Structural insight to elucidate the binding specificity of the anti-cortisol Fab fragment with glucocorticoids.

Cortisol is a steroid hormone that is produced by the adrenal gland. It is a primary stress hormone that increases glucose levels in the blood stream. High concentrations of cortisol in the body can be used as a biomarker for acute and chronic stress and related mental and physiological disorders. Therefore, the accurate quantification of cortisol levels in body fluids is essential for clinical diagnosis. In this article, we describe the isolation of recombinant anti-cortisol antibodies with high affinity for cortisol and discover their cross-reactivity with other glucocorticoids. To describe the cortisol binding site and elucidate the structural basis for the binding specificity, the high-resolution crystal structures of the anti-cortisol (17) Fab fragment in the absence of glucocorticoid (2.00 Å) and the presence of cortisol (2.26 Å), corticosterone (1.86 Å), cortisone (1.85 Å) and prednisolone (2.00 Å) were determined. To our knowledge, this is the first determined crystal structure of a cortisol-specific antibody. The recognition of cortisol is driven by hydrophobic interactions and hydrogen bonding at the protein-ligand interface coupled with a conformational transition. Comparison of ligand-free and ligand-bound structures showed that the side chains of residues Tyr58-H and Arg56-H can undergo local conformational changes at the binding site, most likely prior to the binding event via a conformational selection mechanism. Compared to other anti-steroid antibody-antigen complexes, (17) Fab possesses a structurally unique steroid binding site, as the H3 loop from the CDR area has only a minor contribution, but framework residues have a prominent contribution to hapten binding.

Functionalisation of Graphene Sensor Surfaces for the Specific Detection of Biomarkers.

We report on a controllable and specific functionalisation route for graphene field-effect transistors (GFETs) for the recognition of small physiologically active molecules. Key element is the noncovalent functionalisation of the graphene surface with perylene bisimide (PBI) molecules directly on the growth substrate. This Functional Layer Transfer enables the homogeneous self-assembly of PBI molecules on graphene, onto which antibodies are subsequently immobilised. The sensor surface was characterised by atomic force microscopy, Raman spectroscopy and electrical measurements, showing superior performance over conventional functionalisation after transfer. Specific sensing of small molecules was realised by monitoring the electrical property changes of functionalised GFET devices upon the application of methamphetamine and cortisol. The concentration dependent electrical response of our sensors was determined down to a concentration of 300 ng ml-1 for methamphetamine.

Recombinant Peptide Mimetic NanoLuc Tracer for Sensitive Immunodetection of Mycophenolic Acid.

Mycophenolic acid (MPA) is an immunosuppressant drug commonly used to prevent organ rejection in transplanted patients. MPA monitoring is of great interest due to its small therapeutic window. In this work, a phage-displayed peptide library was used to select cyclic peptides that bind to the MPA-specific recombinant antibody fragment (Fab) and mimic the behavior of MPA. After biopanning, several phage-displayed peptides were isolated and tested to confirm their epitope-mimicking nature in phage-based competitive immunoassays. After identifying the best MPA mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL-1 and an IC50 of 2.9 ± 0.5 ng mL-1. The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection.

Androgen receptor-interacting protein HSPBAP1 facilitates growth of prostate cancer cells in androgen-deficient conditions.

