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Supplementary Data from CD151 Regulates Tumorigenesis by Modulating the Communication between Tumor Cells and Endothelium

The tetraspanin CD151 forms stoichiometric complexes with laminin-binding integrins (e.g., α3β1, α6β1, and α6β4) and regulates their ligand-binding and signaling functions. We have found that high expression of CD151 in breast cancers is associated with decreased overall survival (3.44-fold higher risk of death). Five-year estimated survival rates were 45.8% (95% confidence interval, 16.4-71.4%) for CD151-positive patients and 79.9% (95% confidence interval, 62.2-90.0%) for CD151-negative patients. Furthermore, CD151 was positively associated with axillary lymph node involvement. To study the biological significance of this observation, we investigated the contribution of CD151 in breast cancer tumorigenesis using MDA-MB-231 cells as a model system. Stable down-regulation of this tetraspanin by short-hairpin RNA decreased the tumorigenicity of these cells in mice. Detailed immunohistologic analysis of CD151(+) and CD151(−) xenografts showed differences in tumor vascular pattern. Vascularization observed at the subcutaneous border of the CD151(+) tumors was less pronounced or absent in the CD151(−) xenografts. In vitro experiments have established that depletion of CD151 did not affect the inherent proliferative capacity of breast cancer cells in three-dimensional extracellular matrices, but modified their responses to endothelial cells in coculture experiments. The modulatory activity of CD151 was dependent on its association with both α3β1 and α6β4 integrins. These data point to a new role of CD151 in tumorigenesis, whereby it functions as an important regulator of communication between tumor cells and endothelial cells. These results also identify CD151 as a potentially novel prognostic marker and target for therapy in breast cancer. (Mol Cancer Res 2009;7(6):787–98)

PDF file - 120K, C57Bl/6 mice bearing B16-Ova tumors were injected intratumorally with the respective viruses as described in materials and methods. Lymph nodes, spleens and tumors were collected 8 days after first virus injection, smashed and cultured for 24 hours. Analysis of T-cell markers was performed by flow cytometry for CD3, CD4 and CD8 and plotted as mean +SEM. *, P

PDF file - 91K, Nude mice bearing MB49 tumors were injected with Ad5/3-CMV-mCD40L, Ad5/3-Luc1 or PBS (Mock) on days 0,2 and 4. Eight days after first virus injection mice were killed and spleens and tumors were collected, smashed and cultured for 24 hours. Homogenates were analyzed for NK+ cells (left side) and CD19+ (right side). Results are presented as mean from 6 mice + SEM. *, P

PDF file - 2.2MB, (A) Cell monolayers were infected with 100 viral paticles /cell (VP/cell) and 24 hours later cells were analyzed by flow-cytometry measuring Annexin-V expression. Results are presented as percentage of positive cells relative to Mock uninfected cells. **, P

Supplementary Figure Legends 1-5, Table Legend from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Supplementary Figure 2 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Supplementary Figure 3 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

PDF file - 85K

PDF file - 128K, T-cell analyses in C57Bl/6 mice bearing B16-Ova tumors C57Bl/6 mice bearing B16-Ova tumors were injected intratumorally. Lymph nodes, spleens and tumors were collected 8 days after first virus injection, homogenized and cultured for 24 hours. Analysis of Ova-specific CD8+CD19- was performed by flow cytometry gating the CD19- population and plotting SIINFEKEL marker of Ova with CD8. Results are represented as percentage of double positive CD8 and SIINFEKEL peptide and plotted as mean +SEM. *, P

Supplementary Table 1 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Supplementary Figure 5 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Supplementary Figure 1 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

PDF file - 75K, (A) MB49 cells were infected with 100 viral particles/cell with Ad5/3-CMV-mCD40L and Ad5/3-Luc1 and Annexin-V was assessed 24 hours post-infection by flow cytometry. (B) Replication of Ad5/3-CMV-mCD40L and Ad5/3-Luc1 over time in MB49 cell line. Cell monolayers were infected at doses specified ranging from 101 to 108 viral particles and assays were stopped at different time points (days 2, 3, 4 and 8). Cell viability was measured and expressed overtime. (C) MB49 cells were infected with 100 viral particles and samples were collected at 0, 24, 48 and 72 hours post infection. Quantitative PCR was performed for E4 sequence and values were normalized to mouse beta-actin gene.

