Assessing the Antitumor Potential of Variants of the Extracellular Carbohydrate Polymer from Synechocystis ΔsigF Mutant.

Cancer is a leading cause of death worldwide with a huge societal and economic impact. Clinically effective and less expensive anticancer agents derived from natural sources can help to overcome limitations and negative side effects of chemotherapy and radiotherapy. Previously, we showed that the extracellular carbohydrate polymer of a Synechocystis ΔsigF overproducing mutant displayed a strong antitumor activity towards several human tumor cell lines, by inducing high levels of apoptosis through p53 and caspase-3 activation. Here, the ΔsigF polymer was manipulated to obtain variants that were tested in a human melanoma (Mewo) cell line. Our results demonstrated that high molecular mass fractions were important for the polymer bioactivity, and that the reduction of the peptide content generated a variant with enhanced in vitro antitumor activity. This variant, and the original ΔsigF polymer, were further tested in vivo using the chick chorioallantoic membrane (CAM) assay. Both polymers significantly decreased xenografted CAM tumor growth and affected tumor morphology, by promoting less compact tumors, validating their antitumor potential in vivo. This work contributes with strategies for the design and testing tailored cyanobacterial extracellular polymers and further strengths the relevance of evaluating this type of polymers for biotechnological/biomedical applications.

Helicobacter pylori infection induces abnormal expression of pro-angiogenic gene ANGPT2 and miR-203a in AGS gastric cell line.

Helicobacter pylori colonizes the stomach and induces an inflammatory response that can develop into gastric pathologies including cancer. The infection can alter the gastric vasculature by the deregulation of angiogenic factors and microRNAs. In this study, we investigate the expression level of pro-angiogenic genes (ANGPT2, ANGPT1, receptor TEK), and microRNAs (miR-135a, miR-200a, miR-203a) predicted to regulate those genes, using H. pylori co-cultures with gastric cancer cell lines. In vitro infections of different gastric cancer cell lines with H. pylori strains were performed, and the expression of ANGPT1, ANGPT2, and TEK genes, and miR-135a, miR-200a, and miR-203a, was quantified after 24 h of infection (h.p.i.). We performed a time course experiment of H. pylori 26695 infections in AGS cells at 6 different time points (3, 6, 12, 28, 24, and 36 h.p.i.). The angiogenic response induced by supernatants of non-infected and infected cells at 24 h.p.i. was evaluated in vivo, using the chicken chorioallantoic membrane (CAM) assay. In response to infection, ANGPT2 mRNA was upregulated at 24 h.p.i, and miR-203a was downregulated in AGS cells co-cultured with different H. pylori strains. The time course of H. pylori 26695 infection in AGS cells showed a gradual decrease of miR-203a expression concomitant with an increase of ANGPT2 mRNA and protein expression. Expression of ANGPT1 and TEK mRNA or protein could not be detected in any of the infected or non-infected cells. CAM assays showed that the supernatants of AGS-infected cells with 26695 strain induced a significantly higher angiogenic and inflammatory response. Our results suggest that H. pylori could contribute to the process of carcinogenesis by downregulating miR-203a, which further promotes angiogenesis in gastric mucosa by increasing ANGPT2 expression. Further investigation is needed to elucidate the underlying molecular mechanisms.

The Extracellular Small Leucine-Rich Proteoglycan Biglycan Is a Key Player in Gastric Cancer Aggressiveness.

Biglycan (BGN gene), an extracellular proteoglycan, has been described to be associated with cancer aggressiveness. The purpose of this study was to clarify the clinical value of biglycan as a biomarker in multiple independent GC cohorts and determine the in vitro and in vivo role of biglycan in GC malignant features. We found that BGN is commonly over-expressed in all analyzed cohorts, being associated with disease relapse and poor prognosis in patients with advanced stages of disease. In vitro and in vivo experiments demonstrated that biglycan knock-out GC cells display major phenotypic changes with a lower cell survival, migration, and angiogenic potential when compared with biglycan expressing cells. Biglycan KO GC cells present increased levels of PARP1 and caspase-3 cleavage and a decreased expression of mesenchymal markers. Importantly, biglycan deficient GC cells that were supplemented with exogenous biglycan were able to restore biological features, such as survival, clonogenic and migratory capacities. Our in vitro and in vivo findings were validated in human GC samples, where BGN expression was associated with several oncogenic gene signatures that were associated with apoptosis, cell migration, invasion, and angiogenesis. This study provided new insights on biglycan role in GC that should be taken in consideration as a key cellular regulator with major impact in tumor progression and patients' clinical outcome.

