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Cerebral cavernous malformations associated with loss of function of Ccm1 are shown to be formed by endothelial cells undergoing endothelial-to-mesenchymal transition (EndMT) induced by TGF-β and BMP signalling; inhibition of TGF-β and BMP signalling prevents EndMT and the appearance of CCM lesions. CCM disease pathology Cerebral cavernous malformation (CCM) is a genetic disease causing lesions of the brain vasculature that can lead to seizures and stroke. Neurosurgery is the only treatment offered today. CCM lesions are caused by loss-of-function mutations in one of three genes: CCM1 , CCM2 and CCM3 . Elisabetta Dejana and colleagues show that lesions associated with loss of function of CCM1 are formed by endothelial cells undergoing endothelial-to-mesenchymal transition (EndMT), induced by activation of transforming growth factor-β (TGF-β) signalling. They show that inhibition of TGF-β signalling prevents EndMT and the appearance of CCM lesions in vivo . Inhibitors of various steps of the TGF-β and bone morphogenetic protein signalling pathways might therefore be candidates to reduce or reverse the development of CCM lesions. Cerebral cavernous malformation (CCM) is a vascular dysplasia, mainly localized within the brain and affecting up to 0.5% of the human population. CCM lesions are formed by enlarged and irregular blood vessels that often result in cerebral haemorrhages. CCM is caused by loss-of-function mutations in one of three genes, namely CCM1 (also known as KRIT1 ), CCM2 ( OSM ) and CCM3 ( PDCD10 ), and occurs in both sporadic and familial forms 1 . Recent studies 2 , 3 , 4 , 5 , 6 , 7 have investigated the cause of vascular dysplasia and fragility in CCM, but the in vivo functions of this ternary complex remain unclear 8 . Postnatal deletion of any of the three Ccm genes in mouse endothelium results in a severe phenotype, characterized by multiple brain vascular malformations that are markedly similar to human CCM lesions 9 . Endothelial-to-mesenchymal transition (EndMT) has been described in different pathologies, and it is defined as the acquisition of mesenchymal- and stem-cell-like characteristics by the endothelium 10 , 11 , 12 . Here we show that endothelial-specific disruption of the Ccm1 gene in mice induces EndMT, which contributes to the development of vascular malformations. EndMT in CCM1-ablated endothelial cells is mediated by the upregulation of endogenous BMP6 that, in turn, activates the transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signalling pathway. Inhibitors of the TGF-β and BMP pathway prevent EndMT both in vitro and in vivo and reduce the number and size of vascular lesions in CCM1-deficient mice. Thus, increased TGF-β and BMP signalling, and the consequent EndMT of CCM1-null endothelial cells, are crucial events in the onset and progression of CCM disease. These studies offer novel therapeutic opportunities for this severe, and so far incurable, pathology.

Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell–selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor–independent stimulation of β-catenin transcription activity in CCM3 -deficient endothelial cells both in vitro and in vivo and a triggering of a β-catenin–driven transcription program that leads to endothelial-to-mesenchymal transition. TGF-β/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate β-catenin transcription activity, reduce vascular malformations in endothelial CCM3 -deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas.

