Tumor microenvironment-induced FOXM1 regulates ovarian cancer stemness.

In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.

Patient-Derived In Vitro Models of Ovarian Cancer: Powerful Tools to Explore the Biology of the Disease and Develop Personalized Treatments.

Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy worldwide due to a late diagnosis caused by the lack of specific symptoms and rapid dissemination into the peritoneal cavity. The standard of care for OC treatment is surgical cytoreduction followed by platinum-based chemotherapy. While a response to this frontline treatment is common, most patients undergo relapse within 2 years and frequently develop a chemoresistant disease that has become unresponsive to standard treatments. Moreover, also due to the lack of actionable mutations, very few alternative therapeutic strategies have been designed as yet for the treatment of recurrent OC. This dismal clinical perspective raises the need for pre-clinical models that faithfully recapitulate the original disease and therefore offer suitable tools to design novel therapeutic approaches. In this regard, patient-derived models are endowed with high translational relevance, as they can better capture specific aspects of OC such as (i) the high inter- and intra-tumor heterogeneity, (ii) the role of cancer stem cells (a small subset of tumor cells endowed with tumor-initiating ability, which can sustain tumor spreading, recurrence and chemoresistance), and (iii) the involvement of the tumor microenvironment, which interacts with tumor cells and modulates their behavior. This review describes the different in vitro patient-derived models that have been developed in recent years in the field of OC research, focusing on their ability to recapitulate specific features of this disease. We also discuss the possibilities of leveraging such models as personalized platforms to design new therapeutic approaches and guide clinical decisions.

Rational design, synthesis, and pharmacological evaluation of a cohort of novel beta-adrenergic receptors ligands enables an assessment of structure-activity relationships.

Biomedical applications of molecules that are able to modulate ß-adrenergic signaling have become increasingly attractive over the last decade, revealing that ß-adrenergic receptors (ß-ARs) are key targets for a plethora of therapeutic interventions, including cancer. Despite successes in ß-AR drug discovery, identification of ß-AR ligands that are useful as selective chemical tools in pharmacological studies of the three ß-AR subtypes, or lead compounds for drug development is still a highly challenging task. This is mainly due to the intrinsic plasticity of ß-ARs as G protein-coupled receptors in conjunction with the requirement for functional receptor subtype selectivity, tissue specificity and minimal off-target effects. With the aim to provide insight into structure-activity relationships for the three ß-AR subtypes, we have synthesized and obtained the pharmacological profile of a series of structurally diverse compounds (named MC) that were designed based on the aryloxy-propanolamine scaffold of SR59230A. Comparative analysis of their predicted binding mode within the active and inactive states of the receptors in combination with their pharmacological profile revealed key structural elements that control their activity as agonists or antagonists, in addition to clues about substituents that mediate selectivity for one receptor subtype over the others. We anticipate that these results will facilitate selective ß-AR drug development efforts.

Development of a Multiprotein Classifier for the Detection of Early Stage Ovarian Cancer.

BACKGROUND: Individual serum biomarkers are neither adequately sensitive nor specific for use in screening the general population for ovarian cancer. The purpose of this study was to develop a multiprotein classifier to detect the early stages of ovarian cancer, when it is most treatable. METHODS: The Olink Proseek Multiplex Oncology II panel was used to simultaneously quantify the expression levels of 92 cancer-related proteins in sera. RESULTS: In the discovery phase, we generated a multiprotein classifier that included CA125, HE4, ITGAV, and SEZ6L, based on an analysis of sera from 116 women with early stage ovarian cancer and 336 age-matched healthy women. CA125 alone achieved a sensitivity of 87.9% at a specificity of 95%, while the multiprotein classifier resulted in an increased sensitivity of 91.4%, while holding the specificity fixed at 95%. The performance of the multiprotein classifier was validated in a second cohort comprised of 192 women with early stage ovarian cancer and 467 age-matched healthy women. The sensitivity at 95% specificity increased from 74.5% (CA125 alone) to 79.2% with the multiprotein classifier. In addition, the multiprotein classifier had a sensitivity of 95.1% at 98% specificity for late stage ovarian cancer samples and correctly classified 80.5% of the benign samples using the 98% specificity cutpoint. CONCLUSIONS: The inclusion of the proteins HE4, ITGAV, and SEZ6L improved the sensitivity and specificity of CA125 alone for the detection of early stages of ovarian cancer in serum samples. Furthermore, we identified several proteins that may be novel biomarkers of early stage ovarian cancer.

Antagonistic L1 Adhesion Molecule Mimetic Compounds Inhibit Glioblastoma Cell Migration In Vitro.

