Cinnamomum zeylanicum Blume Essential Oil Inhibits Metastatic Melanoma Cell Proliferation by Triggering an Incomplete Tumour Cell Stress Response.

Given the known pro-oxidant status of tumour cells, the development of anti-proliferative strategies focuses on products with both anti- and pro-oxidant properties that can enhance antitumour drug cytotoxicity. We used a C. zeylanicum essential oil (CINN-EO) and assessed its effect on a human metastatic melanoma cell line (M14). Human PBMCs and MDMs from healthy donors were used as normal control cells. CINN-EO induced cell growth inhibition, cell cycle perturbation, ROS and Fe(II) increases, and mitochondrial membrane depolarization. To assess whether CINN-EO could affect the stress response, we analysed iron metabolism and stress response gene expression. CINN-EO increased HMOX1, FTH1, SLC7A11, DGKK, and GSR expression but repressed OXR1, SOD3, Tf, and TfR1 expression. HMOX1, Fe(II), and ROS increases are associated with ferroptosis, which can be reversed by SnPPIX, an HMOX1 inhibitor. Indeed, our data demonstrated that SnPPIX significantly attenuated the inhibition of cell proliferation, suggesting that the inhibition of cell proliferation induced by CINN-EO could be related to ferroptosis. Concurrent treatment with CINN-EO enhanced the anti-melanoma effect of two conventional antineoplastic drugs: the mitochondria-targeting tamoxifen and the anti-BRAF dabrafenib. We demonstrate that CINN-EO-mediated induction of an incomplete stress response specifically in cancer cells affects the proliferation of melanoma cells and can enhance drug cytotoxicity.

Astrocytes-Derived Small Extracellular Vesicles Hinder Glioma Growth.

All cells are capable of secreting extracellular vesicles (EVs), which are not a means to eliminate unneeded cellular compounds but represent a process to exchange material (nucleic acids, lipids and proteins) between different cells. This also happens in the brain, where EVs permit the crosstalk between neuronal and non-neuronal cells, functional to homeostatic processes or cellular responses to pathological stimuli. In brain tumors, EVs are responsible for the bidirectional crosstalk between glioblastoma cells and healthy cells, and among them, astrocytes, that assume a pro-tumoral or antitumoral role depending on the stage of the tumor progression. In this work, we show that astrocyte-derived small EVs (sEVs) exert a defensive mechanism against tumor cell growth and invasion. The effect is mediated by astrocyte-derived EVs (ADEVs) through the transfer to tumor cells of factors that hinder glioma growth. We identified one of these factors, enriched in ADEVs, that is miR124. It reduced both the expression and function of the volume-regulated anion channel (VRAC), that, in turn, decreased the cell migration and invasion of murine glioma GL261 cells.

miRNome and Proteome Profiling of Small Extracellular Vesicles Secreted by Human Glioblastoma Cell Lines and Primary Cancer Stem Cells.

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. Despite available therapeutic interventions, it is very difficult to treat, and a cure is not yet available. The intra-tumoral GBM heterogeneity is a crucial factor contributing to poor clinical outcomes. GBM derives from a small heterogeneous population of cancer stem cells (CSCs). In cancer tissue, CSCs are concentrated within the so-called niches, where they progress from a slowly proliferating phase. CSCs, as most tumor cells, release extracellular vesicles (EVs) into the surrounding microenvironment. To explore the role of EVs in CSCs and GBM tumor cells, we investigated the miRNA and protein content of the small EVs (sEVs) secreted by two GBM-established cell lines and by GBM primary CSCs using omics analysis. Our data indicate that GBM-sEVs are selectively enriched for miRNAs that are known to display tumor suppressor activity, while their protein cargo is enriched for oncoproteins and tumor-associated proteins. Conversely, among the most up-regulated miRNAs in CSC-sEVs, we also found pro-tumor miRNAs and proteins related to stemness, cell proliferation, and apoptosis. Collectively, our findings support the hypothesis that sEVs selectively incorporate different miRNAs and proteins belonging both to fundamental processes (e.g., cell proliferation, cell death, stemness) as well as to more specialized ones (e.g., EMT, membrane docking, cell junction organization, ncRNA processing).

Glioma extracellular vesicles for precision medicine: prognostic and theragnostic application.

