New Insights in ATP Synthesis as Therapeutic Target in Cancer and Angiogenic Ocular Diseases.

Lactate and ATP formation by aerobic glycolysis, the Warburg effect, is considered a hallmark of cancer. During angiogenesis in non-cancerous tissue, proliferating stalk endothelial cells (ECs) also produce lactate and ATP by aerobic glycolysis. In fact, all proliferating cells, both non-cancer and cancer cells, need lactate for the biosynthesis of building blocks for cell growth and tissue expansion. Moreover, both non-proliferating cancer stem cells in tumors and leader tip ECs during angiogenesis rely on glycolysis for pyruvate production, which is used for ATP synthesis in mitochondria through oxidative phosphorylation (OXPHOS). Therefore, aerobic glycolysis is not a specific hallmark of cancer but rather a hallmark of proliferating cells and limits its utility in cancer therapy. However, local treatment of angiogenic eye conditions with inhibitors of glycolysis may be a safe therapeutic option that warrants experimental investigation. Most types of cells in the eye such as photoreceptors and pericytes use OXPHOS for ATP production, whereas proliferating angiogenic stalk ECs rely on glycolysis for lactate and ATP production. (J Histochem Cytochem XX.XXX-XXX, XXXX).

Anti-Vimentin Nanobody Decreases Glioblastoma Cell Invasion In Vitro and In Vivo.

PURPOSE: Glioblastoma (GBM) is the most common primary brain tumour and one of the deadliest cancers. In addition to late diagnosis and inadequate treatment, the extremely low survival rate is also due to the lack of appropriate therapeutic biomarkers and corresponding therapeutic agents. One of the potential therapeutic biomarkers is the intermediate filament vimentin, which is associated with epithelial-mesenchymal transition (EMT). The purpose of this study was to analyse the effect of the anti-vimentin nanobody Nb79 on cell invasion in vitro and in vivo. To further our understanding of the mechanism of action, we investigated the association between Nb79 and EMT in GBM and GBM stem cells by analysing the expression levels of key EMT-related proteins. METHODS: The expression of vimentin in glioma tissues and cells was determined by RT-qPCR. An invasion assay was performed on differentiated glioblastoma cell line U-87 MG and stem cell line NCH421k in vitro as well as in vivo in zebrafish embryos. The effect of Nb79 on expression of EMT biomarkers beta-catenin, vimentin, ZEB-1 and ZO1 was determined by Western blot and immunocytochemistry. RESULTS: Our study shows that vimentin is upregulated in glioblastoma tissue compared to lower grade glioma and non-tumour brain tissue. We demonstrated that treatment with Nb79 reduced glioblastoma cell invasion by up to 64% in vitro and up to 21% in vivo. In addition, we found that the tight junction protein ZO-1 had higher expression on the cell membrane, when treated with inhibitory anti-vimentin Nb79 compared to control. CONCLUSION: In conclusion, our results suggest that anti-vimentin nanobody Nb79 is a promising tool to target glioblastoma cell invasion.

The Cytotoxic Effects of Cannabidiol and Cannabigerol on Glioblastoma Stem Cells May Mostly Involve GPR55 and TRPV1 Signalling.

Glioblastoma (GBM) is one of the most aggressive cancers, comprising 60-70% of all gliomas. The large G-protein-coupled receptor family includes cannabinoid receptors CB1, CB2, GPR55, and non-specific ion receptor protein transporters TRPs. First, we found up-regulated CNR1, GPR55, and TRPV1 expression in glioma patient-derived tissue samples and cell lines compared with non-malignant brain samples. CNR1 and GPR55 did not correlate with glioma grade, whereas TRPV1 negatively correlated with grade and positively correlated with longer overall survival. This suggests a tumour-suppressor role of TRPV1. With respect to markers of GBM stem cells, preferred targets of therapy, TRPV1 and GPR55, but not CNR1, strongly correlated with different sets of stemness gene markers: NOTCH, OLIG2, CD9, TRIM28, and TUFM and CD15, SOX2, OCT4, and ID1, respectively. This is in line with the higher expression of TRPV1 and GPR55 genes in GSCs compared with differentiated GBM cells. Second, in a panel of patient-derived GSCs, we found that CBG and CBD exhibited the highest cytotoxicity at a molar ratio of 3:1. We suggest that this mixture should be tested in experimental animals and clinical studies, in which currently used Δ9-tetrahydrocannabinol (THC) is replaced with efficient and non-psychoactive CBG in adjuvant standard-of-care therapy.

