Induction of the sphingosine-1-phosphate signaling pathway by TGF-ß1 during Langerhans-type dendritic cell differentiation.

Gene profiling revealed that the S1P signaling pathway is induced by TGF-ß1 during LC commitment of monocytopoietic cells. Constitutive-active TGF-ß1-S1P signaling seems to elevate the activation threshold of LCs and thereby prevent inappropriate and overshooting immune responses to microbial and physicochemical environmental signals. In turn, signals that lead to LC migration may disrupt this pathway via inhibiting S1P bioavailability.

Singularity and Commonality in Response to SARS-CoV-2 in Lung and Colon Cell Models.

The systemic nature of COVID-19 with multiple extrapulmonary manifestations of disease, largely due to the wide tissue expression of SARS-CoV-2 major entry factors, as well as the patient-specific features of COVID-19 pathobiology, determine important directions for basic and translational research. In the current study, we addressed the questions of singularities and commonalities in cellular responses to SARS-CoV-2 and related SARS-CoV on the basis of compendium-wide analysis of publicly available transcriptomic datasets as part of the herein implemented multi-modular UNCOVIDING approach. We focused on cellular models attributed to the epithelial cells of the respiratory system, the Calu-3 cell line, and epithelial cells of the gastrointestinal tract, the Caco-2 cell line, infected with either SARS-CoV-2 or SARS-CoV. Here, we report the outcome of a comparative analysis based on differentially expressed genes in terms of perturbations and diseases, Canonical pathways, and Upstream Regulators. We furthermore performed compendium-wide analysis across more than 19,000 mRNASeq datasets and dissected the condition-specific gene signatures. Information was gained with respect to common and unique cellular responses and molecular events. We identified that in cell lines of colon or lung origin, both viruses show similarities in cellular responses; by contrast, there are cell type-specific regulators that differed for Calu-3 and Caco-2 cells. Among the major findings is the impact of the interferon system for lung Calu-3 cells and novel links to the liver- and lipid-metabolism-associated responses for colon Caco-2 cells as part of the extrapulmonary pathomechanisms in the course of COVID-19. Among differently expressed genes, we specifically dissected the expression pattern of the APOBEC family members and propose APOBEC3G as a promising intrinsic antiviral factor of the host response to SARS-CoV-2. Overall, our study provides gene expression level evidence for the cellular responses attributed to pulmonary and gastrointestinal manifestations of COVID-19.

Human skin dendritic cell fate is differentially regulated by the monocyte identity factor Kruppel-like factor 4 during steady state and inflammation.

BACKGROUND: Langerhans cell (LC) networks play key roles in immunity and tolerance at body surfaces. LCs are established prenatally and can be replenished from blood monocytes. Unlike skin-resident dermal DCs (dDCs)/interstitial-type DCs and inflammatory dendritic epidermal cells appearing in dermatitis/eczema lesions, LCs lack key monocyte-affiliated markers. Inversely, LCs express various epithelial genes critical for their long-term peripheral tissue residency. OBJECTIVE: Dendritic cells (DCs) are functionally involved in inflammatory diseases; however, the mechanisms remained poorly understood. METHODS: In vitro differentiation models of human DCs, gene profiling, gene transduction, and immunohistology were used to identify molecules involved in DC subset specification. RESULTS: Here we identified the monocyte/macrophage lineage identity transcription factor Kruppel-like factor 4 (KLF4) to be inhibited during LC differentiation from human blood monocytes. Conversely, KLF4 is maintained or induced during dermal DC and monocyte-derived dendritic cell/inflammatory dendritic epidermal cell differentiation. We showed that in monocytic cells KLF4 has to be repressed to allow their differentiation into LCs. Moreover, respective KLF4 levels in DC subsets positively correlate with proinflammatory characteristics. We identified epithelial Notch signaling to repress KLF4 in monocytes undergoing LC commitment. Loss of KLF4 in monocytes transcriptionally derepresses Runt-related transcription factor 3 in response to TGF-ß1, thereby allowing LC differentiation marked by a low cytokine expression profile. CONCLUSION: Monocyte differentiation into LCs depends on activation of Notch signaling and the concomitant loss of KLF4.

AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer.

BACKGROUND: Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells. RESULTS: We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID/APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling-based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC-related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background. CONCLUSIONS: The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue.

Immunology of Osteoporosis: A Mini-Review.

Osteoporosis is a major cause of fractures and associated morbidity in the aged population. The pathogenesis of osteoporosis is multifactorial; whereas traditional pathophysiological concepts emphasize endocrine mechanisms, it has been recognized that also components of the immune system have a significant impact on bone. Since 2000, when the term 'osteoimmunology' was coined, novel insights into the role of inflammatory cytokines by influencing the fine-tuned balance between bone resorption and bone formation have helped to explain the occurrence of osteoporosis in conjunction with chronic inflammatory reactions. Moreover, the phenomenon of a low-grade, chronic, systemic inflammatory state associated with aging has been defined as 'inflamm-aging' by Claudio Franceschi and has been linked to age-related diseases such as osteoporosis. Given the tight anatomical and physiological coexistence of B cells and the bone-forming units in the bone marrow, a role of B cells in osteoimmunological interactions has long been suspected. Recent findings of B cells as active regulators of the RANK/RANKL/OPG axis, of altered RANKL/OPG production by B cells in HIV-associated bone loss or of a modulated expression of genes linked to B-cell biology in response to estrogen deficiency support this assumption. Furthermore, oxidative stress and the generation of advanced glycation end products have emerged as links between inflammation and bone destruction.

The calcium-sensing receptor suppresses epithelial-to-mesenchymal transition and stem cell- like phenotype in the colon.

BACKGROUND: The calcium sensing receptor (CaSR), a calcium-binding G protein-coupled receptor is expressed also in tissues not directly involved in calcium homeostasis like the colon. We have previously reported that CaSR expression is down-regulated in colorectal cancer (CRC) and that loss of CaSR provides growth advantage to transformed cells. However, detailed mechanisms underlying these processes are largely unknown. METHODS AND RESULTS: In a cohort of 111 CRC patients, we found significant inverse correlation between CaSR expression and markers of epithelial-to-mesenchymal transition (EMT), a process involved in tumor development in CRC. The colon of CaSR/PTH double-knockout, as well as the intestine-specific CaSR knockout mice showed significantly increased expression of markers involved in the EMT process. In vitro, stable expression of the CaSR (HT29(CaSR)) gave a more epithelial-like morphology to HT29 colon cancer cells with increased levels of E-Cadherin compared with control cells (HT29(EMP)). The HT29(CaSR) cells had reduced invasive potential, which was attributed to the inhibition of the Wnt/ß-catenin pathway as measured by a decrease in nuclear translocation of ß-catenin and transcriptional regulation of genes like GSK-3ß and Cyclin D1. Expression of a spectrum of different mesenchymal markers was significantly down-regulated in HT29(CaSR) cells. The CaSR was able to block upregulation of mesenchymal markers even in an EMT-inducing environment. Moreover, overexpression of the CaSR led to down-regulation of stem cell-like phenotype. CONCLUSIONS: The results from this study demonstrate that the CaSR inhibits epithelial-to-mesenchymal transition and the acquisition of a stem cell-like phenotype in the colon of mice lacking the CaSR as well as colorectal cancer cells, identifying the CaSR as a key molecule in preventing tumor progression. Our results support the rationale to develop new strategies either preventing CaSR loss or reversing its silencing.

Eicosanoid modulation by the short-chain fatty acid n-butyrate in human monocytes.