The antibacterial and angiogenic effect of magnesium oxide in a hydroxyapatite bone substitute.

Bone graft infections are serious complications in orthopaedics and the growing resistance to antibiotics is increasing the need for antibacterial strategies. The use of magnesium oxide (MgO) is an interesting alternative since it possesses broad-spectrum antibacterial activity. Additionally, magnesium ions also play a role in bone regeneration, which makes MgO more appealing than other metal oxides. Therefore, a bone substitute composed of hydroxyapatite and MgO (HAp/MgO) spherical granules was developed using different sintering heat-treatment cycles to optimize its features. Depending on the sintering temperature, HAp/MgO spherical granules exhibited distinct surface topographies, mechanical strength and degradation profiles, that influenced the in vitro antibacterial activity and cytocompatibility. A proper balance between antibacterial activity and cytocompatibility was achieved with HAp/MgO spherical granules sintered at 1100 ºC. The presence of MgO in these granules was able to significantly reduce bacterial proliferation and simultaneously provide a suitable environment for osteoblasts growth. The angiogenic and inflammation potentials were also assessed using the in vivo chicken embryo chorioallantoic membrane (CAM) model and the spherical granules containing MgO stimulated angiogenesis without increasing inflammation. The outcomes of this study evidence a dual effect of MgO for bone regenerative applications making this material a promising antibacterial bone substitute.

BR-BCSC Signature: The Cancer Stem Cell Profile Enriched in Brain Metastases that Predicts a Worse Prognosis in Lymph Node-Positive Breast Cancer.

Brain metastases remain an unmet clinical need in breast oncology, being frequently found in HER2-overexpressing and triple-negative carcinomas. These tumors were reported to be highly cancer stem-like cell-enriched, suggesting that brain metastases probably arise by the seeding of cancer cells with stem features. Accordingly, we found that brain-tropic breast cancer cells show increased stem cell activity and tumorigenic capacity in the chick embryo choriallantoic membrane when compared to the parental cell line. These observations were supported by a significant increase in their stem cell frequency and by the enrichment for the breast cancer stem cell (BCSC) phenotype CD44+CD24-/low. Based on this data, the expression of BCSC markers (CD44, CD49f, P-cadherin, EpCAM, and ALDH1) was determined and found to be significantly enriched in breast cancer brain metastases when compared to primary tumors. Therefore, a brain (BR)-BCSC signature was defined (3-5 BCSC markers), which showed to be associated with decreased brain metastases-free and overall survival. Interestingly, this signature significantly predicted a worse prognosis in lymph node-positive patients, acting as an independent prognostic factor. Thus, an enrichment of a BCSC signature was found in brain metastases, which can be used as a new prognostic factor in clinically challenging breast cancer patients.

Helicobacter Pylori Targets the EPHA2 Receptor Tyrosine Kinase in Gastric Cells Modulating Key Cellular Functions.

Helicobacter pylori, a stomach-colonizing Gram-negative bacterium, is the main etiological factor of various gastroduodenal diseases, including gastric adenocarcinoma. By establishing a life-long infection of the gastric mucosa, H. pylori continuously activates host-signaling pathways, in particular those associated with receptor tyrosine kinases. Using two different gastric epithelial cell lines, we show that H. pylori targets the receptor tyrosine kinase EPHA2. For long periods of time post-infection, H. pylori induces EPHA2 protein downregulation without affecting its mRNA levels, an effect preceded by receptor activation via phosphorylation. EPHA2 receptor downregulation occurs via the lysosomal degradation pathway and is independent of the H.pylori virulence factors CagA, VacA, and T4SS. Using small interfering RNA, we show that EPHA2 knockdown affects cell-cell and cell-matrix adhesion, invasion, and angiogenesis, which are critical cellular processes in early gastric lesions and carcinogenesis mediated by the bacteria. This work contributes to the unraveling of the underlying mechanisms of H. pylori-host interactions and associated diseases. Additionally, it raises awareness for potential interference between H. pylori infection and the efficacy of gastric cancer therapies targeting receptors tyrosine kinases, given that infection affects the steady-state levels and dynamics of some receptor tyrosine kinases (RTKs) and their signaling pathways.