Hormonal therapies targeting androgen receptor (AR) are effective in prostate cancer (PCa), but often the cancers progress to fatal castrate-resistant disease. Improved understanding of the cellular events during androgen deprivation would help to identify survival and stress pathways whose inhibition could synergize with androgen deprivation. Toward this aim, we performed an RNAi screen on 2,068 genes, including kinases, phosphatases, epigenetic enzymes and other druggable gene targets. High-content cell spot microarray (CSMA) screen was performed in VCaP cells in the presence and absence of androgens with detection of Ki67 and cleaved ADP-ribose polymerase (cPARP) as assays for cell proliferation and apoptosis. Thirty-nine candidate genes were identified, whose silencing inhibited proliferation or induced apoptosis of VCaP cells exclusively under androgen-deprived conditions. One of the candidates, HSPB (heat shock 27 kDa)-associated protein 1 (HSPBAP1), was confirmed to be highly expressed in tumor samples and its mRNA expression levels increased with the Gleason grade. We found that strong HSPBAP1 immunohistochemical staining (IHC) was associated with shorter disease-specific survival of PCa patients compared with negative to moderate staining. Furthermore, we demonstrate that HSPBAP1 interacts with AR in the nucleus of PCa cells specifically during androgen-deprived conditions, occupies chromatin at PSA/klk3 and TMPRSS2/tmprss2 enhancers and regulates their expression. In conclusion, we suggest that HSPBAP1 aids in sustaining cell viability by maintaining AR signaling during androgen-deprived conditions.

Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

BACKGROUND: Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. METHODS: Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. RESULTS: The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. CONCLUSIONS: Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen-food syndrome and to study the IgG epitope of the allergens.

Robust Approach for Quantifying Glucocorticoid Binding to the Anti-Cortisol Fab Fragment via Native Mass Spectrometry.

In the development of proteins, aptamers, and molecular imprints for diagnostic purposes, a major goal is to obtain a molecule with both a high binding affinity and specificity for the target ligand. Cushing syndrome or Addison's disease can be diagnosed by cortisol level tests. We have previously characterized and solved the crystal structure of an anti-cortisol (17) Fab fragment having a high affinity to cortisol but also significant cross-reactivity to other glucocorticoids, especially the glucocorticoid drug prednisolone. We used native mass spectrometry (MS) to determine the binding affinities of nine steroid hormones to anti-cortisol (17) Fab, including steroidogenic precursors of cortisol. Based on the results, the number of hydroxyl groups in the structure of a steroid ligand plays a key role in the antigen recognition by the Fab fragment as the ligands with three hydroxyl groups, cortisol and prednisolone, had the highest affinities. The antibody affinity toward steroid hormones often decreases with a decrease in the number of hydroxyl groups in the structure. The presence of the hydroxyl group at position C11 increased the affinity more than did the other hydroxyl groups at positions C17 or C21. The binding affinities obtained by native MS were compared to the values determined by surface plasmon resonance (SPR), and the affinities were found to correlate well between these two techniques. Our study demonstrates that native MS with a large dynamic range and high sensitivity is a versatile tool for ligand binding studies of proteins.

Interaction with ErbB4 promotes hypoxia-inducible factor-1α signaling.

The receptor-tyrosine kinase ErbB4 was identified as a direct regulator of hypoxia-inducible factor-1α (HIF-1α) signaling. Cleaved intracellular domain of ErbB4 directly interacted with HIF-1α in the nucleus, and stabilized HIF-1α protein in both normoxic and hypoxic conditions by blocking its proteasomal degradation. The mechanism of HIF stabilization was independent of VHL and proline hydroxylation but dependent on RACK1. ErbB4 activity was necessary for efficient HRE-driven promoter activity, transcription of known HIF-1α target genes, and survival of mammary carcinoma cells in vitro. In addition, mammary epithelial specific targeting of Erbb4 in the mouse significantly reduced the amount of HIF-1α protein in vivo. ERBB4 expression also correlated with the expression of HIF-regulated genes in a series of 4552 human normal and cancer tissue samples. These data demonstrate that soluble ErbB4 intracellular domain promotes HIF-1α stability and signaling via a novel mechanism.

Differentiation-promoting culture of competent and noncompetent keratinocytes identifies biomarkers for head and neck cancer.

Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.

15-Hydroxyprostaglandin dehydrogenase associates with poor prognosis in breast cancer, induces epithelial-mesenchymal transition, and promotes cell migration in cultured breast cancer cells.