Cell adhesion molecule L1 is a cell surface glycoprotein that promotes neuronal cell migration, fosters regeneration after spinal cord injury and ameliorates the consequences of neuronal degeneration in mouse and zebrafish models. Counter-indicative features of L1 were found in tumor progression: the more L1 is expressed, the more tumor cells migrate and increase their metastatic potential. L1's metastatic potential is further evidenced by its promotion of epithelial-mesenchymal transition, endothelial cell transcytosis and resistance to chemo- and radiotherapy. These unfortunate features are indicated by observations that cells that normally do not express L1 are induced to express it when becoming malignant. With the aim to ameliorate the devastating functions of L1 in tumors, we designed an alternative approach to counteract tumor cell migration. Libraries of small organic compounds were screened using the ELISA competition approach similar to the one that we used for identifying L1 agonistic mimetics. Whereas in the former approach, a function-triggering monoclonal antibody was used for screening libraries, we here used the function-inhibiting monoclonal antibody 324 that reduces the migration of neurons. We now show that the L1 antagonistic mimetics anagrelide, 2-hydroxy-5-fluoropyrimidine and mestranol inhibit the migration of cultured tumor cells in an L1-dependent manner, raising hopes for therapy.

Integrated molecular profiling of patient-derived ovarian cancer models identifies clinically relevant signatures and tumor vulnerabilities.

High-grade serous ovarian carcinoma (HGSOC) is a highly aggressive and intractable neoplasm, mainly because of its rapid dissemination into the abdominal cavity, a process that is favored by tumor-associated peritoneal ascites. The precise molecular alterations involved in HGSOC onset and progression remain largely unknown due to the high biological and genetic heterogeneity of this tumor. We established a set of different tumor samples (termed the As11-set) derived from a single HGSOC patient, consisting of peritoneal ascites, primary tumor cells, ovarian cancer stem cells (OCSC) and serially propagated tumor xenografts. The As11-set was subjected to an integrated RNA-seq and DNA-seq analysis which unveiled molecular alterations that marked the different types of samples. Our profiling strategy yielded a panel of signatures relevant in HGSOC and in OCSC biology. When such signatures were used to interrogate the TCGA dataset from HGSOC patients, they exhibited prognostic and predictive power. The molecular alterations also identified potential vulnerabilities associated with OCSC, which were then tested functionally in stemness-related assays. As a proof of concept, we defined PI3K signaling as a novel druggable target in OCSC.

Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer.

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients' metastatic ascites, establishing the conditions for propagating them as 3D cultures that we refer to as single cell-derived metastatic ovarian cancer spheroids (sMOCS). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that sMOCS retain and amplify key subpopulations from the original patients' samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients' specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method provides a powerful instrument for precision oncology in ovarian cancer.

VEGF pathway inhibition potentiates PARP inhibitor efficacy in ovarian cancer independent of BRCA status.

Poly ADP-ribose polymerase inhibitors (PARPi) have transformed ovarian cancer (OC) treatment, primarily for tumours deficient in homologous recombination repair. Combining VEGF-signalling inhibitors with PARPi has enhanced clinical benefit in OC. To study drivers of efficacy when combining PARP inhibition and VEGF-signalling, a cohort of patient-derived ovarian cancer xenografts (OC-PDXs), representative of the molecular characteristics and drug sensitivity of patient tumours, were treated with the PARPi olaparib and the VEGFR inhibitor cediranib at clinically relevant doses. The combination showed broad anti-tumour activity, reducing growth of all OC-PDXs, regardless of the homologous recombination repair (HRR) mutational status, with greater additive combination benefit in tumours poorly sensitive to platinum and olaparib. In orthotopic models, the combined treatment reduced tumour dissemination in the peritoneal cavity and prolonged survival. Enhanced combination benefit was independent of tumour cell expression of receptor tyrosine kinases targeted by cediranib, and not associated with change in expression of genes associated with DNA repair machinery. However, the combination of cediranib with olaparib was effective in reducing tumour vasculature in all the OC-PDXs. Collectively our data suggest that olaparib and cediranib act through complementary mechanisms affecting tumour cells and tumour microenvironment, respectively. This detailed analysis of the combined effect of VEGF-signalling and PARP inhibitors in OC-PDXs suggest that despite broad activity, there is no dominant common mechanistic inter-dependency driving therapeutic benefit.

L1CAM promotes ovarian cancer stemness and tumor initiation via FGFR1/SRC/STAT3 signaling.