O-glycans truncation modulates gastric cancer cell signaling and transcription leading to a more aggressive phenotype.

BACKGROUND: Changes in glycosylation are known to play critical roles during gastric carcinogenesis. Expression of truncated O-glycans, such as the Sialyl-Tn (STn) antigen, is a common feature shared by many cancers and is associated with cancer aggressiveness and poor-prognosis. METHODS: Glycoengineered cell lines were used to evaluate the impact of truncated O-glycans in cancer cell biology using in vitro functional assays, transcriptomic analysis and in vivo models. Tumor patients 'samples and datasets were used for clinical translational significance evaluation. FINDINGS: In the present study, we demonstrated that gastric cancer cells expressing truncated O-glycans display major phenotypic alterations associated with higher cell motility and cell invasion. Noteworthy, the glycoengineered cancer cells overexpressing STn resulted in tumor xenografts with less cohesive features which had a critical impact on mice survival. Furthermore, truncation of O-glycans induced activation of EGFR and ErbB2 receptors and a transcriptomic signature switch of gastric cancer cells. The disclosed top activated genes were further validated in gastric tumors, revealing that SRPX2 and RUNX1 are concomitantly overexpressed in gastric carcinomas and its expression is associated with patients' poor-survival, highlighting their prognosis potential in clinical practice. INTERPRETATION: This study discloses novel molecular links between O-glycans truncation frequently observed in cancer and key cellular regulators with major impact in tumor progression and patients' clinical outcome.

A New Role for Helicobacter pylori Urease: Contributions to Angiogenesis.

Helicobacter pylori is a pathogen involved in gastric diseases such as ulcers and carcinomas. H. pylori's urease is an important virulence factor produced in large amounts by this bacterium. In previous studies, we have shown that this protein is able to activate several cell types like neutrophils, monocytes, platelets, endothelial cells, and gastric epithelial cells. Angiogenesis is a physiological process implicated in growth, invasion and metastization of tumors. Here, we have analyzed the angiogenic potential of H. pylori urease (HPU) in gastric epithelial cells. No cytotoxicity was observed in AGS, Kato-III, and MKN28 gastric cell lines treated with 300 nM HPU, as evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As we previously reported in neutrophils, treatment with 300 nM HPU also had an anti-apoptotic effect in gastric epithelial cells leading to a 2.2-fold increase in the levels of Bcl-XL after 6 h, and a decrease of 80% in the content of BAD, after 48 h, two mitochondrial proteins involved in regulation of apoptosis. Within 10 min of exposure, HPU is rapidly internalized by gastric epithelial cells. Treatment of the gastric cells with methyl-ß-cyclodextrin abolished HPU internalization suggesting a cholesterol-dependent process. HPU induces the expression of pro-angiogenic factors and the decrease of expression of anti-angiogenic factors by AGS cells. The angiogenic activity of HPU was analyzed using in vitro and in vivo models. HPU induced formation of tube-like structures by human umbilical vascular endothelial cells in a 9 h experiment. In the chicken embryo chorioallantoic membrane model, HPU induced intense neo-vascularization after 3 days. In conclusion, our results indicate that besides allowing bacterial colonization of the gastric mucosa, H. pylori's urease triggers processes that initiate pro-angiogenic responses in different cellular models. Thus, this bacterial urease, a major virulence factor, may also play a role in gastric carcinoma development.

Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis.

AIMS: Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). METHODS AND RESULTS: Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. CONCLUSIONS: This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply.

Intricate Macrophage-Colorectal Cancer Cell Communication in Response to Radiation.

