Patient-Reported Quality of Life 6 Years After Breast Cancer.

This cohort study assesses quality-of-life trajectories up to 6 years after breast cancer diagnosis among individuals in France.

Regulation of eIF4F Translation Initiation Complex by the Peptidyl Prolyl Isomerase FKBP7 in Taxane-resistant Prostate Cancer.

PURPOSE: Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets for overcoming chemoresistance.Experimental Design: We established 4 chemoresistant prostate cancer cell lines and used image-based high-content siRNA functional screening, based on gene-expression signature, to explore mechanisms of chemoresistance and identify new potential targets with potential roles in taxane resistance. The functional role of a new target was assessed by in vitro and in vivo silencing, and mass spectrometry analysis was used to identify its downstream effectors. RESULTS: We identified FKBP7, a prolyl-peptidyl isomerase overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. This is the first study to characterize the function of human FKBP7 and explore its role in cancer. We discovered that FKBP7 was upregulated in human prostate cancers and its expression correlated with the recurrence observed in patients receiving docetaxel. FKBP7 silencing showed that FKBP7 is required to maintain the growth of chemoresistant cell lines and chemoresistant tumors in mice. Mass spectrometry analysis revealed that FKBP7 interacts with eIF4G, a component of the eIF4F translation initiation complex, to mediate the survival of chemoresistant cells. Using small-molecule inhibitors of eIF4A, the RNA helicase component of eIF4F, we were able to kill docetaxel- and cabazitaxel-resistant cells. CONCLUSIONS: Targeting FKBP7 or the eIF4G-containing eIF4F translation initiation complex could be novel therapeutic strategies to eradicate taxane-resistant prostate cancer cells.

Accuracy of several maternal seric markers for predicting histological chorioamnionitis after preterm premature rupture of membranes: a prospective and multicentric study.

OBJECTIVE: To assess and compare several maternal seric markers for the prediction of histological chorioamnionitis (HCA) after preterm premature rupture of membranes (PPROM). Study design A prospective and multicentric observational study was undertaken, including six French tertiary referral centres. Pregnant women over 18 years, with PPROM between 22+0 and 36+6 WG were enrolled. A blood sample was obtained before delivery and analysed for C-Reactive Protein (CRP), InterCellular Adhesion Molecule-1 (ICAM-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Matrix-Metalloproteinase 8 and 9 (MMP-8, MMP-9), Triggering receptor on myeloid cells (TREM-1), and Human Neutrophile Peptides (HNP). HCA was determined by histological examination distinguishing maternal from fetal inflammatory response. Placental analyses and biological assays were performed in duplicate. Comparison of maternal seric markers levels in women with or vs. without HCA was performed, using a non-parametric Receiver Operating Characteristic. RESULTS: 295 women were kept for analysis. The prevalence of HCA was 42.7% (126/295). The concentrations of MMP-8, MMP-9, HNP and CRP were higher in HCA vs. the non-HCA group (P<0.05) whereas the concentrations of ICAM- 1, IL-6, IL-8 were not different (P>0.05). The ROC curve with the largest AUC was for CRP (AUC; 0.70; 95% CI; 0.64-0.77) and it was significantly higher than those for MMP-8, MMP-9, or HNP (P<0.03). CONCLUSION: CRP was the best maternal marker for predicting HCA in women with PPROM.

Targeting CDC25C, PLK1 and CHEK1 to overcome Docetaxel resistance induced by loss of LZTS1 in prostate cancer.

Docetaxel is used as a standard treatment in patients with metastatic castration-resistant prostate cancer. However, a large subset of patients develops resistance. Understanding resistance mechanisms, which are largely unknown, will allow identification of predictive biomarkers and therapeutic targets. We established resistant IGR-CaP1 prostate cancer cell lines for different doses of Docetaxel. We investigated gene expression profiles by microarray analyses in these cell lines and generated a signature of 99 highly differentially expressed genes potentially implicated in chemoresistance. We focused on the role of the cell cycle regulator LZTS1, which was under-expressed in the Docetaxel-resistant cell lines, its inhibition resulting from the promoter methylation. Knockdown of LZTS1 in parental cells with siRNA showed that LZTS1 plays a role in the acquisition of the resistant phenotype. Furthermore, we observed that targeting CDC25C, a partner of LZTS1, with the NSC663284 inhibitor specifically killed the Docetaxel-resistant cells. To further investigate the role of CDC25C, we used inhibitors of the mitotic kinases that regulate CDC25C. Inhibition of CHEK1 and PLK1 induced growth arrest and cell death in the resistant cells. Our findings identify an important role of LZTS1 through its regulation of CDC25C in Docetaxel resistance in prostate cancer and suggest that CDC25C, or the mitotic kinases CHEK1 and PLK1, could be efficient therapeutic targets to overcome Docetaxel resistance.