Bioactive peptides from venoms against glioma progression.

Venoms are complex mixtures of different molecules and ions. Among them, bioactive peptides have been found to affect cancer hallmarks, such as cell proliferation, cell invasion, cell migration, and can also modulate the immune response of normal and cancer-bearing organisms. In this article, we review the mechanisms of action on these cancer cell features, focusing on bioactive peptides being developed as potential therapeutics for one of the most aggressive and deadly brain tumors, glioblastoma (GB). Novel therapeutic approaches applying bioactive peptides may contribute to multiple targeting of GB and particularly of GB stem cells. Bioactive peptides selectively target cancer cells without harming normal cells. Various molecular targets related to the effects of bioactive peptides on GB have been proposed, including ion channels, integrins, membrane phospholipids and even immunomodulatory treatment of GB. In addition to therapy, some bioactive peptides, such as disintegrins, can also be used for diagnostics or are used as labels for cytotoxic drugs to specifically target cancer cells. Given the limitations described in the last section, successful application in cancer therapy is rather low, as only 3.4% of such peptides have been included in clinical trials and have passed successfully phases I to III. Combined approaches of added bioactive peptides to standard cancer therapies need to be explored using advanced GB in vitro models such as organoids. On the other hand, new methods are also being developed to improve translation from research to practice and provide new hope for GB patients and their families.

Infiltrating natural killer cells bind, lyse and increase chemotherapy efficacy in glioblastoma stem-like tumorospheres.

Glioblastomas remain the most lethal primary brain tumors. Natural killer (NK) cell-based therapy is a promising immunotherapeutic strategy in the treatment of glioblastomas, since these cells can select and lyse therapy-resistant glioblastoma stem-like cells (GSLCs). Immunotherapy with super-charged NK cells has a potential as antitumor approach since we found their efficiency to kill patient-derived GSLCs in 2D and 3D models, potentially reversing the immunosuppression also seen in the patients. In addition to their potent cytotoxicity, NK cells secrete IFN-γ, upregulate GSLC surface expression of CD54 and MHC class I and increase sensitivity of GSLCs to chemotherapeutic drugs. Moreover, NK cell localization in peri-vascular regions in glioblastoma tissues and their close contact with GSLCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSLCs. Due to GSLC heterogeneity and plasticity in regards to their stage of differentiation personalized immunotherapeutic strategies should be designed to effectively target glioblastomas.

Upregulation of Cathepsin X in Glioblastoma: Interplay with γ-Enolase and the Effects of Selective Cathepsin X Inhibitors.

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Understanding GBM pathobiology and discovering novel therapeutic targets are critical to finding efficient treatments. Upregulation of the lysosomal cysteine carboxypeptidase cathepsin X has been linked to immune dysfunction and neurodegenerative diseases, but its role in cancer and particularly in GBM progression in patients is unknown. In this study, cathepsin X expression and activity were found to be upregulated in human GBM tissues compared to low-grade gliomas and nontumor brain tissues. Cathepsin X was localized in GBM cells as well as in tumor-associated macrophages and microglia. Subsequently, potent irreversible (AMS36) and reversible (Z7) selective cathepsin X inhibitors were tested in vitro. Selective cathepsin X inhibitors decreased the viability of patient-derived GBM cells as well as macrophages and microglia that were cultured in conditioned media of GBM cells. We next examined the expression pattern of neuron-specific enzyme γ-enolase, which is the target of cathepsin X. We found that there was a correlation between high proteolytic activity of cathepsin X and C-terminal cleavage of γ-enolase and that cathepsin X and γ-enolase were colocalized in GBM tissues, preferentially in GBM-associated macrophages and microglia. Taken together, our results on patient-derived material suggest that cathepsin X is involved in GBM progression and is a potential target for therapeutic approaches against GBM.