EV produced by tumour cells carry a diverse population of proteins, lipids, DNA, and RNA molecules throughout the body and appear to play an important role in the overall development of the disease state, according to growing data. Gliomas account for a sizable fraction of all primary brain tumours and the vast majority of brain malignancies. Glioblastoma multiforme (GBM) is a kind of grade IV glioma that has a very dismal prognosis despite advancements in diagnostic methods and therapeutic options. The authors discuss advances in understanding the function of extracellular vesicles (EVs), in overall glioma growth, as well as how recent research is uncovering the utility of EVs in glioma diagnostics, prognostic and therapeutics approaches.

Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy.

Glioblastomas (GBM) are the most aggressive tumors originating in the brain. Histopathologic features include circuitous, disorganized, and highly permeable blood vessels with intermittent blood flow. These features contribute to the inability to direct therapeutic agents to tumor cells. Known targets for anti-angiogenic therapies provide minimal or no effect in overall survival of 12-15 months following diagnosis. Identification of novel targets therefore remains an important goal for effective treatment of highly vascularized tumors such as GBM. We previously demonstrated in zebrafish that a balanced level of expression of the transmembrane protein TMEM230/C20ORF30 was required to maintain normal blood vessel structural integrity and promote proper vessel network formation. To investigate whether TMEM230 has a role in the pathogenesis of GBM, we analyzed its prognostic value in patient tumor gene expression datasets and performed cell functional analysis. TMEM230 was found necessary for growth of U87-MG cells, a model of human GBM. Downregulation of TMEM230 resulted in loss of U87 migration, substratum adhesion, and re-passaging capacity. Conditioned media from U87 expressing endogenous TMEM230 induced sprouting and tubule-like structure formation of HUVECs. Moreover, TMEM230 promoted vascular mimicry-like behavior of U87 cells. Gene expression analysis of 702 patients identified that TMEM230 expression levels distinguished high from low grade gliomas. Transcriptomic analysis of patients with gliomas revealed molecular pathways consistent with properties observed in U87 cell assays. Within low grade gliomas, elevated TMEM230 expression levels correlated with reduced overall survival independent from tumor subtype. Highest level of TMEM230 correlated with glioblastoma and ATP-dependent microtubule kinesin motor activity, providing a direction for future therapeutic intervention. Our studies support that TMEM230 has both glial tumor and endothelial cell intracellular and extracellular functions. Elevated levels of TMEM230 promote glial tumor cell migration, extracellular scaffold remodeling, and hypervascularization and abnormal formation of blood vessels. Downregulation of TMEM230 expression may inhibit both low grade glioma and glioblastoma tumor progression and promote normalization of abnormally formed blood vessels. TMEM230 therefore is both a promising anticancer and antiangiogenic therapeutic target for inhibiting GBM tumor cells and tumor-driven angiogenesis.

Role of Lamin A/C as Candidate Biomarker of Aggressiveness and Tumorigenicity in Glioblastoma Multiforme.

Nuclear lamina components have long been regarded as scaffolding proteins, forming a dense fibrillar structure necessary for the maintenance of the nucleus shape in all the animal kingdom. More recently, mutations, aberrant localisation and deregulation of these proteins have been linked to several diseases, including cancer. Using publicly available data we found that the increased expression levels of the nuclear protein Lamin A/C correlate with a reduced overall survival in The Cancer Genome Atlas Research Network (TCGA) patients affected by glioblastoma multiforme (GBM). We show that the expression of the LMNA gene is linked to the enrichment of cancer-related pathways, particularly pathways related to cell adhesion and cell migration. Mimicking the modulation of LMNA in a GBM preclinical cancer model, we confirmed both in vitro and in vivo that the increased expression of LMNA is associated with an increased aggressiveness and tumorigenicity. In addition, delving into the possible mechanism behind LMNA-induced GBM aggressiveness and tumorigenicity, we found that the mTORC2 component, Rictor, plays a central role in mediating these effects.

Microglia-Derived Small Extracellular Vesicles Reduce Glioma Growth by Modifying Tumor Cell Metabolism and Enhancing Glutamate Clearance through miR-124.

