Revealing the Ferroptotic Phenotype of Medulloblastoma.

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named "ferroptosis." The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic "bubbling" phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

Integrin-αvß3 is a Therapeutically Targetable Fundamental Factor in Medulloblastoma Tumorigenicity and Radioresistance.

Medulloblastoma is one of the most prevalent solid tumors found in children, occurring in the brain's posterior fossa. The standard treatment protocol involves maximal resection surgery followed by craniospinal irradiation and chemotherapy. Despite a long-term survival rate of 70%, wide disparities among patients have been observed. The identification of pertinent targets for both initial and recurrent medulloblastoma cases is imperative. Both primary and recurrent medulloblastoma are marked by their aggressive infiltration into surrounding brain tissue, robust angiogenesis, and resistance to radiotherapy. While the significant role of integrin-αvß3 in driving these characteristics has been extensively documented in glioblastoma, its impact in the context of medulloblastoma remains largely unexplored. Integrin-αvß3 was found to be expressed in a subset of patients with medulloblastoma. We investigated the role of integrin-αvß3 using medulloblastoma-derived cell lines with ß3-subunit depletion or overexpression both in vitro and in vivo settings. By generating radioresistant medulloblastoma cell lines, we uncovered an increased integrin-αvß3 expression, which correlated with increased susceptibility to pharmacologic integrin-αvß3 inhibition with cilengitide, a competitive ligand mimetic. Finally, we conducted single-photon emission computed tomography (SPECT)/MRI studies on orthotopic models using a radiolabeled integrin-αvß3 ligand (99mTc-RAFT-RGD). This innovative approach presents the potential for a novel predictive imaging technique in the realm of medulloblastoma. Altogether, our findings lay the foundation for employing SPECT/MRI to identify a specific subset of patients with medulloblastoma eligible for integrin-αvß3-directed therapies. This breakthrough offers a pathway toward more targeted and effective interventions in the treatment of medulloblastoma. SIGNIFICANCE: This study demonstrates integrin-αvß3's fundamental role in medulloblastoma tumorigenicity and radioresistance and the effect of its expression on cilengitide functional activity.

Recent Pre-Clinical Advancements in Nuclear Medicine: Pioneering the Path to a Limitless Future.

The theranostic approach in oncology holds significant importance in personalized medicine and stands as an exciting field of molecular medicine. Significant achievements have been made in this field in recent decades, particularly in treating neuroendocrine tumors using 177-Lu-radiolabeled somatostatin analogs and, more recently, in addressing prostate cancer through prostate-specific-membrane-antigen targeted radionuclide therapy. The promising clinical results obtained in these indications paved the way for the further development of this approach. With the continuous discovery of new molecular players in tumorigenesis, the development of novel radiopharmaceuticals, and the potential combination of theranostics agents with immunotherapy, nuclear medicine is poised for significant advancements. The strategy of theranostics in oncology can be categorized into (1) repurposing nuclear medicine agents for other indications, (2) improving existing radiopharmaceuticals, and (3) developing new theranostics agents for tumor-specific antigens. In this review, we provide an overview of theranostic development and shed light on its potential integration into combined treatment strategies.

Targeting of the ELR+CXCL/CXCR1/2 Pathway Is a Relevant Strategy for the Treatment of Paediatric Medulloblastomas.

Medulloblastoma (MB) is the most common and aggressive paediatric brain tumour. Although the cure rate can be as high as 70%, current treatments (surgery, radio- and chemotherapy) excessively affect the patients' quality of life. Relapses cannot be controlled by conventional or targeted treatments and are usually fatal. The strong heterogeneity of the disease (four subgroups and several subtypes) is related to innate or acquired resistance to reference treatments. Therefore, more efficient and less-toxic therapies are needed. Here, we demonstrated the efficacy of a novel inhibitor (C29) of CXCR1/2 receptors for ELR+CXCL cytokines for the treatment of childhood MB. The correlation between ELR+CXCL/CXCR1/2 expression and patient survival was determined using the R2: Genomics Analysis and Visualization platform. In vitro efficacy of C29 was evaluated by its ability to inhibit proliferation, migration, invasion, and pseudo-vessel formation of MB cell lines sensitive or resistant to radiotherapy. The growth of experimental MB obtained by MB spheroids on organotypic mouse cerebellar slices was also assayed. ELR+CXCL/CXCR1/2 levels correlated with shorter survival. C29 inhibited proliferation, clone formation, CXCL8/CXCR1/2-dependent migration, invasion, and pseudo-vessel formation by sensitive and radioresistant MB cells. C29 reduced experimental growth of MB in the ex vivo organotypic mouse model and crossed the blood-brain barrier. Targeting CXCR1/2 represents a promising therapeutic strategy for the treatment of paediatric MB in first-line treatment or after relapse following conventional therapy.