Cell Biology Meets Cell Metabolism: Energy Production Is Similar in Stem Cells and in Cancer Stem Cells in Brain and Bone Marrow.

Energy production by means of ATP synthesis in cancer cells has been investigated frequently as a potential therapeutic target in this century. Both (an)aerobic glycolysis and oxidative phosphorylation (OXPHOS) have been studied. Here, we review recent literature on energy production in glioblastoma stem cells (GSCs) and leukemic stem cells (LSCs) versus their normal counterparts, neural stem cells (NSCs) and hematopoietic stem cells (HSCs), respectively. These two cancer stem cell types were compared because their niches in glioblastoma tumors and in bone marrow are similar. In this study, it became apparent that (1) ATP is produced in NSCs and HSCs by anaerobic glycolysis, whereas fatty acid oxidation (FAO) is essential for their stem cell fate and (2) ATP is produced in GSCs and LSCs by OXPHOS despite the hypoxic conditions in their niches with FAO and amino acids providing its substrate. These metabolic processes appeared to be under tight control of cellular regulation mechanisms which are discussed in depth. However, our conclusion is that systemic therapeutic targeting of ATP production via glycolysis or OXPHOS is not an attractive option because of its unwanted side effects in cancer patients.

TRIM28 Selective Nanobody Reduces Glioblastoma Stem Cell Invasion.

Glioblastoma (GB), is the most common and aggressive malignant primary brain tumour in adults. Intra- and inter-tumour heterogeneity, infiltrative GB cell invasion and presence of therapy-resistant GB stem cells (GSCs) represent major obstacles to favourable prognosis and poor therapy response. Identifying the biomarkers of the most aggressive tumour cells and their more efficient targeting strategies are; therefore, crucial. Recently, transcription factor TRIM28 has been identified as a GB biomarker and, in this study, we have shown high expression of TRIM28 in GB and in low grade gliomas as well as higher expression in GSCs vs. differentiated GB cells, although in both cases not significant. We demonstrated significant in vitro inhibition of GB cells and GSCs invasiveness and spread in zebrafish brains in vivo by anti-TRIM28 selective nanobody NB237. TRIM28 was also enriched in GB (tumour) core and associated with the expression of stem cell genes, but was not prognostic for overall survival. However, based on the above results, we conclude that TRIM28 nanobody NB237 offers a new opportunity as a GB therapeutic tool.

Proteases Regulate Cancer Stem Cell Properties and Remodel Their Microenvironment.

Proteolytic activity is perturbed in tumors and their microenvironment, and proteases also affect cancer stem cells (CSCs). CSCs are the therapy-resistant subpopulation of cancer cells with tumor-initiating capacity that reside in specialized tumor microenvironment niches. In this review, we briefly summarize the significance of proteases in regulating CSC activities with a focus on brain tumor glioblastoma. A plethora of proteases and their inhibitors participate in CSC invasiveness and affect intercellular interactions, enhancing CSC immune, irradiation, and chemotherapy resilience. Apart from their role in degrading the extracellular matrix enabling CSC migration in and out of their niches, we review the ability of proteases to modulate CSC properties, which prevents their elimination. When designing protease-oriented therapies, the multifaceted roles of proteases should be thoroughly investigated.

Energy Metabolism in IDH1 Wild-Type and IDH1-Mutated Glioblastoma Stem Cells: A Novel Target for Therapy?

