Interleukin-30 subverts prostate cancer-endothelium crosstalk by fostering angiogenesis and activating immunoregulatory and oncogenic signaling pathways.

BACKGROUND: Cancer-endothelial interplay is crucial for tumor behavior, yet the molecular mechanisms involved are largely unknown. Interleukin(IL)-30, which is expressed as a membrane-anchored cytokine by human prostate cancer (PC) cells, promotes PC vascularization and progression, but the underlying mechanisms have yet to be fully explored. METHODS: PC-endothelial cell (EC) interactions were investigated, after coculture, by flow cytometry, transcriptional profiling, western blot, and ELISA assays. Proteome profiler phospho-kinase array unveiled the molecular pathways involved. The role of tumor-derived IL30 on the endothelium's capacity to generate autocrine circuits and vascular budding was determined following IL30 overexpression, by gene transfection, or its deletion by CRISPR/Cas9 genome editing. Clinical value of the experimental findings was determined through immunopathological study of experimental and patient-derived PC samples, and bioinformatics of gene expression profiles from PC patients. RESULTS: Contact with PC cells favors EC proliferation and production of angiogenic and angiocrine factors, which are boosted by PC expression of IL30, that feeds autocrine loops, mediated by IGF1, EDN1, ANG and CXCL10, and promotes vascular budding and inflammation, via phosphorylation of multiple signaling proteins, such as Src, Yes, STAT3, STAT6, RSK1/2, c-Jun, AKT and, primarily CREB, GSK-3α/ß, HSP60 and p53. Deletion of the IL30 gene in PC cells inhibits endothelial expression of IGF1, EDN1, ANG and CXCL10 and substantially impairs tumor angiogenesis. In its interaction with IL30-overexpressing PC cells the endothelium boosts their expression of a wide range of immunity regulatory genes, including CCL28, CCL4, CCL5, CCR2, CCR7, CXCR4, IL10, IL13, IL17A, FASLG, IDO1, KITLG, TNFA, TNFSF10 and PDCD1, and cancer driver genes, including BCL2, CCND2, EGR3, IL6, VEGFA, KLK3, PTGS1, LGALS4, GNRH1 and SHBG. Immunopathological analyses of PC xenografts and in silico investigation of 1116 PC cases, from the Prostate Cancer Transcriptome Atlas, confirmed the correlation between the expression of IL30 and that of both pro-inflammatory genes, NOS2, TNFA, CXCR5 and IL12B, and cancer driver genes, LGALS4, GNRH1 and SHBG, which was validated in a cohort of 80 PC patients. CONCLUSIONS: IL30 regulates the crosstalk between PC and EC and reshapes their transcriptional profiles, triggering angiogenic, immunoregulatory and oncogenic gene expression programs. These findings highlight the angiostatic and oncostatic efficacy of targeting IL30 to fight PC.

Galectin-4 is associated with diabetes and obesity in a heart failure population.

An association between high Galectin-4 (Gal-4) and prevalence of diabetes in subjects with heart failure (HF) has previously been reported. The purpose of this study was to confirm these findings, as well as to further investigate this association, in a Swedish HF population. In addition, a second aim was to explore Gal-4's association with obesity and biomarkers of metabolism and heart failure. Gal-4 was measured using a proximity extension array technique in 324 hospitalized HF patients within the Swedish HeArt and bRain failure investigation trial cohort. Obesity was defined as BMI ≥ 30. Multivariable logistic regression models were used to explore associations between Gal-4 and diabetes/obesity, and linear regression models were used to explore the associations between Gal-4 and biomarkers. A total of 309 participants (29.1% female; mean age 74.8 years) provided complete data for the analysis of associations between Gal-4 and diabetes. Additionally, for the analysis of heart failure phenotype, complete data was available for 230 subjects. Gal-4 was positively associated with prevalent diabetes (OR 2.60; CI 95% 1.56-4.32). In multivariable models, Gal-4 levels were significantly associated with obesity, but only for subjects with diabetes (OR 2.48; 1.09-5.62). Additionally, Gal-4 demonstrated a significant association with the incretin Glucose-dependent insulinotropic polypeptide (GIP), as well as with biomarkers of HF. In the stratified analyses, the association between Gal-4 and diabetes was prominent in patients with reduced ejection fraction (n = 160, OR 3.26; 95%CI 1.88-5.66), while it was not observed in those without (n = 70, 1.96 (0.75-5.10)). In this cross-sectional, observational study, higher Gal-4 levels in HF patients were associated with higher GIP levels. Further, increased levels of Gal-4 were associated with increased likelihood of diabetes, and obesity. This association was particularly pronounced in individuals with HF characterized by reduced ejection fraction. Additionally, Gal-4 levels were significantly elevated in heart failure patients with diabetes and obesity.