Both cancer and tumour-associated host cells are exposed to ionizing radiation when a tumour is subjected to radiotherapy. Macrophages frequently constitute the most abundant tumour-associated immune population, playing a role in tumour progression and response to therapy. The present work aimed to evaluate the importance of macrophage-cancer cell communication in the cellular response to radiation. To address this question, we established monocultures and indirect co-cultures of human monocyte-derived macrophages with RKO or SW1463 colorectal cancer cells, which exhibit higher and lower radiation sensitivity, respectively. Mono- and co-cultures were then irradiated with 5 cumulative doses, in a similar fractionated scheme to that used during cancer patients' treatment (2 Gy/fraction/day). Our results demonstrated that macrophages sensitize RKO to radiation-induced apoptosis, while protecting SW1463 cells. Additionally, the co-culture with macrophages increased the mRNA expression of metabolism- and survival-related genes more in SW1463 than in RKO. The presence of macrophages also upregulated glucose transporter 1 expression in irradiated SW1463, but not in RKO cells. In addition, the influence of cancer cells on the expression of pro- and anti-inflammatory macrophage markers, upon radiation exposure, was also evaluated. In the presence of RKO or SW1463, irradiated macrophages exhibit higher levels of pro-inflammatory TNF, IL6, CCL2 and CCR7, and of anti-inflammatory CCL18. However, RKO cells induce an increase of macrophage pro-inflammatory IL1B, while SW1463 cells promote higher pro-inflammatory CXCL8 and CD80, and also anti-inflammatory VCAN and IL10 levels. Thus, our data demonstrated that macrophages and cancer cells mutually influence their response to radiation. Notably, conditioned medium from irradiated co-cultures increased non-irradiated RKO cell migration and invasion and did not impact on angiogenesis in a chicken embryo chorioallantoic membrane assay. Overall, the establishment of primary human macrophage-cancer cell co-cultures revealed an intricate cell communication in response to ionizing radiation, which should be considered when developing therapies adjuvant to radiotherapy.

Modelling the tumour microenvironment in long-term microencapsulated 3D co-cultures recapitulates phenotypic features of disease progression.

3D cell tumour models are generated mainly in non-scalable culture systems, using bioactive scaffolds. Many of these models fail to reflect the complex tumour microenvironment and do not allow long-term monitoring of tumour progression. To overcome these limitations, we have combined alginate microencapsulation with agitation-based culture systems, to recapitulate and monitor key aspects of the tumour microenvironment and disease progression. Aggregates of MCF-7 breast cancer cells were microencapsulated in alginate, either alone or in combination with human fibroblasts, then cultured for 15 days. In co-cultures, the fibroblasts arranged themselves around the tumour aggregates creating distinct epithelial and stromal compartments. The presence of fibroblasts resulted in secretion of pro-inflammatory cytokines and deposition of collagen in the stromal compartment. Tumour cells established cell-cell contacts and polarised around small lumina in the interior of the aggregates. Over the culture period, there was a reduction in oestrogen receptor and membranous E-cadherin alongside loss of cell polarity, increased collective cell migration and enhanced angiogenic potential in co-cultures. These phenotypic alterations, typical of advanced stages of cancer, were not observed in the mono-cultures of MCF-7 cells. The proposed model system constitutes a new tool to study tumour-stroma crosstalk, disease progression and drug resistance mechanisms.

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade.

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and ßPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), ß1 and ß4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression.

Matrix metalloproteases as maestros for the dual role of LPS- and IL-10-stimulated macrophages in cancer cell behaviour.

BACKGROUND: The interactions established between macrophages and cancer cells are largely dependent on instructions from the tumour microenvironment. Macrophages may differentiate into populations with distinct inflammatory profiles, but knowledge on their role on cancer cell activities is still very scarce. In this work, we investigated the influence of pro-inflammatory (LPS-stimulated) and anti-inflammatory (IL-10-stimulated) macrophages on gastric and colorectal cancer cell invasion, motility/migration, angiogenesis and proteolysis, and the associated molecular mechanisms. METHODS: Following exposure of gastric and colon cancer cell lines to LPS- and IL-10-stimulated human macrophages, either by indirect contact or conditioned media, we analyzed the effect of the different macrophage populations on cancer cell invasion, migration, motility and phosphorylation status of EGFR and several interacting partners. Cancer-cell induced angiogenesis upon the influence of conditioned media from both macrophage populations was assessed using the chick embryo chorioallantoic membrane assay. MMP activities were evaluated by gelatin zymograhy. RESULTS: Our results show that IL-10-stimulated macrophages are more efficient in promoting in vitro cancer cell invasion and migration. In addition, soluble factors produced by these macrophages enhanced in vivo cancer cell-induced angiogenesis, as opposed to their LPS-stimulated counterparts. We further demonstrate that differences in the ability of these macrophage populations to stimulate invasion or angiogenesis cannot be explained by the EGFR-mediated signalling, since both LPS- and IL-10-stimulated macrophages similarly induce the phosphorylation of cancer cell EGFR, c-Src, Akt, ERK1/2, and p38. Interestingly, both populations exert distinct proteolytic activities, being the IL-10-stimulated macrophages the most efficient in inducing matrix metalloprotease (MMP)-2 and MMP-9 activities. Using a broad-spectrum MMP inhibitor, we demonstrated that proteolysis was essential for macrophage-mediated cancer cell invasion and angiogenesis. CONCLUSIONS: We propose that IL-10- and LPS-stimulated macrophages distinctly modulate gastric and colorectal cancer cell behaviour, as result of distinct proteolytic profiles that impact cell invasion and angiogenesis.