n-Butyrate deriving from bacterial fermentation in the mammalian intestine is a key determinant in gastrointestinal homeostasis. We examined the effects of this short-chain fatty acid and Toll-like receptor 2 (TLR) and TLR4 engagement on inflammatory/immunity-associated genes, cyclo-oxygenases (COXs), prostaglandins (PGs) and leukotrienes (LTs) in human monocytes. Before RNA isolation, freshly isolated human monocytes were co-incubated for different time-points with 1 mm n-butyrate alone or in combination with bacterial stimuli. Based on a knowledge-driven approach, a signature of 180 immunity/inflammation-associated genes was picked and real-time PCR analysis was performed. Pathway analysis was carried out using a web-based database analysing program. Based on these gene expression studies the findings were evaluated at the protein/mediator level by Western blot analysis, FACS and ELISA. Following co-incubation with n-butyrate and lipopolysaccharide, key enzymes of the eicosanoid pathway, like PTGS2 (COX-2), TXS, ALOX5, LTA4H and LTC4S, were significantly up-regulated compared with stimulation with lipopolysaccharide alone. Furthermore, release of the lipid mediators PGE(2), 15d-PGJ(2), LTB(4) and thromboxane B(2) was increased by n-butyrate. Regarding signalling, n-butyrate had no additional effect on mitogen-activated protein kinase and interfered differently with early and late phases of nuclear factor-κB signalling. Our results suggest that among many other mediators of eicosanoid signalling n-butyrate massively induces PGE(2) production by increasing the expression of PTGS2 (COX-2) in monocytes following TLR4 and TLR2 activation and induces secretion of LTB(4) and thromboxane B(2). This underscores the role of n-butyrate as a crucial mediator of gut-specific immunity.

Microenvironmental and cell intrinsic factors governing human cDC2 differentiation and monocyte reprogramming.

cDC2s occur abundantly in peripheral tissues and arise from circulating blood cDC2s. However, the factors governing cDC2 differentiation in tissues, especially under inflammatory conditions, remained poorly defined. We here found that psoriatic cDC2s express the efferocytosis receptor Axl and exhibit a bone morphogenetic protein (BMP) and p38MAPK signaling signature. BMP7, strongly expressed within the lesional psoriatic epidermis, cooperates with canonical TGF-ß1 signaling for inducing Axl+cDC2s from blood cDC2s in vitro. Moreover, downstream induced p38MAPK promotes Axl+cDC2s at the expense of Axl+CD207+ Langerhans cell differentiation from blood cDC2s. BMP7 supplementation allowed to model cDC2 generation and their further differentiation into LCs from CD34+ hematopoietic progenitor cells in defined serum-free medium. Additionally, p38MAPK promoted the generation of another cDC2 subset lacking Axl but expressing the non-classical NFkB transcription factor RelB in vitro. Such RelB+cDC2s occurred predominantly at dermal sites in the inflamed skin. Finally, we found that cDC2s can be induced to acquire high levels of the monocyte lineage identity factor kruppel-like-factor-4 (KLF4) along with monocyte-derived DC and macrophage phenotypic characteristics in vitro. In conclusion, inflammatory and psoriatic epidermal signals instruct blood cDC2s to acquire phenotypic characteristics of several tissue-resident cell subsets.

Activation-induced cytidine deaminase (AID) linking immunity, chronic inflammation, and cancer.

Activation-induced cytidine deaminase (AID) is critically involved in class switch recombination and somatic hypermutation of Ig loci resulting in diversification of antibodies repertoire and production of high-affinity antibodies and as such represents a physiological tool to introduce DNA alterations. These processes take place within germinal centers of secondary lymphoid organs. Under physiological conditions, AID is expressed predominantly in activated B lymphocytes. Because of the mutagenic and recombinogenic potential of AID, its expression and activity is tightly regulated on different levels to minimize the risk of unwanted DNA damage. However, chronic inflammation and, probably, combination of other not-yet-identified factors are able to create a microenvironment sufficient for triggering an aberrant AID expression in B cells and, importantly, in non-B-cell background. Under these circumstances, AID may target also non-Ig genes, including cancer-related genes as oncogenes, tumor suppressor genes, and genomic stability genes, and modulate both genetic and epigenetic information. Despite ongoing progress, the complete understanding of fundamental aspects is still lacking as (1) what are the crucial factors triggering an aberrant AID expression/activity including the impact of Th2-driven inflammation and (2) to what extent may aberrant AID in human non-B cells lead to abnormal cell state associated with an increased rate of genomic alterations as point mutations, small insertions or deletions, and/or recurrent chromosomal translocations during solid tumor development and progression.

SLCO4A1 expression is associated with activated inflammatory pathways in high-grade serous ovarian cancer.