Galectin-4 Is Involved in the Structural Changes of Glycosphingolipid Glycans in Poorly Differentiated Gastric Cancer Cells with High Metastatic Potential.

Gastric cancer with peritoneal dissemination is difficult to treat surgically, and frequently recurs and metastasizes. Currently, there is no effective treatment for this disease, and there is an urgent need to elucidate the molecular mechanisms underlying peritoneal dissemination and metastasis. Our previous study demonstrated that galectin-4 participates in the peritoneal dissemination of poorly differentiated gastric cancer cells. In this study, the glycan profiles of cell surface proteins and glycosphingolipids (GSLs) of the original (wild), galectin-4 knockout (KO), and rescue cells were investigated to understand the precise mechanisms involved in the galectin-4-mediated regulation of associated molecules, especially with respect to glycosylation. Glycan analysis of the NUGC4 wild type and galectin-4 KO clones with and without peritoneal metastasis revealed a marked structural change in the glycans of neutral GSLs, but not in N-glycan. Furthermore, mass spectrometry (MS) combined with glycosidase digestion revealed that this structural change was due to the presence of the lacto-type (ß1-3Galactosyl) glycan of GSL, in addition to the neolacto-type (ß1-4Galactosyl) glycan of GSL. Our results demonstrate that galectin-4 is an important regulator of glycosylation in cancer cells and galectin-4 expression affects the glycan profile of GSLs in malignant cancer cells with a high potential for peritoneal dissemination.

TGM2, HMGA2, FXYD3, and LGALS4 genes as biomarkers in acquired oxaliplatin resistance of human colorectal cancer: A systems biology approach.

Acquired resistance to oxaliplatin is considered as the primary reason for failure in colorectal cancer (CRC) therapy. Identifying the underlying resistance mechanisms may improve CRC treatment. The present study aims to identify the key genes involved in acquired oxaliplatin-resistant in CRC by confirming the oxaliplatin resistance index (OX-RI). To this aim, two public microarray datasets regarding oxaliplatin-resistant CRC cells with different OX-RI, GSE42387, and GSE76092 were downloaded from GEO database to identify differentially expressed genes (DEGs). The results indicated that the OX-RI affects the gene expression pattern significantly. Then, 54 common DEGs in both datasets including 18 up- and 36 down-regulated genes were identified. Protein-protein interaction (PPI) analysis revealed 13 up- (MAGEA6, TGM2, MAGEA4, SCHIP1, ECI2, CD33, AKAP12, MAGEA12, CALD1, WFDC2, VSNL1, HMGA2, and MAGEA2B) and 12 down-regulated (PDZK1IP1, FXYD3, ALDH2, CEACAM6, QPRT, GRB10, TM4SF4, LGALS4, ALDH3A1, USH1C, KCNE3, and CA12) hub genes. In the next step, two novel up-regulated hub genes including ECI2 and SCHIP1 were identified to be related to oxaliplatin resistance. Functional enrichment and pathway analysis indicated that metabolic pathways, proliferation, and epithelial-mesenchymal transition may play dominant roles in CRC progression and oxaliplatin resistance. In the next procedure, two in vitro oxaliplatin-resistant sub-lines including HCT116/OX-R4.3 and HCT116/OX-R10 cells with OX-IR 3.93 and 10.06 were established, respectively. The results indicated the up-regulation of TGM2 and HMGA2 in HCT116/OX-R10 cells with high OX-RI and down-regulation of FXYD3, LGALS4, and ECI2 in both cell types. Based on the results, TGM2, HMGA2, FXYD3, and LGALS4 genes are related to oxaliplatin-resistant CRC and may serve as novel therapeutic targets.