Breast cancer is the most frequent cancer and the leading cause of cancer-related deaths in women worldwide. The prognosis of breast cancer is tightly correlated with the degree of spread beyond the primary tumour. Arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) are known to regulate tumour metastasis enabling epithelial-mesenchymal transition (EMT). However, the detailed role of 15-hydroxyprostaglandin dehydrogenase (HPGD), the key enzyme degrading prostaglandin E(2) , remains unclear in breast cancer. Here, we show that HPGD mRNA is overexpressed in a subset of clinical breast cancers compared to normal breast tissue samples and that high HPGD mRNA expression associates with poor prognosis. Immunohistochemical staining of primary breast cancer and lymph node metastasis tissue samples confirmed high HPGD protein expression in 20% of the samples, as well as associated HPGD expression with aggressive characteristics, such as increased risk of disease relapse and shorter disease-free survival. Results from cultured cells indicated abundant HPGD expression in highly metastatic breast cancer cells, and impairment of HPGD expression using RNA interference led to a significant decrease in transforming growth factor-ß signalling, in cellular arachidonic acid levels as well as in cell migration. Furthermore, gene expression microarray analysis followed by quantitative RT-PCR validation showed that HPGD silencing decreased aryl hydrocarbon receptor signalling and induced mesenchymal-epithelial transition. In conclusion, our results indicate that HPGD is highly expressed in metastatic and aggressive breast cancer and promotes EMT and migration in breast cancer cells.

Integration of metabolomics and expression of glycerol-3-phosphate acyltransferase (GPAM) in breast cancer-link to patient survival, hormone receptor status, and metabolic profiling.

Changes in lipid metabolism are an important but not well-characterized hallmark of cancer. On the basis of our recent findings of lipidomic changes in breast cancer, we investigated glycerol-3-phosphate acyltransferase (GPAM), a key enzyme in the lipid biosynthesis of triacylglycerols and phospholipids. GPAM protein expression was evaluated and linked to metabolomic and lipidomic profiles in a cohort of human breast carcinomas. In addition, GPAM mRNA expression was analyzed using the GeneSapiens in silico transcriptiomics database. High cytoplasmic GPAM expression was associated with hormone receptor negative status (p = 0.013). On the protein (p = 0.048) and mRNA (p = 0.001) levels, increased GPAM expression was associated with a better overall survival. Metabolomic analysis by GC-MS showed that sn-glycerol-3-phosphate, the substrate of GPAM, was elevated in breast cancer compared to normal breast tissue. LC-MS based lipidomic analysis identified significantly higher levels of phospholipids, especially phosphatidylcholines in GPAM protein positive tumors. In conclusion, our results suggest that GPAM is expressed in human breast cancer with associated changes in the cellular metabolism, in particular an increased synthesis of phospholipids, the major structural component of cellular membranes.

Integrative genomic, transcriptomic, and RNAi analysis indicates a potential oncogenic role for FAM110B in castration-resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) represents a therapeutic challenge for current medications. METHODS: In order to explore the molecular mechanisms involved in CRPC progression and to identify new therapeutic targets, we analyzed a unique sample set of 11 CRPCs and 7 advanced tumors by array-CGH and gene expression microarrays. The genome-wide DNA and RNA data were integrated to identify genes whose overexpression was driven by their amplification. To assess the functional role of these genes, their expression was analyzed in a transcriptional data set of 329 clinical prostate cancers and the corresponding gene products were silenced using RNA interference in prostate cancer cells. RESULTS: Six recurrent genetic targets were identified in the CRPCs; ATP1B1, AR, FAM110B, LAS1L, MYC, and YIPF6. In addition to AR and MYC, FAM110B emerged as a potential key gene involved in CRPC progression in a subset of the tumors. FAM110B was able to regulate AR signaling in prostate cancer cells and FAM110B itself was regulated by androgens. FAM110B siRNA inhibited the growth of prostate cancer cells in vitro, and this effect was substantially enhanced in androgen deficient conditions. Ectopic FAM110B expression in non-cancerous epithelial prostate cells induced aneuploidy and impaired antigen presentation. CONCLUSIONS: The DNA/RNA gene outlier detection combined with siRNA cell proliferation assay identified FAM110B as a potential growth promoting key gene for CRPC. FAM110B appears to have a key role in the androgen signaling and progression of CRPC impacting multiple cancer hallmarks and therefore highlighting a potential therapeutic target.

