Delineating three-dimensional behavior of uveal melanoma cells under anchorage independent or dependent conditions.

BACKGROUND: Although rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention. METHODS: Six UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis. RESULTS: UM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions. CONCLUSIONS: Under an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

Filtering blue light mitigates the deleterious effects induced by the oxidative stress in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a major cause of blindness in elderly. It is characterized by the loss of central vision due to damaged retinal pigment epithelial (RPE) cells and photoreceptors. Blue Light (BL) exposure was proposed as a risk factor for AMD progression. We undertook this study to determine the effects of BL on the behaviour of RPE cells and their potential mitigation by BL-filtering intraocular lenses (IOL). Human RPE cells were exposed or not to BL, with the absence or presence of either a clear ultraviolet (UV)-filtering IOL (CIOL), or a yellow UV- and BL-filtering IOL (YIOL). Cells were analyzed for their oxidative stress by measuring the levels of reactive oxygen species (ROS), and their viability. BL exposure significantly increased the levels of both total cellular and mitochondrial ROS. While this increase was not affected by placing the CIOL in the BL beam, YIOL decreased the levels of both ROS reservoirs. Increased ROS production was accompanied by increased cell death which was similarly decreased when cells were protected with the YIOL. Pre-treatment of cells with N-acetylcycteine (NAC) abolished the increased cell death, suggesting that the effects of BL on cell viability were mainly due to increased levels of ROS. BL is deleterious to RPE cells due to increased oxidative stress and cell death. These effects were mitigated by filtering these radiations. The use of BL-filtering devices may represent a strategy to reduce these effects on RPE cells and delay the onset of AMD.

Loss of Bmi1 causes anomalies in retinal development and degeneration of cone photoreceptors.

Retinal development occurs through the sequential but overlapping generation of six types of neuronal cells and one glial cell type. Of these, rod and cone photoreceptors represent the functional unit of light detection and phototransduction and are frequently affected in retinal degenerative diseases. During mouse development, the Polycomb group protein Bmi1 is expressed in immature retinal progenitors and differentiated retinal neurons, including cones. We show here that Bmi1 is required to prevent post natal degeneration of cone photoreceptors and bipolar neurons and that inactivation of Chk2 or p53 could improve but not overcome cone degeneration in Bmi1(-/-) mice. The retinal phenotype of Bmi1(-/-) mice was also characterized by loss of heterochromatin, activation of tandem repeats, oxidative stress and Rip3-associated necroptosis. In the human retina, BMI1 was preferentially expressed in cones at heterochromatic foci. BMI1 inactivation in human embryonic stem cells was compatible with retinal induction but impaired cone terminal differentiation. Despite this developmental arrest, BMI1-deficient cones recapitulated several anomalies observed in Bmi1(-/-) photoreceptors, such as loss of heterochromatin, activation of tandem repeats and induction of p53, revealing partly conserved biological functions between mouse and man.

Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFß and Wnt signaling.

Cone photoreceptors are required for color discrimination and high-resolution central vision and are lost in macular degenerations, cone and cone/rod dystrophies. Cone transplantation could represent a therapeutic solution. However, an abundant source of human cones remains difficult to obtain. Work performed in model organisms suggests that anterior neural cell fate is induced 'by default' if BMP, TGFß and Wnt activities are blocked, and that photoreceptor genesis operates through an S-cone default pathway. We report here that Coco (Dand5), a member of the Cerberus gene family, is expressed in the developing and adult mouse retina. Upon exposure to recombinant COCO, human embryonic stem cells (hESCs) differentiated into S-cone photoreceptors, developed an inner segment-like protrusion, and could degrade cGMP when exposed to light. Addition of thyroid hormone resulted in a transition from a unique S-cone population toward a mixed M/S-cone population. When cultured at confluence for a prolonged period of time, COCO-exposed hESCs spontaneously developed into a cellular sheet composed of polarized cone photoreceptors. COCO showed dose-dependent and synergistic activity with IGF1 at blocking BMP/TGFß/Wnt signaling, while its cone-inducing activity was blocked in a dose-dependent manner by exposure to BMP, TGFß or Wnt-related proteins. Our work thus provides a unique platform to produce human cones for developmental, biochemical and therapeutic studies and supports the hypothesis that photoreceptor differentiation operates through an S-cone default pathway during human retinal development.