BACKGROUND: Cancer stem cells (CSC) have been implicated in tumor progression. In ovarian carcinoma (OC), CSC drive tumor formation, dissemination and recurrence, as well as drug resistance, thus contributing to the high death-to-incidence ratio of this disease. However, the molecular basis of such a pathogenic role of ovarian CSC (OCSC) has been elucidated only to a limited extent. In this context, the functional contribution of the L1 cell adhesion molecule (L1CAM) to OC stemness remains elusive. METHODS: The expression of L1CAM was investigated in patient-derived OCSC. The genetic manipulation of L1CAM in OC cells provided gain and loss-of-function models that were then employed in cell biological assays as well as in vivo tumorigenesis experiments to assess the role of L1CAM in OC cell stemness and in OCSC-driven tumor initiation. We applied antibody-mediated neutralization to investigate L1CAM druggability. Biochemical approaches were then combined with functional in vitro assays to study the molecular mechanisms underlying the functional role of L1CAM in OCSC. RESULTS: We report that L1CAM is upregulated in patient-derived OCSC. Functional studies showed that L1CAM promotes several stemness-related properties in OC cells, including sphere formation, tumor initiation and chemoresistance. These activities were repressed by an L1CAM-neutralizing antibody, pointing to L1CAM as a druggable target. Mechanistically, L1CAM interacted with and activated fibroblast growth factor receptor-1 (FGFR1), which in turn induced the SRC-mediated activation of STAT3. The inhibition of STAT3 prevented L1CAM-dependent OC stemness and tumor initiation. CONCLUSIONS: Our study implicate L1CAM in the tumorigenic function of OCSC and point to the L1CAM/FGFR1/SRC/STAT3 signaling pathway as a novel driver of OC stemness. We also provide evidence that targeting this pathway can contribute to OC eradication.

A cell-of-origin epigenetic tracer reveals clinically distinct subtypes of high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) is a major unmet need in oncology. The remaining uncertainty on its originating tissue has hampered the discovery of molecular oncogenic pathways and the development of effective therapies. METHODS: We used an approach based on the retention in tumors of a DNA methylation trace (OriPrint) that distinguishes the two putative tissues of origin of HGSOC, the fimbrial (FI) and ovarian surface epithelia (OSE), to stratify HGSOC by several clustering methods, both linear and non-linear. The identified tumor subtypes (FI-like and OSE-like HGSOC) were investigated at the RNAseq level to stratify an in-house cohort of macrodissected HGSOC FFPE samples to derive overall and disease-free survival and identify specific transcriptional alterations of the two tumor subtypes, both by classical differential expression and weighted correlation network analysis. We translated our strategy to published datasets and verified the co-occurrence of previously described molecular classification of HGSOC. We performed cytokine analysis coupled to immune phenotyping to verify alterations in the immune compartment associated with HGSOC. We identified genes that are both differentially expressed and methylated in the two tumor subtypes, concentrating on PAX8 as a bona fide marker of FI-like HGSOC. RESULTS: We show that: - OriPrint is a robust DNA methylation tracer that exposes the tissue of origin of HGSOC. - The tissue of origin of HGSOC is the main determinant of DNA methylation variance in HGSOC. - The tissue of origin is a prognostic factor for HGSOC patients. - FI-like and OSE-like HGSOC are endowed with specific transcriptional alterations that impact patients' prognosis. - OSE-like tumors present a more invasive and immunomodulatory phenotype, compatible with its worse prognostic impact. - Among genes that are differentially expressed and regulated in FI-like and OSE-like HGSOC, PAX8 is a bona fide marker of FI-like tumors. CONCLUSIONS: Through an integrated approach, our work demonstrates that both FI and OSE are possible origins for human HGSOC, whose derived subtypes are both molecularly and clinically distinct. These results will help define a new roadmap towards rational, subtype-specific therapeutic inroads and improved patients' care.

JAM-A Acts via C/EBP-α to Promote Claudin-5 Expression and Enhance Endothelial Barrier Function.

RATIONALE: Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. OBJECTIVE: We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. METHODS AND RESULTS: Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of ß-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A-C/EBP-α-mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. CONCLUSIONS: We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic Abstract: An graphic abstract is available for this article.

Different Shades of L1CAM in the Pathophysiology of Cancer Stem Cells.

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in several tumor types where it is causally linked to malignancy and therapy resistance, acting also as a poor prognosis factor. Accordingly, several approaches have been developed to interfere with L1CAM function or to deliver cytotoxic agents to L1CAM-expressing tumors. Metastatic dissemination, tumor relapse and drug resistance can be fueled by a subpopulation of neoplastic cells endowed with peculiar biological properties that include self-renewal, efficient DNA repair, drug efflux machineries, quiescence, and immune evasion. These cells, known as cancer stem cells (CSC) or tumor-initiating cells, represent, therefore, an ideal target for tumor eradication. However, the molecular and functional traits of CSC have been unveiled only to a limited extent. In this context, it appears that L1CAM is expressed in the CSC compartment of certain tumors, where it plays a causal role in stemness itself and/or in biological processes intimately associated with CSC (e.g., epithelial-mesenchymal transition (EMT) and chemoresistance). This review summarizes the role of L1CAM in cancer focusing on its functional contribution to CSC pathophysiology. We also discuss the clinical usefulness of therapeutic strategies aimed at targeting L1CAM in the context of anti-CSC treatments.