Cerebral cavernous malformations (CCMs) are vascular malformations located within the central nervous system often resulting in cerebral hemorrhage. Pharmacological treatment is needed, since current therapy is limited to neurosurgery. Familial CCM is caused by loss‐of‐function mutations in any of Ccm1 , Ccm2, and Ccm3 genes. CCM cavernomas are lined by endothelial cells (ECs) undergoing endothelial‐to‐mesenchymal transition (EndMT). This switch in phenotype is due to the activation of the transforming growth factor beta/bone morphogenetic protein (TGFβ/BMP) signaling. However, the mechanism linking Ccm gene inactivation and TGFβ/BMP‐dependent EndMT remains undefined. Here, we report that Ccm1 ablation leads to the activation of a MEKK3‐MEK5‐ERK5‐MEF2 signaling axis that induces a strong increase in Kruppel‐like factor 4 (KLF4) in ECs in vivo . KLF4 transcriptional activity is responsible for the EndMT occurring in CCM1‐null ECs. KLF4 promotes TGFβ/BMP signaling through the production of BMP6. Importantly, in endothelial‐specific Ccm1 and Klf4 double knockout mice, we observe a strong reduction in the development of CCM and mouse mortality. Our data unveil KLF4 as a therapeutic target for CCM. Synopsis Current therapy for cerebral cavernous malformation (CCM) therapy is limited to neurosurgery. Transcription factor KLF4 is found to be a crucial determinant for the development of cavernomas and thus a future therapeutic target. KLF4 is strongly upregulated in endothelial cells in the absence of any of the three CCM genes. The endothelial‐to‐mesenchymal transition observed in endothelial cells null for CCM1 is induced by KLF4. KLF4 activates TGFβ/BMP signaling by increasing Bmp6 expression in endothelial cells in the absence of CCM1. The development and progression of cavernomas is strongly reduced upon genetic Klf4 inactivation. KLF4 is a strong candidate as a novel target for the pharmacological treatment of CCM, since its inactivation reduces mouse mortality associated to this disease by 75%. Graphical Abstract Current therapy for cerebral cavernous malformation (CCM) therapy is limited to neurosurgery. Transcription factor KLF4 is found to be a crucial determinant for the development of cavernomas and thus a future therapeutic target.

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

We retrospectively investigated in women treated with fulvestrant for HR+/HER2 negative advanced breast cancer clinical, pathological and molecular features associated with long-term benefit from treatment defined as being progression-free at 18 months. Specifically, we analyzed on formalin-fixed paraffin-embedded tumor samples ESR1 and PI3KCA mutations and miRNAs profiles. 59 patients were evaluable (median age of 67 years, range 32–92). 18-month PFS rate was 27%; the lack of visceral metastases significantly predicted the likelihood of being progression-free at 18 months, while PI3KCA mutations, found in 36% of patients, were not associated with 18-month PFS. As of miRNAs, miR-549a , miR-644a, miR-16-5p were negatively while let-7c-5p was positively associated with 18-month PFS. In addition , miR-520d-3p and miR-548g-3p values were significantly lower while miR-603, miR-181a-5p and miR-199a-miR-199b-3p values were significantly higher in patients achieving 18-month PFS. In silico analysis of targets modulated by these two latter groups of miRNAs show that in patients achieving 18-month PFS the Hippo and Wnt signaling pathways were predicted to be upregulated while endocrine resistance was potentially repressed by miR-603, miR-181a-5p and miR-199a-miR-199b-3p. Our results provide additional clues on the molecular mechanisms involved in fulvestrant activity and resistance. Underlying pathways should be further elucidated and confirmed in larger cohorts.

While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6-mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular-specific therapies.

Neurotrophic tyrosine receptor kinase (NTRK) fusions are infrequent genetic events that can occur in various tumor types. Specifically, NTRK-rearranged sarcoma has been observed in pediatric mesenchymal tumors and, to a lesser extent, in adult mesenchymal tumors like fibrosarcoma. Recently, NTRK-rearranged uterine sarcoma (US) has been identified as a rare entity characterized by constitutive activation or overexpression of the TRK receptor, which plays a role in cell proliferation and differentiation. Since its initial description in 2018, only 46 cases of NTRK-rearranged US have been reported. In this context, herein we describe an exceptional case of an STRN3::NTRK3 fused US with histologically confirmed splenic metastasis. Notably, such localization has not been previously associated with pure uterine sarcomas in the literature. The fusion involved STRN3 (exon-3) and NTRK3 (exon-14) genes and was identified through next-generation sequencing analysis. Recognizing this specific molecular rearrangement is crucial, as it not only enables targeted therapy but also holds diagnostic significance in specific clinical scenarios.