Oligosaccharide Ligands of Galectin-4 and Its Subunits: Multivalency Scores Highly.

Galectins are carbohydrate-binding lectins that modulate the proliferation, apoptosis, adhesion, or migration of cells by cross-linking glycans on cell membranes or extracellular matrix components. Galectin-4 (Gal-4) is a tandem-repeat-type galectin expressed mainly in the epithelial cells of the gastrointestinal tract. It consists of an N- and a C-terminal carbohydrate-binding domain (CRD), each with distinct binding affinities, interconnected with a peptide linker. Compared to other more abundant galectins, the knowledge of the pathophysiology of Gal-4 is sparse. Its altered expression in tumor tissue is associated with, for example, colon, colorectal, and liver cancers, and it increases in tumor progression, and metastasis. There is also very limited information on the preferences of Gal-4 for its carbohydrate ligands, particularly with respect to Gal-4 subunits. Similarly, there is virtually no information on the interaction of Gal-4 with multivalent ligands. This work shows the expression and purification of Gal-4 and its subunits and presents a structure-affinity relationship study with a library of oligosaccharide ligands. Furthermore, the influence of multivalency is demonstrated in the interaction with a model lactosyl-decorated synthetic glycoconjugate. The present data may be used in biomedical research for the design of efficient ligands of Gal-4 with diagnostic or therapeutic potential.

Multifaceted role of galectin-4 in cancer: A systematic review.

BACKGROUND: Galectins are ß-galactoside-binding proteins. Galectin-4 has shown an effect on cancer progression/metastasis, especially in cancers of the digestive system. This can be attributed to altered glycosylation pattern of cell membrane molecules, which is a characteristic attribute of oncogenesis. The aim of this paper is to systematically review galectin-4 in different cancers and its role in disease progression. METHODS: The study was designed on the basis of Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines. PubMed, Scopus, Web of Science, and Science Direct were used to search relevant literature with keywords "galectin-4 AND cancer", "galectin-4", "LGALS4", and "LGALS4 AND cancer". Inclusion criteria for study selection were availability of full-text articles, articles in English language and articles relevant to current topic, that is, galectin-4 and cancer. Exclusion criteria were studies that investigated other disease conditions, interventions unrelated to cancer or galectin-4 and bias outcome. RESULTS: A total of 73 articles were retrieved after removing duplication from databases, out of which 40 studies were included in the review that followed the inclusion criteria, including low to moderate bias. These included 23 studies in digestive system, 5 in reproductive system, 4 in respiratory system, and 2 in brain and urothelial cancers. CONCLUSIONS: A differential expression of galectin-4 was observed in different cancer stages/ and types. Furthermore, galectin-4 was found to modulate disease progression. A meta-analysis and comprehensive mechanistic studies, pertaining to different aspects of galectin-4 biology, could give statistically driven correlations, elucidating multifaceted role of galectin-4 in cancer.

Seven bacterial response-related genes are biomarkers for colon cancer.

