FOXP3 EXPRESSION IN BREAST CARCINOMA CELLS AND METASTATIC SPREAD

Forkhead box P3 (FOXP3), a gene member of the forkhead/winged-helix family of transcription regulators, is implicated in regulating immune system development and function. This gene has been found to be of crucial importance for the generation of CD4+CD25+ regulatory T cells (Tregs). Tregs express both the full-length FOXP3 and \u3942FOXP3 natural splice variant. In addition to its expression in the lymphocyte lineage, studies have recently described FOXP3 expression in non-hematopoietic-derived cells, including cancerous or normal epithelial cells of multiple lineages and origins. The role of FOXP3 in cancer cells is still unclear. Our immunohistochemical (IHC) and statistical analyses of archival material from two old series of breast cancer patients indicated that the expression of FOXP3 in tumor cells is an independent strong prognostic factor for distant metastases. The impact of FOXP3 on patient survival has been confirmed by our IHC analysis on chemotherapy-treated breast cancer patients. In fact, FOXP3 positive patients had poorer disease-free survival compared to FOXP3-negative patients. To investigate FOXP3 role in breast cancer, its expression was assessed in a panel of breast carcinoma cell lines and in human primary breast carcinoma samples by Western blot analyses. Full lenght FOXP3 was detected in all human breast cancer samples and breast cancer cell lines analyzed, whereas the \u3942FOXP3 isoform was visible solely in human breast tumors. The involvement of 062FOXP3 in breast cancer and the possibility that this isoform could have a different role from that of full length FOXP3 in breast cancer progression has been investigated. WTFOXP3 or 062FOXP3 overexpression was induced in MDA-MB-231 breast cancer cell line. Both WTFOXP3 and \u3942FOXP3 overexpression significantly increased in vitro migration and invasion capability of breast cancer cells, whilst inhibiting breast cancer cell proliferation. Taking advantage of these in vitro results and to further investigate in vivo role of FOXP3 in breast cancer metastasis, the metastatic capability of WTFOXP3- or \u3942FOXP3-overexpressing MDA-MB-231 breast cancer cells was investigated. The mean number of spontaneous lung metastases was superimposable in WTFOXP3- and \u3942FOXP3-overexpressing tumor bearing mice (mean\ub1SD: 12.5\ub127.4 and 10.3\ub118.1, for WTFOXP3- and \u3942FOXP3-MDA-MB-231-injected mice, respectively). These findings do not support a role of \u3942FOXP3 isoform in promoting breast cancer metastasis. In our IHC analyses of breast carcinoma specimens subcellular staining of FOXP3 was observed to be cytoplasmic or cytoplasmic/nuclear. Since the role of FOXP3 is transcription regulation, which mainly occurs in the nucleus, a cytoplasmic FOXP3 localization could affect its biological role. Thus the hypothesis that FOXP3 in tumor cells may have distinct biological activities and prognostic values according to its subcellular localization was investigated. Metastatic capability of two breast cancer cell clones with inducible FOXP3 expression and with different FOXP3 subcellular localization was evaluated. FOXP3 overexpression in breast cancer cells with a predominantly nuclear FOXP3 localization led to a significant reduction in the number of both spontaneous and experimental lung metastases compared to controls (mice in which FOXP3 overexpression was not induced). Contrarily, in mice injected with breast cancer cells which showed a predominantly cytoplasmic FOXP3 localization, FOXP3 overexpression in tumor cells led to a significant increase in the number of lung metastatic lesions compared to control group. These results suggested that nuclear FOXP3 localization enable its transcriptional activity, resulting in an onco-suppressive effect, while its cytoplasmic localization unable this transcription factor to perform its biological functions, resulting in an opposite in vivo effect. Our findings indicate that FOXP3 subcellular localization in breast tumor cells is an important determinant of prognosis, supporting the involvement of this transcription factor in breast cancer metastasis. Additional studies are in progress to confirm these data and to better understand the molecular mechanisms involved in FOXP3 role in driving breast cancer metastasis

Decision Support System for Vulnerability Assessment of Masonry Churches Including Architectural and Artistic Assets

