Polyploid Giant Cancer Cells Are Frequently Found in the Urine of Prostate Cancer Patients.

Prostate cancer is the third cause of cancer-related deaths in men. Its early and reliable diagnosis is still a public health issue, generating many useless prostate biopsies. Prostate cancer cells detected in urine could be the target of a powerful test but they are considered too rare. By using an approach targeting rare cells, we have analyzed urine from 45 patients with prostate cancer and 43 healthy subjects under 50 y.o. We observed a relevant number of giant cells in patients with cancer. Giant cells, named Polyploid Giant Cancer Cells (PGCC), are thought to be involved in tumorigenesis and treatment resistance. We thus performed immune-morphological studies with cancer-related markers such as α-methylacyl-CoA racemase (AMACR), prostate-specific membrane antigen (PSMA), and telomerase reverse transcriptase (TERT) to understand if the giant cells we found are PGCC or other urinary cells. We found PGCC in the urine of 22 patients, including those with early-stage prostate cancer, and one healthy subject. Although these results are preliminary, they provide, for the first time, clinical evidence that prostate cancers release PGCC into the urine. They are expected to stimulate further studies aimed at understanding the role of urinary PGCC and their possible use as a diagnostic tool and therapeutic target.

Circulating cancer giant cells with unique characteristics frequently found in patients with myelodysplastic syndromes (MDS).

Myelodysplastic syndromes (MDS) are incurable diseases characterized by dysplastic hematopoietic cells, cytopenias in the blood and an inherent tendency for transformation to secondary acute myeloid leukemia (AML). Since most therapies fail to prevent rapid clonal evolution and disease resistance, new and non-invasive predictive markers are needed to monitor patients and adapt the therapeutic strategy. By using ISET, a very sensitive approach to isolate cells larger than mature leukocytes from peripheral blood samples, we looked for cellular markers in 99 patients (158 samples) with MDS and 66 healthy individuals (76 samples) used as controls. We found a total of 680 Giant Cells, defined as cells having a size of 40 microns or larger in 46 MDS patients (80 samples) and 28 Giant Cells in 11 healthy individuals (11 samples). In order to understand if we had enriched from peripheral blood atypical cells of the megakaryocyte line, we studied the Giant Cells using immunolabeling with megakaryocytes and tumor-specific markers. We report that the Giant Cells we found in the peripheral blood of MDS patients primarily express tumor markers. Our results show that Polyploid Giant Cancer Cells (PGCC), similar to those described in solid tumors, are found in the peripheral blood of patients with MDS and suggest the working hypothesis that they could play a role in hematological malignancies.

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion.

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.

Toll-like receptor 9 deficiency protects mice against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. While a number of studies have demonstrated the roles of TLR2, TLR4 and TLR5 in host defense againt P. aeruginosa infection, the implication of TLR9 in this process has been overlooked. Here, we show that P. aeruginosa DNA stimulates the inflammatory response through TLR9 pathway in both a cell line and primary alveolar macrophages (AMs). This activation requires asparagine endopeptidase- and endosomal acidification. Interestingly, TLR9-/- mice resisted to lethal lung infection by P. aeruginosa, compared to WT C57BL/6 mice. The resistance of TLR9-/- mice to P. aeruginosa infection was associated with: (i) a higher ability of TLR9-/- AMs to kill P. aeruginosa; (ii) a rapid increase in the pro-inflammatory cytokines such as TNFα, IL-1ß and IL-6 production; and (iii) an increase in nitric oxide (NO) production and inductible NO synthase expression in AMs. In addition, inhibition of both IL-1ß and NO production resulted in a significant decrease of P. aeruginosa clearance by AMs. Altogether these results indicate that TLR9 plays a detrimental role in pulmonary host defense toward P. aeruginosa by reducing the AMs clearance activity and production of IL-1ß and NO necessary for bacteria killing.

A role for 12R-lipoxygenase in MUC5AC expression by respiratory epithelial cells.

Eicosanoids are metabolites of arachidonic acid produced by cyclooxygenases (COXs) or lipoxygenases (LOXs). They mediate inflammation and mucus secretion in chronic pulmonary inflammatory diseases. The gel-forming mucin MUC5AC is over-expressed in the airways of patients with these diseases. MUC5AC expression is mediated by an extracellular signal-regulated kinase (ERK)/Sp1 dependent mechanism. Our aim was to study the role of eicosanoids and their signalling pathways in MUC5AC expression. Inhibitors of 12-LOX, but not those of COX, 5-LOX or 15-LOX, reduce MUC5AC expression induced by phorbol myristate acetate (PMA) in the bronchial epithelial cell line NCI-H292. These inhibitors also abrogate the production of whole mucus by cell monolayers. Two forms of 12-LOX (R and S) exist in mammals. Using siRNAs we show that 12R-LOX but not 12S-LOX is involved in MUC5AC expression induced by PMA, lipopolysaccharide or transforming growth factor-α. 12R-LOX also participates in MUC2 and MUC5B expression, although to a lesser extent than for MUC5AC. Contrarily, 12R-LOX silencing does not modify interleukin-8 production. 12-LOX inhibitors reduce ERK activation and Sp1 translocation induced by PMA. Moreover, the 12R-LOX product 12(R)-hydroxyeicosatetraenoic acid, induces MUC5AC expression, ERK activation and Sp1 translocation. 12R-LOX is involved in MUC5AC expression. This occurs via ERK- and Sp1-signalling pathways.

c-mip impairs podocyte proximal signaling and induces heavy proteinuria.