[Last updates about amniotic fluid embolism and a case report]. / L'embolie amniotique : données récentes et illustration.

Amniotic fluid embolism (AFE) is a rare complication of pregnancy, which, in France, is the second leading cause of maternal mortality. It results from the passage of amniotic fluid into the maternal circulation, but mechanisms leading to clinical signs are not yet clearly identified. After a literature review, we report on the case of a 34 years old patient occurred during labor. Laboratory tests were performed during acute phase to diagnose coagulopathy and to monitor vital parameters. Further laboratory tests were conducted to support the diagnosis: cytology, study of insulin-like growth factor-binding protein 1, tryptase and alpha-foetoprotein. Performances and place of these laboratory tests must be precised as no specific test can attest the diagnosis.

The IGR-CaP1 xenograft model recapitulates mixed osteolytic/blastic bone lesions observed in metastatic prostate cancer.

Bone metastases have a devastating impact on quality of life and bone pain in patients with prostate cancer and decrease survival. Animal models are important tools in investigating the pathogenesis of the disease and in developing treatment strategies for bone metastases, but few animal models recapitulate spontaneous clinical bone metastatic spread. In the present study, IGR-CaP1, a new cell line derived from primary prostate cancer, was stably transduced with a luciferase-expressing viral vector to monitor tumor growth in mice using bioluminescence imaging. The IGR-CaP1 tumors grew when subcutaneously injected or when orthotopically implanted, reconstituted the prostate adenocarcinoma with glandular acini-like structures, and could disseminate to the liver and lung. Bone lesions were detected using bioluminescence imaging after direct intratibial or intracardiac injections. Anatomic bone structure assessed using high-resolution computed tomographic scans showed both lytic and osteoblastic lesions. Technetium Tc 99m methylene diphosphonate micro single-photon emission computed tomography confirmed the mixed nature of the lesions and the intensive bone remodeling. We also identified an expression signature for responsiveness of IGR-CaP1 cells to the bone microenvironment, namely expression of CXCR4, MMP-9, Runx2, osteopontin, osteoprotegerin, ADAMTS14, FGFBP2, and HBB. The IGR-CaP1 cell line is a unique model derived from a primary tumor, which can reconstitute human prostate adenocarcinoma in animals and generate experimental bone metastases, providing a novel means for understanding the mechanisms of bone metastasis progression and allowing preclinical testing of new therapies.

Unique heme-iron coordination by the hemoglobin receptor IsdB of Staphylococcus aureus.

Iron is an essential requirement for life for nearly all organisms. The human pathogen Staphylococcus aureus is able to acquire iron from the heme cofactor of hemoglobin (Hb) released from lysed erythrocytes. IsdB, the predominant Hb receptor of S. aureus, is a cell wall-anchored protein that is composed of two NEAT domains. The N-terminal NEAT domain (IsdB-N1) binds Hb, and the C-terminal NEAT domain (IsdB-N2) relays heme to IsdA for transport into the cell. Here we present the 1.45 Å resolution X-ray crystal structure of the IsdB-N2-heme complex. While the structure largely conforms to the eight-strand ß-sandwich fold seen in other NEAT domains such as IsdA-N and uses a conserved Tyr residue to coordinate heme-iron, a Met residue is also involved in iron coordination, resulting in a novel Tyr-Met hexacoordinate heme-iron state. The kinetics of the transfer of heme from IsdB-N2 to IsdA-N can be modeled as a two-step process. The rate of transfer of heme between the isolated NEAT domains (82 s(-1)) was found to be similar to that measured for the full-length proteins. Replacing the iron coordinating Met with Leu did not abrogate high-affinity heme binding but did reduce the heme transfer rate constant by more than half. This unusual Met-Tyr heme coordination may also bestow properties on IsdB that help it to bind heme in different oxidation states or extract heme from hemoglobin.

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer.

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2ß1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.

Structural biology of heme binding in the Staphylococcus aureus Isd system.

Iron is an absolute requirement for nearly all organisms, but most bacterial pathogens are faced with extreme iron-restriction within their host environments. To overcome iron limitation pathogens have evolved precise mechanisms to steal iron from host supplies. Staphylococcus aureus employs the iron-responsive surface determinant (Isd) system as its primary heme-iron uptake pathway. Hemoglobin or hemoglobin-haptoglobin complexes are bound by Near iron-Transport (NEAT) domains within cell surface anchored proteins IsdB or IsdH. Heme is stripped from the host proteins and transferred between NEAT domains through IsdA and IsdC to the membrane transporter IsdEF for internalization. Once internalized, heme can be degraded by IsdG or IsdI, thereby liberating iron for the organism. Most components of the Isd system have been structurally characterized to provide insight into the mechanisms of heme binding and transport. This review summarizes recent research on the Isd system with a focus on the structural biology of heme recognition.

