Innate Immune Responses of Drosophila melanogaster Are Altered by Spaceflight

Alterations and impairment of immune responses in humans present a health risk for space exploration missions. The molecular mechanisms underpinning innate immune defense can be confounded by the complexity of the acquired immune system of humans. Drosophila (fruit fly) innate immunity is simpler, and shares many similarities with human innate immunity at the level of molecular and genetic pathways. The goals of this study were to elucidate fundamental immune processes in Drosophila affected by spaceflight and to measure host-pathogen responses post-flight. Five containers, each containing ten female and five male fruit flies, were housed and bred on the space shuttle (average orbit altitude of 330.35 km) for 12 days and 18.5 hours. A new generation of flies was reared in microgravity. In larvae, the immune system was examined by analyzing plasmatocyte number and activity in culture. In adults, the induced immune responses were analyzed by bacterial clearance and quantitative real-time polymerase chain reaction (qPCR) of selected genes following infection with E. coli. The RNA levels of relevant immune pathway genes were determined in both larvae and adults by microarray analysis. The ability of larval plasmatocytes to phagocytose E. coli in culture was attenuated following spaceflight, and in parallel, the expression of genes involved in cell maturation was downregulated. In addition, the level of constitutive expression of pattern recognition receptors and opsonins that specifically recognize bacteria, and of lysozymes, antimicrobial peptide (AMP) pathway and immune stress genes, hallmarks of humoral immunity, were also reduced in larvae. In adults, the efficiency of bacterial clearance measured in vivo following a systemic infection with E. coli post-flight, remained robust. We show that spaceflight altered both cellular and humoral immune responses in Drosophila and that the disruption occurs at multiple interacting pathways

Antibody targeting of claudin-1 as a potential colorectal cancer therapy

Abstract Background Metastatic colorectal cancer (mCRC) is one of the major causes of cancer-related death. Despite the substantial progress in mCRC management, it remains important to identify new therapeutic options and biological markers for personalized medicine. Here, we investigated the expression of claudin-1 (CLDN1), a major tight junction transmembrane protein, in the different colorectal cancer (CRC) molecular subtypes and then assessed the anti-tumor effect of a new anti-CLDN1 monoclonal antibody (mAb). Methods Gene expression profiling and immunochemistry analysis of normal and tumor tissue samples from patients with stage IV CRC were used to determine CLDN1 gene expression. Then, the 6F6 mAb against CLDN1 extracellular part was generated. Its effect on CRC cell cycle, proliferation, survival and migration was assessed in vitro, using a 3D cell culture system, flow cytometry, clonogenic and migration assays. In vivo, 6 F6 mAb efficacy was evaluated in nude mice after subcutaneous xenografts or intrasplenic injection of CRC cells. Results Compared with normal mucosa where it was almost exclusively cytoplasmic, in CRC samples CLDN1 was overexpressed (p < 0.001) and mainly localized at the membrane. Moreover, it was differentially expressed in the various CRC molecular subtypes. The strongest expressions were found in the consensus molecular subtype CMS2 (p < 0.001), the transit-ampliflying (p < 0.001) and the C5 subtypes (p < 0.001). Lower CLDN1 expression predicted a better outcome in the molecular subtypes C3 and C5 (p = 0.012 and p = 0.004, respectively). CLDN1 targeting with the 6 F6 mAb led to reduction of survival, growth and migration of CLDN1-positive cells. In preclinical mouse models, the 6F6 mAb decreased tumor growth and liver metastasis formation. Conclusion Our data indicate that CLDN1 targeting with an anti-CLDN1 mAb results in decreased growth and survival of CRC cells. This suggests that CLDN1 could be a new potential therapeutic target

A calcium/cAMP signaling loop at the ORAI1 mouth drives channel inactivation to shape NFAT induction

ORAI1 constitutes the store-operated Ca2+ release-activated Ca2+ (CRAC) channel, but how this channel is turned off through Ca2+-dependent inactivation (CDI) remained unclear. Here the authors identify a spatially-restricted Ca2+/cAMP signaling crosstalk critical for mediating CDI which in turn regulates cellular Ca2+ signals and NFAT activation

STAT3 localizes to the ER, acting as a gatekeeper for ER-mitochondrion Ca2+ fluxes and apoptotic responses

STAT3 is an oncogenic transcription factor exerting its functions both as a canonical transcriptional activator and as a non-canonical regulator of energy metabolism and mitochondrial functions. While both activities are required for cell transformation downstream of different oncogenic stimuli, they rely on different post-translational activating events, namely phosphorylation on either Y705 (nuclear activities) or S727 (mitochondrial functions). Here, we report the discovery of the unexpected STAT3 localization to the endoplasmic reticulum (ER), from where it modulates ER-mitochondria Ca2+ release by interacting with the Ca2+ channel IP3R3 and facilitating its degradation. The release of Ca2+ is of paramount importance for life/death cell decisions, as excessive Ca2+ causes mitochondrial Ca2+ overload, the opening of the mitochondrial permeability transition pore, and the initiation of the intrinsic apoptotic program. Indeed, STAT3 silencing enhances ER Ca2+ release and sensitivity to apoptosis following oxidative stress in STAT3-dependent mammary tumor cells, correlating with increased IP3R3 levels. Accordingly, basal-like mammary tumors, which frequently display constitutively active STAT3, show an inverse correlation between IP3R3 and STAT3 protein levels. These results suggest that STAT3-mediated IP3R3 downregulation in the ER crucially contributes to its anti-apoptotic functions via modulation of Ca2+ fluxes