DNAJB4 molecular chaperone distinguishes WT from mutant E-cadherin, determining their fate in vitro and in vivo.

E-cadherin (Ecad) is a well-known invasion suppressor and its loss of expression is common in invasive carcinomas. Germline Ecad mutations are the only known genetic cause of hereditary diffuse gastric cancer (HDGC), demonstrating the causative role of Ecad impairment in gastric cancer. HDGC-associated Ecad missense mutations can lead to folding defects and premature proteasome-dependent endoplasmic reticulum-associated degradation (ERAD), but the molecular determinants for this fate were unidentified. Using a Drosophila-based genetic screen, we found that Drosophila DnaJ-1 interacts with wild type (WT) and mutant human Ecad in vivo. DnaJ (Hsp40) homolog, subfamily B, member 4 (DNAJB4), the human homolog of DnaJ-1, influences Ecad localization and stability even in the absence of Ecad endogenous promoter, suggesting a post-transcriptional level of regulation. Increased expression of DNAJB4 leads to stabilization of WT Ecad in the plasma membrane, while it induces premature degradation of unfolded HDGC mutants in the proteasome. The interaction between DNAJB4 and Ecad is direct, and is increased in the context of the unfolded mutant E757K, especially when proteasome degradation is inhibited, suggesting that DNAJB4 is a molecular mediator of ERAD. Post-translational regulation of native Ecad by DNAJB4 molecular chaperone is sufficient to influence cell adhesion in vitro. Using a chick embryo chorioallantoic membrane assay with gastric cancer derived cells, we demonstrate that DNAJB4 stimulates the anti-invasive function of WT Ecad in vivo. Additionally, the expression of DNAJB4 and Ecad is concomitantly decreased in human gastric carcinomas. Altogether, we demonstrate that DNAJB4 is a sensor of Ecad structural features that might contribute to gastric cancer progression.

CPEB1, a novel gene silenced in gastric cancer: a Drosophila approach.

BACKGROUND: Gastric cancer (GC) is a highly prevalent disease, being the fourth most common cancer and the second leading cause of cancer-associated deaths worldwide. Although many genes have been implicated in its development, many cases remain genetically unexplained. Hence, there is an urgent need to find new disease-related genes. METHODS: A transgenic Drosophila model was used to screen for novel genes putatively involved in GC. The authors evaluated the expression of the most interesting candidates in GC cell lines and primary tumours by semi-quantitative reverse transcription PCR, dissected the molecular mechanisms responsible for the deregulation of the most relevant one, and analysed its functional role in vitro and in a chicken embryo model. RESULTS: Six candidate genes were identified, of which cytoplasmic polyadenylation element binding protein 1 (CPEB1) was downregulated in all GC cell lines and in 11 of 12 primary GC tumours. The pivotal CPEB1 promoter CpG site was determined, and it was found that methylation at this 79th CpG site was associated with CPEB1 silencing in GC cell lines and primary tumours. It was also discovered that methylation of this site was significantly more prevalent in diffuse type GC (p=0.007) and in cases with lymph node metastases (p=0.042). In vitro, CPEB1 impaired invasion. Its antiangiogenic role was also discovered, which was associated with downregulation of MMP14 and VEGFA. CONCLUSIONS: The first evidence of CPEB1 involvement in GC is presented, along with the molecular mechanism underlying the regulation of its expression and its potential role in invasion and angiogenesis.