Hypoxia-induced switch in SNAT2/SLC38A2 regulation generates endocrine resistance in breast cancer.

Tumor hypoxia is associated with poor patient outcomes in estrogen receptor-α-positive (ERα+) breast cancer. Hypoxia is known to affect tumor growth by reprogramming metabolism and regulating amino acid (AA) uptake. Here, we show that the glutamine transporter, SNAT2, is the AA transporter most frequently induced by hypoxia in breast cancer, and is regulated by hypoxia both in vitro and in vivo in xenografts. SNAT2 induction in MCF7 cells was also regulated by ERα, but it became predominantly a hypoxia-inducible factor 1α (HIF-1α)-dependent gene under hypoxia. Relevant to this, binding sites for both HIF-1α and ERα overlap in SNAT2's cis-regulatory elements. In addition, the down-regulation of SNAT2 by the ER antagonist fulvestrant was reverted in hypoxia. Overexpression of SNAT2 in vitro to recapitulate the levels induced by hypoxia caused enhanced growth, particularly after ERα inhibition, in hypoxia, or when glutamine levels were low. SNAT2 up-regulation in vivo caused complete resistance to antiestrogen and, partially, anti-VEGF therapies. Finally, high SNAT2 expression levels correlated with hypoxia profiles and worse outcome in patients given antiestrogen therapies. Our findings show a switch in the regulation of SNAT2 between ERα and HIF-1α, leading to endocrine resistance in hypoxia. Development of drugs targeting SNAT2 may be of value for a subset of hormone-resistant breast cancer.

Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery.

Cancer cells can switch between signaling pathways to regulate growth under different conditions. In the tumor microenvironment, this likely helps them evade therapies that target specific pathways. We must identify all possible states and utilize them in drug screening programs. One such state is characterized by expression of the transcription factor Hairy and Enhancer of Split 3 (HES3) and sensitivity to HES3 knockdown, and it can be modeled in vitro. Here, we cultured 3 primary human brain cancer cell lines under 3 different culture conditions that maintain low, medium, and high HES3 expression and characterized gene regulation and mechanical phenotype in these states. We assessed gene expression regulation following HES3 knockdown in the HES3-high conditions. We then employed a commonly used human brain tumor cell line to screen Food and Drug Administration (FDA)-approved compounds that specifically target the HES3-high state. We report that cells from multiple patients behave similarly when placed under distinct culture conditions. We identified 37 FDA-approved compounds that specifically kill cancer cells in the high-HES3-expression conditions. Our work reveals a novel signaling state in cancer, biomarkers, a strategy to identify treatments against it, and a set of putative drugs for potential repurposing.-Poser, S. W., Otto, O., Arps-Forker, C., Ge, Y., Herbig, M., Andree, C., Gruetzmann, K., Adasme, M. F., Stodolak, S., Nikolakopoulou, P., Park, D. M., Mcintyre, A., Lesche, M., Dahl, A., Lennig, P., Bornstein, S. R., Schroeck, E., Klink, B., Leker, R. R., Bickle, M., Chrousos, G. P., Schroeder, M., Cannistraci, C. V., Guck, J., Androutsellis-Theotokis, A. Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery.

IGF1R signalling in testicular germ cell tumour cells impacts on cell survival and acquired cisplatin resistance.