Brain homeostasis needs continuous exchange of intercellular information among neurons, glial cells, and immune cells, namely microglial cells. Extracellular vesicles (EVs) are active players of this process. All the cells of the body, including the brain, release at least two subtypes of EVs, the medium/large EVs (m/lEVs) and small EVs (sEVs). sEVs released by microglia play an important role in brain patrolling in physio-pathological processes. One of the most common and malignant forms of brain cancer is glioblastoma. Altered intercellular communications constitute a base for the onset and the development of the disease. In this work, we used microglia-derived sEVs to assay their effects in vitro on murine glioma cells and in vivo in a glioma model on C57BL6/N mice. Our findings indicated that sEVs carry messages to cancer cells that modify glioma cell metabolism, reducing lactate, nitric oxide (NO), and glutamate (Glu) release. sEVs affect Glu homeostasis, increasing the expression of Glu transporter Glt-1 on astrocytes. We demonstrated that these effects are mediated by miR-124 contained in microglia-released sEVs. The in vivo benefit of microglia-derived sEVs results in a significantly reduced tumor mass and an increased survival of glioma-bearing mice, depending on miR-124.

Expression profiling of microRNAs and isomiRs in conventional central chondrosarcoma.

Conventional central chondrosarcoma (CCC) is a malignant bone tumor that is characterized by the production of chondroid tissue. Since radiation therapy and chemotherapy have limited effects on CCC, treatment of most patients depends on surgical resection. This study aimed to identify the expression profiles of microRNAs (miRNAs) and isomiRs in CCC tissues to highlight their possible participation to the regulation of pathways critical for the formation and growth of this type of tumor. Our study analyzed miRNAs and isomiRs from Grade I (GI), Grade II (GII), and Grade III (GIII) histologically validated CCC tissue samples. While the different histological grades shared a similar expression profile for the top abundant miRNAs, we found several microRNAs and isomiRs showing a strong different modulation in GII + GIII vs GI grade samples and their involvement in tumor biology could be consistently hypothesized. We then in silico validated these differently expressed miRNAs in a larger chondrosarcoma public dataset and confirmed the expression trend for 17 out of 34 miRNAs. Our results clearly suggests that the contribution of miRNA deregulation, and their targeted pathways, to the progression of CCC could be relevant and strongly indicates that when studying miRNA deregulation in tumors, not only the canonical miRNAs, but the whole set of corresponding isomiRs should be taken in account. Improving understanding of the precise roles of miRNAs and isomiRs over the course of central chondrosarcoma progression could help identifying possible targets for precision medicine therapeutic intervention.

Circulating miRNAs in Small Extracellular Vesicles Secreted by a Human Melanoma Xenograft in Mouse Brains.

The identification of liquid biomarkers remains a major challenge to improve the diagnosis of melanoma patients with brain metastases. Circulating miRNAs packaged into tumor-secreted small extracellular vesicles (sEVs) contribute to tumor progression. To investigate the release of tumor-secreted miRNAs by brain metastasis, we developed a xenograft model where human metastatic melanoma cells were injected intracranially in nude mice. The comprehensive profiles of both free miRNAs and those packaged in sEVs secreted by the melanoma cells in the plasma demonstrated that most (80%) of the sEV-associated miRNAs were also present in serum EVs from a cohort of metastatic melanomas, included in a publicly available dataset. Remarkably, among them, we found three miRNAs (miR-224-5p, miR-130a-3p and miR-21-5p) in sEVs showing a trend of upregulation during melanoma progression. Our model is proven to be valuable for identifying miRNAs in EVs that are unequivocally secreted by melanoma cells in the brain and could be associated to disease progression.

Extracellular Vesicles, A Possible Theranostic Platform Strategy for Hepatocellular Carcinoma-An Overview.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third highest cause of mortality from cancer, largely because of delays in diagnosis. There is currently no effective therapy for advanced stage HCC, although sorafenib, the standard treatment for HCC, systemic therapy (including tyrosine kinase inhibitors and anti-angiogenesis agents), and more recently, immunotherapy, have demonstrated some survival benefit. The measurement and modification of extracellular vesicle (EVs) cargoes-composed of nucleic acids, including miRNAs, proteins, and lipids-holds great promise for future HCC diagnosis, prognosis, and treatment. This review will provide an overview of the most recent findings regarding EVs in HCC, and the possible future use of EVs as "liquid biopsy"-based biomarkers for early diagnosis and as a vehicle for targeted drug-delivery.

