BMI1 induces an invasive signature in melanoma that promotes metastasis and chemoresistance.

Melanoma can switch between proliferative and invasive states, which have identifying gene expression signatures that correlate with good and poor prognosis, respectively. However, the mechanisms controlling these signatures are poorly understood. In this study, we identify BMI1 as a key determinant of melanoma metastasis by which its overexpression enhanced and its deletion impaired dissemination. Remarkably, in this tumor type, BMI1 had no effect on proliferation or primary tumor growth but enhanced every step of the metastatic cascade. Consistent with the broad spectrum of effects, BMI1 activated widespread gene expression changes, which are characteristic of melanoma progression and also chemoresistance. Accordingly, we showed that up-regulation or down-regulation of BMI1 induced resistance or sensitivity to BRAF inhibitor treatment and that induction of noncanonical Wnt by BMI1 is required for this resistance. Finally, we showed that our BMI1-induced gene signature encompasses all of the hallmarks of the previously described melanoma invasive signature. Moreover, our signature is predictive of poor prognosis in human melanoma and is able to identify primary tumors that are likely to become metastatic. These data yield key insights into melanoma biology and establish BMI1 as a compelling drug target whose inhibition would suppress both metastasis and chemoresistance of melanoma.

Targeting the cyclin-dependent kinase 5 in metastatic melanoma.

The cyclin-dependent kinase 5 (CDK5), originally described as a neuronal-specific kinase, is also frequently activated in human cancers. Using conditional CDK5 knockout mice and a mouse model of highly metastatic melanoma, we found that CDK5 is dispensable for the growth of primary tumors. However, we observed that ablation of CDK5 completely abrogated the metastasis, revealing that CDK5 is essential for the metastatic spread. In mouse and human melanoma cells CDK5 promotes cell invasiveness by directly phosphorylating an intermediate filament protein, vimentin, thereby inhibiting assembly of vimentin filaments. Chemical inhibition of CDK5 blocks the metastatic spread of patient-derived melanomas in patient-derived xenograft (PDX) mouse models. Hence, inhibition of CDK5 might represent a very potent therapeutic strategy to impede the metastatic dissemination of malignant cells.

Integration of Multiple Platforms for the Analysis of Multifluorescent Marking Technology Applied to Pediatric GBM and DIPG.

The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.

Cost-Effective Technologies to Study the Arctic Ocean Environment †.

The Arctic region is known to be severely affected by climate change, with evident alterations in both physical and biological processes. Monitoring the Arctic Ocean ecosystem is key to understanding the impact of natural and human-induced change on the environment. Large data sets are required to monitor the Arctic marine ecosystem and validate high-resolution satellite observations (e.g., Sentinel), which are necessary to feed climatic and biogeochemical forecasting models. However, the Global Observing System needs to complete its geographic coverage, particularly for the harsh, extreme environment of the Arctic Region. In this scenario, autonomous systems are proving to be valuable tools for increasing the resolution of existing data. To this end, a low-cost, miniaturized and flexible probe, ArLoC (Arctic Low-Cost probe), was designed, built and installed on an innovative unmanned marine vehicle, the PROTEUS (Portable RObotic TEchnology for Unmanned Surveys), during a preliminary scientific campaign in the Svalbard Archipelago within the UVASS project. This study outlines the instrumentation used and its design features, its preliminary integration on PROTEUS and its test results.

Morgana acts as an oncosuppressor in chronic myeloid leukemia.

We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana (+/-) mice spontaneously develop a lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph(+) CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph(+) CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph(+) CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, can overcome suboptimal responses in patients in which morgana is underexpressed.

Morgana acts as a proto-oncogene through inhibition of a ROCK-PTEN pathway.

Morgana/CHP-1 is a ubiquitously expressed protein able to inhibit ROCK II kinase activity. We have previously demonstrated that morgana haploinsufficiency leads to multiple centrosomes, genomic instability, and higher susceptibility to tumour development. While a large fraction of human cancers has shown morgana down-regulation, a small subset of tumours was shown to express high morgana levels. Here we demonstrate that high morgana expression in different breast cancer subtypes correlates with high tumour grade, mitosis number, and lymph node positivity. Moreover, morgana overexpression induces transformation in NIH-3T3 cells and strongly protects them from various apoptotic stimuli. From a mechanistic point of view, we demonstrate that morgana causes PTEN destabilization, by inhibiting ROCK activity, hence triggering the PI3K/AKT survival pathway. In turn, morgana down-regulation in breast cancer cells that express high morgana levels increases PTEN expression and leads to sensitization of cells to chemotherapy.

Elevated neck circumference and associated factors in adolescents.