Testicular germ cell tumours (TGCTs) are the most frequent malignancy and cause of death from solid tumours in the 20- to 40-year age group. Although most cases show sensitivity to cis-platinum-based chemotherapy, this is associated with long-term toxicities and chemo-resistance. Roles for receptor tyrosine kinases other than KIT are largely unknown in TGCT. We therefore conducted a phosphoproteomic screen and identified the insulin growth factor receptor-1 (IGF1R) as both highly expressed and activated in TGCT cell lines representing the nonseminomatous subtype. IGF1R was also frequently expressed in tumour samples from patients with nonseminomas. Functional analysis of cell line models showed that long-term shRNA-mediated IGF1R silencing leads to apoptosis and complete ablation of nonseminoma cells with active IGF1R signalling. Cell lines with high levels of IGF1R activity also showed reduced AKT signalling in response to decreased IGF1R expression as well as sensitivity to the small-molecule IGF1R inhibitor NVP-AEW541. These results were in contrast to those in the seminoma cell line TCAM2 that lacked IGF1R signalling via AKT and was one of the two cell lines least sensitive to the IGF1R inhibitor. The dependence on IGF1R activity in the majority of nonseminomas parallels the known role of IGF signalling in the proliferation, migration, and survival of primordial germ cells, the putative cell of origin for TGCT. Upregulation of IGF1R expression and signalling was also found to contribute to acquired cisplatin resistance in an in vitro nonseminoma model, providing a rationale for targeting IGF1R in cisplatin-resistant disease. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Altered glutamine metabolism in breast cancer; subtype dependencies and alternative adaptations.

Cancer cells must alter their metabolism in order to satisfy the demands of necessary energy and cellular building blocks. These metabolic alterations are mediated by many oncogenic changes that affect cellular signalling pathways, which result in sustained cell growth and proliferation. Recently, metabolomics has received great attention in the field of cancer research, and as the essential metabolic pathways that drive tumour growth and progression are determined the possibilities of new targets for therapeutic intervention are opened. More specifically, as breast cancer is a heterogeneous disease, there is growing evidence that differences in metabolic changes exist between molecular subtypes. In this review, the most recent findings in breast cancer cell metabolism are discussed, with particular emphasis on glutamine and its transporters, which is considered one of the key amino acids fuelling cancer growth. Furthermore, the metabolic differences between the molecular subtypes of breast cancer are examined, highlighting the clinical utility for breast cancer diagnosis and treatment.

The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions.

Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using (31)P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications.

The Role of pH Regulation in Cancer Progression.

Frequently observed phenotypes of tumours include high metabolic activity, hypoxia and poor perfusion; these act to produce an acidic microenvironment. Cellular function depends on pH homoeostasis, and thus, tumours become dependent on pH regulatory mechanisms. Many of the proteins involved in pH regulation are highly expressed in tumours, and their expression is often of prognostic significance. The more acidic tumour microenvironment also has important implications with regard to chemotherapeutic and radiotherapeutic interventions. In addition, we review pH-sensing mechanisms, the role of pH regulation in tumour phenotype and the use of pH regulatory mechanisms as therapeutic targets.

Methods: Using Three-Dimensional Culture (Spheroids) as an In Vitro Model of Tumour Hypoxia.

Regions of hypoxia in tumours can be modelled in vitro in 2D cell cultures with a hypoxic chamber or incubator in which oxygen levels can be regulated. Although this system is useful in many respects, it disregards the additional physiological gradients of the hypoxic microenvironment, which result in reduced nutrients and more acidic pH. Another approach to hypoxia modelling is to use three-dimensional spheroid cultures. In spheroids, the physiological gradients of the hypoxic tumour microenvironment can be inexpensively modelled and explored. In addition, spheroids offer the advantage of more representative modelling of tumour therapy responses compared with 2D culture. Here, we review the use of spheroids in hypoxia tumour biology research and highlight the different methodologies for spheroid formation and how to obtain uniformity. We explore the challenge of spheroid analyses and how to determine the effect on the hypoxic versus normoxic components of spheroids. We discuss the use of high-throughput analyses in hypoxia screening of spheroids. Furthermore, we examine the use of mathematical modelling of spheroids to understand more fully the hypoxic tumour microenvironment.

Fatty acid-binding protein 4, a point of convergence for angiogenic and metabolic signaling pathways in endothelial cells.

Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment.

NANOG controls testicular germ cell tumour stemness through regulation of MIR9-2.

