interaction entre la protéine CYR61 et ses récepteurs aVb5/aVb3 comme un mécanisme important du processus de métastatisation et en font une cible thérapeutique potentielle pour le traitement de patients souffrant d'une récidive tumorale après un traitement de radiothérapie. Finalement, bien que l'inhibition de l'angiogenèse soit locale dans ce cas particulier, nos résultats justifient une surveillance particulière des patients souffrant d'une pathologie tumorale et étant au bénéfice d'un traitement inhibiteur de l'angiogenèse. SUMMARY Radiotherapy is successfully used to treat a large variety of tumours (1 ). However, cancer patients experiencing local recurrent disease after radiation therapy are at increased risk of developing regional and distant metastasis (2, 3). The clinical management of this condition represents a difficult and challenging issue, mainly because the underlying physio-pathological mechanisms remain poorly understood. Given the well established role of the tumour stroma in promoting cancer progression (4) and since radiotherapy is known to persistently alter the tumour microenvironment (5), we hypothesized that ionising radiations may generate stromal modifications contributing to the metastatic spread of relapsing tumours. Here, we report that irradiation of the prospective tumour microenvironment promotes tumour invasion and metastasis through a mechanism of local and sustained impairment of angiogenesis leading to both HIF-1 dependent activation of pro-metastatic genes and hypoxia-mediated selection of highly metastatic tumour cell variants. Through gene expression profiling and functional experiments, we identified the matricellular signalling protein CYR61 and its interaction partners aVb5/ aVb3 integrins as critical mediators of these effects. Furthermore, we found a significant correlation between CYR61 expression and the hypoxic status of a large number of human mammary carcinomas. A positive correlation between increased levels of CYR61 expression and shorter relapse free survival was also identified in breast cancer patients treated with adjuvant chemotherapy. Together, these results identify CYR61 and aVb5/aVb3 integrins as critical mediators of metastasis and potential therapeutic targets to improve outcome in patients with post-radiation tumour recurrences. Finally, although inhibition of angiogenesis is local in this setting, our data warrant close monitoring of tumour progression in patients under anti-angiogenic therapy.

Inter- and intra-individual variation of oxytocin secretion in oestrous mares exposed to stallions

The neuro-peptide hormone oxytocin regulates several reproductive mechanisms in mammals, such as uterine contractions during parturition and milk ejection in the lactating mammary gland. Oxytocin may also influence behavior and behavioral strategies, e.g. pair bonding, social recognition, maternal behavior, trust building, or anxiety. Teasing oestrous mares by a stallion provokes the release of oxytocin. We therefore tested whether such elevated oxytocin levels reveal possible mate preferences as determined in typical preference tests.

Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape.

Malignant melanoma accounts for most of the increasing mortality from skin cancer. Melanoma cells were found to express Fas (also called Apo-1 or CD95) ligand (FasL). In metastatic lesions, Fas-expressing T cell infiltrates were proximal to FasL+ tumor cells. In vitro, apoptosis of Fas-sensitive target cells occurred upon incubation with melanoma tumor cells; and in vivo, injection of FasL+ mouse melanoma cells in mice led to rapid tumor formation. In contrast, tumorigenesis was delayed in Fas-deficient lpr mutant mice in which immune effector cells cannot be killed by FasL. Thus, FasL may contribute to the immune privilege of tumors.

Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumor spheroids.

The binding and penetration of two 125I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) and their F(ab')2 and Fab fragments were measured in multicellular spheroids of poorly (HT29) and moderately well differentiated (Co112) human colon adenocarcinomas which express different amounts of CEA. Spheroids cultured in vitro model tumor microenvironments where poor vascular supply may modulate antigen expression and accessibility. The two MAb studied, 202 and 35, were shown previously to react with different CEA epitopes and to have high affinities of 1.2 and 5.8 X 10(9) M-1, respectively. MAb 202 has also been shown to cross-react with antigens present on human granulocytes and normal epithelial cells from human lung and pancreas. Specific binding of intact MAb and fragments of both antibodies was demonstrated for both types of human colon carcinoma spheroids compared to mouse colon carcinoma (CL26) and mammary tumor (EMT6/Ro) spheroids. Total binding of MAb and fragments was greater (1.5- to 2.5-fold) after 4 h compared to 1 h of exposure; the amount of binding compared to control IgG1 was 5- to 30-fold greater after 1-h incubation and 15 to 200 times greater after 4 h. This binding was stable as demonstrated by short and long wash experiments at 37 degrees and 4 degrees C. The binding of F(ab')2 and Fab fragments of the anti-CEA MAb 35 to spheroids of human colon Co112 was almost 2-fold greater than that of the intact MAb. However, for MAb 202, the binding of intact MAb and F(ab')2 was greater than that of Fab fragments. In addition the binding of both intact and F(ab')2 fragments of MAb 202 was greater than that obtained with MAb 35. Specific binding of both antibodies to HT29 spheroids, which express less CEA, was decreased for MAb and fragments of both 202 and 35. Autoradiography and immunoperoxidase experiments were performed to determine the penetration of MAb and fragments after incubation with intact spheroids. Comparisons were made with labeled MAb directly applied to frozen sections of spheroids. F(ab')2 and Fab fragments of both antibodies were bound at the surface of intact spheroids and penetrated to eight to ten cells, but the intact MAb were localized mainly at the spheroid surface and the outer one to three cell layers. There was much less binding at the surfaces of HT29 compared to Co112 spheroids. An enzyme immunoassay using MAb 35 and 202 demonstrated that Co112 spheroids produced about 8-fold more CEA/mg of cell protein than did monolayer cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Subversion of host immune responses by viral superantigens.

Recent experiments with mouse mammary tumor virus indicate that expression of a virally encoded superantigen by B cells and its subsequent recognition by T cells are essential steps for amplification of infection and virus transmission. Preliminary results suggest that superantigens may also be expressed during retroviral infection in humans.

Distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features.

BACKGROUND: Differences exist between the proximal and distal colon in terms of developmental origin, exposure to patterning genes, environmental mutagens, and gut flora. Little is known on how these differences may affect mechanisms of tumorigenesis, side-specific therapy response or prognosis. We explored systematic differences in pathway activation and their clinical implications. MATERIALS AND METHODS: Detailed clinicopathological data for 3045 colon carcinoma patients enrolled in the PETACC3 adjuvant chemotherapy trial were available for analysis. A subset of 1404 samples had molecular data, including gene expression and DNA copy number profiles for 589 and 199 samples, respectively. In addition, 413 colon adenocarcinoma from TCGA collection were also analyzed. Tumor side-effect on anti-epidermal growth factor receptor (EGFR) therapy was assessed in a cohort of 325 metastatic patients. Outcome variables considered were relapse-free survival and survival after relapse (SAR). RESULTS: Proximal carcinomas were more often mucinous, microsatellite instable (MSI)-high, mutated in key tumorigenic pathways, expressed a B-Raf proto-oncogene, serine/threonine kinase (BRAF)-like and a serrated pathway signature, regardless of histological type. Distal carcinomas were more often chromosome instable and EGFR or human epidermal growth factor receptor 2 (HER2) amplified, and more frequently overexpressed epiregulin. While risk of relapse was not different per side, SAR was much poorer for proximal than for distal stage III carcinomas in a multivariable model including BRAF mutation status [N = 285; HR 1.95, 95% CI (1.6-2.4), P < 0.001]. Only patients with metastases from a distal carcinoma responded to anti-EGFR therapy, in line with the predictions of our pathway enrichment analysis. CONCLUSIONS: Colorectal carcinoma side is associated with differences in key molecular features, some immediately druggable, with important prognostic effects which are maintained in metastatic lesions. Although within side significant molecular heterogeneity remains, our findings justify stratification of patients by side for retrospective and prospective analyses of drug efficacy and prognosis.

CD93 is required for maintenance of antibody secretion and persistence of plasma cells in the bone marrow niche.

Plasma cells represent the end stage of B-cell development and play a key role in providing an efficient antibody response, but they are also involved in numerous pathologies. Here we show that CD93, a receptor expressed during early B-cell development, is reinduced during plasma-cell differentiation. High CD93/CD138 expression was restricted to antibody-secreting cells both in T-dependent and T-independent responses as naive, memory, and germinal-center B cells remained CD93-negative. CD93 was expressed on (pre)plasmablasts/plasma cells, including long-lived plasma cells that showed decreased cell cycle activity, high levels of isotype-switched Ig secretion, and modification of the transcriptional network. T-independent and T-dependent stimuli led to re-expression of CD93 via 2 pathways, either before or after CD138 or Blimp-1 expression. Strikingly, while humoral immune responses initially proceeded normally, CD93-deficient mice were unable to maintain antibody secretion and bone-marrow plasma-cell numbers, demonstrating that CD93 is important for the maintenance of plasma cells in bone marrow niches.