BACKGROUND: Colon cancer (CC) is a common tumor that causes significant harm to human health. Bacteria play a vital role in cancer biology, particularly the biology of CC. Genes related to bacterial response were seldom used to construct prognosis models. We constructed a bacterial response-related risk model based on three Molecular Signatures Database gene sets to explore new markers for predicting CC prognosis. METHODS: The Cancer Genome Atlas (TCGA) colon adenocarcinoma samples were used as the training set, and Gene Expression Omnibus (GEO) databases were used as the test set. Differentially expressed bacterial response-related genes were identified for prognostic gene selection. Univariate Cox regression analysis, least absolute shrinkage and selection operator-penalized Cox regression analysis, and multivariate Cox regression analysis were performed to construct a prognostic risk model. The individual diagnostic effects of genes in the prognostic model were also evaluated. Moreover, differentially expressed long noncoding RNAs (lncRNAs) were identified. Finally, the expression of these genes was validated using quantitative polymerase chain reaction (qPCR) in cell lines and tissues. RESULTS: A prognostic signature was constructed based on seven bacterial response genes: LGALS4, RORC, DDIT3, NSUN5, RBCK1, RGL2, and SERPINE1. Patients were assigned a risk score based on the prognostic model, and patients in the TCGA cohort with a high risk score had a poorer prognosis than those with a low risk score; a similar finding was observed in the GEO cohort. These seven prognostic model genes were also independent diagnostic factors. Finally, qPCR validated the differential expression of the seven model genes and two coexpressed lncRNAs (C6orf223 and SLC12A9-AS1) in 27 pairs of CC and normal tissues. Differential expression of LGALS4 and NSUN5 was also verified in cell lines (FHC, COLO320DM, SW480). CONCLUSIONS: We created a seven-gene bacterial response-related gene signature that can accurately predict the outcomes of patients with CC. This model can provide valuable insights for personalized treatment.

Galectin-4 increases the ability of M2 macrophages to enhance antiviral CD4+ T-cell responses.

Galectin-4 (Gal-4) is a ß-galactoside-binding protein belonging to the galectin family. Although Gal-4 is known to be involved in several physiologic processes of the gastrointestinal tract, its immunomodulatory roles remain unclear. In this study, we investigated whether Gal-4 influences the function of M1 and M2 macrophages. Gal-4 treatment drove more robust changes in the gene expression of M2 macrophages compared to M1 macrophages. Antiviral immune response-related genes were significantly upregulated in Gal-4-treated M2 macrophages. Gal-4 significantly enhanced the immunostimulatory activity of M2 macrophages upon Toll-like receptor 7 stimulation or infection with lymphocytic choriomeningitis virus (LCMV). Moreover, the antibody production against LCMV infection and the antiviral CD4+ T-cell responses, but not the antiviral CD8+ T-cell responses, were greatly increased by Gal-4-treated M2 macrophages in vivo. The present results indicate that Gal-4 enhances the ability of M2 macrophages to promote antiviral CD4+ T-cell responses. Thus, Gal-4 could be used to boost antiviral immune responses.

Identification of potential biomarkers in Barrett's esophagus derived esophageal adenocarcinoma.

Almost 50% of esophageal adenocarcinoma (EAC) patients progressed from Barrett's esophagus (BE). EAC is often diagnosed at late stages and is related to dismal prognosis. However, there are still no effective methods for stratification and therapy in BE and EAC. Two public datasets (GSE26886 and GSE37200) were analyzed to identify differentially expressed genes (DEGs) between BE and EAC. Then, a series of bioinformatics analyses were performed to explore potential biomarkers associated with BE-EAC. 27 up- and 104 down-regulated genes were observed between GSE26886 and GSE37200. The GO and KEGG enrichment analysis indicated that the DEGs were highly involved in tumorigenesis. Subsequently, Weighted Gene Co-Expression Network Analysis (WGCNA) were performed to explore the potential genes related to BE-EAC, which were validated in The Cancer Genome Atlas (TCGA) database, and 5 up-regulated genes (MYO1A, ACE2, COL1A1, LGALS4, and ADRA2A) and 3 down-regulated genes (AADAC, RAB27A, and P2RY14) were found in EAC. Meanwhile, ADRA2A and AADAC could contribute to EAC pathogenesis and progression. MYO1A, ACE2, COL1A1, LGALS4, ADRA2A, AADAC, RAB27A, and P2RY14 could be potential novel diagnostic and prognostic biomarkers in BE-EAC.

Cytosolic galectin-4 enchains bacteria, restricts their motility, and promotes inflammasome activation in intestinal epithelial cells.