Conditioned medium of primary lung cancer cells induces EMT in A549 lung cancer cell line by TGF-ß1 and miRNA21 cooperation.

The epithelial-mesenchymal transition (EMT) plays a key role in tumor progression, drug resistance and metastasis. Recently, numerous microRNA (miRNA) have been described to regulate EMT in tumor progression. In this study, we found that conditioned medium from the LC212 non-small-cell lung cancer (NSCLC) cell line (LC212-CM) induces morphological changes and overexpression of Vimentin, CD90, SMAD 2/3, SLUG and TWIST in A549 NSCLC cells, consistent with a mesenchymal phenotype. To identify the soluble mediators in LC212-CM involved in this phenomenon, we performed miRNA profiling and TGF-ß1 quantification. We found that LC212-CM contains high levels of TGF-ß1 as well as different secreted miRNAs. We focused our attention on Homo sapiens-microRNA21 (hsa-miR21), one of most relevant miRNA associated with lung cancer progression, metastasis and EMT. An hsa-miR21 antagomiR was able to prevent the LC212-CM-induced EMT phenotype in A549 cells. Furthermore, we found that TGF-ß1 and hsa-miR21 cooperate in the induction of EMT in A549 cells. Intriguingly, TGF-ß1 was found to induce hsa-miR21 expression in A549 cell, thus suggesting that the hsa-miR21 mediates at least in part the pro-EMT effects of TGF-ß1. In conclusion, hsa-miR21 and TGF-ß1 are involved in autocrine and paracrine circuits that regulate the EMT status of lung cancer cells.

RNA-seq reveals distinctive RNA profiles of small extracellular vesicles from different human liver cancer cell lines.

Liver cancer (LC) is one of the most common cancers and represents the third highest cause of cancer-related deaths worldwide. Extracellular vesicle (EVs) cargoes, which are selectively enriched in RNA, offer great promise for the diagnosis, prognosis and treatment of LC. Our study analyzed the RNA cargoes of EVs derived from 4 liver-cancer cell lines: HuH7, Hep3B, HepG2 (hepato-cellular carcinoma) and HuH6 (hepatoblastoma), generating two different sets of sequencing libraries for each. One library was size-selected for small RNAs and the other targeted the whole transcriptome. Here are reported genome wide data of the expression level of coding and non-coding transcripts, microRNAs, isomiRs and snoRNAs providing the first comprehensive overview of the extracellular-vesicle RNA cargo released from LC cell lines. The EV-RNA expression profiles of the four liver cancer cell lines share a similar background, but cell-specific features clearly emerge showing the marked heterogeneity of the EV-cargo among the individual cell lines, evident both for the coding and non-coding RNA species.

Lamin A/C Is Required for ChAT-Dependent Neuroblastoma Differentiation.

The mouse neuroblastoma N18TG2 clone is unable to differentiate and is defective for the enzymes of the biosynthesis of neurotransmitters. The forced expression of choline acetyltransferase (ChAT) in these cells results in the synthesis and release of acetylcholine (Ach) and hence in the expression of neurospecific features and markers. To understand how the expression of ChAT triggered neuronal differentiation, we studied the differences in genome-wide transcription profiles between the N18TG2 parental cells and its ChAT-expressing 2/4 derived clone. The engagement of the 2/4 cells in the neuronal developmental program was confirmed by the increase of the expression level of several differentiation-related genes and by the reduction of the amount of transcripts of cell cycle genes. At the same time, we observed a massive reorganization of cytoskeletal proteins in terms of gene expression, with the accumulation of the nucleoskeletal lamina component Lamin A/C in differentiating cells. The increase of the Lmna transcripts induced by ChAT expression in 2/4 cells was mimicked treating the parental N18TG2 cells with the acetylcholine receptor agonist carbachol, thus demonstrating the direct role played by this receptor in neuron nuclei maturation. Conversely, a treatment of 2/4 cells with the muscarinic receptor antagonist atropine resulted in the reduction of the amount of Lmna RNA. Finally, the hypothesis that Lmna gene product might play a crucial role in the ChAT-dependent molecular differentiation cascade was strongly supported by Lmna knockdown in 2/4 cells leading to the downregulation of genes involved in differentiation and cytoskeleton formation and to the upregulation of genes known to regulate self-renewal and stemness.