BACKGROUND: Upper body subcutaneous fat, estimated by neck circumference (NC), may present greater metabolic risk than visceral fat. The aim of this study was to determine cutoff values for NC in adolescents that identify overweight and obesity, the prevalence of elevated NC, and its association with associated factors. METHODS: Cross-sectional study with adolescents from public schools in São Paulo. Anthropometric variables, blood pressure and pubertal stage were collected. Cutoff values for NC were determined by Receiver Operating Characteristic curves. A binary logistic regression was used to determine relationships between NC and associated factors. RESULTS: Among 1668 adolescents studied, 54.92% were female. The cutoff values of NC in girls and boys that identified overweight were 31.25 and 34.25 cm, and obesity, 32.65 and 37.95 cm, respectively, and the prevalence of adolescents with high NC was 32.63% in females and 37.63% among males. NC for overweight was observed that there was an association with sex, weight, body mass index, arm, waist and thigh circumferences, pubertal stages and body fat percent (BF%). NC for obesity was found association with gender, weight, arm and thigh circumferences, and BF% (p < 0.001). CONCLUSION: It was concluded that there is high prevalence of elevated NC and higher risks for this outcome considering overweight and obesity, sex, weight, arm and thigh circumferences, BF%, besides being an easy and simple measure for use in clinical practice.

Cyclin D3 critically regulates the balance between self-renewal and differentiation in skeletal muscle stem cells.

Satellite cells are mitotically quiescent myogenic stem cells resident beneath the basal lamina surrounding adult muscle myofibers. In response to injury, multiple extrinsic signals drive the entry of satellite cells into the cell cycle and then to proliferation, differentiation, and self-renewal of their downstream progeny. Because satellite cells must endure for a lifetime, their cell cycle activity must be carefully controlled to coordinate proliferative expansion and self-renewal with the onset of the differentiation program. In this study, we find that cyclin D3, a member of the family of mitogen-activated D-type cyclins, is critically required for proper developmental progression of myogenic progenitors. Using a cyclin D3-knockout mouse we determined that cyclin D3 deficiency leads to reduced myofiber size and impaired establishment of the satellite cell population within the adult muscle. Cyclin D3-null myogenic progenitors, studied ex vivo on isolated myofibers and in vitro, displayed impaired cell cycle progression, increased differentiation potential, and reduced self-renewal capability. Similarly, silencing of cyclin D3 in C2 myoblasts caused anticipated exit from the cell cycle and precocious onset of terminal differentiation. After induced muscle damage, cyclin D3-null myogenic progenitors exhibited proliferation deficits, a precocious ability to form newly generated myofibers and a reduced capability to repopulate the satellite cell niche at later stages of the regeneration process. These results indicate that cyclin D3 plays a cell-autonomous and nonredundant function in regulating the dynamic balance between proliferation, differentiation, and self-renewal that normally establishes an appropriate pool size of adult satellite cells.

Neck Circumference for Identification of Excess Adiposity in Adolescents with Cancer: A Better Parameter for Use in Clinical Practice?

Purpose: Excess weight in adolescents with cancer during treatment does affect cancer outcomes. Neck circumference (NC), an easygoing anthropometric measure, may present greater metabolic risk, and is associated with excess adiposity. The aim of this study was to identify the prevalence of elevated NC in adolescents with cancer and associated factors. Methods: Cross-sectional study with adolescents aged 10-19 years, under antineoplastic treatment, evaluated from 2015 to 2017, at a Pediatric Oncology Institute's outpatient clinic. Anthropometric parameters were collected, besides diagnosis, sex, and age. The classification of elevated NC was carried out considering cutoff values for adolescents, according to sex and age group. A binary logistic regression was used to determine relationships between NC and associated factors. Results: Among 496 eligible cases, most were male (n = 299, 60.3%). A total of 31.9% of cases had high NC. There is significant and moderate correlation between skinfold thickness (TS) and NC (ρ = 0.6; p = 0.000), and a significant but weak correlation between TS and body mass index (ρ = 0.267; p = 0.000). The adjusted analysis for sex, age group, and type of tumor showed that females are more likely to belong to the high NC category, to have excess adiposity. The age group between 10 and 12 years was the most associated with this outcome (2.795 [0.979-7.977]; p < 0.05). TS is also associated with high NC (1.114 [1.050-1.182]; p < 0.05). Conclusion: It was concluded that there is high prevalence of elevated NC and higher risks for this outcome considering type of tumor, sex, age group, besides being an easy and simple measure for use in clinical practice.

BMI1 is required for melanocyte stem cell maintenance and hair pigmentation.