Cancer is a redox disease. Low levels of reactive oxygen species (ROS) are beneficial for cells and have anti-cancer effects. ROS are produced in the mitochondria during ATP production by oxidative phosphorylation (OXPHOS). In the present review, we describe ATP production in primary brain tumors, glioblastoma, in relation to ROS production. Differentiated glioblastoma cells mainly use glycolysis for ATP production (aerobic glycolysis) without ROS production, whereas glioblastoma stem cells (GSCs) in hypoxic periarteriolar niches use OXPHOS for ATP and ROS production, which is modest because of the hypoxia and quiescence of GSCs. In a significant proportion of glioblastoma, isocitrate dehydrogenase 1 (IDH1) is mutated, causing metabolic rewiring, and all cancer cells use OXPHOS for ATP and ROS production. Systemic therapeutic inhibition of glycolysis is not an option as clinical trials have shown ineffectiveness or unwanted side effects. We argue that systemic therapeutic inhibition of OXPHOS is not an option either because the anti-cancer effects of ROS production in healthy cells is inhibited as well. Therefore, we advocate to remove GSCs out of their hypoxic niches by the inhibition of their binding to niches to enable their differentiation and thus increase their sensitivity to radiotherapy and/or chemotherapy.

An Update on Glioblastoma Biology, Genetics, and Current Therapies: Novel Inhibitors of the G Protein-Coupled Receptor CCR5.

The mechanisms governing therapeutic resistance of the most aggressive and lethal primary brain tumor in adults, glioblastoma, have increasingly focused on tumor stem cells. These cells, protected by the periarteriolar hypoxic GSC niche, contribute to the poor efficacy of standard of care treatment of glioblastoma. Integrated proteogenomic and metabolomic analyses of glioblastoma tissues and single cells have revealed insights into the complex heterogeneity of glioblastoma and stromal cells, comprising its tumor microenvironment (TME). An additional factor, which isdriving poor therapy response is the distinct genetic drivers in each patient's tumor, providing the rationale for a more individualized or personalized approach to treatment. We recently reported that the G protein-coupled receptor CCR5, which contributes to stem cell expansion in other cancers, is overexpressed in glioblastoma cells. Overexpression of the CCR5 ligand CCL5 (RANTES) in glioblastoma completes a potential autocrine activation loop to promote tumor proliferation and invasion. CCL5 was not expressed in glioblastoma stem cells, suggesting a need for paracrine activation of CCR5 signaling by the stromal cells. TME-associated immune cells, such as resident microglia, infiltrating macrophages, T cells, and mesenchymal stem cells, possibly release CCR5 ligands, providing heterologous signaling between stromal and glioblastoma stem cells. Herein, we review current therapies for glioblastoma, the role of CCR5 in other cancers, and the potential role for CCR5 inhibitors in the treatment of glioblastoma.

EPHA2 Interacts with DNA-PKcs in Cell Nucleus and Controls Ionizing Radiation Responses in Non-Small Cell Lung Cancer Cells.

Ephrin (EFN)/ Erythropoietin-producing human hepatocellular receptors (Eph) signaling has earlier been reported to regulate non-small cell lung cancer (NSCLC) cell survival and cell death as well as invasion and migration. Here, the role of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) signaling and ionizing radiation (IR) cellular effect was studied in NSCLC cells. Silencing of EphA2 resulted in IR sensitization, with increased activation of caspase-3, PARP-1 cleavage and reduced clonogenic survival. Profiling of EphA2 expression in a NSCLC cell line panel showed a correlation to an IR refractory phenotype. EphA2 was found to be transiently and rapidly phosphorylated at Ser897 in response to IR, which was paralleled with the activation of ribosomal protein S6 kinase (RSK). Using cell fractionation, a transient increase in both total and pSer897 EphA2 in the nuclear fraction in response to IR was revealed. By immunoprecipitation and LC-MS/MS analysis of EphA2 complexes, nuclear localized EphA2 was found in a complex with DNA-PKcs. Such complex formation rapidly increased after IR but returned back to basal level within an hour. Targeting EphA2 with siRNA or by treatment with EFNA1 ligand partly reduced phosphorylation of DNA-PKcs at S2056 at early time points after IR. Thus, we report that EphA2 interacts with DNA-PKcs in the cell nucleus suggesting a novel mechanism involving the EphA2 receptor in DDR signaling and IR responsiveness.

Immunotherapy of Glioblastoma: Current Strategies and Challenges in Tumor Model Development.