Arachidonic acid pathway members PLA2G7, HPGD, EPHX2, and CYP4F8 identified as putative novel therapeutic targets in prostate cancer.

The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress.

Characterization of Diaphanous-related formin FMNL2 in human tissues.

BACKGROUND: Diaphanous-related formins govern actin-based processes involved in many cellular functions, such as cell movement and invasion. Possible connections to developmental processes and cellular changes associated with malignant phenotype make them interesting study targets. In spite of this, very little is known of the tissue distribution and cellular location of any mammalian formin. Here we have carried out a comprehensive analysis of the formin family member formin -like 2 (FMNL2) in human tissues. RESULTS: An FMNL2 antibody was raised and characterized. The affinity-purified FMNL2 antibody was validated by Western blotting, Northern blotting, a peptide competition assay and siRNA experiments. Bioinformatics-based mRNA profiling indicated that FMNL2 is widely expressed in human tissues. The highest mRNA levels were seen in central and peripheral nervous systems. Immunohistochemical analysis of 26 different human tissues showed that FMNL2 is widely expressed, in agreement with the mRNA profile. The widest expression was detected in the central nervous system, since both neurons and glial cells expressed FMNL2. Strong expression was also seen in many epithelia. However, the expression in different cell types was not ubiquitous. Many mesenchymal cell types showed weak immunoreactivity and cells lacking expression were seen in many tissues. The subcellular location of FMNL2 was cytoplasmic, and in some tissues a strong perinuclear dot was detected. In cultured cells FMNL2 showed mostly a cytoplasmic localization with perinuclear accumulation consistent with the Golgi apparatus. Furthermore, FMNL2 co-localized with F-actin to the tips of cellular protrusions in WM164 human melanoma cells. This finding is in line with FMNL2's proposed function in the formation of actin filaments in cellular protrusions, during amoeboid cellular migration. CONCLUSION: FMNL2 is expressed in multiple human tissues, not only in the central nervous system. The expression is especially strong in gastrointestinal and mammary epithelia, lymphatic tissues, placenta, and in the reproductive tract. In cultured melanoma cells, FMNL2 co-localizes with F-actin dots at the tips of cellular protrusions.

Defining the molecular action of HDAC inhibitors and synergism with androgen deprivation in ERG-positive prostate cancer.

Gene fusions between prostate-specific, androgen responsive TMPRSS2 gene and oncogenic ETS factors, such as ERG, occur in up to 50% of all prostate cancers. We recently defined a gene signature that was characteristic to prostate cancers with ERG activation. This suggested epigenetic reprogramming, such as upregulation of histone deactylase 1 (HDAC1) gene and downregulation of its target genes. We then hypothesized that patients with ERG-positive prostate cancers may benefit from epigenetic therapy such as HDAC inhibition (HDACi), especially in combination with antiandrogens. Here, we exposed ERG-positive prostate cancer cell lines to HDAC inhibitors Trichostatin A (TSA), MS-275 and suberoylanilide hydroxamic acid (SAHA) with or without androgen deprivation. We explored the effects on cell phenotype, gene expression as well as ERG and androgen receptor (AR) signaling. When compared with 5 other prostate cell lines, ERG-positive VCaP and DuCap cells were extremely sensitive to HDACi, in particular TSA, showing synergy with concomitant androgen deprivation increasing apoptosis. Both of the HDAC inhibitors studied caused repression of the ERG-fusion gene, whereas the pan-HDAC inhibitor TSA prominently repressed the ERG-associated gene signature. Additionally, HDACi and flutamide caused retention of AR in the cytoplasm, indicating blockage of androgen signaling. Our results support the hypothesis that HDACi, especially in combination with androgen deprivation, is effective against TMPRSS2-ERG-fusion positive prostate cancer in vitro. Together with our previous in vivo observations of an "epigenetic reprogramming gene signature" in clinical ERG-positive prostate cancers, these studies provide mechanistic insights to ERG-associated tumorigenesis and suggest therapeutic paradigms to be tested in vivo.