Down-regulation of the Lamin A/C in neuroblastoma triggers the expansion of tumor initiating cells.

Tumor-initiating cells constitute a population within a tumor mass that shares properties with normal stem cells and is considered responsible for therapy failure in many cancers. We have previously demonstrated that knockdown of the nuclear envelope component Lamin A/C in human neuroblastoma cells inhibits retinoic acid-mediated differentiation and results in a more aggressive phenotype. In addition, Lamin A/C is often lost in advanced tumors and changes in the nuclear envelope composition occur during tumor progression. Based on our previous data and considering that Lamin A/C is expressed in differentiated tissues, we hypothesize that the lack of Lamin A/C could predispose cells toward a stem-like phenotype, thus influencing the development of tumor-initiating cells in neuroblastoma. This paper demonstrates that knockdown of Lamin A/C triggers the development of a tumor-initiating cell population with self-renewing features in human neuroblastoma cells. We also demonstrates that the development of TICs is due to an increased expression of MYCN gene and that in neuroblastoma exists an inverse relationship between LMNA and MYCN expression.

Signaling by exosomal microRNAs in cancer.

A class of small non-coding RNAs, the microRNAs (miRNAs), have recently attracted great attention in cancer research since they play a central role in regulation of gene-expression and miRNA aberrant expression is found in almost all types of human cancer. The discovery of circulating miRNAs in body fluids and the finding that they are often tumor specific and can be detected early in tumorigenesis has soon led to the evaluation of their possible use as cancer biomarkers and treatment-response predictors. The evidence that tumor cells communicate via the secretion and delivery of miRNAs packed into tumor-released microvesicles has prompted to investigate miRNA contribution as signaling molecules to the establishment and maintenance of the tumor microenvironment and the metastatic niche in cancer. In this review we highlight the recent advances on the role of exosomal miRNAs as mediators of cancer cell-to-cell communication.

Human placenta-derived neurospheres are susceptible to transformation after extensive in vitro expansion.

INTRODUCTION: The cancer stem cell model links neoplastic cells with normal stem cell biology, but little is known on how normal stem cells are transformed into cancer stem cells. METHODS: To investigate the processes underlying the transformation of normal stem cells we developed in vitro a cancer stem cell model from human amniotic and chorionic placenta membranes. In this model we studied the expression of specific stem cell molecules by flow cytometry, and genes, by real time RT-PCR. Microscopy immunfluorescence was employed to investigate the proliferative and differentiation patterns. Fluorescence microscopy and FACS were employed to investigate the proliferative and differentiation patterns. To evaluate the tumorigenic potential of our model we injected the cells into NOD.CB17-Prkdcscid/NCrHsd mice. RESULTS: Normal human stem cells from amniotic and chorionic placenta membranes were converted into neural cell lineages, under specific conditions, to form secondary neurospheres with a capacity for self-renewal. After extensive in vitro culture, these cells underwent spontaneous transformations and acquired a neuroblastoma (NB)-like phenotype with an elevated proliferative potential that is comparable to established neuroblastoma cell lines. The ability of these cells to transform their phenotype was evidenced by increased clonogenic ability in vitro; by augmented expression level of certain proliferation- and transformation-related genes (e.g., CCNA2, MYCN, ENPP2, GRIA3, and KIT); by the presence of multinucleated and hyperdiploid cells. We further demonstrated that the transformed phenotype is an NB by measuring the expression of NB-specific markers, disialoganglioside GD2 and N-Myc proteins. CONCLUSIONS: We have developed a cancer stem cell model starting from normal human stem cells derived from amniotic and chorionic placenta membranes. These cells are able to differentiate into neural cell lineages and to undergo spontaneous transformations and acquire an NB-like phenotype.

LMNA knock-down affects differentiation and progression of human neuroblastoma cells.