Glioblastoma is the most common brain malignant tumor in the adult population, and immunotherapy is playing an increasingly central role in the treatment of many cancers. Nevertheless, the search for effective immunotherapeutic approaches for glioblastoma patients continues. The goal of immunotherapy is to promote tumor eradication, boost the patient's innate and adaptive immune responses, and overcome tumor immune resistance. A range of new, promising immunotherapeutic strategies has been applied for glioblastoma, including vaccines, oncolytic viruses, immune checkpoint inhibitors, and adoptive cell transfer. However, the main challenges of immunotherapy for glioblastoma are the intracranial location and heterogeneity of the tumor as well as the unique, immunosuppressive tumor microenvironment. Owing to the lack of appropriate tumor models, there are discrepancies in the efficiency of various immunotherapeutic strategies between preclinical studies (with in vitro and animal models) on the one hand and clinical studies (on humans) on the other hand. In this review, we summarize the glioblastoma characteristics that drive tolerance to immunotherapy, the currently used immunotherapeutic approaches against glioblastoma, and the most suitable tumor models to mimic conditions in glioblastoma patients. These models are improving and can more precisely predict patients' responses to immunotherapeutic treatments, either alone or in combination with standard treatment.

Cannabigerol Is a Potential Therapeutic Agent in a Novel Combined Therapy for Glioblastoma.

Glioblastoma is the most aggressive cancer among primary brain tumours. As with other cancers, the incidence of glioblastoma is increasing; despite modern therapies, the overall mean survival of patients post-diagnosis averages around 16 months, a figure that has not changed in many years. Cannabigerol (CBG) has only recently been reported to prevent the progression of certain carcinomas and has not yet been studied in glioblastoma. Here, we have compared the cytotoxic, apoptotic, and anti-invasive effects of the purified natural cannabinoid CBG together with CBD and THC on established differentiated glioblastoma tumour cells and glioblastoma stem cells. CBG and THC reduced the viability of both types of cells to a similar extent, whereas combining CBD with CBG was more efficient than with THC. CBD and CBG, both alone and in combination, induced caspase-dependent cell apoptosis, and there was no additive THC effect. Of note, CBG inhibited glioblastoma invasion in a similar manner to CBD and the chemotherapeutic temozolomide. We have demonstrated that THC has little added value in combined-cannabinoid glioblastoma treatment, suggesting that this psychotropic cannabinoid should be replaced with CBG in future clinical studies of glioblastoma therapy.

CCR5-Mediated Signaling Is Involved in Invasion of Glioblastoma Cells in Its Microenvironment.

The chemokine CCL5/RANTES is a versatile inflammatory mediator, which interacts with the receptor CCR5, promoting cancer cell interactions within the tumor microenvironment. Glioblastoma is a highly invasive tumor, in which CCL5 expression correlates with shorter patient survival. Using immunohistochemistry, we identified CCL5 and CCR5 in a series of glioblastoma samples and cells, including glioblastoma stem cells. CCL5 and CCR5 gene expression were significantly higher in a cohort of 38 glioblastoma samples, compared to low-grade glioma and non-cancerous tissues. The in vitro invasion of patients-derived primary glioblastoma cells and glioblastoma stem cells was dependent on CCL5-induced CCR5 signaling and is strongly inhibited by the small molecule CCR5 antagonist maraviroc. Invasion of these cells, which was enhanced when co-cultured with mesenchymal stem cells (MSCs), was inhibited by maraviroc, suggesting that MSCs release CCR5 ligands. In support of this model, we detected CCL5 and CCR5 in MSC monocultures and glioblastoma-associated MSC in tissue sections. We also found CCR5 expressing macrophages were in close proximity to glioblastoma cells. In conclusion, autocrine and paracrine cross-talk in glioblastoma and, in particular, glioblastoma stem cells with its stromal microenvironment, involves CCR5 and CCL5, contributing to glioblastoma invasion, suggesting the CCL5/CCR5 axis as a potential therapeutic target that can be targeted with repositioned drug maraviroc.

Brain malignancies: Glioblastoma and brain metastases.