Integrin-αvß3 as a Therapeutic Target in Glioblastoma: Back to the Future?

Glioblastoma (GBM), the most common primary malignant brain tumor, is associated with a dismal prognosis. Standard therapies including maximal surgical resection, radiotherapy, and temozolomide chemotherapy remain poorly efficient. Improving GBM treatment modalities is, therefore, a paramount challenge for researchers and clinicians. GBMs exhibit the hallmark feature of aggressive invasion into the surrounding tissue. Among cell surface receptors involved in this process, members of the integrin family are known to be key actors of GBM invasion. Upregulation of integrins was reported in both tumor and stromal cells, making them a suitable target for innovative therapies targeting integrins in GBM patients, as their impairment disrupts tumor cell proliferation and invasive capacities. Among them, integrin-αvß3 expression correlates with high-grade GBM. Driven by a plethora of preclinical biological studies, antagonists of αvß3 rapidly became attractive therapeutic candidates to impair GBM tumorigenesis. In this perspective, the advent of nuclear medicine is currently one of the greatest components of the theranostic concept in both preclinical and clinical research fields. In this review, we provided an overview of αvß3 expression in GBM to emphasize the therapeutic agents developed. Advanced current and future developments in the theranostic field targeting αvß3 are finally discussed.

Antiangiogenic Compound Axitinib Demonstrates Low Toxicity and Antitumoral Effects against Medulloblastoma.

BACKGROUND: Despite the improvement of medulloblastoma (MB) treatments, survivors face severe long-term adverse effects and associated morbidity following multimodal treatments. Moreover, relapses are fatal within a few months. Therefore, chemotherapies inducing fewer adverse effects and/or improving survival at relapse are key for MB patients. Our purpose was to evaluate the last-generation antiangiogenic drugs for their relevance in the therapeutic arsenal of MB. METHODS: We screened three EMA- and FDA-approved antiangiogenic compounds (axitinib, cabozantinib and sunitinib) for their ability to reduce cell viability of five MB cell lines and their low toxicity towards two normal cell lines in vitro. Based on this screening, single-agent and combination therapies were designed for in vivo validation. RESULTS: Axitinib, cabozantinib and sunitinib decreased viability of all the tested tumor cells. Although sunitinib was the most efficient in tumor cells, it also impacted normal cells. Therefore, axitinib showed the highest selectivity index for MB cells as compared to normal cells. The compound did not lead to acute toxicity in juvenile rats and crossed the blood-brain barrier. Moreover, axitinib efficiently reduced the growth rate of experimental brain tumors. Analysis of public databases showed that high expression of axitinib targets correlates with poor prognosis. CONCLUSION: Our results suggest that axitinib is a compelling candidate for MB treatment.

Author Correction: VEGFC negatively regulates the growth and aggressiveness of medulloblastoma cells.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-020-01502-2.

VEGFC negatively regulates the growth and aggressiveness of medulloblastoma cells.

Medulloblastoma (MB), the most common brain pediatric tumor, is a pathology composed of four molecular subgroups. Despite a multimodal treatment, 30% of the patients eventually relapse, with the fatal appearance of metastases within 5 years. The major actors of metastatic dissemination are the lymphatic vessel growth factor, VEGFC, and its receptors/co-receptors. Here, we show that VEGFC is inversely correlated to cell aggressiveness. Indeed, VEGFC decreases MB cell proliferation and migration, and their ability to form pseudo-vessel in vitro. Irradiation resistant-cells, which present high levels of VEGFC, lose the ability to migrate and to form vessel-like structures. Thus, irradiation reduces MB cell aggressiveness via a VEGFC-dependent process. Cells intrinsically or ectopically overexpressing VEGFC and irradiation-resistant cells form smaller experimental tumors in nude mice. Opposite to the common dogma, our results give strong arguments in favor of VEGFC as a negative regulator of MB growth.

Inhibition of the amino-acid transporter LAT1 demonstrates anti-neoplastic activity in medulloblastoma.

Most cases of medulloblastoma (MB) occur in young children. While the overall survival rate can be relatively high, current treatments combining surgery, chemo- and radiotherapy are very destructive for patient development and quality of life. Moreover, aggressive forms and recurrences of MB cannot be controlled by classical therapies. Therefore, new therapeutic approaches yielding good efficacy and low toxicity for healthy tissues are required to improve patient outcome. Cancer cells sustain their proliferation by optimizing their nutrient uptake capacities. The L-type amino acid transporter 1 (LAT1) is an essential amino acid carrier overexpressed in aggressive human cancers that was described as a potential therapeutic target. In this study, we investigated the therapeutic potential of JPH203, a LAT1-specific pharmacological inhibitor, on two independent MB cell lines belonging to subgroups 3 (HD-MB03) and Shh (DAOY). We show that while displaying low toxicity towards normal cerebral cells, JPH203 disrupts AA homeostasis, mTORC1 activity, proliferation and survival in MB cells. Moreover, we demonstrate that a long-term treatment with JPH203 does not lead to resistance in MB cells. Therefore, this study suggests that targeting LAT1 with JPH203 is a promising therapeutic approach for MB treatment.