Renal medullary carcinoma (RMC) is a rare entity with poor prognosis bearing inactivating genomic alterations in SMARCB1/INI1 resulting in the loss of expression of INI1 and occurring in young patients with sickle cell trait or sickle cell disease. Recently, rare examples with histological characteristics of RMC have been described in older patients without hemoglobinopathies and provisionally termed “Renal cell carcinoma unclassified with medullary phenotype” (RCCU-MP). Fluorescence in situ Hybridization (FISH) can detect alterations in SMARCB1/INI1 consisting mostly in inactivating translocation of one allele and deletion of the second. To date, only seven further cases of RCCU-MP have been described in the literature. Here we report the second Italian case of RCCU-MP, a 62-year-old man presenting with persistent dull back pain and incidentally discovering a 13 cm mass in the right kidney. The nomenclature of this entity is still debated and might be updated as a variant of medullary carcinoma in the upcoming WHO classification. In the meantime, we encourage awareness of these extraordinarily rare neoplasms with poor outcomes.

VE‐cadherin is an endothelial‐specific transmembrane protein concentrated at cell‐to‐cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE‐cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE‐cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE‐cadherin expression and junctional clustering are required for optimal transforming growth factor‐β (TGF‐β) signalling in endothelial cells (ECs). TGF‐β antiproliferative and antimigratory responses are increased in the presence of VE‐cadherin. ECs lacking VE‐cadherin are less responsive to TGF‐β/ALK1‐ and TGF‐β/ALK5‐induced Smad phosphorylation and target gene transcription. VE‐cadherin coimmunoprecipitates with all the components of the TGF‐β receptor complex, TβRII, ALK1, ALK5 and endoglin. Clustered VE‐cadherin recruits TβRII and may promote TGF‐β signalling by enhancing TβRII/TβRI assembly into an active receptor complex. Taken together, our data indicate that VE‐cadherin is a positive and EC‐specific regulator of TGF‐β signalling. This suggests that reduction or inactivation of VE‐cadherin may contribute to progression of diseases where TGF‐β signalling is impaired.

In recent years, immunohistochemical protein expression was studied as a surrogate to the molecular classification of bladder cancer, although no tissue biomarkers are available for clinical use to predict survival or the response to neoadjuvant chemotherapy (CT) in UC, as the literature produced conflicting results. This retrospective study included TURB specimens harboring foci of HG pT2 muscle-invasive bladder carcinoma (MIBC) from 251 patients who subsequently underwent radical cystectomy. We performed immunohistochemical analysis on tumor samples, for relevant gene-expression-based markers for basal type (CD44, CK5/6) and luminal type (CK20 and pPARγ). Piescore, investigated in both non-muscle-invasive (NMI) and muscle-invasive (MI) components of the tumor, divided basal and luminal UC-types when at least three of the four markers were consistent with a specific phenotype, mixed types if one/two luminal and basal markers were present simultaneously, and neu-like types when all four markers investigated were negative. Eighteen selected cases were also investigated with RT-PCR to validate, and to increase the specificity of, the immunohistochemical results. We observe an immunophenotypical difference in the NMI and MI components in 96/251 UC patients (38.25%): half of tumors (44/96 cases) have a transition to basal, 36.46% (35/96 cases) to neu-like, 12.5% (12/96 cases) to mixed, and 5.2% (5/96 cases) to luminal phenotypes. Mixed tumors in the NMI component are more likely to change phenotype than other groups, particularly compared with basal tumors, which demonstrate greater stability (only 8/96 cases, p < 0.00001). The transition of luminal tumors to basal display a better OS compared with the transition toward neu-like tumors (p = 0.027). Overall, the phenotypical switch does not affect lymphovascular invasion, pT, DFS, or OS compared with non-switched cases. In the MI component, the presence of CD44 expression, irrespective of score-related phenotype, shows a protective effect in papillary-type UC (OS p = 0.008, HR 0.453, PFS p = 0.07, HR 0.599), and in UC naïve for CT (p = 0.0479). Piescore immunophenotyping reveals an intratumoral phenotypical transition between the NMI and MI components of the same tumor. The molecular change is a common event in the mixed and luminal categories, but not in basal tumors, which show better phenotypical stability. This phenomenon could partially explain the sensitivity of a subset of luminal UC to chemotherapy: good responders could be “non-real” luminal UC, which acquire nasal markers, such as CD44.