Brain, the major organ of the central nervous system controls and processes most of body activities. Therefore, the most aggressive brain tumor - glioblastoma and metastases from other organs to the brain are lethal leaving the patients with very short time of survival. The brain tissue landscape is very different from any other tissues and the specific microenvironment, comprising stem cells niches and blood-brain barrier, significantly influences the low rate of glioblastoma metastasis out of the brain, but better accommodates brain-invading cancer. In contrast to low frequency (0.5%) of all glioblastoma metastases, 10%-45% of other primary cancers do metastasize to the brain. This review addresses general cellular and molecular pathways that are to some extent similar in both types of metastases, involving circulating tumor cells (CTCs) with cancer stem cells (CSCs) characteristics, and metastatic niches. The invasion is a dynamic process involving reversible epithelial-to-mesenchymal (EMT) cell process, creating a transient gradient state that is inter-connected with epigenetic plasticity of the metastasizing (m)CSCs. These cells can switch between stationary, low proliferating/dormant state to a migratory, mesenchymal-like state. Settling in their respective niches as dormant CSCs in the secondary organ is a common feature in all types of metastases. In glioblastoma metastasis, the malignant mGSC cells express markers of mesenchymal GSC subtype (MES-GSC), such as CD44 and YK-40 and their major obstacle seems to be propagating in the in various organs' microenvironments, different from the niches that home GSCs in the primary glioblastoma. Focusing on one stromal component in the glioblastoma niches, the mesenchymal stem cells (MSCs), we report herein on their differential effects on glioblastoma cells, highly depending on their genetic subtype. On the other hand, in brain metastases, the major hindrance to metastatic progression of mCSCs seem to be crossing the blood-brain-barrier. Novel therapeutic approaches for brain metastases from various cancer types are advancing slowly, and the general trends involve targeting metastatic sub-clones and selective determinants of their niches. The update on the four most common brain metastases from lung, breast, melanoma and colorectal carcinoma is presented.

Cytokine CCL5 and receptor CCR5 axis in glioblastoma multiforme.

Background Glioblastoma is the most frequent and aggressive brain tumour in humans with median survival from 12 to 15 months after the diagnosis. This is mostly due to therapy resistant glioblastoma stem cells in addition to intertumour heterogeneity that is due to infiltration of a plethora of host cells. Besides endothelial cells, mesenchymal stem cells and their differentiated progenies, immune cells of various differentiation states, including monocytes, comprise resident, brain tumour microenvironment. There are compelling evidence for CCL5/CCR5 in the invasive and metastatic behaviour of many cancer types. CCR5, a G-protein coupled receptor, known to function as an essential co-receptor for HIV entry, is now known to participate in driving tumour heterogeneity, the formation of cancer stem cells and the promotion of cancer invasion and metastasis. Clinical trials have recently opened targeting CCR5 using a humanized monoclonal antibody (leronlimab) for metastatic triple negative breast cancer (TNBC) or a small molecule inhibitor (maraviroc) for metastatic colon cancer. There are important CCL5 and CCR5 structure and signalling mechanisms in glioblastoma. In addition, the CCL5/CCR5 axis directs infiltration and interactions with monocytes/macrophages and mesenchymal stem cells, comprising glioblastoma stem cell niches. Conclusions CCR5 is highly expressed in glioblastoma and is associated with poor prognosis of patients. CCL5/CCR5 is suggested to be an excellent new target for glioblastoma therapy. The molecular mechanisms, by which chemoattractant and receptor respond within the complex tissue microenvironment to promote cancer stem cells and tumour heterogeneity, should be considered in forthcoming studies.

Exosomal RNA-profiling of pleural effusions identifies adenocarcinoma patients through elevated miR-200 and LCN2 expression.