Patients with high-grade serous ovarian cancer (HGSOC) have a very poor overall survival. Current therapeutic approaches do not bring benefit to all patients. Although genetic alterations and molecular mechanisms are well characterized, the molecular pathological conditions are poorly investigated. Solute carrier organic anion transporter family member 4A1 (SLCO4A1) encodes OATP4A1, which is an uptake membrane transporter of metabolic products. Its expression may influence various signaling pathways associated with the molecular pathophysiological conditions of HGSOC and consequently tumor progression. RNA sequencing of 33 patient-derived HGSOC cell lines showed that SLCO4A1 expression was diverse by individual tumors, which was further confirmed by RT-qPCR, Western blotting and immunohistochemistry. Gene Set Enrichment Analysis revealed that higher SLCO4A1 level was associated with inflammation-associated pathways including NOD-like receptor, adipocytokine, TALL1, CD40, NF-κB, and TNF-receptor 2 signaling cascades, while low SLCO4A1 expression was associated with the mitochondrial electron transport chain pathway. The overall gene expression pattern in all cell lines was specific to each patient and remained largely unchanged during tumor progression. In addition, genes encoding ABCC3 along with SLCO4A1-antisense RNA 1, were associated with higher expression of the SLCO4A1, indicating their possible involvement in inflammation-associated pathways that are downstream to the prostaglandin E2/cAMP axis. Taken together, increased SLCO4A1/OATP4A1 expression is associated with the upregulation of specific inflammatory pathways, while the decreased level is associated with mitochondrial dysfunction. These molecular pathophysiological conditions are tumor specific and should be taken into consideration by the development of therapies against HGSOC.

AID and APOBECs as Multifaceted Intrinsic Virus-Restricting Factors: Emerging Concepts in the Light of COVID-19.

The AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme catalytic subunit) family with its multifaceted mode of action emerges as potent intrinsic host antiviral system that acts against a variety of DNA and RNA viruses including coronaviruses. All family members are cytosine-to-uracil deaminases that either have a profound role in driving a strong and specific humoral immune response (AID) or restricting the virus itself by a plethora of mechanisms (APOBECs). In this article, we highlight some of the key aspects apparently linking the AID/APOBECs and SARS-CoV-2. Among those is our discovery that APOBEC4 shows high expression in cell types and anatomical parts targeted by SARS-CoV-2. Additional focus is given by us to the lymphoid structures and AID as the master regulator of germinal center reactions, which result in antibody production by plasma and memory B cells. We propose the dissection of the AID/APOBECs gene signature towards decisive determinants of the patient-specific and/or the patient group-specific antiviral response. Finally, the patient-specific mapping of the AID/APOBEC polymorphisms should be considered in the light of COVID-19.

Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains.

Ceramide kinase (CERK) and the ceramide kinase-like protein (CERKL), two related members of the diacylglycerol kinase family, are ill-defined at the molecular level. In particular, what determines their distinctive subcellular localization is not well understood. Here we show that the Pleckstrin Homology (PH) domain of CERK, which is required for Golgi complex localization, can substitute for the N-terminal region of CERKL and allow for wild-type CERKL localization, which is typified by nucleolar accumulation. This demonstrates that determinants for localization of these two enzymes do not lie solely in their PH domain-containing N-terminal regions. Moreover, we present evidence for a previously unrecognized participation of CERK distal sequences in structural stability, localization and activity of the full-length protein. Progressive deletion of CERK and CERKL from the C-terminus revealed similar sequential organization in both proteins, with nuclear import signals in their N-terminal part, and nuclear export signals in their C-terminal part. Furthermore, mutagenesis of individual cysteine residues of a CERK-specific CXXXCXXC motif severely compromised both exportation of CERK from the nucleus and its association with the Golgi complex. Altogether, this work identifies conserved domains in CERK and CERKL as well as new determinants for their subcellular localization. It further suggests a nucleocytoplasmic shuttling mechanism for both proteins that may be defective in CERKL mutant proteins responsible for retinal degenerative diseases.

Two common polymorphic variants of OATP4A1 as potential risk factors for colorectal cancer.