Idiopathic nephrotic syndrome comprises several podocyte diseases of unknown origin that affect the glomerular podocyte, which controls the permeability of the filtration barrier in the kidney to proteins. It is characterized by the daily loss of more than 3 g of protein in urine and the lack of inflammatory lesions or cell infiltration. We found that the abundance of c-mip (c-maf inducing protein) was increased in the podocytes of patients with various acquired idiopathic nephrotic syndromes in which the podocyte is the main target of injury. Mice engineered to have excessive c-mip in podocytes developed proteinuria without morphological alterations, inflammatory lesions, or cell infiltration. Excessive c-mip blocked podocyte signaling by preventing the interaction of the slit diaphragm transmembrane protein nephrin with the tyrosine kinase Fyn, thereby decreasing phosphorylation of nephrin in vitro and in vivo. Moreover, c-mip inhibited interactions between Fyn and the cytoskeletal regulator N-WASP (neural Wiskott-Aldrich syndrome protein) and between the adaptor protein Nck and nephrin, potentially accounting for cytoskeletal disorganization and the effacement of foot processes seen in idiopathic nephrotic syndromes. The intravenous injection of small interfering RNA targeting c-mip prevented lipopolysaccharide-induced proteinuria in mice. Together, these results identify c-mip as a key component in the molecular pathogenesis of acquired podocyte diseases.

C-mip interacts with the p85 subunit of PI3 kinase and exerts a dual effect on ERK signaling via the recruitment of Dip1 and DAP kinase.

In naive T cells, Lck exerts a negative control on the ERK/MAPK pathway. We show that c-mip (c-maf inducing protein) interacts with the p85 subunit of PI3 kinase and inactivates Lck, which results in Erk1/2 and p38 MAPK activation. This effect is not enough to activate AP1 given the inability of ERK to migrate into the nucleus and to transactivate its target genes. We demonstrate that c-mip interacts with Dip1 and upregulates DAPK, which blocks the nuclear translocation of ERK1/2. This dual effect of c-mip is unique and might represent a potential mechanism to prevent the development of an immune response.

Identification of functional corticosteroid response elements involved in regulation of Cacna1g expression in cardiac myocytes.

We recently reported that corticosteroids increase the expression of the T-type channel Ca(v)3.1 through a transcriptional up-regulation of the Ca(v)3.1 encoding gene cacna1g. The nucleotide sequence analysis of cacna1g promoter revealed putative glucocorticoid response elements (GREs). However, the functional GREs involved in the regulation of cacna1g expression in neonatal cardiac myocytes are unknown. In the present study we have investigated the nuclear targets responsible for the transcriptional regulation of cacna1g. We identified five GREs from the nucleotide sequence of cacna1g promoter. Additionally, using punctual mutagenesis approach, three functional categories of GREs have been identified: (i) GRE-1 involved in promoter activity induced by aldosterone (Aldo, 1 microM); (ii) GRE-4 and GRE-5 involved in promoter activity induced by dexamethasone (Dex, 1 microM); and (iii) GRE-2 and GRE-3 involved in the basal level of neonatal promoter activity. The data presented here lead to better understanding of the molecular mechanisms underlying the regulation of Ca(v)3.1 channel expression by corticosteroids. These new findings have attractive physiological features during cardiac development and pathology such as arrhythmias.

Regulation of T-type Cav3.1 channels expression by synthetic glucocorticoid dexamethasone in neonatal cardiac myocytes.

The effect of the dexamethasone (Dex) on the regulation of the T-type Ca(2+) channel expressions was investigated in primary cultures of neonatal rat ventricular myocytes. We found that Dex (1 microM) increases the T-type Ca(2+) current (I(CaT)) associated with an increase in Ca(v)3.1 mRNA amount. We isolated the upstream region from Ca(v)3.1 encoding gene and tested the activity of the promoter in transfected ventricular myocytes. We found a minimal Dex-responsive region that displayed putative glucocorticoid receptor (GR) and nuclear factor kappa-B (NFkappaB) targets. The GR selective antagonist, RU38486 (10 microM), nearly turned off the transcriptional activity of Ca(v)3.1 encoding gene, and an NFkappaB inhibitor, pyrrolodine dithiocarbonate (10 microM), completely abolished the Dex-induced mRNA increase. However, Dex-induced GR and NFkappaB synthesis and nuclear translocation were not timely related to Ca(v)3.1 mRNA increase. These results indicate that both GR and NFkappaB were necessary, but not sufficient, to trigger the increase in Ca(v)3.1 mRNA amount. This study showed the relationship between glucocorticoid and T-type channels up-regulation that may be involved in cardiac development and pathology.