The Polycomb Repressive Complex 1 Protein BMI1 Is Required for Constitutive Heterochromatin Formation and Silencing in Mammalian Somatic Cells.

The polycomb repressive complex 1 (PRC1), containing the core BMI1 and RING1A/B proteins, mono-ubiquitinylates histone H2A (H2A(ub)) and is associated with silenced developmental genes at facultative heterochromatin. It is, however, assumed that the PRC1 is excluded from constitutive heterochromatin in somatic cells based on work performed on mouse embryonic stem cells and oocytes. We show here that BMI1 is required for constitutive heterochromatin formation and silencing in human and mouse somatic cells. BMI1 was highly enriched at intergenic and pericentric heterochromatin, co-immunoprecipitated with the architectural heterochromatin proteins HP1, DEK1, and ATRx, and was required for their localization. In contrast, BRCA1 localization was BMI1-independent and partially redundant with that of BMI1 for H2A(ub) deposition, constitutive heterochromatin formation, and silencing. These observations suggest a dynamic and developmentally regulated model of PRC1 occupancy at constitutive heterochromatin, and where BMI1 function in somatic cells is to stabilize the repetitive genome.

ASPP1/2 regulate p53-dependent death of retinal ganglion cells through PUMA and Fas/CD95 activation in vivo.

The transcription factor p53 mediates neuronal death in a variety of stress-related and neurodegenerative conditions. The proapoptotic activity of p53 is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP) family members: ASPP1 and ASPP2. However, whether ASPP1/2 play a role in the regulation of p53-dependent neuronal death in the CNS is currently unknown. To address this, we asked whether ASPP1/2 contribute to the death of retinal ganglion cells (RGCs) using in vivo models of acute optic nerve damage in mice and rats. Here, we show that p53 is activated in RGCs soon after injury and that axotomy-induced RGC death is attenuated in p53 heterozygote and null mice. We demonstrate that ASPP1/2 proteins are abundantly expressed by injured RGCs, and that short interfering (si)RNA-based ASPP1 or ASPP2 knockdown promotes robust RGC survival. Comparative gene expression analysis revealed that siASPP-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA), Fas/CD95, and Noxa depends on p53 transcriptional activity. Furthermore, siRNA against PUMA or Fas/CD95 confers neuroprotection, demonstrating a functional role for these p53 targets in RGC death. Our study demonstrates a novel role for ASPP1 and ASPP2 in the death of RGCs and provides evidence that blockade of the ASPP-p53 pathway is beneficial for central neuron survival after axonal injury.

Horizontal Transfer of Malignant Traits and the Involvement of Extracellular Vesicles in Metastasis.

Metastases are responsible for the vast majority of cancer deaths, yet most therapeutic efforts have focused on targeting and interrupting tumor growth rather than impairing the metastatic process. Traditionally, cancer metastasis is attributed to the dissemination of neoplastic cells from the primary tumor to distant organs through blood and lymphatic circulation. A thorough understanding of the metastatic process is essential to develop new therapeutic strategies that improve cancer survival. Since Paget's original description of the "Seed and Soil" hypothesis over a hundred years ago, alternative theories and new players have been proposed. In particular, the role of extracellular vesicles (EVs) released by cancer cells and their uptake by neighboring cells or at distinct anatomical sites has been explored. Here, we will outline and discuss these alternative theories and emphasize the horizontal transfer of EV-associated biomolecules as a possibly major event leading to cell transformation and the induction of metastases. We will also highlight the recently discovered intracellular pathway used by EVs to deliver their cargoes into the nucleus of recipient cells, which is a potential target for novel anti-metastatic strategies.

EV-ADD, a database for EV-associated DNA in human liquid biopsy samples.