The heritage building stock represents a significant element at risk from earthquakes, as recent seismic events have shown, especially in the Mediterranean area. In fact, in the last few years, the issue of assessing its seismic vulnerability has been widely discussed by the scientific community. The vulnerability assessment procedures involve many critical points related to the complexity and uncertainty of the parameters involved. If a detailed analysis of the individual buildings is to be performed this of course requires a great effort in both the data retrieval, modelling and analysis phases. In particular, historical masonry churches have been studied in detail in Italy and empirical approaches have been proposed in which a vulnerability index based on the classification of recurrent failure mechanisms is defined, exploiting a macro-elements approach to identify the parameters that influence the index. On the other hand, intangible aspects related to the architectural, historical and artistic value are not included in the Index, either in the structural parts themselves or in additional non-structural elements or contents. This paper proposes a procedure that combines the well-known vulnerability analysis based on the macro-elements approach and classification of recurrent failure mechanisms with an evaluation of the church’s architectural and artistic assets, such as frescoes, statues and paintings, by applying the Analytic Hierarchy Process. The novel procedure is integrated in a useful Decision Support System to provide a complete overview of a church’s structural condition, including its artworks, in order to create a priority scale for the assessment, retrofitting and protection of existing masonry churches

Gut microbiota markers associated with obesity and overweight in Italian adults

In the present study, we characterized the distinctive signatures of the gut microbiota (GM) from overweight/obese patients (OB), and normal-weight controls (NW), both of Sardinian origin. Fecal bacterial composition of 46 OB patients (BMI = 36.6 ± 6.0; F/M = 40/6) was analyzed and compared to that of 46 NW subjects (BMI = 21.6 ± 2.1; F/M = 41/5), matched for sex, age and smoking status, by using 16S rRNA gene sequencing on MiSeq Illumina platform. The gut microbial community of OB patients exhibited a significant decrease in the relative abundance of several Bacteroidetes taxa (i.e. Flavobacteriaceae, Porphyromonadaceae, Sphingobacteriaceae, Flavobacterium, Rikenella spp., Pedobacter spp., Parabacteroides spp., Bacteroides spp.) when compared to NW; instead, several Firmicutes taxa were significantly increased in the same subjects (Lachnospiraceae, Gemellaceae, Paenibacillaceae, Streptococcaceae, Thermicanaceae, Gemella, Mitsuokella, Streptococcus, Acidaminococcus spp., Eubacterium spp., Ruminococcus spp., Megamonas spp., Streptococcus, Thermicanus, Megasphaera spp. and Veillonella spp.). Correlation analysis indicated that body fatness and waist circumference negatively correlated with Bacteroidetes taxa, while Firmicutes taxa positively correlated with body fat and negatively with muscle mass and/or physical activity level. Furthermore, the relative abundance of several bacterial taxa belonging to Enterobacteriaceae family, known to exhibit endotoxic activity, was increased in the OB group compared to NW. The results extend our knowledge on the GM profiles in Italian OB, identifying novel taxa linking obesity and intestine

Molecular evolution of the proopiomelanocortin system in Barn owl species.

Examination of genetic polymorphisms in outbred wild-living species provides insights into the evolution of complex systems. In higher vertebrates, the proopiomelanocortin (POMC) precursor gives rise to α-, β-, and γ-melanocyte-stimulating hormones (MSH), which are involved in numerous physiological aspects. Genetic defects in POMC are linked to metabolic disorders in humans and animals. In the present study, we undertook an evolutionary genetic approach complemented with biochemistry to investigate the functional consequences of genetic polymorphisms in the POMC system of free-living outbred barn owl species (family Tytonidae) at the molecular level. Our phylogenetic studies revealed a striking correlation between a loss-of-function H9P mutation in the β-MSH receptor-binding motif and an extension of a poly-serine stretch in γ3-MSH to ≥7 residues that arose in the barn owl group 6-8 MYA ago. We found that extension of the poly-serine stretches in the γ-MSH locus affects POMC precursor processing, increasing γ3-MSH production at the expense of γ2-MSH and resulting in an overall reduction of γ-MSH signaling, which may be part of a negative feedback mechanism. Extension of the γ3-MSH poly-serine stretches ≥7 further markedly increases peptide hormone stability in plasma, which is conserved in humans, and is likely relevant to its endocrine function. In sum, our phylogenetic analysis of POMC in wild living owls uncovered a H9P β-MSH mutation subsequent to serine extension in γ3-MSH to 7 residues, which was then followed by further serine extension. The linked MSH mutations highlight the genetic plasticity enabled by the modular design of the POMC gene

Impact of a Moderately Hypocaloric Mediterranean Diet on the Gut Microbiota Composition of Italian Obese Patients