Galectin-4, a member of the galectin family of animal glycan-binding proteins (GBPs), is specifically expressed in gastrointestinal epithelial cells and is known to be able to bind microbes. However, its function in host-gut microbe interactions remains unknown. Here, we show that intracellular galectin-4 in intestinal epithelial cells (IECs) coats cytosolic Salmonella enterica serovar Worthington and induces the formation of bacterial chains and aggregates. Galectin-4 enchains bacteria during their growth by binding to the O-antigen of lipopolysaccharides. Furthermore, the binding of galectin-4 to bacterial surfaces restricts intracellular bacterial motility. Galectin-4 enhances caspase-1 activation and mature IL-18 production in infected IECs especially when autophagy is inhibited. Finally, orally administered S. enterica serovar Worthington, which is recognized by human galectin-4 but not mouse galectin-4, translocated from the intestines to mesenteric lymph nodes less effectively in human galectin-4-transgenic mice than in littermate controls. Our results suggest that galectin-4 plays an important role in host-gut microbe interactions and prevents the dissemination of pathogens. The results of the study revealed a novel mechanism of host-microbe interactions that involves the direct binding of cytosolic lectins to glycans on intracellular microbes.

Suppression of galectin-4 attenuates peritoneal metastasis of poorly differentiated gastric cancer cells.

BACKGROUND: Peritoneal dissemination, most often seen in metastatic and/or recurrent gastric cancer, is an inoperable condition that lacks effective treatment. The use of molecular targeted drugs is also limited; therefore, identifying novel therapeutic targets and improving our understanding of this metastatic cancer are an urgent requirement. In this study, we focused on galectin-4, which is specifically expressed in poorly differentiated cells with high potential for peritoneal dissemination. METHODS: We knocked out the galectin-4 gene in NUGC4 cells using CRISPR/Cas9-mediated genome editing. Proliferation and peritoneal cancer formation in knockout cells were compared with those in wild-type and galectin-4 re-expressing cells. Western blotting and proximity ligation assays were performed to identify associated molecules affected by the expression of galectin-4. The effect of galectin-4 knockdown on cell proliferation and peritoneal metastasis was studied using a specific siRNA. Expression of galectin-4 in peritoneal metastatic tumors from 10 patients with gastric cancer was examined by immunohistochemistry. RESULTS: Suppression of galectin-4 expression reduced proliferation and peritoneal metastasis of malignant gastric cancer cells. Galectin-4 knockout and knockdown reduced the expression of activated c-MET and CD44. Galectin-4 was found to interact with several proteins on the cell surface, including CD44 and c-MET, via its carbohydrate-binding ability. Immunohistochemistry showed galectin-4 expression in peritoneal metastatic tumor cells in all patients examined. CONCLUSIONS: We clarified the role of galectin-4 in the development of peritoneal dissemination of poorly differentiated gastric cancer cells. Our data highlight the diagnostic and therapeutic potential of galectin-4 in the peritoneal dissemination of gastric cancer.

Extracellular Galectin 4 Drives Immune Evasion and Promotes T-cell Apoptosis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell-produced protein that was deposited into the ECM of PDAC tumors and detected high-circulating levels of gal 4 in patients with PDAC. In orthotopic transplantation experiments, we observed increased infiltration of T cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1-/- mice, demonstrating that the effect was mediated by the adaptive immune system. By performing single-cell RNA-sequencing, we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression associated with a higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T cells, and antigen-presenting dendritic cells in tumors with reduced gal 4 expression. Using a coculture system, we observed that extracellular gal 4 induced apoptosis in T cells by binding N-glycosylation residues on CD3ε/δ. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC.

Construction and validation of an immune-related genes prognostic index (IRGPI) model in colon cancer.