TMPRSS2 fusions with oncogenic ETS factors in prostate cancer involve unbalanced genomic rearrangements and are associated with HDAC1 and epigenetic reprogramming.

Translocations fusing the strong androgen-responsive gene, TMPRSS2, with ERG or other oncogenic ETS factors may facilitate prostate cancer development. Here, we studied 18 advanced prostate cancers for ETS factor alterations, using reverse transcription-PCR and DNA and RNA array technologies, and identified putative ERG downstream gene targets from the microarray data of 410 prostate samples. Out of the 27 ETS factors, ERG was most frequently overexpressed. Seven cases showed TMPRSS2:ERG gene fusions, whereas the TMPRSS2:ETV4 fusion was seen in one case. In five out of six tumors with high ERG expression, array-CGH analysis revealed interstitial 2.8 Mb deletions between the TMPRSS2 and ERG loci, or smaller, unbalanced rearrangements. In silico analysis of the ERG gene coexpression patterns revealed an association with high expression of the histone deacetylase 1 gene, and low expression of its target genes. Furthermore, we observed increased expression of WNT-associated pathways and down-regulation of tumor necrosis factor and cell death pathways. In summary, our data indicate that the TMPRSS2:ERG translocation is common in advanced prostate cancer and occurs by virtue of unbalanced genomic rearrangements. Activation of ERG by fusion with TMPRSS2 may lead to epigenetic reprogramming, WNT signaling, and down-regulation of cell death pathways, implicating ERG in several hallmarks of cancer with potential therapeutic importance.

Identifying the druggable interactome of EWS-FLI1 reveals MCL-1 dependent differential sensitivities of Ewing sarcoma cells to apoptosis inducers.

Ewing sarcoma (EwS) is an aggressive pediatric bone cancer in need of more effective therapies than currently available. Most research into novel targeted therapeutic approaches is focused on the fusion oncogene EWSR1-FLI1, which is the genetic hallmark of this disease. In this study, a broad range of 3,325 experimental compounds, among them FDA approved drugs and natural products, were screened for their effect on EwS cell viability depending on EWS-FLI1 expression. In a network-based approach we integrated the results from drug perturbation screens and RNA sequencing, comparing EWS-FLI1-high (normal expression) with EWS-FLI1-low (knockdown) conditions, revealing novel interactions between compounds and EWS-FLI1 associated biological processes. The top candidate list of druggable EWS-FLI1 targets included genes involved in translation, histone modification, microtubule structure, topoisomerase activity as well as apoptosis regulation. We confirmed our in silico results using viability and apoptosis assays, underlining the applicability of our integrative and systemic approach. We identified differential sensitivities of Ewing sarcoma cells to BCL-2 family inhibitors dependent on the EWS-FLI1 regulome including altered MCL-1 expression and subcellular localization. This study facilitates the selection of effective targeted approaches for future combinatorial therapies of patients suffering from Ewing sarcoma.

Retinoic acid and TGF-ß signalling cooperate to overcome MYCN-induced retinoid resistance.