The decreased susceptibility of metastatic melanoma cells to killing involves an alteration of CTL reactivity.

Metastases are known to be more resistant to therapy than matching primary tumors, in particular they are less prone to apoptosis. In this study we investigated the functional interaction of a CTL clone (LT12) specific for a melanoma TA with the primary tumor (T1) versus its metastatic counterpart (G1). The CTL clone (LT12) was shown to lyse the primary T1 cells more efficiently in a classical cytotoxicity test. This differential susceptibility was not associated with MHC class I down-regulation and conjugate formation but correlated with a differential increase in Ca++ flux in the LT12 CTL when stimulated with the primary versus the metastatic tumor cells. Since LT12 uses perforin/granzyme B to kill its autologous target we analysed perforin and granzyme B mRNA expression in the CTL in the presence of either primary and metastatic melanoma cells. Quantitative PCR analysis showed an increased expression of granzyme B and perforin mRNA levels in LT12 when cocultured in the presence of the primary tumor. However, a similar level of (cytotoxic molecule) degranulation as revealed by CD107 expression was observed when LT12 was stimulated with T1 or G1 cells. These data suggest that the differential susceptibility of primary and metastatic melanoma cells involves at least in part their distinct potential to induce autologous CTL reactivity and the subsequent triggering of granzyme B and perforin in these cells.

Identification of target actin content and polymerization status as a mechanism of tumor resistance after cytolytic T lymphocyte pressure.

To investigate tumor resistance to T cell lysis, a resistant variant was selected after specific cytolytic T lymphocytes (CTL) selection pressure. Although the resistant variant triggered perforin and granzyme B transcription in specific CTLs, as well as their degranulation, it exhibited a dramatic resistance to cytotoxic T cell killing. It also displayed strong morphological changes with alterations of the actin cytoskeleton. Electron microscopy analysis revealed a loosen interaction between CTLs and the resistant variant despite the formation of apparently normal conjugates. Transcriptional profiling identified a gene expression signature that distinguished sensitive from resistant tumor targets. More notably, we found that actin-related genes ephrin-A1 and scinderin were overexpressed in resistant target. Silencing of these genes using RNA interference resulted in a restoration of normal cell morphology and a significant attenuation of variant resistance to CTL killing. Our present study shows that a shift in cytoskeletal organization can be used, by tumor cells, as a strategy to promote their resistance after CTL selection pressure.

Human NK cells lyse organ-specific endothelial cells: analysis of adhesion and cytotoxic mechanisms.

Human organ-specific microvascular endothelial cells (ECs) were established and used in the present study to investigate their susceptibility to natural killer cell line (NKL)-induced lysis. Our data indicate that although IL-2-stimulated NKL (NKL2) cells adhered to the human peripheral (HPLNEC.B3), mesenteric lymph node (HMLNEC), brain (HBrMEC), and lung (HLMEC) and skin (HSkMEC.2) ECs, they significantly killed these cells quite differently. A more pronounced lysis of OSECs was also observed when IL-2-stimulated, purified peripheral blood NK cells were used as effector cells. In line with the correlation observed between adhesion pattern and the susceptibility to NKL2-mediated killing, we demonstrated using different chelators that the necessary adhesion step was governed by an Mg(2+)-dependent, but Ca(2+)-independent, mechanism as opposed to the subsequent Ca(2+)-dependent killing. To identify the cytotoxic pathway used by NKL2 cells, the involvement of the classical and alternate pathways was examined. Blocking of the Ca(2+)-dependent cytotoxicity pathway by EGTA/MgCl(2) significantly inhibited endothelial target cell killing, suggesting a predominant role for the perforin/granzyme pathway. Furthermore, using confocal microscopy, we demonstrated that the interaction between NKL2 effectors and ECs induced cytochrome c release and Bid translocation in target cells, indicating an involvement of the mitochondrial pathway in NKL2-induced EC death. In addition, although all tested cells were sensitive to the cytotoxic action of TNF, no susceptibility to TRAIL or anti-Fas mAb was observed. The present studies emphasize that human NK cell cytotoxicity toward ECs may be a potential target to block vascular injury.

Prion protein prevents human breast carcinoma cell line from tumor necrosis factor alpha-induced cell death.

To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor alpha (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrP(c)), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrP(c) is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrP(c,) our data demonstrate that PrP(c) overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.