The epigenetic repressor BMI1 plays an integral role in promoting the self-renewal and proliferation of many adult stem cell populations, and also tumor types, primarily through silencing the Cdkn2a locus, which encodes the tumor suppressors p16Ink4a and p19Arf . However, in cutaneous melanoma, BMI1 drives epithelial-mesenchymal transition programs, and thus metastasis, while having little impact on proliferation or primary tumor growth. This raised questions about the requirement and role for BMI1 in melanocyte stem cell (McSC) biology. Here, we demonstrate that murine melanocyte-specific Bmi1 deletion causes premature hair greying and gradual loss of melanocyte lineage cells. Depilation enhances this hair greying defect, accelerating depletion of McSCs in early hair cycles, suggesting that BMI1 acts to protect McSCs against stress. RNA-seq of McSCs, harvested before onset of detectable phenotypic defects, revealed that Bmi1 deletion derepresses p16Ink4a and p19Arf , as observed in many other stem cell contexts. Additionally, BMI1 loss downregulated the glutathione S-transferase enzymes, Gsta1 and Gsta2, which can suppress oxidative stress. Accordingly, treatment with the antioxidant N-acetyl cysteine (NAC) partially rescued melanocyte expansion. Together, our data establish a critical function for BMI1 in McSC maintenance that reflects a partial role for suppression of oxidative stress, and likely transcriptional repression of Cdkn2a.

A framework for FAIR robotic datasets.

It is essential to publish and make available environmental data gathered by emerging robotic platforms to contribute to the Global Ocean Observing System (GOOS), supported by the United Nations - Decade of Ocean Science for Sustainable Development (2021-2030). The transparency of these unique observational datasets needs to be supported by the corresponding robotic records. The data describing the observational platform behaviour and its performance are necessary to validate the environmental data and repeat consistently the in-situ robotic deployment. The Free and Open Source Software (FOSS), proposed in this manuscript, describes how, using the established approach in Earth Sciences, the data characterising marine robotic missions can be formatted and shared following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. The manuscript is a step-by-step guide to render marine robotic telemetry FAIR and publishable. State-of-the-art protocols for metadata and data formatting are proposed, applied and integrated automatically using Jupyter Notebooks to maximise visibility and ease of use. The method outlined here aims to be a first fundamental step towards FAIR interdisciplinary observational science.

Inhibition of exosome biogenesis affects cell motility in heterogeneous sub-populations of paediatric-type diffuse high-grade gliomas.

BACKGROUND: Paediatric-type diffuse High-Grade Gliomas (PDHGG) are highly heterogeneous tumours which include distinct cell sub-populations co-existing within the same tumour mass. We have previously shown that primary patient-derived and optical barcoded single-cell-derived clones function as interconnected networks. Here, we investigated the role of exosomes as a route for inter-clonal communication mediating PDHGG migration and invasion. RESULTS: A comprehensive characterisation of seven optical barcoded single-cell-derived clones obtained from two patient-derived cell lines was performed. These analyses highlighted extensive intra-tumour heterogeneity in terms of genetic and transcriptional profiles between clones as well as marked phenotypic differences including distinctive motility patterns. Live single-cell tracking analysis of 3D migration and invasion assays showed that the single-cell-derived clones display a higher speed and longer travelled distance when in co-culture compared to mono-culture conditions. To determine the role of exosomes in PDHGG inter-clonal cross-talks, we isolated exosomes released by different clones and characterised them in terms of marker expression, size and concentration. We demonstrated that exosomes are actively internalized by the cells and that the inhibition of their biogenesis, using the phospholipase inhibitor GW4689, significantly reduced the cell motility in mono-culture and more prominently when the cells from the clones were in co-culture. Analysis of the exosomal miRNAs, performed with a miRNome PCR panel, identified clone-specific miRNAs and a set of miRNA target genes involved in the regulation of cell motility/invasion/migration. These genes were found differentially expressed in co-culture versus mono-culture conditions and their expression levels were significantly modulated upon inhibition of exosome biogenesis. CONCLUSIONS: In conclusion, our study highlights for the first time a key role for exosomes in the inter-clonal communication in PDHGG and suggests that interfering with the exosome biogenesis pathway may be a valuable strategy to inhibit cell motility and dissemination for these specific diseases.

MCT4 blockade increases the efficacy of immune checkpoint blockade.

BACKGROUND & AIMS: Intratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be prognostic for the survival of patients with rectal cancer, indicating that single MCT4 blockade might be a promising strategy to overcome glycolysis-related therapy resistance. METHODS: To determine whether blockade of MCT4 alone is sufficient to improve the efficacy of immune checkpoint blockade (ICB) therapy, we examined the effects of the selective MCT1 inhibitor AZD3965 and a novel MCT4 inhibitor in a colorectal carcinoma (CRC) tumor spheroid model co-cultured with blood leukocytes in vitro and the MC38 murine CRC model in vivo in combination with an antibody against programmed cell death ligand-1(PD-L1). RESULTS: Inhibition of MCT4 was sufficient to reduce lactate efflux in three-dimensional (3D) CRC spheroids but not in two-dimensional cell-cultures. Co-administration of the MCT4 inhibitor and ICB augmented immune cell infiltration, T-cell function and decreased CRC spheroid viability in a 3D co-culture model of human CRC spheroids with blood leukocytes. Accordingly, combination of MCT4 and ICB increased intratumoral pH, improved leukocyte infiltration and T-cell activation, delayed tumor growth, and prolonged survival in vivo. MCT1 inhibition exerted no further beneficial impact. CONCLUSIONS: These findings demonstrate that single MCT4 inhibition represents a novel therapeutic approach to reverse lactic-acid driven immunosuppression and might be suitable to improve ICB efficacy.