HYPOTHESIS: The inherent challenges associated with tissue biopsies from lung have spurred an interest in the use of liquid biopsies. Pleural effusions are one source of liquid biopsy. Recently, extracellular vesicles of endocytic origin, exosomes, have attracted interest as liquid biopsy of tumors as they are thought to be a mirror of their tumor of origin. Here, we aimed to analyze if RNA profiling of exosomes isolated from pleural effusions could differentiate patients with lung adenocarcinoma from patients with benign inflammatory processes. METHODS: Exosomes were isolated from 36 pleural effusions from patients with adenocarcinoma (n = 18) and patients with benign inflammatory processes (n = 18). The two groups were balanced with respect to age and smoking history but with a gender bias towards males in the benign group. Profiling was conducted using RT-qPCR arrays covering 754 microRNAs and 624 mRNAs followed by statistical ranking of differentially regulated transcripts between the two patient cohorts. RESULTS: RNA profiling revealed differential expression of 17 microRNAs and 71 mRNAs in pleural effusions collected from patients with lung adenocarcinoma compared to pleural effusions from benign lung disease. Overall, top differentially expressed microRNAs, including miR-200 family microRNAs, provided a stronger diagnostic power compared to top differentially expressed mRNAs. However, the mRNA transcript encoding Lipocalin-2 (LCN2) displayed the strongest diagnostic power of all analyzed transcripts (AUC: 0.9916). CONCLUSIONS: Our study demonstrates that exosomal RNA profiling from pleural effusions can be used to identify patients with lung adenocarcinoma from individuals with benign processes and further proposes miR-200 microRNAs and LCN2 as diagnostic markers in lung cancer liquid biopsies.

Ephrin B3 interacts with multiple EphA receptors and drives migration and invasion in non-small cell lung cancer.

Ephrin receptors (Ephs) are reported to control metastatic signaling of non-small cell lung cancer (NSCLC) and other tumors. Here we show for the first time that blocking expression of the Eph ligand Ephrin B3 inhibits NSCLC cell migration and invasion. We demonstrate that Ephrin B3 directly binds the EphAs EphA2, EphA3, EphA4, and EphA5. EphA2 Ser897 was previously shown to drive migration propensity of tumor cells and our study reveals that EphA2 stays phosphorylated on Ser897 in the Ephrin B3/EphA2 complex in NSCLC cells of different histology. Moreover, we report that within such Ephrin B3/EphA2 complex both Akt Ser 129 and p38MAPK are found indicating a potential to drive migration/proliferation. We also found the EMT marker E-cadherin expression to be maintained or increased upon Ephrin B3 blockade in NSCLC cells. Expression of Ephrin B3 was furthermore analyzed in a cohort of NSCLC stage IA-IB cases (n=200) alongside EphA2 and Ephrin A1. We found that Ephrin B3 was concomitantly expressed with EphA2 and Ephrin A1 with higher Ephrin B3 levels found in non-squamous than in squamous tumors, whereas EphA2 was higher expressed in well-differentiated than in low-differentiated tumors. In the entire NSCLC cohort, Ephrin B3 expression was not linked to patient survival, whereas a high EphA2 expression was associated with improved survival (p=0.03). In conclusion, we show that blocking Ephrin B3 expression inhibits NSCLC proliferation-, migration- and invasion capacity which calls for further studies on interference with Ephrin B3 as a possible therapeutic avenue in this tumor malignancy.

Compounds from the marine sponge Cribrochalina vasculum offer a way to target IGF-1R mediated signaling in tumor cells.

In this work two acetylene alcohols, compound 1 and compound 2, which were isolated and identified from the sponge Cribrochalina vasculum, and which showed anti-tumor effects were further studied with respect to targets and action mechanisms. Gene expression analyses suggested insulin like growth factor receptor (IGF-1R) signaling to be instrumental in controlling anti-tumor efficacy of these compounds in non-small cell lung cancer (NSCLC). Indeed compounds 1 and 2 inhibited phosphorylation of IGF-1Rß as well as reduced its target signaling molecules IRS-1 and PDK1 allowing inhibition of pro-survival signaling. In silico docking indicated that compound 1 binds to the kinase domain of IGF-1R at the same binding site as the well known tyrosine kinase inhibitor AG1024. Indeed, cellular thermal shift assay (CETSA) confirmed that C. vasculum compound 1 binds to IGF-1R but not to the membrane localized tyrosine kinase receptor EGFR. Importantly, we demonstrate that compound 1 causes IGF-1Rß but not Insulin Receptor degradation specifically in tumor cells with no effects seen in normal diploid fibroblasts. Thus, these compounds hold potential as novel therapeutic agents targeting IGF-1R signaling for anti-tumor treatment.