Extracellular vesicles (EVs) play a key role in cellular communication both in physiological conditions and in pathologies such as cancer. Emerging evidence has shown that EVs are active carriers of molecular cargo (e.g. protein and nucleic acids) and a powerful source of biomarkers and targets. While recent studies on EV-associated DNA (EV-DNA) in human biofluids have generated a large amount of data, there is currently no database that catalogues information on EV-DNA. To fill this gap, we have manually curated a database of EV-DNA data derived from human biofluids (liquid biopsy) and in-vitro studies, called the Extracellular Vesicle-Associated DNA Database (EV-ADD). This database contains validated experimental details and data extracted from peer-reviewed published literature. It can be easily queried to search for EV isolation methods and characterization, EV-DNA isolation techniques, quality validation, DNA fragment size, volume of starting material, gene names and disease context. Currently, our database contains samples representing 23 diseases, with 13 different types of EV isolation techniques applied on eight different human biofluids (e.g. blood, saliva). In addition, EV-ADD encompasses EV-DNA data both representing the whole genome and specifically including oncogenes, such as KRAS, EGFR, BRAF, MYC, and mitochondrial DNA (mtDNA). An EV-ADD data metric system was also integrated to assign a compliancy score to the MISEV guidelines based on experimental parameters reported in each study. While currently available databases document the presence of proteins, lipids, RNA and metabolites in EVs (e.g. Vesiclepedia, ExoCarta, ExoBCD, EVpedia, and EV-TRACK), to the best of our knowledge, EV-ADD is the first of its kind to compile all available EV-DNA datasets derived from human biofluid samples. We believe that this database provides an important reference resource on EV-DNA-based liquid biopsy research, serving as a learning tool and to showcase the latest developments in the EV-DNA field. EV-ADD will be updated yearly as newly published EV-DNA data becomes available and it is freely available at www.evdnadatabase.com.

BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery.

Glioblastoma multiforme (GBM) is an aggressive brain tumor that is resistant to all known therapies. Within these tumors, a CD133-positive cancer-initiating neural stem cell (NSC) population was shown to be resistant to gamma radiation through preferential activation of the DNA double-strand break (DSB) response machinery, including the ataxia-telangiectasia-mutated (ATM) kinase. The polycomb group protein BMI1 is enriched in CD133-positive GBM cells and required for their self-renewal in an INK4A/ARF-independent manner through transcriptional repression of alternate tumor suppressor pathways. We report here that BMI1 copurifies with DNA DSB response and nonhomologous end joining (NHEJ) repair proteins in GBM cells. BMI1 was enriched at the chromatin after irradiation and colocalized and copurified with ATM and the histone gammaH2AX. BMI1 also preferentially copurified with NHEJ proteins DNA-PK, PARP-1, hnRNP U, and histone H1 in CD133-positive GBM cells. BMI1 deficiency in GBM cells severely impaired DNA DSB response, resulting in increased sensitivity to radiation. In turn, BMI1 overexpression in normal NSCs enhanced ATM recruitment to the chromatin, the rate of gammaH2AX foci resolution, and resistance to radiation. BMI1 thus displays a previously uncharacterized function in controlling DNA DSB response and repair. Pharmacological inhibition of BMI1 combined with radiation therapy may provide an effective mean to target GBM stem cells.

Bmi1 distinguishes immature retinal progenitor/stem cells from the main progenitor cell population and is required for normal retinal development.

The developing mammalian retina is generated by the proliferation and differentiation of multipotent retinal progenitor cells (RPCs) giving rise to neuronal and glial lineages. Whether an immature progenitor/stem cell subpopulation is present in the developing mammalian retina remains undefined. Deficiency in the polycomb group gene Bmi1 results in reduced proliferation and postnatal depletion of neural and hematopoietic stem cells. Here, we show that Bmi1 is required for the self-renewal of most immature RPCs and for postnatal retinal development. In the embryo, Bmi1 is highly enriched in a rare stage-specific embryonic antigen-1-positive RPC subpopulation expressing the stem cell markers Sox2, Lhx2, and Musashi. Gain-of-function experiments revealed that Bmi1 overexpression could convert RPCs having limited proliferation capacity into RPCs showing extensive proliferation and multiple differentiation capacities over time. At all developmental stages analyzed using the neurosphere assay, Bmi1 deficiency resulted in reduced proliferation and self-renewal of most immature RPCs. Reduced RPCs proliferation was also observed in the peripheral retina of Bmi1(-/-) fetus and newborn mice. The biological impact of these developmental anomalies was revealed by the reduced retinal diameter of Bmi1-deficient pups. P19(Arf) and p16(Ink4a) were upregulated in vivo and in vitro and coinactivation of p53, which lies downstream of p19(Arf), partially restored Bmi1-deficient RPCs self-renewal phenotype. Bmi1 thus distinguishes immature RPCs from the main RPC population and is required for normal retinal development.