The SigmaR1 chaperone drives breast and colorectal cancer cell migration by tuning SK3-dependent Ca2+ homeostasis

International audienc

Potassium and Calcium Channel Complexes as Novel Targets for Cancer Research

International audienceThe intracellular Ca2+ concentration is mainly controlled by Ca2+ channels. These channels form complexes with K+ channels, which function to amplify Ca2+ flux. In cancer cells, voltage-gated/voltage-dependent Ca2+ channels and non-voltage-gated/voltage-independent Ca2+ channels have been reported to interact with K+ channels such as Ca2+-activated K+ channels and voltage-gated K+ channels. These channels are activated by an increase in cytosolic Ca2+ concentration or by membrane depolarization, which induces membrane hyperpolarization, increasing the driving force for Ca2+ flux. These complexes, composed of K+ and Ca2+ channels, are regulated by several molecules including lipids (ether lipids and cholesterol), proteins (e.g. STIM), receptors (e.g. S1R/SIGMAR1), and peptides (e.g. LL-37) and can be targeted by monoclonal antibodies, making them novel targets for cancer research

Additional file 2: Figure S1. of Antibody targeting of claudin-1 as a potential colorectal cancer therapy

CLDN1 gene (222549_at.) expression. a, in 17 normal colorectal mucosa (NM), 20 primary tumor (PT) samples and 19 hepatic metastases (HM); *** = p < 0.0001 (Kruskall Wallis/Dunn’s test). b, Ratio between CLDN1 expression in PT and CLDN1 expression in NM for the 15 paired NM and PT samples from patients with mCRC. Data from the Affymetrix GeneChip Human Genome U133 Array Set (GSE 62322). Figure S2. The 6F6 mAb is specific for CLDN1. a, Reactivity of the hybridoma supernatant 6F6 against CLDN1. Western blotting of protein extracts from SW480 cells stably transfected with CLDN1 and from SW620 cells transduced with shLUC (control) or ShCLDN1. FACS histograms show the binding of the hybridoma supernatant to CLDN1-positive cell lines (SW480-CLDN1 and SW620shLUC) (■), negative control (-----), CLDN1-negative cell lines (―). b, Immunofluorescence experiments in cells that express CLDN1 (SW480-CLDN1) or transfected with empty vector (SW480-pcDNA) using the 6 F6 mAb as primary antibody (green). Images were recorded using a 63X NA objective on a Leica inverted microscope. c, Surface plasmon resonance measurements of the interaction of 6F6 or of an irrelevant mAb (Irr) with membrane extracts from SW620 cells that express CLDN1. d, Cross-reactivity analysis of the 6F6 mAb towards other CLDN proteins. Top: The expression of the various CLDN proteins (as indicated) in cell lysates from parental or CLDN-transfected SW480 cells was tested by western blotting using the relevant antibodies; Bottom: FACS histograms of 6 F6 binding (10 μg/mL) to parental or CLDN-transfected SW480 cells. Gray, 6 F6 mAb; dotted line, no antibody; black line, irrelevant mAb. Figure S3. CLDN1 is expressed in various cancer cell lines a, FACS histograms of the 6F6 mAb binding (gray histogram) to different cancer cell lines (pancreatic cancer: PANC-1, BXPC-3; ovarian cancer: SKOV-3, IGROV-1; hepatocarcinoma: HUH7). b, Quantification of total CLDN1 expression in the cell lines used in a by western blotting using the anti-CLDN1 polyclonal antibody JAY-8. c, CLDN1 mRNA expression in cell lines from the Cancer Cell Line Encyclopedia ( http://www.broadinstitute.org/ccle ). Figure S4. Detection of apoptosis in Difi spheroids using the Celigo™ imaging system and the NucView™ 488 cell membrane-permeable fluorogenic caspase-3 substrate. Difi cells were seeded at a density of 104/ml in FluoroBrite™ DMEM supplemented with 10% fetal bovine serum and incubated or not (NT) with 100 μg/ml of the 6 F6 mAb, the anti-EGFR cetuximab (cetux) or an irrelevant mAb (IRR). The caspase-3 substrate was added (5 μM) at the same time. Images were acquired at day 5. The bright-field and caspase 3 (green) images were merged (top panels) and the histogram (lower panel) represents the mean fluorescence intensity; * = p < 0.05 (t-test). Figure S5. Effects of the 6F6 mAb on cancer cell migration in vitro. a, Wound healing assay: confluent SW620 cell monolayers were scratched and then grown in the presence or not (NT) of 100 μg/ml of the 6 F6 mAb or irrelevant antibody (IRR). Images were captured at day 0 (D0) and day 5 (D5) after wounding. b, Cell migration assay in Boyden chambers. Caco2 cells were pre-incubated or not (NT) with 100 μg/ml of 6F6 or irrelevant (IRR) mAb. Data used for statistical analysis were from at least three independent experiments; **p < 0.01 (Kruskall Wallis/Dunn’s test). (PPTX 2370 kb

Additional file 1: Table S1. of Antibody targeting of claudin-1 as a potential colorectal cancer therapy

Distribution of patients with mCRC according to the tumor molecular subtype. (DOCX 33 kb

Additional file 3: of Antibody targeting of claudin-1 as a potential colorectal cancer therapy

Supplementary methods: Flow cytometry experiments. Immunofluorescence studies. Surface plasmon resonance measurements. Cell migration assays. Apoptosis assay. (DOCX 37 kb