Supplementary Figure 4 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Integrin receptors for cell adhesion to extracellular matrix have important roles in promoting tumor growth and progression. Integrin α3β1 is highly expressed in breast cancer cells in which it is thought to promote invasion and metastasis; however, its roles in regulating malignant tumor cell behavior remain unclear. In the current study, we used short-hairpin RNA (shRNA) to show that suppression of α3β1 in a human breast cancer cell line, MDA-MB-231, leads to decreased tumorigenicity, reduced invasiveness, and decreased production of factors that stimulate endothelial cell migration. Real-time PCR revealed that suppression of α3β1 caused a dramatic reduction in expression of the cyclooxygenase-2 (COX-2) gene, which is frequently overexpressed in breast cancers and has been exploited as a therapeutic target. Decreased COX-2 was accompanied by reduced prostaglandin E2 (PGE2), a major prostanoid produced downstream of COX-2 and an important effector of COX-2 signaling. shRNA-mediated suppression of COX-2 showed that it has a role in tumor cell invasion and cross-talk to endothelial cells. Furthermore, treatment with PGE2 restored these functions in α3β1-deficient MDA-MB-231 cells. These findings identify a role for α3β1 in regulating two properties of tumor cells that facilitate cancer progression: invasiveness and ability to stimulate endothelial cells. They also reveal a novel role for COX-2 as a downstream effector of α3β1 in tumor cells, thereby identifying α3β1 as a potential therapeutic target to inhibit breast cancer. Cancer Res; 70(15); 6359–67. ©2010 AACR.

PDF file - 881K, (A) Oncolytic potency of Ad5/3-hTERT-E1A-hCD40L and (B) Ad5/3-hTERT-E1A on EJ (CD40+) and A549 (CD40-). Cell lines were infected with Ad5/3Luc1, Ad5/3-hTERT-E1A, Ad5/3-hTERT-E1A-hCD40L and Ad5/3-CMV-hCD40L and cell viability was measured by MTS assay The assay was stopped 7 days after infection and the data was plotted to compare efficacy on CD40+ versus CD40- cells; Ad5/3-hTERT-E1A-hCD40L was more effective than the positive control on the former while the opposite was true for the latter. . VP=viral particles. ***, P

Background/objectives Evidence regarding the influence of coffee on appetite and weight control is equivocal and the influence of covariates, such as genetic variation in caffeine metabolism, remains unknown. Herein, we addressed the novel hypothesis that genetic variation in CYP1A2, a gene responsible for more than 95% of caffeine metabolism, differentially impacts the association of coffee consumption with appetite and BMI among individuals with different genetic predispositions to obesity. Subjects/methods A cross-over randomized intervention study involving 18 volunteers assessed the effects of coffee consumption on dietary intake, appetite, and levels of the appetite-controlling hormones asprosin and leptin. Data on habitual coffee intake, BMI, and perceived appetite were obtained from an observational cohort of 284 volunteers using validated questionnaires. Participants were stratified according to a validated genetic risk score (GRS) for obesity and to the -163C > A (rs762551) polymorphism of CYP1A2 as rapid (AA), intermediate (AC), or slow (CC) caffeine metabolizers. Results Coffee consumption led to lower energy and dietary fat intake and circulating asprosin levels (P for interaction of rs762551 genotype*coffee consumption=0.056, 0.039, and 0.043, respectively) as compared to slow/intermediate metabolizers. High coffee consumption was more prevalent in rapid compared to slow metabolizers (P = 0.008 after adjustment for age, sex, and BMI) and was associated with lower appetite perception and lower BMI only in rapid metabolizers (P for interaction of rs762551 genotype*coffee consumption = 0.002 and 0.048, respectively). This differential association of rs762551 genotype and coffee consumption with BMI was more evident in individuals at higher genetic risk of obesity (mean adjusted difference in BMI = -5.82 kg/m2 for rapid versus slow/intermediate metabolizers who consumed more than 14 cups of coffee per week). Conclusions CYP1A2 rs762551 polymorphism modifies the association of habitual coffee consumption with BMI, in part by influencing appetite, energy intake and circulating levels of the orexigenic hormone asprosin. This association is more evident in subjects with high genetic predisposition to obesity. ClinicalTrials.gov: registered Clinical Trial NCT04514588.