Oncosuppressor-Mutated Cell-Based Diagnostic Platform for Liquid Biopsy Diagnoses Benign Head and Neck Masses and Predicts Malignancy in Thyroid Nodules: Results from a Consecutive Cohort of Patients.

BACKGROUND: We reported that a novel oncosuppressor-mutated cell (OMC)-based platform has the potential for early cancer detection in healthy individuals and for identification of cancer patients at risk of developing metachronous metastases. OBJECTIVE: Herein, we sought to determine the diagnostic accuracy of this novel OMC-based platform in a consecutive cohort of patients operated for suspicious head and neck masses. METHODS: OMCs (BRCA1-deficient fibroblasts) were exposed to blood serum from patients with head and neck nodules before surgical removal. These cells were analyzed for their proliferation and survival. Treated OMCs were inoculated subcutaneously in NOD/SCID mice, and tumor growth was monitored over time. RESULTS: OMCs exposed to serum from patients with malignant lesions displayed increased proliferation compared to those exposed to serum from patients with benign lesions. Only OMCs exposed to serum from patients diagnosed with malignant thyroid neoplasia generated a cancerous mass. The sensitivity of the test was 92%, with only 1 false negative out of 34 patients. Immunohistochemical staining showed that the cancerous masses were poorly differentiated adenocarcinomas with high proliferative index. CONCLUSIONS: These data show that liquid biopsy combined with an OMC-based in vivo platform has the potential to diagnose benign head and neck masses and predict whether a thyroid nodule is malignant. These results strengthen the concept that OMCs can be used to detect circulating malignant factors in cancer patients.

Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. METHODS: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCß4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). RESULTS: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. CONCLUSIONS: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

The polycomb group gene Bmi1 regulates antioxidant defenses in neurons by repressing p53 pro-oxidant activity.

Aging may be determined by a genetic program and/or by the accumulation rate of molecular damages. Reactive oxygen species (ROS) generated by the mitochondrial metabolism have been postulated to be the central source of molecular damages and imbalance between levels of intracellular ROS and antioxidant defenses is a characteristic of the aging brain. How aging modifies free radicals concentrations and increases the risk to develop most neurodegenerative diseases is poorly understood, however. Here we show that the Polycomb group and oncogene Bmi1 is required in neurons to suppress apoptosis and the induction of a premature aging-like program characterized by reduced antioxidant defenses. Before weaning, Bmi1(-/-) mice display a progeroid-like ocular and brain phenotype, while Bmi1(+/-) mice, although apparently normal, have reduced lifespan. Bmi1 deficiency in neurons results in increased p19(Arf)/p53 levels, abnormally high ROS concentrations, and hypersensitivity to neurotoxic agents. Most Bmi1 functions on neurons' oxidative metabolism are genetically linked to repression of p53 pro-oxidant activity, which also operates in physiological conditions. In Bmi1(-/-) neurons, p53 and corepressors accumulate at antioxidant gene promoters, correlating with a repressed chromatin state and antioxidant gene downregulation. These findings provide a molecular mechanism explaining how Bmi1 regulates free radical concentrations and reveal the biological impact of Bmi1 deficiency on neuronal survival and aging.

BMI1 sustains human glioblastoma multiforme stem cell renewal.

Glioblastoma multiforme (GBM) is one of the most common and aggressive types of brain tumors. In GBM, a subpopulation of CD133-positive cancer initiating cells displays stem cell characteristics. The Polycomb group (PcG) and oncogene BMI1 is part of the Polycomb repressive complex 1 (PRC1) that regulates gene expression by modifying chromatin organization. Here we show that BMI1 is expressed in human GBM tumors and highly enriched in CD133-positive cells. Stable BMI1 knockdown using short hairpin RNA-expressing lentiviruses resulted in inhibition of clonogenic potential in vitro and of brain tumor formation in vivo. Cell biology studies support the notion that BMI1 prevents CD133-positive cell apoptosis and/or differentiation into neurons and astrocytes, depending on the cellular context. Gene expression analyses suggest that BMI1 represses alternate tumor suppressor pathways that attempt to compensate for INK4A/ARF/P53 deletion and PI(3)K/AKT hyperactivity. Inhibition of EZH2, the main component of the PRC2, also impaired GBM tumor growth. Our results reveal that PcG proteins are involved in GBM tumor growth and required to sustain cancer initiating stem cell renewal.