Although it is known that the gut microbiota (GM) can be modulated by diet, the efficacy of specific dietary interventions in determining its composition and diversity in obese patients remains to be ascertained. The present work aims to evaluate the impact of a moderately hypocaloric Mediterranean diet on the GM of obese and overweight patients (OB). The GM of 23 OB patients (F/M = 20/3) was compared before (T0) and after 3 months (T3) of nutritional intervention (NI). Fecal samples were analyzed by Illumina MiSeq sequencing of the 16S rRNA gene. At baseline, GM characterization confirmed typical obesity-associated dysbiosis. After 3 months of NI, patients presented a statistically significant reduction in body weight and fat mass, along with changes in the relative abundance of many microbial patterns. In fact, an increase in the abundance of several Bacteroidetes taxa (i.e., Sphingobacteriaceae, Sphingobacterium, Bacteroides spp., Prevotella stercorea) and a depletion of many Firmicutes taxa (i.e., Lachnospiraceae members, Ruminococcaceae and Ruminococcus, Veillonellaceae, Catenibacterium, Megamonas) were observed. In addition, the phylum Proteobacteria showed an increased abundance, while the genus Sutterella, within the same phylum, decreased after the intervention. Metabolic pathways, predicted by bioinformatic analyses, showed a decrease in membrane transport and cell motility after NI. The present study extends our knowledge of the GM profiles in OB, highlighting the potential benefit of moderate caloric restriction in counteracting the gut dysbiosis

Clinical phenotypes of Parkinson’s disease associate with distinct gut microbiota and metabolome enterotypes

Parkinson’s disease (PD) is a clinically heterogenic disorder characterized by distinct clinical entities. Most studies on motor deficits dichotomize PD into tremor dominant (TD) or non-tremor dominant (non-TD) with akinetic-rigid features (AR). Different pathophysiological mechanisms may affect the onset of motor manifestations. Recent studies have suggested that gut microbes may be involved in PD pathogenesis. The aim of this study was to investigate the gut microbiota and metabolome composition in PD patients in relation to TD and non-TD phenotypes. In order to address this issue, gut microbiota and the metabolome structure of PD patients were determined from faecal samples using 16S next generation sequencing and gas chromatography–mass spectrometry approaches. The results showed a reduction in the relative abundance of Lachnospiraceae, Blautia, Coprococcus, Lachnospira, and an increase in Enterobacteriaceae, Escherichia and Serratia linked to non-TD subtypes. Moreover, the levels of important molecules (i.e., nicotinic acid, cadaverine, glucuronic acid) were altered in relation to the severity of phenotype. We hypothesize that the microbiota/metabolome enterotypes associated to non-TD subtypes may favor the development of gut inflammatory environment and gastrointestinal dysfunctions and therefore a more severe α-synucleinopathy. This study adds important information to PD pathogenesis and emphasizes the potential pathophysiological link between gut microbiota/metabolites and PD motor subtypes

FOXP3 expression in tumor cells and its role in cancer progression

Deep insight on FOXP3 expression in tumor cells and its role in cancer progression

Gut Microbiota and Metabolome Alterations Associated with Parkinson's Disease.

Parkinson's disease is a neurodegenerative disorder characterized by the accumulation of intracellular aggregates of misfolded alpha-synuclein along the cerebral axis. Several studies report the association between intestinal dysbiosis and Parkinson's disease, although a cause-effect relationship remains to be established. Herein, the gut microbiota composition of 64 Italian patients with Parkinson's disease and 51 controls was determined using a next-generation sequencing approach. A real metagenomics shape based on gas chromatography-mass spectrometry was also investigated. The most significant changes within the Parkinson's disease group highlighted a reduction in bacterial taxa, which are linked to anti-inflammatory/neuroprotective effects, particularly in the Lachnospiraceae family and key members, such as Butyrivibrio, Pseudobutyrivibrio, Coprococcus, and Blautia The direct evaluation of fecal metabolites revealed changes in several classes of metabolites. Changes were seen in lipids (linoleic acid, oleic acid, succinic acid, and sebacic acid), vitamins (pantothenic acid and nicotinic acid), amino acids (isoleucine, leucine, phenylalanine, glutamic acid, and pyroglutamic acid) and other organic compounds (cadaverine, ethanolamine, and hydroxy propionic acid). Most modified metabolites strongly correlated with the abundance of members belonging to the Lachnospiraceae family, suggesting that these gut bacteria correlate with altered metabolism rates in Parkinson's disease.IMPORTANCE To our knowledge, this is one of the few studies thus far that correlates the composition of the gut microbiota with the direct analysis of fecal metabolites in patients with Parkinson's disease. Overall, our data highlight microbiota modifications correlated with numerous fecal metabolites. This suggests that Parkinson's disease is associated with gut dysregulation that involves a synergistic relationship between gut microbes and several bacterial metabolites favoring altered homeostasis. Interestingly, a reduction of short-chain fatty acid (SCFA)-producing bacteria influenced the shape of the metabolomics profile, affecting several metabolites with potential protective effects in the Parkinson group. On the other hand, the extensive impact that intestinal dysbiosis has at the level of numerous metabolic pathways could encourage the identification of specific biomarkers for the diagnosis and treatment of Parkinson's disease, also in light of the effect that specific drugs have on the composition of the intestinal microbiota