BACKGROUND: Testicular germ cell tumours (TGCTs) represent a clinical challenge; they are most prevalent in young individuals and are triggered by molecular mechanisms that are not fully understood. The origin of TGCTs can be traced back to primordial germ cells that fail to mature during embryonic development. These cells express high levels of pluripotency factors, including the transcription factor NANOG which is highly expressed in TGCTs. Gain or amplification of the NANOG locus is common in advanced tumours, suggesting a key role for this master regulator of pluripotency in TGCT stemness and malignancy. METHODS: In this study, we analysed the expression of microRNAs (miRNAs) that are regulated by NANOG in TGCTs via integrated bioinformatic analyses of data from The Cancer Genome Atlas and NANOG chromatin immunoprecipitation in human embryonic stem cells. Through gain-of-function experiments, MIR9-2 was further investigated as a novel tumour suppressor regulated by NANOG. After transfection with MIR9-2 mimics, TGCT cells were analysed for cell proliferation, invasion, sensitivity to cisplatin, and gene expression signatures by RNA sequencing. RESULTS: For the first time, we identified 86 miRNAs regulated by NANOG in TGCTs. Among these, 37 miRNAs were differentially expressed in NANOG-high tumours, and they clustered TGCTs according to their subtypes. Binding of NANOG within 2 kb upstream of the MIR9-2 locus was associated with a negative regulation. Low expression of MIR9-2 was associated with tumour progression and MIR9-2-5p was found to play a role in the control of tumour stemness. A gain of function of MIR9-2-5p was associated with reduced proliferation, invasion, and sensitivity to cisplatin in both embryonal carcinoma and seminoma tumours. MIR9-2-5p expression in TGCT cells significantly reduced the expression of genes regulating pluripotency and cell division, consistent with its functional effect on reducing cancer stemness. CONCLUSIONS: This study provides new molecular insights into the role of NANOG as a key determinant of pluripotency in TGCTs through the regulation of MIR9-2-5p, a novel epigenetic modulator of cancer stemness. Our data also highlight the potential negative feedback mediated by MIR9-2-5p on NANOG expression, which could be exploited as a therapeutic strategy for the treatment of TGCTs.

Protein Oxidative Modifications in Neurodegenerative Diseases: From Advances in Detection and Modelling to Their Use as Disease Biomarkers.

Oxidation-reduction post-translational modifications (redox-PTMs) are chemical alterations to amino acids of proteins. Redox-PTMs participate in the regulation of protein conformation, localization and function, acting as signalling effectors that impact many essential biochemical processes in the cells. Crucially, the dysregulation of redox-PTMs of proteins has been implicated in the pathophysiology of numerous human diseases, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. This review aims to highlight the current gaps in knowledge in the field of redox-PTMs biology and to explore new methodological advances in proteomics and computational modelling that will pave the way for a better understanding of the role and therapeutic potential of redox-PTMs of proteins in neurodegenerative diseases. Here, we summarize the main types of redox-PTMs of proteins while providing examples of their occurrence in neurodegenerative diseases and an overview of the state-of-the-art methods used for their detection. We explore the potential of novel computational modelling approaches as essential tools to obtain insights into the precise role of redox-PTMs in regulating protein structure and function. We also discuss the complex crosstalk between various PTMs that occur in living cells. Finally, we argue that redox-PTMs of proteins could be used in the future as diagnosis and prognosis biomarkers for neurodegenerative diseases.

Drug-resilient Cancer Cell Phenotype Is Acquired via Polyploidization Associated with Early Stress Response Coupled to HIF2α Transcriptional Regulation.