Uveal Melanoma-Derived Extracellular Vesicles Display Transforming Potential and Carry Protein Cargo Involved in Metastatic Niche Preparation.

Extracellular vesicles (EVs) carry molecules derived from donor cells and are able to alter the properties of recipient cells. They are important players during the genesis and progression of tumors. Uveal melanoma (UM) is the most common primary intraocular tumor in adults and is associated with a high rate of metastasis, primarily to the liver. However, the mechanisms underlying this process are poorly understood. In the present study, we analyzed the oncogenic potential of UM-derived EVs and their protein signature. We isolated and characterized EVs from five UM cell lines and from normal choroidal melanocytes (NCMs). BRCA1-deficient fibroblasts (Fibro-BKO) were exposed to the EVs and analyzed for their growth in vitro and their reprograming potential in vivo following inoculation into NOD-SCID mice. Mass spectrometry of proteins from UM-EVs and NCM-EVs was performed to determine a protein signature that could elucidate potential key players in UM progression. In-depth analyses showed the presence of exosomal markers, and proteins involved in cell-cell and focal adhesion, endocytosis, and PI3K-Akt signaling pathway. Notably, we observed high expression levels of HSP90, HSP70 and integrin V in UM-EVs. Our data bring new evidence on the involvement of UM-EVs in cancer progression and metastasis.

Off-target effect of the BMI1 inhibitor PTC596 drives epithelial-mesenchymal transition in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an incurable primary brain tumor containing a sub-population of cancer stem cells (CSCs). Polycomb Repressive Complex (PRC) proteins BMI1 and EZH2 are enriched in CSCs, promoting clonogenic growth and resistance to genotoxic therapies. We report here that when used at appropriate concentrations, pharmaceutical inhibitors of BMI1 could efficiently prevent GBM colony growth and CSC self-renewal in vitro and significantly extend lifespan in terminally ill tumor-bearing mice. Notably, molecular analyses revealed that the commonly used PTC596 molecule targeted both BMI1 and EZH2, possibly providing beneficial therapeutic effects in some contexts. On the other hand, treatment with PTC596 resulted in instant reactivation of EZH2 target genes and induction of a molecular program of epithelial-mesenchymal transition (EMT), possibly explaining the modified phenotype of some PTC596-treated tumors. Treatment with a related but more specific BMI1 inhibitor resulted in tumor regression and maintenance of cell identity. We conclude that inhibition of BMI1 alone is efficient at inducing GBM regression, and that dual inhibition of BMI1 and EZH2 using PTC596 may be also beneficial but only in specific contexts.

Acetylsalicylic Acid Exerts Potent Antitumor and Antiangiogenic Effects in Cutaneous and Uveal Melanoma Cell Lines.

INTRODUCTION: Acetylsalicylic acid (ASA) has been investigated for a potential anticancer role in several cancers, such as colorectal, ovarian, and endometrial cancer. Moreover, ASA has been shown to abrogate various processes that contribute to tumor growth and progression. OBJECTIVE: The aim of this study was to evaluate the effects of ASA on cutaneous melanoma (CM) and uveal melanoma (UM). METHODS: Human CM and UM cells were treated with 5 mM ASA and assessed for changes in cellular functions. Antiangiogenic effects of ASA were determined using an ELISA-based assay for 10 proangiogenic cytokines, and then validated by Western blot. Finally, proteomic analysis of ASA-treated cells was performed to elucidate the changes that may be responsible for ASA-mediated effects in melanoma cells. RESULTS: Treatment with ASA significantly inhibited the proliferation, invasion, and migration capabilities, and caused a significant decrease in angiogenin and PIGF secretion in both CM and UM. Mass spectrometry revealed 179 protein changes associated with ASA in the CM and UM cell lines. CONCLUSIONS: These results suggest that ASA may be effective as an adjuvant therapy in metastatic CM and UM. Future studies are needed to determine the regulating targets that are responsible for the antitumor effects of ASA.