Hepatocellular carcinoma (HCC) occurs in patients with chronic liver damage, inflammation and cirrhosis. The facilitators involved in increasing the HCC risk in the damaged liver are yet to be discovered. Diet and lifestyle have a profound effect on the liver inflammation and HCC. The term “gut liver axis” describes the bidirectional relationship between the liver and the gut, which are both anatomically and functionally related. Chronic liver damage is characterised by increased intestinal permeability that allows the translocation of various components and metabolites from the gut microbiota to the liver, resulting in liver inflammation and fibrosis. In this review, we discuss how diet-induced changes in gut microbiome composition, such as lipopolysaccharide and lipoteichoic acid, and its metabolites, such as bile acids, play a role in the pathogenesis of liver fibrosis and HCC.

The oncogenic pathways activated by the NPM-ALK chimeric kinase of ALK+ anaplastic large cell lymphoma (ALCL) are well characterized; however, the potential interactions of ALK signaling with the microenvironment are not yet known. Here we report that ALK+ ALCL-derived exosomes contain critical components of ALK signaling as well as CD30, and that exosome uptake by lymphoid cells led to increased proliferation and expression of critical antiapoptotic proteins by the recipient cells. The bone marrow fibroblasts highly uptake ALK+ ALCL-derived exosomes and acquire a cancer-associated fibroblast (CAF) phenotype. Moreover, exosome-mediated activation of stromal cells altered the cytokine profile of the microenvironment. These interactions may contribute to tumor aggressiveness and possibly resistance to treatment.

Nutritional and lifestyle changes remain at the core of healthy aging and disease prevention. Accumulating evidence underscores the impact of genetic, metabolic, and host gut microbial factors on individual responses to nutrients, paving the way for the stratification of nutritional guidelines. However, technological advances that incorporate biological, nutritional, lifestyle, and health data at an unprecedented scale and depth conceptualize a future where preventative dietary interventions will exceed stratification and will be highly individualized. We herein discuss how genetic information combined with longitudinal metabolomic, immune, behavioral, and gut microbial parameters, and bioclinical variables could define a digital replica of oneself, a "virtual digital twin," which could serve to guide nutrition in a personalized manner. Such a model may revolutionize the management of obesity and its comorbidities, and provide a pillar for healthy aging.

The oncogenic pathways activated by the NPM-ALK chimeric kinase of ALK+ anaplastic large cell lymphoma (ALCL) are well characterized; however, the potential interactions of ALK signaling with the microenvironment are not yet known. Here we report that ALK+ ALCL-derived exosomes contain critical components of ALK signaling as well as CD30, and that exosome uptake by lymphoid cells led to increased proliferation and expression of critical antiapoptotic proteins by the recipient cells. The bone marrow fibroblasts highly uptake ALK+ ALCL-derived exosomes and acquire a cancer-associated fibroblast (CAF) phenotype. Moreover, exosome-mediated activation of stromal cells altered the cytokine profile of the microenvironment. These interactions may contribute to tumor aggressiveness and possibly resistance to treatment.