Genetic variations in the organic-anion-transporting polypeptide (OATP)-encoding solute carrier of organic anions (SLCO) genes can promote cancer development and progression. The overexpression of solute carrier organic anion transporter family member 4A1 (OATP4A1), a transporter for steroid hormones, prostaglandins, and bile acids, has been previously associated with tumor recurrence and progression in colorectal cancer (CRC). Therefore, the present study aimed to investigate the association between 2 frequent single nucleotide polymorphisms (SNPs) in SLCO4A1 (rs34419428, R70Q; rs1047099G, V78I) and CRC predisposition. Following restriction fragment length polymorphism-PCR analysis in 178 patients with CRC [Union for International Cancer Control (UICC) stage I/II] and 65 healthy controls, no significant difference was observed in allele frequency and the number of heterozygous/homozygous individuals between the groups. Notably, the R70Q minor allele was identified to be associated with the V78I minor allele in the genome. Comparing of the individual genotypes of CRC patients to clinical data, including sex, UICC-stage and relapse revealed no increased risk for CRC. In addition, the OATP4A1 immunoreactivity assay in paraffin-embedded CRC and adjacent non-tumorous mucosa sections, examined using quantitative microscopy image analysis, did not reveal any association with these polymorphisms. No significant differences were observed in the expression levels, localization, and sodium fluorescein transport capacity among the OATP4A1 variants, which was studied using functional assays in Sf9-insect and A431 tumor cells overexpressing the 2 single and a double mutant OATP4A1 SNP variants. These results suggested that the 2 most frequent polymorphisms located in the first intracellular loop of OATP4A1 do not associate with CRC predisposition and tumor recurrence. They are unlikely to affect the outcome of CRC in patients.

The Immune Phenotype of Isolated Lymphoid Structures in Non-Tumorous Colon Mucosa Encrypts the Information on Pathobiology of Metastatic Colorectal Cancer.

The gut-associated lymphoid tissue represents an integral part of the immune system. Among the powerful players of the mucosa-associated lymphoid tissue are isolated lymphoid structures (ILSs), which as information centers, drive the local (and systemic) adaptive immune responses. Germinal center reactions, taking place within ILSs, involve the coordinated action of various immune cell types with a central role given to B cells. In the current study, we aimed at dissecting the impact of ILSs within non-tumorous colon tissue (NT) on the pathobiology of colorectal cancer (CRC) with metastasis in the liver (CRCLM). In particular, we focused on the immune phenotypes of ILSs and ectopic lymphoid structures (ELSs), built up at matching primary and metastatic tumor sites. We implemented an integrative analysis strategy on the basis of tissue image cytometry and clonality assessment to explore the immune phenotype of ILS/ELS at three tissue entities: NT, CRC, and CRCLM (69 specimens in total). Applying a panel of lineage markers used for immunostaining, we characterized and compared the anatomical features, the cellular composition, the activation, and proliferation status of ILSs and ELSs, and assessed the clinical relevance of staining-derived data sets. Our major discovery was that ILS characteristics at the NT site predefine the immune phenotype of ELSs at CRC and CRCLM. Thereby, B-cell-enriched (CD20) and highly proliferative (Ki67) ILSs and ELSs were found to be associated with improved clinical outcome in terms of survival and enabled patient stratification into risk groups. Moreover, the data revealed a linkage between B-cell clonality at the NT site and the metastatic characteristics of the tumor in the distant liver tissue. Consolidation of immunostaining-based findings with the results of compendium-wide transcriptomic analysis furthermore proposed CD27 as a novel marker of T follicular helper cells within lymphoid structures. Overall, the study nominates the ILS immune phenotype as a novel prognostic marker for patients with metastatic CRC.

Metabolism of Estrogens: Turnover Differs Between Platinum-Sensitive and -Resistant High-Grade Serous Ovarian Cancer Cells.