Colorectal cancer-derived extracellular vesicles induce transformation of fibroblasts into colon carcinoma cells.

BACKGROUND: We reported that horizontal transfer of malignant traits to target cells is a potential pathway to explain cancer dissemination. Although these results were encouraging, they were never corroborated by data showing the molecular mechanisms responsible for the observed phenomenon. METHODS: In the present study, we exposed BRCA1-KO fibroblasts to extracellular vesicles (EVs) isolated from a colon cancer cell line (HT29) and from sera of patients with colorectal cancer. Three weeks after exposure, fibroblasts were injected subcutaneously into NOD-SCID mice. Whole genome sequencing, transcriptome analysis and RNA sequencing of cancer EVs and fibroblasts prior and after exposure to cancer EVs were performed. RESULTS: Phenotypical transformation of the fibroblasts into colon cancer cells was confirmed by histopathological study of the xenotransplants. We observed that EV-mediated transfer of cancer microRNAs was responsible for the transition from a mesenchymal to an epithelial phenotype (MET) in the treated fibroblasts as well as activation of cell cycle progression and cell survival pathways. DNA and RNA sequencing suggested that cancer DNA was transferred and possibly transcribed in target cells. Furthermore, injection of colon cancer EVs in the tail vein of NOD-SCID mice determined neoplastic transformation and metastases in the lungs of the mice confirming for the first time the hypothesis that transfer of malignant epithelial cancer traits to distant target cells is a concept applicable to in vivo models. CONCLUSIONS: These discoveries shed new light into the molecular mechanisms behind the horizontal transfer of malignant traits and confirm the notion that metastatic disease might be reproduced through transfer of circulating genetic material.

Oncosuppressor-Mutated Cells as a Liquid Biopsy Test for Cancer-Screening.

We reported on the ability of immortalized or oncosuppressor-mutated cells (OMCs) to uptake circulating cancer-factors and give tumors when transplanted into mice. This led to the first biological based liquid biopsy test, which we called MATER-D platform. In the present study, we showed for the first time that a different type of OMCs (PTEN-deficient human epithelial MCF10A cells) turn malignant when exposed to cancer patient's sera, confirming the concept that different cells with diverse oncosuppressor mutations can uptake cancer factors and be used in biological based liquid biopsy tests. Our observations were confirmed in a large variety of solid and haematological malignancies. This test was able to detect dysplasia and carcinomas in situ lesions in different organs and circulating factors in cancer patients years after the removal of their lesions. To our knowledge, this ability is unique and not shared by other liquid biopsy platforms. Immunohistochemistry analysis of the xenotransplants revealed identical patterns of differentiation regardless of the cancer type, showing that differentiation through horizontal transfer might be dependent on the nature of the target cells rather than the type of cancer factors. These data strengthen the notion that OMC-based liquid biopsy tests might be promising platforms for cancer screening.

Heterochromatic genome instability and neurodegeneration sharing similarities with Alzheimer's disease in old Bmi1+/- mice.

Sporadic Alzheimer's disease (AD) is the most common cause of dementia. However, representative experimental models of AD have remained difficult to produce because of the disease's uncertain origin. The Polycomb group protein BMI1 regulates chromatin compaction and gene silencing. BMI1 expression is abundant in adult brain neurons but down-regulated in AD brains. We show here that mice lacking one allele of Bmi1 (Bmi1+/-) develop normally but present with age cognitive deficits and neurodegeneration sharing similarities with AD. Bmi1+/- mice also transgenic for the amyloid beta precursor protein died prematurely and present aggravated disease. Loss of heterochromatin and DNA damage response (DDR) at repetitive DNA sequences were predominant in Bmi1+/- mouse neurons and inhibition of the DDR mitigated the amyloid and Tau phenotype. Heterochromatin anomalies and DDR at repetitive DNA sequences were also found in AD brains. Aging Bmi1+/- mice may thus represent an interesting model to identify and study novel pathogenic mechanisms related to AD.