Therapeutic resistance and recurrence remain core challenges in cancer therapy. How therapy resistance arises is currently not fully understood with tumors surviving via multiple alternative routes. Here, we demonstrate that a subset of cancer cells survives therapeutic stress by entering a transient state characterized by whole-genome doubling. At the onset of the polyploidization program, we identified an upregulation of key transcriptional regulators, including the early stress-response protein AP-1 and normoxic stabilization of HIF2α. We found altered chromatin accessibility, ablated expression of retinoblastoma protein (RB1), and enrichment of AP-1 motif accessibility. We demonstrate that AP-1 and HIF2α regulate a therapy resilient and survivor phenotype in cancer cells. Consistent with this, genetic or pharmacologic targeting of AP-1 and HIF2α reduced the number of surviving cells following chemotherapy treatment. The role of AP-1 and HIF2α in stress response by polyploidy suggests a novel avenue for tackling chemotherapy-induced resistance in cancer. SIGNIFICANCE: In response to cisplatin treatment, some surviving cancer cells undergo whole-genome duplications without mitosis, which represents a mechanism of drug resistance. This study presents mechanistic data to implicate AP-1 and HIF2α signaling in the formation of this surviving cell phenotype. The results open a new avenue for targeting drug-resistant cells.

Classification of cancer cells at the sub-cellular level by phonon microscopy using deep learning.

There is a consensus about the strong correlation between the elasticity of cells and tissue and their normal, dysplastic, and cancerous states. However, developments in cell mechanics have not seen significant progress in clinical applications. In this work, we explore the possibility of using phonon acoustics for this purpose. We used phonon microscopy to obtain a measure of the elastic properties between cancerous and normal breast cells. Utilising the raw time-resolved phonon-derived data (300 k individual inputs), we employed a deep learning technique to differentiate between MDA-MB-231 and MCF10a cell lines. We achieved a 93% accuracy using a single phonon measurement in a volume of approximately 2.5 µm3. We also investigated means for classification based on a physical model that suggest the presence of unidentified mechanical markers. We have successfully created a compact sensor design as a proof of principle, demonstrating its compatibility for use with needles and endoscopes, opening up exciting possibilities for future applications.

Minimum regions of genomic imbalance in stage I testicular embryonal carcinoma and association of 22q loss with relapse.

Testicular germ cell tumors (TGCT) are the most frequent solid tumor to affect young adult males and are histologically divided into seminomas and nonseminomas (NS). NS comprise undifferentiated embryonal carcinoma (EC) and differentiated tumors with embryonic (teratoma) or extra-embryonic (choriocarcinoma, yolk sac tumor) features. In contrast to other subtypes, EC have uniform cellular morphology and lack normal cell infiltrates, ideal for nucleic acid profiling. EC are under-represented in previous studies due to their relative rarity. To gain insights into NS tumorigenesis, metastatic dissemination and potential markers of relapse, a full tiling path BAC platform was used to obtain array comparative genomic hybridization (aCGH) profiles from 32 formalin fixed paraffin embedded stage I EC samples from patients with follow-up data. In addition to identifying regions previously described in TGCT, novel minimum overlapping regions of gain at 6p21.33, 10q11.21, and 22q13.32 and loss at 22q12.2 were defined and confirmed by fluorescence in situ hybridization analyses. Specifically, the region at 6p21.33 included OCT3/4, the expression of which is involved in the maintenance of pluripotency and the 10q11.21 region contains the gene encoding the RAS activating factor RASGEF1A, the expression of which was demonstrably increased in RNA extracted from these samples. The region of loss at 22q12.2 was more frequently seen in tumors that relapsed and protein expression of genes from 22q12.2 included PIK3IP1, a negative regulator of PI3 kinase signaling was reduced. These data support the role for genes involved in pluripotency and RAS/PI3K signaling in EC development and progression.

Targeting hypoxia regulated sodium driven bicarbonate transporters reduces triple negative breast cancer metastasis.