AKT1 restricts the invasive capacity of head and neck carcinoma cells harboring a constitutively active PI3 kinase activity.

BACKGROUND: In mammals, the AKT/PKB protein kinase family comprises three members (AKT1-3). PI3-Kinase (PI3K), a key oncogene involved in a wide variety of cancers, drives AKT activity. Constitutive activation of the PI3K/AKT pathway has been associated with tumorigenic properties including uncontrolled cell proliferation and survival, angiogenesis, promotion of cellular motility, invasiveness and metastasis. However, AKT1 activity has also been recently shown to repress the invasive properties of breast cancer cells in specific contexts. METHODS: This study used both pharmacological and shRNA approaches to inhibit AKT function, microscopy to characterize the cellular morphology, 3D spheroid models to assess migratory and invasive cellular capacities and a phenotypic screening approach based on electrical properties of the cells. RESULTS: Here we demonstrate that the alternative action of AKT1 on invasive properties of breast cancers can be extended to head and neck carcinomas, which exhibit constitutive activation of the PI3K/AKT pathway. Indeed, inhibition of AKT1 function by shRNA or a specific pharmacological inhibitor resulted in cellular spreading and an invasive phenotype. A phenotypic screening approach based on cellular electrical properties corroborated microscopic observations and provides a foundation for future high-throughput screening studies. This technique further showed that the inhibition of AKT1 signaling is phenocopied by blocking the mTORC1 pathway with rapamycin. CONCLUSION: Our study suggests that the repressive action of PI3K/AKT1 on cellular invasive properties may be a mechanism common to several cancers. Current and future studies involving AKT inhibitors must therefore consider this property to prevent metastases and consequently to improve survival.

Validation of commercial ERK antibodies against the ERK orthologue of the scleractinian coral Stylophora pistillata.

The extracellular signal-regulated protein kinase (ERK) signalling pathway controls key cellular processes, such as cell cycle regulation, cell fate determination and the response to external stressors. Although ERK functions are well studied in a variety of living organisms ranging from yeast to mammals, its functions in corals are still poorly known. The present work aims to give practical tools to study the expression level of ERK protein and the activity of the ERK signalling pathway in corals. The antibody characterisation experiment was performed five times and identical results were obtained. The present study validated the immune-reactivity of commercially available antibodies directed against ERK and its phosphorylated/activated forms on protein extracts of the reef-building coral Stylophora pistillata.

The c-Jun N-terminal kinase prevents oxidative stress induced by UV and thermal stresses in corals and human cells.

Coral reefs are of major ecological and socio-economic interest. They are threatened by global warming and natural pressures such as solar ultraviolet radiation. While great efforts have been made to understand the physiological response of corals to these stresses, the signalling pathways involved in the immediate cellular response exhibited by corals remain largely unknown. Here, we demonstrate that c-Jun N-terminal kinase (JNK) activation is involved in the early response of corals to thermal and UV stress. Furthermore, we found that JNK activity is required to repress stress-induced reactive oxygen species (ROS) accumulation in both the coral Stylophora pistillata and human skin cells. We also show that inhibiting JNK activation under stress conditions leads to ROS accumulation, subsequent coral bleaching and cell death. Taken together, our results suggest that an ancestral response, involving the JNK pathway, is remarkably conserved from corals to human, protecting cells from the adverse environmental effects.

ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2.

Telomere stability is a hallmark of immortalized cells, including cancer cells. While the telomere length is maintained in most cases by the telomerase, the activity of a protein complex called Shelterin is required to protect telomeres against unsuitable activation of the DNA damage response pathway. Within this complex, telomeric repeat binding factor 2 (TRF2) plays an essential role by blocking the ataxia telangiectasia-mutated protein (ATM) signaling pathway at telomeres and preventing chromosome end fusion. We showed that TRF2 was phosphorylated in vitro and in vivo on serine 323 by extracellular signal-regulated kinase (ERK1/2) in both normal and cancer cells. Moreover, TRF2 and activated ERK1/2 unexpectedly interacted in the cytoplasm of tumor cells and human tumor tissues. The expression of non-phosphorylatable forms of TRF2 in melanoma cells induced the DNA damage response, leading to growth arrest and tumor reversion. These findings revealed that the telomere stability is under direct control of one of the major pro-oncogenic signaling pathways (RAS/RAF/MEK/ERK) via TRF2 phosphorylation.