Dual IGF1R/IR inhibitors in combination with GD2-CAR T-cells display a potent anti-tumor activity in diffuse midline glioma H3K27M-mutant.

BACKGROUND: Diffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy. METHODS: Immunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function. RESULTS: GD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo. CONCLUSION: Our study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.

Morgana/CHP-1 is a novel chaperone able to protect cells from stress.

Morgana/CHP-1 (CHORD containing protein-1) has been recently shown to be necessary for proper cell divisions. However, the presence of the protein in postmitotic tissues such as brain and striated muscle suggests that morgana/CHP-1 has additional cellular functions. Here we show that morgana/CHP-1 behaves like an HSP90 co-chaperone and possesses an independent molecular chaperone activity towards denatured proteins. The expression time profile of morgana/Chp-1 in NIH3T3 cells in response to heat stress is similar to that of Hsp70, a classical effector of Heat Shock Factor-1 mediated stress response. Moreover, overexpression of morgana/CHP-1 in NIH3T3 cells leads to the increased stress resistance of the cells. Interestingly, morgana/Chp-1 upregulation in response to transient global brain ischemia lasts longer in ischemia-resistant regions of the gerbil hippocampus than in vulnerable ones, suggesting the involvement of morgana/CHP-1 in natural protective mechanisms in vivo.

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

Tumor cell invasion into Matrigel: optimized protocol for RNA extraction.

3D models are increasingly used to study mechanisms driving tumor progression and mimicking in vitro processes such as invasion and migration. However, there is a need to establish more protocols based on 3D culture systems that allow for downstream molecular biology investigations. Materials & methods: Here we present a method for optimal RNA extraction from highly aggressive primary glioma cells invading into Matrigel. The method has been established by comparing previously reported protocols, available commercial kits and optimizing specific steps for matrix dissociation, RNA separation and purification. Results and conclusion: The protocol allows RNA extraction from cells embedded into Matrigel, with optimal yield, purity and integrity suitable for subsequent sequencing analysis of both high and low molecular weight RNA.

Discovery of 5-{2-[5-Chloro-2-(5-ethoxyquinoline-8-sulfonamido)phenyl]ethynyl}-4-methoxypyridine-2-carboxylic Acid, a Highly Selective in Vivo Useable Chemical Probe to Dissect MCT4 Biology.

Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.

Remodeling of striatal NMDA receptors by chronic A(2A) receptor blockade in Huntington's disease mice.

Excitotoxicity plays a major role in the pathogenesis of Huntington disease (HD), a fatal neurodegenerative disorder. Adenosine A(2A) receptors (A(2A)Rs) modulate excitotoxicity and have been suggested to play a pathogenetic role in HD. The main aim of this study was to evaluate the effect of A(2A)R blockade on the expression and functions of NMDA receptors in the striatum of HD mice (R6/2). We found that 3 weeks' treatment with SCH 58261 (0.01 mg/kg/day i.p. from the 8th week of age) modified NR1 and NR2A/NR2B expression in the striatum of R6/2 (Western blotting) while had no effect on NMDA-induced toxicity in corticostriatal slices (electrophysiological experiments). In conclusion, in vivo A(2A)R blockade induced a remodeling of NMDA receptors in the striatum of HD mice. Even though the functional relevance of the above effect remains to be fully elucidated, these results add further evidence to the modulatory role of A(2A)Rs in HD.

The mammalian CHORD-containing protein melusin is a stress response protein interacting with Hsp90 and Sgt1.

Melusin is a mammalian muscle specific CHORD containing protein capable of activating signal transduction pathways leading to cardiomyocytes hypertrophy in response to mechanical stress. To define melusin function we searched for molecular partners possibly involved in melusin dependent signal transduction. Here we show that melusin and heat shock proteins are co-regulated. Moreover, melusin directly binds to Hsp90, a ubiquitous chaperone involved in regulating several signaling pathways. In addition, melusin interacts with Sgt1, an Hsp90 binding molecule. Melusin does not behave as an Hsp90 substrate but rather as a chaperone capable to protect citrate synthase from heat induced aggregation. These results describe melusin as a new component of the Hsp90 chaperone machinery.