BACKGROUND: Retinoid therapy is widely employed in clinical oncology to differentiate malignant cells into their more benign counterparts. However, certain high-risk cohorts, such as patients with MYCN-amplified neuroblastoma, are innately resistant to retinoid therapy. Therefore, we employed a precision medicine approach to globally profile the retinoid signalling response and to determine how an excess of cellular MYCN antagonises these signalling events to prevent differentiation and confer resistance. METHODS: We applied RNA sequencing (RNA-seq) and interaction proteomics coupled with network-based systems level analysis to identify targetable vulnerabilities of MYCN-mediated retinoid resistance. We altered MYCN expression levels in a MYCN-inducible neuroblastoma cell line to facilitate or block retinoic acid (RA)-mediated neuronal differentiation. The relevance of differentially expressed genes and transcriptional regulators for neuroblastoma outcome were then confirmed using existing patient microarray datasets. RESULTS: We determined the signalling networks through which RA mediates neuroblastoma differentiation and the inhibitory perturbations to these networks upon MYCN overexpression. We revealed opposing regulation of RA and MYCN on a number of differentiation-relevant genes, including LMO4, CYP26A1, ASCL1, RET, FZD7 and DKK1. Furthermore, we revealed a broad network of transcriptional regulators involved in regulating retinoid responsiveness, such as Neurotrophin, PI3K, Wnt and MAPK, and epigenetic signalling. Of these regulators, we functionally confirmed that MYCN-driven inhibition of transforming growth factor beta (TGF-ß) signalling is a vulnerable node of the MYCN network and that multiple levels of cross-talk exist between MYCN and TGF-ß. Co-targeting of the retinoic acid and TGF-ß pathways, through RA and kartogenin (KGN; a TGF-ß signalling activating small molecule) combination treatment, induced the loss of viability of MYCN-amplified retinoid-resistant neuroblastoma cells. CONCLUSIONS: Our approach provides a powerful precision oncology tool for identifying the driving signalling networks for malignancies not primarily driven by somatic mutations, such as paediatric cancers. By applying global omics approaches to the signalling networks regulating neuroblastoma differentiation and stemness, we have determined the pathways involved in the MYCN-mediated retinoid resistance, with TGF-ß signalling being a key regulator. These findings revealed a number of combination treatments likely to improve clinical response to retinoid therapy, including co-treatment with retinoids and KGN, which may prove valuable in the treatment of high-risk MYCN-amplified neuroblastoma.

High-throughput RNAi screen in Ewing sarcoma cells identifies leucine rich repeats and WD repeat domain containing 1 (LRWD1) as a regulator of EWS-FLI1 driven cell viability.

A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.

PLA2G7 associates with hormone receptor negativity in clinical breast cancer samples and regulates epithelial-mesenchymal transition in cultured breast cancer cells.

Breast cancer is the leading cause of cancer-related deaths in women due to distinct cancer subtypes associated with early recurrence and aggressive metastatic progression. High lipoprotein-associated phospholipase A2 (PLA2G7) expression has previously been associated with aggressive disease and metastasis in prostate cancer. Here, we explore the expression pattern and functional role of PLA2G7 in breast cancer. First, a bioinformatic analysis of genome-wide gene expression data from 970 breast samples was carried out to evaluate the expression pattern of PLA2G7 mRNA in breast cancer. Second, the expression profile of PLA2G7 was studied in 1042 breast cancer samples including 89 matched lymph node metastasis samples using immunohistochemistry. Third, the effect of PLA2G7 silencing on genome-wide gene expression profile was studied and validated in cultured breast cancer cells expressing PLA2G7 at high level. Last, the expression pattern of PLA2G7 mRNA was investigated in 24 nonmalignant tissue samples and 65 primary and 7 metastatic tumour samples derived from various organs using qRT-PCR. The results from clinical breast cancer samples indicated that PLA2G7 is overexpressed in a subset of breast cancer samples compared to its expression in benign breast tissue samples and that high PLA2G7 expression associated with hormone receptor negativity as well as with poor prognosis in a subset of breast cancer samples. In vitro functional studies highlighted the putative role of PLA2G7 in the regulation of epithelial-mesenchymal transition (EMT)-related signalling pathways, vimentin and E-cadherin protein expression as well as cell migration in cultured breast cancer cells. Furthermore, supporting the findings in breast and prostate cancer, high PLA2G7 mRNA expression was associated with metastatic cancer in four additional organs of origin. In conclusion, our results indicate that PLA2G7 is highly expressed in a subset of metastatic and aggressive breast cancers and in metastatic samples of various tissues of origin and promotes EMT and migration in cultured breast cancer cells.