High-grade serous ovarian cancer (HGSOC) is currently treated with cytoreductive surgery and platinum-based chemotherapy. The majority of patients show a primary response; however, many rapidly develop drug resistance. Antiestrogens have been studied as low toxic treatment options for HGSOC, with higher response rates in platinum-sensitive cases. Mechanisms for this difference in response remain unknown. Therefore, the present study investigated the impact of platinum resistance on steroid metabolism in six established HGSOC cell lines sensitive and resistant against carboplatin using a high-resolution mass spectrometry assay to simultaneously quantify the ten main steroids of the estrogenic metabolic pathway. An up to 60-fold higher formation of steroid hormones and their sulfated or glucuronidated metabolites was observed in carboplatin-sensitive cells, which was reversible by treatment with interleukin-6 (IL-6). Conversely, treatment of carboplatin-resistant cells expressing high levels of endogenous IL-6 with the monoclonal anti-IL-6R antibody tocilizumab changed their status to "platinum-sensitive", exhibiting a decreased IC50 value for carboplatin, decreased growth, and significantly higher estrogen metabolism. Analysis of these metabolic differences could help to detect platinum resistance in HGSOC patients earlier, thereby allowing more efficient interventions.

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells.

Trastuzumab (Herceptin), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.

Interrelations of Sphingolipid and Lysophosphatidate Signaling with Immune System in Ovarian Cancer.

The sphingolipid and lysophosphatidate regulatory networks impact diverse mechanisms attributed to cancer cells and the tumor immune microenvironment. Deciphering the complexity demands implementation of a holistic approach combined with higher-resolution techniques. We implemented a multi-modular integrative approach consolidating the latest accomplishments in gene expression profiling, prognostic/predictive modeling, next generation digital pathology, and systems biology for epithelial ovarian cancer. We assessed patient-specific transcriptional profiles using the sphingolipid/lysophosphatidate/immune-associated signature. This revealed novel sphingolipid/lysophosphatidate-immune gene-gene associations and distinguished tumor subtypes with immune high/low context. These were characterized by robust differences in sphingolipid-/lysophosphatidate-related checkpoints and the drug response. The analysis also nominates novel survival models for stratification of patients with CD68, LPAR3, SMPD1, PPAP2B, and SMPD2 emerging as the most prognostically important genes. Alignment of proprietary data with curated transcriptomic data from public databases across a variety of malignancies (over 600 categories; over 21,000 arrays) showed specificity for ovarian carcinoma. Our systems approach identified novel sphingolipid-lysophosphatidate-immune checkpoints and networks underlying tumor immune heterogeneity and disease outcomes. This holds great promise for delivering novel stratifying and targeting strategies.

Oxidized phospholipids stimulate angiogenesis via autocrine mechanisms, implicating a novel role for lipid oxidation in the evolution of atherosclerotic lesions.

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2-generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.

Sphingosine 1-phosphate phosphatase 2 is induced during inflammatory responses.

Sphingosine 1-phosphate (S1P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S1P-metabolism (sphingosine kinases including their splice variants, sphingosine 1-phosphate phosphatases, and sphingosine 1-phosphate lyase) under certain inflammatory conditions. We found that sphingosine kinase type 1 (SPHK1) mRNA could be triggered in a cell type-specific manner; individual SPHK1 splice variants were induced with similar kinetics. Remarkably, expression and activity of S1P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-1beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.

Sustained expression of early growth response protein-1 blocks angiogenesis and tumor growth.

Transient induction of the transcription factor early growth response protein-1 (EGR-1) plays a pivotal role in the transcriptional response of endothelial cells to the angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which are produced by most tumors and are involved in the angiogenic switch. We report here that sustained expression of EGR-1 by recombinant adenoviruses in endothelial cells, however, leads to the specific induction of potent feedback inhibitory mechanisms, including strong up-regulation of transcriptional repressors, negative cell cycle check point effectors, proteins with established antiangiogenic activity, and several proapoptotic genes. Sustained EGR-1 expression consistently leads to an antiangiogenic state characterized by an altered responsiveness to VEGF and bFGF and a striking inhibition of sprouting and tubule formation in vitro. Furthermore, EGR-1-expressing viruses potently inhibit cell invasion and vessel formation in the murine Matrigel model and repress tumor growth in a murine fibrosarcoma model. We propose that gene therapy involving sustained EGR-1 expression may constitute a novel therapeutic principle in the treatment of cancer due to the simultaneous induction of multiple pathways of antiangiogenesis, growth arrest, and apoptosis induction in proliferating cells leading to preferential inhibition of angiogenesis and tumor growth.