CD158 receptor controls cytotoxic T-lymphocyte susceptibility to tumor-mediated activation-induced cell death by interfering with Fas signaling.

Renal cell carcinoma-infiltrating lymphocytes express killer cell immunoglobulin-like receptors (KIRs) that inhibit antitumor CD8+ T-cell functions and may contribute to local self-tolerance. In the present study, to better examine the functional consequences of KIR engagement on CTL-tumor interactions, we investigated the influence of KIR2DL1/CD158a on CTL survival. We show that both KIR+ and KIR- antigen-specific CTLs express Fas and Fas ligand and were susceptible to activation-induced cell death (AICD) triggered by coated anti-CD3 monoclonal antibodies. In KIR+ CTLs, anti-CD158a monoclonal antibodies partially inhibited anti-CD3-induced AICD. Interestingly, T-cell receptor activation by cognate tumor cells induced apoptosis in KIR+ CTLs but not in KIR- CTLs. In addition, co-engagement of T-cell receptors and KIRs by tumor cells decreased tumor-mediated CTL apoptosis. Blocking the interaction of KIR/HLA-Cw4 resulted in the restoration of tumor-induced AICD. Most importantly, our data indicate that KIR engagement affected two proximal events of Fas signaling pathway, a sustained c-FLIP-L induction and a decrease in caspase 8 activity. These studies provide evidence that tumor cells selectively favor the local persistence of nonfunctional KIR+ CTLs by promoting their survival.

[Tumor/cytotoxic effector cross-talk in the control of tumor susceptibility to lysis]. / Le dialogue effecteur cytotoxique/tumeur dans le contrôle de la susceptibilité tumorale à la lyse.

During the two least decades, the field of tumor immunology has met an expansion of knowledge about the molecular and cellular bases of immune regulation. The identification of cancer antigens has been of critical importance and cancer vaccine is at present a very fast moving field. However, the immunotherapy approaches in cancer are of modest success. This is mainly due to the capacity of tumor cells to escape from immunological detection and to resist to cell mediated cytotoxicity. We will discuss some mechanisms associated with the acquisition of this tumor resistance and the alteration of T cell function and how cancer profiling through genomics approaches may help to reconceptualize immunotherapy strategies.

Analysis of the mechanisms of human cytotoxic T lymphocyte response inhibition by NO.

NO is a potent cellular mediator which has been shown to modulate several immune mechanisms. Using human T lymphocytes as responder cells in a primary mixed lymphocyte reaction, we demonstrated that, at the initiation of the culture, exogenously provided NO via sodium nitroprusside, in non-toxic concentrations, inhibited both allogeneic proliferative and primary cytotoxic responses in a dose-dependent manner. In contrast, it had no effect on the cytotoxic activity of established human TCR (alpha)beta and TCR (gamma)delta cytotoxic T lymphocyte (CTL) clones. The NO inhibitory effect on primary cytotoxic T cell response correlates with inhibition of T cell blastogenesis. Furthermore, under our stimulation conditions, NO induced an inhibition of IL-2 production, an alteration of IL-2R(alpha) expression, and a down-regulation of NF-AT translocation in CD4(+) and CD8(+)allostimulated T cells. Furthermore, we demonstrate that the inhibition of allospecific CTL activity by the NO donor was at least in part related to an inhibition of granzyme B and Fas ligand transcription as revealed respectively by RNase protection and RT-PCR analysis. These results suggest that NO may function to fine tune human CD3(+) T cell activation and subsequent CTL generation.

Engagement of the inhibitory receptor CD158a interrupts TCR signaling, preventing dynamic membrane reorganization in CTL/tumor cell interaction.

Renal cell carcinoma (RCC) infiltrating lymphocytes (TILs) express killer cell immunoglobulinlike receptors (KIRs) that inhibit the antitumor CD8(+) T-cell lysis. In the present study, to better examine the functional consequences of KIR engagement on cytotoxic T lymphocyte (CTL)/tumor interaction, we have investigated the influence of KIR CD158a on early steps of T-cell activation. We show that coengagement of T-cell receptor (TCR) and CD158a by tumor cells inhibited tyrosine phosphorylation of early signaling proteins ZAP-70 and LAT, lipid raft coalescence, and TCR/CD3 accumulation at the CTL/tumor cell interface. In addition, the guanine exchange factor Vav was not phosphorylated, and no actin cytoskeleton rearrangement was observed. Our data indicate a role of KIR CD158a in the dynamic events induced by TCR triggering, preventing CTL membrane reorganization, and subsequent completion of CTL activation program. Accordingly, the expression of CD158 by TILs may favor tumor cell escape to the immune response.