Background: Though immunotherapy has become one of the standard therapies for colon cancer, the overall effective rate of immunotherapy is very low. Constructing an immune-related genes prognostic index (IRGPI) model may help to predict the response to immunotherapy and clinical outcomes. Methods: Differentially expressed immune-related genes (DEIRGs) between normal tissues and colon cancer tissues were identified and used to construct the co-expression network. Genes in the module with the most significant differences were further analyzed. Independent prognostic immune-related genes (IRGs) were identified by univariate and multivariate cox regression analysis. Independent prognostic IRGs were used to construct the IRGPI model using the multivariate cox proportional hazards regression model, and the IRGPI model was validated by independent dataset. ROC curves were plotted and AUCs were calculated to estimate the predictive power of the IRGPI model to prognosis. Gene set enrichment analysis (GSEA) was performed to screen the enriched KEGG pathways in the high-risk and low-risk phenotype. Correlations between IRGPI and clinical characteristic, immune checkpoint expression, TMB, immune cell infiltration, immune function, immune dysfunction, immune exclusion, immune subtype were analyzed. Results: Totally 680 DEIRGs were identified. Three independent IRGs,NR5A2, PPARGC1A and LGALS4, were independently related to survival. NR5A2, PPARGC1A and LGALS4 were used to establish the IRGPI model. Survival analysis showed that patients with high-risk showed worse survival than patients in the low-risk group. The AUC of the IRGPI model for 1-year, 3-year and 5-year were 0.584, 0.608 and 0.697, respectively. Univariate analysis and multivariate cox regression analysis indicated that IRGPI were independent prognostic factors for survival. Stratified survival analysis showed that patients with IRGPI low-risk and low TMB had the best survival, which suggested that combination of TMB and IRGPI can better predict clinical outcome. Immune cell infiltration, immune function, immune checkpoint expression and immune exclusion were different between IRGPI high-risk and low-risk patients. Conclusion: An immune-related genes prognostic index (IRGPI) was constructed and validated in the current study and the IRGPI maybe a potential biomarker for evaluating response to immunotherapy and clinical outcome for colon cancer patients.

Human Milk Oligosaccharide 2'-Fucosyllactose Modulates Local Viral Immune Defense by Supporting the Regulatory Functions of Intestinal Epithelial and Immune Cells.

Human milk contains bioactive components that provide protection against viral infections in early life. In particular, intestinal epithelial cells (IEC) have key regulatory roles in the prevention of enteric viral infections. Here we established an in vitro model to study the modulation of host responses against enteric viruses mimicked by poly I:C (pIC). The effects of 2'-fucosyllactose (2'FL), abundantly present in human milk, were studied on IEC and/or innate immune cells, and the subsequent functional response of the adaptive immune cells. IEC were pre-incubated with 2'FL and stimulated with naked or Lyovec™-complexed pIC (LV-pIC). Additionally, monocyte-derived dendritic cells (moDC) alone or in co-culture with IEC were stimulated with LV-pIC. Then, conditioned-moDC were co-cultured with naïve CD4+ T helper (Th)-cells. IEC stimulation with naked or LV-pIC promoted pro-inflammatory IL-8, CCL20, GROα and CXCL10 cytokine secretion. However, only exposure to LV-pIC additionally induced IFNß, IFNλ1 and CCL5 secretion. Pre-incubation with 2'FL further increased pIC induced CCL20 secretion and LV-pIC induced CXCL10 secretion. LV-pIC-exposed IEC/moDC and moDC cultures showed increased secretion of IL-8, GROα, IFNλ1 and CXCL10, and in the presence of 2'FL galectin-4 and -9 were increased. The LV-pIC-exposed moDC showed a more pronounced secretion of CCL20, CXCL10 and CCL5. The moDC from IEC/moDC cultures did not drive T-cell development in moDC/T-cell cultures, while moDC directly exposed to LV-pIC secreted Th1 driving IL-12p70 and promoted IFNγ secretion by Th-cells. Hereby, a novel intestinal model was established to study mucosal host-defense upon a viral trigger. IEC may support intestinal homeostasis, regulating local viral defense which may be modulated by 2'FL. These results provide insights regarding the protective capacity of human milk components in early life.

Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses to Inspect Intracellular Functions of the Tumor Suppressor Galectin-4 in Colorectal Cancer Cells.