Signaling through adhesion-related molecules is important for cancer growth and metastasis and cancer cells are resistant to anoikis, a form of cell death ensued by cell detachment from the extracellular matrix. Herein, we report that detached carcinoma cells and immortalized fibroblasts display defects in TNF and CD40 ligand (CD40L)-induced MEK-ERK signaling. Cell detachment results in reduced basal levels of the MEK kinase TPL2, compromises TPL2 activation and sensitizes carcinoma cells to death-inducing receptor ligands, mimicking the synthetic lethal interactions between TPL2 inactivation and TNF or CD40L stimulation. Focal Adhesion Kinase (FAK), which is activated in focal adhesions and mediates anchorage-dependent survival signaling, was found to sustain steady state TPL2 protein levels and to be required for TNF-induced TPL2 signal transduction. We show that when FAK levels are reduced, as seen in certain types of malignancy or malignant cell populations, the formation of cIAP2:RIPK1 complexes increases, leading to reduced TPL2 expression levels by a dual mechanism: first, by the reduction in the levels of NF-κΒ1 which is required for TPL2 stability; second, by the engagement of an RelA NF-κΒ pathway that elevates interleukin-6 production, leading to activation of STAT3 and its transcriptional target SKP2 which functions as a TPL2 E3 ubiquitin ligase. These data underscore a new mode of regulation of TNF family signal transduction on the TPL2-MEK-ERK branch by adhesion-related molecules that may have important ramifications for cancer therapy.

Type 2 diabetes mellitus (T2DM) is a heterogeneous metabolic disorder of multifactorial etiology that includes genetic and dietary influences. By addressing the latter, medical nutrition therapy (MNT) contributes to the management of T2DM or pre-diabetes toward achieving glycaemic control and improved insulin sensitivity. However, the clinical outcomes of MNT vary and may further benefit from personalized nutritional plans that take into consideration genetic variations associated with individual responses to macronutrients. The aim of the present series of

Functional enrichment is a widely used method for interpreting experimental results by identifying classes of proteins/genes associated with certain biological functions, pathways, diseases or phenotypes. Despite the variety of existing tools, most of them can process a single list per time, thus making a more combinatorial analysis more complicated and prone to errors. In this article, we present FLAME, a web tool for combining multiple lists prior to enrichment analysis. Users can upload several lists of preference and use interactive UpSet plots, as an alternative to Venn diagrams, to handle unions or intersections among the given input files. Functional and literature enrichment along with gene conversions are offered by g:Profiler and aGOtool applications for 197 organisms. In its current version, FLAME can analyze genes/proteins for related articles, Gene Ontologies, pathways, annotations, regulatory motifs, domains, diseases, phenotypes while it can also generate protein-protein interactions derived from STRING. We have herein validated FLAME by interrogating gene expression data associated with the sensitivity of the distal part of the large intestine to experimental colitis-propelled colon cancer. The FLAME application comes with an interactive user-friendly interface which allows easy list manipulation and exploration, while results can be visualized as interactive and parameterizable heatmaps, barcharts, Manhattan plots, networks and tables.AvailabilityFLAME application: http://flame.pavlopouloslab.infoCodehttps://github.com/PavlopoulosLab/FLAME

Extracting and processing information from documents is of great importance as lots of experimental results and findings are stored in local files. Therefore, extracting and analysing biomedical terms from such files in an automated way is absolutely necessary. In this article, we present OnTheFly2.0, a web application for extracting biomedical entities from individual files such as plain texts, Office documents, PDF files or images. OnTheFly2.0can generate informative summaries in popup windows containing knowledge related to the identified terms along with links to various databases. It uses the EXTRACT tagging service to perform Named Entity Recognition (NER) for genes/proteins, chemical compounds, organisms, tissues, environments, diseases, phenotypes and Gene Ontology terms. Multiple files can be analysed, whereas identified terms such as proteins or genes can be explored through functional enrichment analysis or be associated with diseases and PubMed entries. Finally, protein-protein and protein-chemical networks can be generated with the use of STRING and STITCH services. To demonstrate its capacity for knowledge discovery, we interrogated published meta-analyses of clinical biomarkers of severe COVID-19 and uncovered inflammatory and senescence pathways that impact disease pathogenesis. OnTheFly2.0currently supports 197 species and is available athttp://onthefly.pavlopouloslab.info.