BACKGROUND: Neuroblastoma (NB) is one of the most aggressive tumors that occur in childhood. Although genes, such as MYCN, have been shown to be involved in the aggressiveness of the disease, the identification of new biological markers is still desirable. The induction of differentiation is one of the strategies used in the treatment of neuroblastoma. A-type lamins are components of the nuclear lamina and are involved in differentiation. We studied the role of Lamin A/C in the differentiation and progression of neuroblastoma. METHODOLOGY/PRINCIPAL FINDINGS: Knock-down of Lamin A/C (LMNA-KD) in neuroblastoma cells blocked retinoic acid-induced differentiation, preventing neurites outgrowth and the expression of neural markers. The genome-wide gene-expression profile and the proteomic analysis of LMNA-KD cells confirmed the inhibition of differentiation and demonstrated an increase of aggressiveness-related genes and molecules resulting in augmented migration/invasion, and increasing the drug resistance of the cells. The more aggressive phenotype acquired by LMNA-KD cells was also maintained in vivo after injection into nude mice. A preliminary immunohistochemistry analysis of Lamin A/C expression in nine primary stages human NB indicated that this protein is poorly expressed in most of these cases. CONCLUSIONS/SIGNIFICANCE: We demonstrated for the first time in neuroblastoma cells that Lamin A/C plays a central role in the differentiation, and that the loss of this protein gave rise to a more aggressive tumor phenotype.

Good Caco-2 cell culture practices.

The human Caco-2 cells differentiate spontaneously in culture forming monolayers of mature intestinal enterocytes which have been used as a model of the intestinal barrier for in vitro toxicology studies. Reproducibility problems often reported in literature have been generally ascribed to different culture-related conditions, such as the type of animal serum used, the supplements added to the culture media, the passage number and the source of cell clones. The Caco-2 cell culture protocol here described has been recently optimized in our laboratory, producing a homogeneous and highly polarized monolayer of cells which display many of the characteristics of the intestinal enterocytes. This protocol differs from standard protocols mainly because Caco-2 cells are subcultured when they reach just 50% of confluence, instead of 80%, retaining a high proliferation potential. When this cell population is seeded at high density on filter inserts differentiates almost synchronously and much more homogenously.

Differentiation of Caco-2 cells requires both transcriptional and post-translational down-regulation of Myc.

Caco-2 cancer cell line is widely used to reproduce in vitro the differentiation of absorptive enterocytes of human intestinal epithelium. This cell line, when cultured over confluence for 21 days, spontaneously undergoes cell cycle arrest and differentiates with the formation of a polarized enterocyte-like monolayer. During this process, Myc protein is completely down-regulated, as occurs in normal enterocytes. Caco-2 cells differ from normal enterocytes for mutations of APC and ß-catenin genes, factors known to be involved in the transcriptional control of MYC gene during enterocyte differentiation. In this paper, we investigated how Myc regulation could be achieved during Caco-2 differentiative process, notwithstanding the APC and ß-catenin mutations. We highlighted the post translational regulation of Myc protein as one of the essential mechanisms that allows the exit from cell cycle and onset of differentiation of Caco-2 cells. Moreover, we found a strong correlation between Myc protein downregulation and the expression of the transcription factor Cdx2, suggesting the existence of a regulative link between these two proteins.

Shotgun proteomics and network analysis of neuroblastoma cell lines treated with curcumin.

Curcumin is a natural compound with recognized anti-inflammatory properties, but its anticancer activity is still object of study. We provided an unsupervised molecular investigation of the main proteome rearrangements involved in the cellular response to curcumin in a human neuroblastoma cell line sensitive to cisplatin and its resistant counterpart by a comparative proteomic approach. Shotgun analysis demonstrated that 66 proteins were differentially expressed in response to 24 h treatment with 40 µM curcumin in sensitive cells, whereas 32 proteins were significantly modulated in treated resistant cells. Functional analysis revealed that proteins involved in cellular assembly and organization, biosynthesis and glycolysis were down-regulated by curcumin treatment. Proteome changes were associated to cell cycle arrest in the G2/M phase and accumulation of polyubiquitinated proteins, also confirmed by flow cytometry and immunoblotting analysis, but not to a significant increment of reactive oxygen species production. Since the polyubiquitination of proteins influences a wide range of cellular pathways, the inhibition of the ubiquitin-proteasome system may be the main way through which curcumin performs its multi-target activity.