Tiziana Guarnieri

IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland

High serum levels of IL-6 correlate with poor outcome in breast cancer patients. However, no data are available on the relationship between IL-6 and mammary stem/progenitor cells, which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in the MCF-7 breast cancer cell line and in primary human mammospheres (MS), multicellular structures enriched in stem/progenitor cells of the mammary gland. MS from node invasive breast carcinoma tissues expressed IL-6 mRNA at higher levels than did MS from matched non-neoplastic mammary glands. In addition, IL-6 mRNA was detected only in basal-like breast carcinoma tissues, an aggressive breast carcinoma variant showing stem cell features. IL-6 treatment triggered Notch-3-dependent upregulation of the Notch ligand Jagged-1 and promotion of MS and MCF-7-derived spheroid growth. Moreover, IL-6 induced Notch-3-dependent upregulation of the carbonic anhydrase IX gene and promoted a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, autocrine IL-6 signaling relied upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, these data support the hypothesis that IL-6 induces malignant features in Notch-3-expressing stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

The basal-like breast carcinoma phenotype is regulated by SLUG gene expression.

Basal‐like breast carcinoma is an aggressive form of breast cancer, characterized by the absence of oestrogen receptor and HER2 expression, the presence of cytokeratin 5 and epidermal growth factor receptor expression, and by the up‐regulation of stem cell regulatory genes. We show here that tumour tissues expressing high levels of SLUG mRNA show a basal‐like breast carcinoma phenotype and that such tumours also express high levels of stem cell‐regulatory genes, ie CD133, Bmi1. Further, we show that stem/progenitor cells, isolated from ductal breast carcinoma and from normal mammary gland as mammospheres, express SLUG, CD133, and Bmi1 mRNA and show a phenotype similar to that of basal‐like breast carcinoma. We also report that SLUG expression in tumour tissues correlates with that of the hypoxia survival gene carbonic anhydrase IX. In this regard, we report that the exposure of SLUG‐negative/luminal‐like MCF‐7 cells to a hypoxic environment promotes the onset of the basal‐like breast carcinoma phenotype, together with up‐regulation of the SLUG gene, which in turn blunts oestrogen receptor‐α and boosts carbonic anhydrase IX gene expression. Finally, we show that SLUG expression promotes the invasiveness of MCF‐7 cells exposed to hypoxia and sustains the in vivo aggressiveness of hypoxia‐selected, MCF‐7‐derived cells in xenografts. These data indicate that SLUG gene expression is part of a hypoxia‐induced genetic programme which sets up a basal/stem cell‐like, aggressive phenotype in breast cancer cells. Copyright

TNFalpha up-regulates SLUG via the NF-kappaB/HIF1alpha axis, which imparts breast cancer cells with a stem cell-like phenotype

Extracellular and intracellular mediators of inflammation, such as tumor necrosis factor alpha (TNFα) and NF‐kappaB (NF‐κB), play major roles in breast cancer pathogenesis, progression and relapse. SLUG, a mediator of the epithelial–mesenchymal transition process, is over‐expressed in CD44+/CD24− tumor initiating breast cancer cells and in basal‐like carcinoma, a subtype of aggressive breast cancer endowed with a stem cell‐like gene expression profile. Cancer stem cells also over‐express members of the pro‐inflammatory NF‐κB network, but their functional relationship with SLUG expression in breast cancer cells remains unclear. Here, we show that TNFα treatment of human breast cancer cells up‐regulates SLUG with a dependency on canonical NF‐κB/HIF1α signaling, which is strongly enhanced by p53 inactivation. Moreover, SLUG up‐regulation engenders breast cancer cells with stem cell‐like properties including enhanced expression of CD44 and Jagged‐1 in conjunction with estrogen receptor alpha down‐regulation, growth as mammospheres, and extracellular matrix invasiveness. Our results reveal a molecular mechanism whereby TNFα, a major pro‐inflammatory cytokine, imparts breast cancer cells with stem cell‐like features, which are connected to increased tumor aggressiveness. J. Cell. Physiol. 225: 682–691, 2010.

Environmental immune disruptors, inflammation and cancer risk

An emerging area in environmental toxicology is the role that chemicals and chemical mixtures have on the cells of the human immune system. This is an important area of research that has been most widely pursued in relation to autoimmune diseases and allergy/asthma as opposed to cancer causation. This is despite the well-recognized role that innate and adaptive immunity play as essential factors in tumorigenesis. Here, we review the role that the innate immune cells of inflammatory responses play in tumorigenesis. Focus is placed on the molecules and pathways that have been mechanistically linked with tumor-associated inflammation. Within the context of chemically induced disturbances in immune function as co-factors in carcinogenesis, the evidence linking environmental toxicant exposures with perturbation in the balance between pro- and anti-inflammatory responses is reviewed. Reported effects of bisphenol A, atrazine, phthalates and other common toxicants on molecular and cellular targets involved in tumor-associated inflammation (e.g. cyclooxygenase/prostaglandin E2, nuclear factor kappa B, nitric oxide synthesis, cytokines and chemokines) are presented as example chemically mediated target molecule perturbations relevant to cancer. Commentary on areas of additional research including the need for innovation and integration of systems biology approaches to the study of environmental exposures and cancer causation are presented.

Nuclear receptors agonists exert opposing effects on the inflammation dependent survival of breast cancer stem cells.

Recent literature highlights the importance of pro-inflammatory cytokines in the biology of breast cancer stem cells (CSCs), unraveling differences with respect to their normal counterparts. Expansion of mammospheres (MS) is a valuable tool for the in vitro study of normal and cancer mammary gland stem cells. Here, we expanded MSs from human breast cancer and normal mammary gland tissues, as well from tumorigenic (MCF7) and non-tumorigenic (MCF10) breast cell lines. We observed that agonists for the retinoid X receptor (6-OH-11-O-hydroxyphenanthrene), retinoic acid receptor (all-trans retinoic acid (RA)) and peroxisome proliferator-activated receptor (PPAR)-γ (pioglitazone (PGZ)), reduce the survival of MS generated from breast cancer tissues and MCF7 cells, but not from normal mammary gland or MCF10 cells. This phenomenon is paralleled by the hampering of pro-inflammatory Nuclear Factor-κB (NF-κB)/Interleukin-6 (IL6) axis that is hyperactive in breast cancer-derived MS. The hindrance of such pathway associates with the downregulation of MS regulatory genes (SLUG, Notch3, Jagged1) and with the upregulation of the differentiation markers estrogen receptor-α and keratin18. At variance, the PPARα agonist Wy14643 promotes MS formation, upregulating NF-κB/IL6 axis and MS regulatory genes. These data reveal that nuclear receptors agonists (6-OH-11-O-hydroxyphenanthrene, RA, PGZ) reduce the inflammation dependent survival of breast CSCs and that PPARα agonist Wy14643 exerts opposite effects on this phenotype.

Localization of NADPH-diaphorase in the goldfish brain.

The distribution of NADPH-diaphorase positive neurons was studied by using the enzyme histochemical method. Numerous neurons were labeled in different brain areas of the goldfish and their distribution showed some differences in comparison with other studied teleosts, indicating a species-specific pattern of NADPH-diaphorase distribution as observed in mammals. The localization of NADPH-diaphorase in the thalamic nuclei, in the paraventricular organ, in the inferior hypothalamic lobe, in the periventricular neurons of the optic tectum, in the nucleus isthmi and in the mesencephalic reticular formation was comparable to the one observed in other teleosts. In addition in the goldfish the telencephalic neurons of the pars centralis and lateralis of the area dorsalis, the habenular neurons, the bipolar neurons of the central grey layer of the optic tectum and the motor neurons of the hypertrophied vagal lobe were labeled. The localization of NADPH-diaphorase positive neurons, compared to the distribution of cholinergic neurons described in fish, indicated that the production of nitric oxide was prevailing in the brain areas where cholinergic circuits are active.

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in goldfish brain

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which selectively damages dopaminergic neurons in mammals, caused a marked depletion of tyrosine hydroxylase (TH) immunoreactivity in the goldfish brain. The concomitant ultrastructural observations showed the neurotoxic effect of MPTP on telencephalic, diencephalic and medullar neurons. The affected neurons revealed darkening of the cytoplasm and swelling of the mitochondria and the endoplasmic reticulum. Concomitant significant decreases in dopamine (DA) and noradrenaline (NA) levels were determined in the brain areas where morphological observations were performed. The loss of catecholamine levels was completely prevented by the treatment with the monoamine oxidase (MAO) inhibitor pargyline to prevent MPTP oxidation. The results indicate that in goldfish brain, acute MPTP administration causes selective catecholamine depletion, without altering the serotoninergic system.

Choline acetyltransferase and NADPH-diaphorase localization in the goldfish habenulo-interpeduncular system

The habenular nuclei are the major sources of projections to the interpeduncular nucleus. The habenular neurons of the goldfish are ChAT and NADPH-diaphorase positive. The localization of these two enzymes in the habenulae suggests the involvement of acetylcholine and nitric oxide (the product of NADPH-diaphorase activity in the nervous tissue) in the habenulo-interpeduncular connection. This finding is supported by the presence of ChAT and NADPH-diaphorase activity in the neuropilar area of the interpeduncular nucleus. This activity was depleted 12 days after habenular ablation. The overlap of ChAT and NADPH-diaphorase localization in goldfish habenular neurons is consistent with previous observations on the co-localization of these two enzymes in some brainstem neurons of other vertebrates. It is, however, not identical to the localization of NADPH-diaphorase in the habenulae and in the interpeduncular nucleus of the rat.

Peroxisome Proliferator Activated Receptor-α/Hypoxia Inducible Factor-1α Interplay Sustains Carbonic Anhydrase IX and Apoliprotein E Expression in Breast Cancer Stem Cells

Aims Cancer stem cell biology is tightly connected to the regulation of the pro-inflammatory cytokine network. The concept of cancer stem cells “inflammatory addiction” leads to envisage the potential role of anti-inflammatory molecules as new anti-cancer targets. Here we report on the relationship between nuclear receptors activity and the modulation of the pro-inflammatory phenotype in breast cancer stem cells. Methods Breast cancer stem cells were expanded as mammospheres from normal and tumor human breast tissues and from tumorigenic (MCF7) and non tumorigenic (MCF10) human breast cell lines. Mammospheres were exposed to the supernatant of breast tumor and normal mammary gland tissue fibroblasts. Results In mammospheres exposed to the breast tumor fibroblasts supernatant, autocrine tumor necrosis factor-α signalling engenders the functional interplay between peroxisome proliferator activated receptor-α and hypoxia inducible factor-1α (PPARα/HIF1α). The two proteins promote mammospheres formation and enhance each other expression via miRNA130b/miRNA17-5p-dependent mechanism which is antagonized by PPARγ. Further, the PPARα/HIF1α interplay regulates the expression of the pro-inflammatory cytokine interleukin-6, the hypoxia survival factor carbonic anhydrase IX and the plasma lipid carrier apolipoprotein E. Conclusion Our data demonstrate the importance of exploring the role of nuclear receptors (PPARα/PPARγ) in the regulation of pro-inflammatory pathways, with the aim to thwart breast cancer stem cells functioning.

The decrease of cell membrane fluidity by the non-steroidal anti-inflammatory drug Licofelone inhibits epidermal growth factor receptor signalling and triggers apoptosis in HCA-7 colon cancer cells

The ability to induce changes in cell membrane properties is nowadays considered an additional mechanism to explain the pharmacological effects of non-steroidal anti-inflammatory drugs (NSAIDs). We previously demonstrated that the NSAID Licofelone, a dual cyclooxygenase/5-lipoxygenase inhibitor, triggers apoptosis in HCA-7 colon cancer cells independently from the inhibition of these enzymes. Here, we provide evidence that, in HCA-7 cells, the pro-apoptotic effect of this drug relies, at least in part, on its ability to inhibit epidermal growth factor receptor (EGFR) signalling by a decrease of cell membrane fluidity. Indeed, Licofelone induced a relevant change in the relative proportions of some saturated, monounsaturated and polyunsaturated fatty acids constituting HCA-7 phospholipid fraction and significantly increased the levels of cholesterol in HCA-7 cell membrane. All of these changes resulted in a remarkable decrease of membrane fluidity. Such phenomenon was associated with the block of EGFR kinase activity and of its downstream targets, the p44-42 mitogen-activated protein kinase (MAPK) and AKT cascades, whose inhibitions were found to induce apoptosis in HCA-7 cells. Overall, these findings provide a new additional mechanism by which NSAIDs are effective toward colon cancer cells.