COVID-19 incidence and case fatality rates (CFR) differ among ethnicities, stimulating efforts to pinpoint genetic factors that could explain these phenomena. In this regard, the multiallelic apolipoprotein E (APOE) gene has recently been interrogated in the UK biobank cohort, demonstrating associations of the APOE e4/e4 genotype with COVID-19 severity and mortality. The frequency of the e4 allele and thus the distribution of APOE e4/e4 genotype may differ among populations. We have assessed APOE genotypes in 1638 Greek individuals, based on haplotypes derived from SNP rs7412 and rs429358 and found reduced frequency of e4/e4 compared to the British cohort. Herein we discuss this finding in relation to CFR and hypothesize on the potential mechanisms linking APOE e4/e4 to severe COVID-19. We postulate that the metabolic deregulation ensued by APOE4, manifested by elevated cholesterol and oxidized lipoprotein levels, may be central to heightened pneumocyte susceptibility to infection and to exaggerated lung inflammation associated with the e4/e4 genotype. We also discuss putative dietary and pharmacological approaches for the prevention and management of COVID-19 in APOE e4/e4 individuals.

Evidence regarding the influence of coffee on appetite and weight control is equivocal and the influence of covariates, such as genetic variation in caffeine metabolism, remains unknown. Herein, we addressed the novel hypothesis that genetic variation in CYP1A2, a gene responsible for more than 95% of caffeine metabolism, differentially impacts the association of coffee consumption with appetite and BMI among individuals with different genetic predispositions to obesity.A cross-over randomized intervention study involving 18 volunteers assessed the effects of coffee consumption on dietary intake, appetite, and levels of the appetite-controlling hormones asprosin and leptin. Data on habitual coffee intake, BMI, and perceived appetite were obtained from an observational cohort of 284 volunteers using validated questionnaires. Participants were stratified according to a validated genetic risk score (GRS) for obesity and to the -163C A (rs762551) polymorphism of CYP1A2 as rapid (AA), intermediate (AC), or slow (CC) caffeine metabolizers.Coffee consumption led to lower energy and dietary fat intake and circulating asprosin levels (P for interaction of rs762551 genotype*coffee consumption=0.056, 0.039, and 0.043, respectively) as compared to slow/intermediate metabolizers. High coffee consumption was more prevalent in rapid compared to slow metabolizers (P = 0.008 after adjustment for age, sex, and BMI) and was associated with lower appetite perception and lower BMI only in rapid metabolizers (P for interaction of rs762551 genotype*coffee consumption = 0.002 and 0.048, respectively). This differential association of rs762551 genotype and coffee consumption with BMI was more evident in individuals at higher genetic risk of obesity (mean adjusted difference in BMI = -5.82 kg/mCYP1A2 rs762551 polymorphism modifies the association of habitual coffee consumption with BMI, in part by influencing appetite, energy intake and circulating levels of the orexigenic hormone asprosin. This association is more evident in subjects with high genetic predisposition to obesity. ClinicalTrials.gov: registered Clinical Trial NCT04514588.

Introduction: Psychological and behavioral interventions, such as Cognitive Behavioral Treatment (CBT) and Tinnitus Retraining Therapy (TRT), are commonly applied either alone or in combination for the management of tinnitus but selection of the intervention model remains arbitrary. Herein we evaluated the hypothesis that genetic markers may guide the choice of tinnitus treatment towards improved therapeutic outcomes.Results: Patients with subjective idiopathic tinnitus were assigned to either a genetically-informed CBT or TRT intervention protocol based on the status of four single nucleotide polymorphisms (SNPs) of the dopamine and serotonin pathways, namely COMT rs4680, HTR2A rs7997012, HTR2A rs6311, and TPH2 rs4570625, that have been associated with behavioral or cognitive responses (Group A), or to a conventional approach (Group B) in which the choice of treatment was not directed by genotypic data. Tinnitus Handicap Index (THI) scores of perceived tinnitus severity were recorded at the initial and at the fourth session of treatment using appropriate questionnaires. A statistically significant reduction in the THI scores was evident only in the genotype-guided group A (t = 3.03, p = 0.01).Conclusion: Individualized incorporation of genetic information may improve the outcome of behavioral and cognitive interventions in tinnitus.