Regions of low oxygen (hypoxia) are found in >50% of breast tumours, most frequently in the more aggressive triple negative breast cancer subtype (TNBC). Metastasis is the cause of 90% of breast cancer patient deaths. Regions of tumour hypoxia tend to be more acidic and both hypoxia and acidosis increase tumour metastasis. In line with this the metastatic process is dependent on pH regulatory mechanisms. We and others have previously identified increased hypoxic expression of Na+ driven bicarbonate transporters (NDBTs) as a major mechanism of tumour pH regulation. Hypoxia induced the expression of NDBTs in TNBC, most frequently SLC4A4 and SLC4A5. NDBT inhibition (S0859) and shRNA knockdown suppressed migration (40% reduction) and invasion (70% reduction) in vitro. Tumour xenograft metastasis in vivo was significantly reduced by NDBT knockdown. To investigate the mechanism by which NDBTs support metastasis, we investigated their role in regulation of phospho-signalling, epithelial-to-mesenchymal transition (EMT) and metabolism. NDBT knockdown resulted in an attenuation in hypoxic phospho-signalling activation; most notably LYN (Y397) reduced by 75%, and LCK (Y394) by 72%. The metastatic process is associated with EMT. We showed that NDBT knockdown inhibited EMT, modulating the expression of key EMT transcription factors and ablating the expression of vimentin whilst increasing the expression of E-cadherin. NDBT knockdown also altered metabolic activity reducing overall ATP and extracellular lactate levels. These results demonstrate that targeting hypoxia-induced NDBT can be used as an approach to modulate phospho-signalling, EMT, and metabolic activity and reduce tumour migration, invasion, and metastasis in vivo.

Endothelial Cell RNA-Seq Data: Differential Expression and Functional Enrichment Analyses to Study Phenotypic Switching.

RNA-seq is a common approach used to explore gene expression data between experimental conditions or cell types and ultimately leads to information that can shed light on the biological processes involved and inform further hypotheses. While the protocols required to generate samples for sequencing can be performed in most research facilities, the resulting computational analysis is often an area in which researchers have little experience. Here we present a user-friendly bioinformatics workflow which describes the methods required to take raw data produced by RNA sequencing to interpretable results. Widely used and well documented tools are applied. Data quality assessment and read trimming were performed by FastQC and Cutadapt, respectively. Following this, STAR was utilized to map the trimmed reads to a reference genome and the alignment was analyzed by Qualimap. The subsequent mapped reads were quantified by featureCounts. DESeq2 was used to normalize and perform differential expression analysis on the quantified reads, identifying differentially expressed genes and preparing the data for functional enrichment analysis. Gene set enrichment analysis identified enriched gene sets from the normalized count data and clusterProfiler was used to perform functional enrichment against the GO, KEGG, and Reactome databases. Example figures of the functional enrichment analysis results were also generated. The example data used in the workflow are derived from HUVECs, an in vitro model used in the study of endothelial cells, published and publicly available for download from the European Nucleotide Archive.

The association of CCND1 overexpression and cisplatin resistance in testicular germ cell tumors and other cancers.

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Doxycycline Attenuates Cancer Cell Growth by Suppressing NLRP3-Mediated Inflammation.

NLR family pyrin domain containing 3 (NLRP3) inflammasome formation is triggered by the damaged mitochondria releasing reactive oxygen species. Doxycycline was shown to regulate inflammation; however, its effect on NLRP3 in cancer remains largely unknown. Therefore, we sought to determine the effect of doxycycline on NLRP3 regulation in cancer using an in vitro model. NLRP3 was activated in a prostate cancer cell line (PC3) and a lung cancer cell line (A549) before treatment with doxycycline. Inflammasome activation was assessed by analyzing RNA expression of NLRP3, Pro-CASP-1, and Pro-IL1ß using RT-qPCR. Additionally, NLPR3 protein expression and IL-1ß secretion were analyzed using Western blot and ELISA, respectively. Tumor cell viability was determined using Annexin V staining and a cell proliferation assay. Cytokine secretion was analyzed using a 41Plex assay for human cytokines. Data were analyzed using one-way ANOVA model with Tukey's post hoc tests. Doxycycline treatment decreased NLRP3 formation in PC3 and A549 cells compared to untreated and LPS only treated cells (p < 0.05). Doxycycline also decreased proliferation and caused cell death through apoptosis, a response that differed to the LPS-Nigericin mediated pyroptosis. Our findings suggest that doxycycline inhibits LPS priming of NLRP3 and reduces tumor progression through early apoptosis in cancer.