Galectin-4 (Gal4) has been suggested to function as a tumor suppressor in colorectal cancer (CRC). In order to systematically explore its function in CRC, we established a CRC cell line where Gal4 expression can be regulated via the doxycycline (dox)-inducible expression of a single copy wildtype LGALS4 transgene generated by recombinase-mediated cassette exchange (RMCE). Using this model and applying in-depth proteomic and phosphoproteomic analyses, we systematically screened for intracellular changes induced by Gal4 expression. Overall, 3083 cellular proteins and 2071 phosphosites were identified and quantified, of which 1603 could be matched and normalized to their protein expression levels. A bioinformatic analysis revealed that most of the regulated proteins and phosphosites can be localized in the nucleus and are categorized as nucleic acid-binding proteins. The top candidates whose expression was modulated by Gal4 are PURB, MAPKAPK3, BTF3 and BCAR1, while the prime candidates with altered phosphorylation included ZBTB7A, FOXK1, PURB and CK2beta. In order to validate the (phospho)proteomic data, we confirmed these candidates by a radiometric metabolic-labelling and immunoprecipitation strategy. All candidates exert functions in the transcriptional or translational control, indicating that Gal4 might be involved in these processes by affecting the expression or activity of these proteins.

Progesterone promotes immunomodulation and tumor development in the murine mammary gland.

BACKGROUND: Clinical studies have linked usage of progestins (synthetic progesterone [P4]) to breast cancer risk. However, little is understood regarding the role of native P4, signaling through the progesterone receptor (PR), in breast tumor formation. Recently, we reported a link between PR and immune signaling pathways, showing that P4/PR can repress type I interferon signaling pathways. Given these findings, we sought to investigate whether P4/PR drive immunomodulation in the mammary gland and promote tumor formation. METHODS: To determine the effect of P4 on immune cell populations in the murine mammary gland, mice were treated with P4 or placebo pellets for 21 days. Immune cell populations in the mammary gland, spleen, and inguinal lymph nodes were subsequently analyzed by flow cytometry. To assess the effect of PR overexpression on mammary gland tumor development as well as immune cell populations in the mammary gland, a transgenic mouse model was used in which PR was overexpressed throughout the entire mouse. Immune cell populations were assessed in the mammary glands, spleens, and inguinal lymph nodes of 6-month-old transgenic and control mice by flow cytometry. Transgenic mice were also monitored for mammary gland tumor development over a 2-year time span. Following development of mammary gland tumors, immune cell populations in the tumors and spleens of transgenic and control mice were analyzed by flow cytometry. RESULTS: We found that mice treated with P4 exhibited changes in the mammary gland indicative of an inhibited immune response compared with placebo-treated mice. Furthermore, transgenic mice with PR overexpression demonstrated decreased numbers of immune cell populations in their mammary glands, lymph nodes, and spleens. On long-term monitoring, we determined that multiparous PR-overexpressing mice developed significantly more mammary gland tumors than control mice. Additionally, tumors from PR-overexpressing mice contained fewer infiltrating immune cells. Finally, RNA sequencing analysis of tumor samples revealed that immune-related gene signatures were lower in tumors from PR-overexpressing mice as compared with control mice. CONCLUSION: Together, these findings offer a novel mechanism of P4-driven mammary gland tumor development and provide rationale in investigating the usage of antiprogestin therapies to promote immune-mediated elimination of mammary gland tumors.

SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.

To elucidate the underlying mechanism of secretory leukocyte protease inhibitor (SLPI)-induced cell migration, we compared SLPI-deleted human gingival carcinoma Ca9-22 (ΔSLPI) cells and original (wild-type: wt) Ca9-22 cells using several microscopic imaging methods and gene expression analysis. Our results indicated reduced migration of ΔSLPI cells compared to wtCa9-22 cells. The lamellipodia/dorsal ruffles were smaller and moved slower in ΔSLPI cells compared to wtCa9-22 cells. Furthermore, well-developed intermediate filament bundles were observed at the desmosome junction of ΔSLPI cells. In addition, Galectin4 was strongly expressed in ΔSLPI cells, and its forced expression suppressed migration of wtCa9-22 cells. Taken together, SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.

A tandem-repeat galectin-4 from Nile tilapia (Oreochromis niloticus) is involved in immune response to bacterial infection via mediating pathogen recognition and opsonization.

Galectins are the family of carbohydrate-binding proteins that participate in host-pathogen interaction. In this study, a galectin-4 homolog (OnGal-4) from Nile tilapia (Oreochromis niloticus) was characterized. The open reading frame of OnGal-4 was 1194 bp, encoding a peptide of 397 amino including two CRD regions and two carbohydrate recognition sites. OnGal-4 mRNA was expressed in all examined tissues with the highest level in spleen. After Streptococcus agalactiae (S.agalactiae) challenge, the OnGal-4 expression was up-regulated in the spleen, head kidney, brain, and monocytes/macrophages (Mo/MΦ). The in vitro experiments showed that recombinant OnGal-4 (rOnGal-4) protein could bind and agglutinate S.agalactiae and A.hydrophila. Also, rOnGal-4 could induce cytokines expressions and increased bactericidal activity of Mo/MΦ. Further in vivo analysis indicated that OnGal-4 overexpression could protect O.niloticus from S.agalactiae infection through modulating inflammation response. Our study suggested that OnGal-4 could improve immune response against bacterial infection by mediating pathogen recognition and opsonization.

LGALS4 as a Prognostic Factor in Urothelial Carcinoma of Bladder Affects Cell Functions.

BACKGROUND: To identify the hub genes related to urothelial carcinoma of the bladder prognosis and to understand their underlying mechanism. METHODS: The expression profiles of 18 pairs of urothelial carcinoma of the bladder patient tissue and paired adjacent tissue obtained from the Cancer Genome Atlas were performed. Weighted gene coexpression network analysis was employed to screen gene modules and hub genes with significant differential expressions in urothelial carcinoma of the bladder. The hub genes expression in urothelial carcinoma of the bladder tissues was validated by reverse transcription-quantitative polymerase chain reaction. The overall survival curve and disease-free survival curve of prognostic factor (LGALS4) were plotted using the Kaplan-Meier method. Furthermore, LGALS4 messenger RNA and protein expression were also assessed in 2 urothelial carcinoma of the bladder cell lines (T24 and 5637) by quantitative reverse transcription-polymerase chain reaction and Western blot. The functions of urothelial carcinoma of the bladder cells with transfected pcDNA3.1-LGALS4 were identified through MTT assay, plate clone formation assay, flow cytometry, and cell migration experiments. RESULTS: LGALS4 was the hub gene of pink module and it was related to prognosis. Higher LGALS4 expression predicted higher probabilities of overall survival and disease-free survival. Overexpression of LGALS4 in urothelial carcinoma of the bladder cells suppressed cell viability and migration but induced apoptosis. CONCLUSION: LGALS4 played a critical role in the progression of urothelial carcinoma of the bladder and held a promise to be the biomarker for diagnosis and treatment of urothelial carcinoma of the bladder. It predicted good prognosis of urothelial carcinoma of the bladder and restrained the growth and migration of urothelial carcinoma of the bladder cells.

Galectin 4 is a biomarker for early recurrence and death after surgical resection for pancreatic ductal adenocarcinoma.

BACKGROUND: Galectins are a group of carbohydrate-binding proteins that are involved in neoplastic development and progression. In a previous mass spectrometry-based study, we identified galectin 4 as a down-regulated protein in short-term survivors of pancreatic cancer. This study was performed to validate the prognostic value of galectin 4 in a larger cohort of pancreatic cancer patients undergoing surgical resection. METHODS: Galectin 4 expression was evaluated by tissue microarrays and immunohistochemistry in 140 patients with surgically resected pancreatic cancer. Kaplan-Meier and Cox proportional hazards modeling were used to explore the association between galectin 4 and survival. RESULTS: Galectin 4 staining expression was positive in 111 cases (79.3%). The expression of galectin 4 was significantly associated with tumor size (p = .008) and differentiation (p = .001). Galectin 4 expression was significantly correlated with disease recurrence within 1 year of surgery (adjusted HR 0.485, p = .027). There was also a significant association between galectin 4 and overall survival at 1 year (adjusted HR 0.482, p = .047) and at 3 years (adjusted HR 0.550, p = .025). CONCLUSION: Galectin 4 expression is a novel biomarker for early recurrence and mortality after surgical resection for pancreatic cancer.