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis.

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis. N&B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.

Number and brightness analysis reveals that NCAM and FGF2 elicit different assembly and dynamics of FGFR1 in live cells.

Both fibroblast growth factor-2 (FGF2) and neural cell adhesion molecule (NCAM) trigger FGF receptor 1 (FGFR1) signaling; however, they induce remarkably distinct receptor trafficking and cellular responses. The molecular basis of such a dichotomy and the role of distinct types of ligand-receptor interaction remain elusive. Number of molecules and brightness (N&B) analysis revealed that FGF2 and NCAM promote different FGFR1 assembly and dynamics at the plasma membrane. NCAM stimulation elicits long-lasting cycles of short-lived FGFR1 monomers and multimers, a behavior that might reflect a rapid FGFR1 internalization and recycling. FGF2, instead, induces stable dimerization at the dose that stimulates cell proliferation. Reducing the occupancy of FGFR1 in response to low FGF2 doses causes a switch towards cyclically exposed and unstable receptor dimers, consistently with previously reported biphasic response to FGF2 and with the divergent signaling elicited by different ligand concentrations. Similar instability was observed upon altering the endocytic pathway. Thus, FGF2 and NCAM induce differential FGFR1 clustering at the cell surface, which might account for the distinct intracellular fate of the receptor and, hence, for the different signaling cascades and cellular responses.

Platinum sensitivity and DNA repair in a recently established panel of patient-derived ovarian carcinoma xenografts.

A xenobank of patient-derived (PDX) ovarian tumor samples has been established consisting of tumors with different sensitivity to cisplatin (DDP), from very responsive to resistant. As the DNA repair pathway is an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in the nucleotide excision repair, fanconi anemia, homologous recombination, base excision repair, mismatch repair and translesion repair pathways and the methylation patterns of some of these genes. We also investigated the correlation with the response to platinum-based therapy. The mRNA levels of the selected genes were evaluated by Real Time-PCR (RT-PCR) with ad hoc validated primers and gene promoter methylation by pyrosequencing. All the DNA repair genes were variably expressed in all 42 PDX samples analyzed, with no particular histotype-specific pattern of expression. In high-grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. High-grade serous/endometrioid PDXs with TP53 mutations had significantly higher levels of POLQ, FANCD2, RAD51 and POLB than high-grade TP53 wild type PDXs. The mRNA levels of CDK12, PALB2 and XPF inversely associated with the in vivo DDP antitumor activity; higher CDK12 mRNA levels were associated with a higher recurrence rate in ovarian patients with low residual tumor. These data support the important role of CDK12 in the response to a platinum based therapy in ovarian patients.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.

CD73 Regulates Stemness and Epithelial-Mesenchymal Transition in Ovarian Cancer-Initiating Cells.

Cancer-initiating cells (CICs) have been implicated in tumor development and aggressiveness. In ovarian carcinoma (OC), CICs drive tumor formation, dissemination, and recurrence, as well as drug resistance, thus accounting for the high death-to-incidence ratio of this neoplasm. However, the molecular mechanisms that underlie such a pathogenic role of ovarian CICs (OCICs) remain elusive. Here, we have capitalized on primary cells either from OC or from its tissues of origin to obtain the transcriptomic profile associated with OCICs. Among the genes differentially expressed in OCICs, we focused on CD73, which encodes the membrane-associated 5'-ectonucleotidase. The genetic inactivation of CD73 in OC cells revealed that this molecule is causally involved in sphere formation and tumor initiation, thus emerging as a driver of OCIC function. Furthermore, functional inhibition of CD73 via either a chemical compound or a neutralizing antibody reduced sphere formation and tumorigenesis, highlighting the druggability of CD73 in the context of OCIC-directed therapies. The biological function of CD73 in OCICs required its enzymatic activity and involved adenosine signaling. Mechanistically, CD73 promotes the expression of stemness and epithelial-mesenchymal transition-associated genes, implying a regulation of OCIC function at the transcriptional level. CD73, therefore, is involved in OCIC biology and may represent a therapeutic target for innovative treatments aimed at OC eradication.

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

RATIONALE: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. OBJECTIVE: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. METHODS AND RESULTS: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and ß-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/ß-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. CONCLUSIONS: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.