Telomeric repeat-binding factor 2: a marker for survival and anti-EGFR efficacy in oral carcinoma.

Oral Squamous Cell Carcinoma (OSCC) is the most common oral cancer worldwide. Treatments including surgery, radio- and chemo-therapies mostly result in debilitating side effects. Thus, a more accurate evaluation of patients at risk of recurrence after radio/chemo treatment is important for preserving their quality of life. We assessed whether the Telomeric Repeat-binding Factor 2 (TERF2) influences tumor aggressiveness and treatment response. TERF2 is over-expressed in many cancers but its correlation to patient outcome remains controversial in OSCC. Our retrospective study on sixty-two patients showed that TERF2 overexpression has a negative impact on survival time. TERF2-dependent survival time was independent of tumor size in a multivariate analysis. In vitro, TERF2 knockdown by RNA interference had no effect on cell proliferation, migration, senescence and apoptosis. Instead, TERF2 knockdown increased the expression of cytokines implicated in inflammation and angiogenesis, except for vascular endothelial growth factor. TERF2 knockdown resulted in a decrease vascularization and growth of xenograft tumors. Finally, response to erlotinib/Tarceva and cetuximab/Erbitux treatment was increased in TRF2 knocked-down cells. Hence, TERF2 may represent an independent marker of survival for OSCC and a predictive marker for cetuximab/Erbitux and erlotinib/Tarceva efficacy.

Linking JNK Activity to the DNA Damage Response.

The activity of c-Jun N-terminal kinase (JNK) was initially described as ultraviolet- and oncogene-induced kinase activity on c-Jun. Shortly after this initial discovery, JNK activation was reported for a wider variety of DNA-damaging agents, including γ-irradiation and chemotherapeutic compounds. As the DNA damage response mechanisms were progressively uncovered, the mechanisms governing the activation of JNK upon genotoxic stresses became better understood. In particular, a recent set of papers links the physical breakage in DNA, the activation of the transcription factor NF-κB, the secretion of TNF-α, and an autocrine activation of the JNK pathway. In this review, we will focus on the pathway that is initiated by a physical break in the DNA helix, leading to JNK activation and the resultant cellular consequences. The implications of these findings will be discussed in the context of cancer therapy with DNA-damaging agents.

p120RasGAP mediates ephrin/Eph-dependent attenuation of FGF/ERK signals during cell fate specification in ascidian embryos.

ERK1/2 MAP kinase exhibits a highly dynamic activation pattern in developing embryos, which largely depends on fibroblast growth factor (FGF) signals. In ascidian embryos, FGF-dependent activation of ERK1/2 occurs differentially between sister cells during marginal zone and neural lineage patterning. Selective attenuation of FGF signals by localised ephrin/Eph signals accounts for this differential ERK activation, which controls the binary fate choice of each sibling cell pair. Here, we show that p120 Ras GTPase-activating protein (p120RasGAP) is a crucial mediator of these ephrin/Eph signals. First, inhibition of p120RasGAP has a similar effect to inhibition of ephrin/Eph function during marginal zone and neural patterning. Second, p120RasGAP acts epistatically to ephrin/Eph signals. Third, p120RasGAP physically associates with Eph3 in an ephrin-dependent manner. This study provides the first in vivo evidence that the functional association between Eph and RasGAP controls the spatial extent of FGF-activated ERK.

TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells.

Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increased levels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. We demonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells. Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results, a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer. By screening for TRF2-bound genes, we found that HS3ST4--a gene encoding for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4--was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2. Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.

Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos.

Asymmetric cell divisions produce two sibling cells with distinct fates, providing an important means of generating cell diversity in developing embryos. Many examples of such cell divisions have been described, but so far only a limited number of the underlying mechanisms have been elucidated. Here, we have uncovered a novel mechanism controlling an asymmetric cell division in the ascidian embryo. This division produces one notochord and one neural precursor. Differential activation of extracellular-signal-regulated kinase (ERK) between the sibling cells determines their distinct fates, with ERK activation promoting notochord fate. We first demonstrate that the segregation of notochord and neural fates is an autonomous property of the mother cell and that the mother cell acquires this functional polarity via interactions with neighbouring ectoderm precursors. We show that these cellular interactions are mediated by the ephrin-Eph signalling system, previously implicated in controlling cell movement and adhesion. Disruption of contacts with the signalling cells or inhibition of the ephrin-Eph signal results in the symmetric division of the mother cell, generating two notochord precursors. Finally, we demonstrate that the ephrin-Eph signal acts via attenuation of ERK activation in the neural-fated daughter cell. We propose a model whereby directional ephrin-Eph signals functionally polarise the notochord/neural mother cell, leading to asymmetric modulation of the FGF-Ras-ERK pathway between the daughter cells and, thus, to their differential fate specification.