Intratumour heterogeneity in microRNAs expression regulates glioblastoma metabolism.

While specific microRNA (miRNA) signatures have been identified in glioblastoma (GBM), the intratumour heterogeneity in miRNA expression has not yet been characterised. In this study, we reveal significant alterations in miRNA expression across three GBM tumour regions: the core, rim, and invasive margin. Our miRNA profiling analysis showed that miR-330-5p and miR-215-5p were upregulated in the invasive margin relative to the core and the rim regions, while miR-619-5p, miR-4440 and miR-4793-3p were downregulated. Functional analysis of newly identified miRNAs suggests their involvement in regulating lipid metabolic pathways. Subsequent liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectroscopy (LC-MS/MS) profiling of the intracellular metabolome and the lipidome of GBM cells with dysregulated miRNA expression confirmed the alteration in the metabolite levels associated with lipid metabolism. The identification of regional miRNA expression signatures may underlie the metabolic heterogeneity within the GBM tumour and understanding this relationship may open new avenues for the GBM treatment.

Therapeutic Potential of Pharmacological Targeting NLRP3 Inflammasome Complex in Cancer.

Introduction: Dysregulation of NLRP3 inflammasome complex formation can promote chronic inflammation by increased release of IL-1ß. However, the effect of NLRP3 complex formation on tumor progression remains controversial. Therefore, we sought to determine the effect of NLRP3 modulation on the growth of the different types of cancer cells, derived from lung, breast, and prostate cancers as well as neuroblastoma and glioblastoma in-vitro. Method: The effect of Caspase 1 inhibitor (VX765) and combination of LPS/Nigericin on NLRP3 inflammasome activity was analyzed in A549 (lung cancer), MCF-7 (breast cancer), PC3 (prostate cancer), SH-SY5Y (neuroblastoma), and U138MG (glioblastoma) cells. Human fibroblasts were used as control cells. The effect of VX765 and LPS/Nigericin on NLRP3 expression was analyzed using western blot, while IL-1ß and IL-18 secretion was detected by ELISA. Tumor cell viability and progression were determined using Annexin V, cell proliferation assay, LDH assay, sphere formation assay, transmission electron microscopy, and a multiplex cytokine assay. Also, angiogenesis was investigated by a tube formation assay. VEGF and MMPs secretion were detected by ELISA and a multiplex assay, respectively. Statistical analysis was done using one-way ANOVA with Tukey's analyses and Kruskal-Wallis one-way analysis of variance. Results: LPS/Nigericin increased NRLP3 protein expression as well as IL-1ß and IL-18 secretion in PC3 and U138MG cells compared to A549, MCF7, SH-SY5Y cells, and fibroblasts. In contrast, MIF expression was commonly found upregulated in A549, PC3, SH-SY5Y, and U138MG cells and fibroblasts after Nigericin treatment. Nigericin and a combination of LPS/Nigericin decreased the cell viability and proliferation. Also, LPS/Nigericin significantly increased tumorsphere size in PC3 and U138MG cells. In contrast, the sphere size was reduced in MCF7 and SH-SY5Y cells treated with LPS/Nigericin, while no effect was detected in A549 cells. VX765 increased secretion of CCL24 in A549, MCF7, PC3, and fibroblasts as well as CCL11 and CCL26 in SH-SY5Y cells. Also, VX765 significantly increased the production of VEGF and MMPs and stimulated angiogenesis in all tumor cell lines. Discussion: Our data suggest that NLRP3 activation using Nigericin could be a novel therapeutic approach to control the growth of tumors producing a low level of IL-1ß and IL-18.

Regulation of cellular sterol homeostasis by the oxygen responsive noncoding RNA lincNORS.

We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance.