Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

OTX2 duplications: a recurrent cause of oculo-auriculo-vertebral spectrum.

BACKGROUND: Oculo-auriculo-vertebral spectrum (OAVS) is the second most common cause of head and neck malformations in children after orofacial clefts. OAVS is clinically heterogeneous and characterised by a broad range of clinical features including ear anomalies with or without hearing loss, hemifacial microsomia, orofacial clefts, ocular defects and vertebral abnormalities. Various genetic causes were associated with OAVS and copy number variations represent a recurrent cause of OAVS, but the responsible gene often remains elusive. METHODS: We described an international cohort of 17 patients, including 10 probands and 7 affected relatives, presenting with OAVS and carrying a 14q22.3 microduplication detected using chromosomal microarray analysis. For each patient, clinical data were collected using a detailed questionnaire addressed to the referring clinicians. We subsequently studied the effects of OTX2 overexpression in a zebrafish model. RESULTS: We defined a 272 kb minimal common region that only overlaps with the OTX2 gene. Head and face defects with a predominance of ear malformations were present in 100% of patients. The variability in expressivity was significant, ranging from simple chondromas to severe microtia, even between intrafamilial cases. Heterologous overexpression of OTX2 in zebrafish embryos showed significant effects on early development with alterations in craniofacial development. CONCLUSIONS: Our results indicate that proper OTX2 dosage seems to be critical for the normal development of the first and second branchial arches. Overall, we demonstrated that OTX2 genomic duplications are a recurrent cause of OAVS marked by auricular malformations of variable severity.

Reprogramming immune cells activity by furin-like enzymes as emerging strategy for enhanced immunotherapy in cancer.

Immunotherapy is becoming an advanced clinical management for various cancers. Rebuilding of aberrant immune surveillance on cancers has achieved notable progress in the past years by either in vivo or ex vivo engineering of efficient immune cells. Immune cells can be programmed with several strategies that improves their therapeutic influence and specificity. It has become noticeable that effective immunotherapy must consider the complete complexity of the immune cell function. However, today, almost all immune cells can be transiently or stably reprogrammed against various cancer cells. As a consequence, investigations have interrogated strategies to improve the efficacy of cancer immunotherapies by enhancing T-cell infiltration into tumour tissues. Here, we review the emerging role of furin-like enzymes work related to T-cell reprogramming, their tumour infiltration and cytotoxic function.

New Insights into the Reparative Angiogenesis after Myocardial Infarction.

Myocardial infarction (MI) causes massive loss of cardiac myocytes and injury to the coronary microcirculation, overwhelming the limited capacity of cardiac regeneration. Cardiac repair after MI is finely organized by complex series of procedures involving a robust angiogenic response that begins in the peri-infarcted border area of the infarcted heart, concluding with fibroblast proliferation and scar formation. Efficient neovascularization after MI limits hypertrophied myocytes and scar extent by the reduction in collagen deposition and sustains the improvement in cardiac function. Compelling evidence from animal models and classical in vitro angiogenic approaches demonstrate that a plethora of well-orchestrated signaling pathways involving Notch, Wnt, PI3K, and the modulation of intracellular Ca2+ concentration through ion channels, regulate angiogenesis from existing endothelial cells (ECs) and endothelial progenitor cells (EPCs) in the infarcted heart. Moreover, cardiac repair after MI involves cell-to-cell communication by paracrine/autocrine signals, mainly through the delivery of extracellular vesicles hosting pro-angiogenic proteins and non-coding RNAs, as microRNAs (miRNAs). This review highlights some general insights into signaling pathways activated under MI, focusing on the role of Ca2+ influx, Notch activated pathway, and miRNAs in EC activation and angiogenesis after MI.

Apelin: an antithrombotic factor that inhibits platelet function.

Apelin peptide and its receptor APJ are directly implicated in various physiological processes ranging from cardiovascular homeostasis to immune signaling. Here, we show that apelin is a key player in hemostasis with an ability to inhibit thrombin- and collagen-mediated platelet activation. Mice lacking apelin displayed a shorter bleeding time and a prothrombotic profile. Their platelets exhibited increased adhesion and a reduced occlusion time in venules, and displayed a higher aggregation rate after their activation by thrombin compared with wild-type platelets. Consequently, human and mouse platelets express apelin and its receptor APJ. Apelin directly interferes with thrombin-mediated signaling pathways and platelet activation, secretion, and aggregation, but not with ADP and thromboxane A2-mediated pathways. IV apelin administration induced excessive bleeding and prevented thrombosis in mice. Taken together, these findings suggest that apelin and/or APJ agonists could potentially be useful adducts in antiplatelet therapies and may provide a promising perspective for patients who continue to display adverse thrombotic events with current antiplatelet therapies.

Apela promotes blood vessel regeneration and remodeling in zebrafish.

In contrast to adult mammals, zebrafish display a high capacity to heal injuries and repair damage to various organs. One of the earliest responses to injury in adult zebrafish is revascularization, followed by tissue morphogenesis. Tissue vascularization entails the formation of a blood vessel plexus that remodels into arteries and veins. The mechanisms that coordinate these processes during vessel regeneration are poorly understood. Hence, investigating and identifying the factors that promote revascularization and vessel remodeling have great therapeutic potential. Here, we revealed that fin vessel remodeling critically depends on Apela peptide. We found that Apela selectively accumulated in newly formed zebrafish fin tissue and vessels. The temporal expression of Apela, Apln, and their receptor Aplnr is different during the regenerative process. While morpholino-mediated knockdown of Apela (Mo-Apela) prevented vessel remodeling, exogenous Apela peptide mediated plexus repression and the development of arteries in regenerated fins. In contrast, Apela enhanced subintestinal venous plexus formation (SIVP). The use of sunitinib completely inhibited vascular plexus formation in zebrafish, which was not prevented by exogenous application. Furthermore, Apela regulates the expression of vessel remolding-related genes including VWF, IGFPB3, ESM1, VEGFR2, Apln, and Aplnr, thereby linking Apela to the vascular plexus factor network as generated by the STRING online database. Together, our findings reveal a new role for Apela in vessel regeneration and remodeling in fin zebrafish and provide a framework for further understanding the cellular and molecular mechanisms involved in vessel regeneration.

Sulconazole inhibits PD-1 expression in immune cells and cancer cells malignant phenotype through NF-κB and calcium activity repression.

The overexpression of the immunoinhibitory receptor programmed death-1 (PD1) on T-cells is involved in immune evasion in cancer. The use of anti-PD-1/PDL-1 strategy has deeply changed the therapies of cancers and patient survival. However, their efficacy diverges greatly along with tumor type and patient populations. Thereby, novel treatments are needed to interfere with the anti-tumoral immune responses and propose an adjunct therapy. In the current study, we found that the antifungal drug Sulconazole (SCZ) inhibits PD-1 expression on activated PBMCs and T cells at the RNA and protein levels. SCZ repressed NF-κB and calcium signaling, both, involved in the induction of PD-1. Further analysis revealed cancer cells treatment with SCZ inhibited their proliferation, and migration and ability to mediate tumor growth in zebrafish embryos. SCZ found also to inhibit calcium mobilization in cancer cells. These results suggest the SCZ therapeutic potential used alone or as adjunct strategy to prevent T-cell exhaustion and promotes cancer cell malignant phenotype repression in order to improve tumor eradication.

Identification and functional analysis of endothelial tip cell-enriched genes.

Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4(+/-) mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4(+/-) and wild-type mice, we identified 3 clusters of tip cell-enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior.

Targeting PCSK9 in Liver Cancer Cells Triggers Metabolic Exhaustion and Cell Death by Ferroptosis.

Deregulated lipid metabolism is a common feature of liver cancers needed to sustain tumor cell growth and survival. We aim at taking advantage of this vulnerability and rewiring the oncogenic metabolic hub by targeting the key metabolic player pro-protein convertase subtilisin/kexin type 9 (PCSK9). We assessed the effect of PCSK9 inhibition using the three hepatoma cell lines Huh6, Huh7 and HepG2 and validated the results using the zebrafish in vivo model. PCSK9 deficiency led to strong inhibition of cell proliferation in all cell lines. At the lipid metabolic level, PCSK9 inhibition was translated by an increase in intracellular neutral lipids, phospholipids and polyunsaturated fatty acids as well as a higher accumulation of lipid hydroperoxide. Molecular signaling analysis involved the disruption of the sequestome 1/Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (p62/Keap1/Nrf2) antioxidative axis, leading to ferroptosis, for which morphological features were confirmed by electron and confocal microscopies. The anti-tumoral effects of PCSK9 deficiency were validated using xenograft experiments in zebrafish. The inhibition of PCSK9 was effective in disrupting the oncometabolic process, inducing metabolic exhaustion and enhancing the vulnerability of cancer cells to iron-triggered lipid peroxidation. We provide strong evidence supporting the drug repositioning of anti-PCSK9 approaches to treat liver cancers.

Selective inhibition of proprotein convertases represses the metastatic potential of human colorectal tumor cells.

The proprotein convertases (PCs) are implicated in the activation of various precursor proteins that play an important role in tumor cell metastasis. Here, we report their involvement in the regulation of the metastatic potential of colorectal tumor cells. PC function in the human and murine colon carcinoma cell lines HT-29 and CT-26, respectively, was inhibited using siRNA targeting the PCs furin, PACE4, PC5, and PC7 or by overexpression of the general PC inhibitor alpha1-antitrypsin Portland (alpha1-PDX). We found that overexpression of alpha1-PDX and knockdown of furin expression inhibited processing of IGF-1 receptor and its subsequent activation by IGF-1 to induce IRS-1 and Akt phosphorylation, all important in colon carcinoma metastasis. These data suggest that the PC furin is a major IGF-1 receptor convertase. Expression of alpha1-PDX reduced the production of TNF-alpha and IL-1alpha by human colon carcinoma cells, and incubation of murine liver endothelial cells with conditioned media derived from these cells failed to induce tumor cell adhesion to activated murine endothelial cells, a critical step in metastatic invasion. Furthermore, colon carcinoma cells in which PC activity was inhibited by overexpression of alpha1-PDX when injected into the portal vein of mice showed a significantly reduced ability to form liver metastases. This suggests that inhibition of PCs is a potentially promising strategy for the prevention of colorectal liver metastasis.

Elabela/toddler: New peptide with a promising future in cancer diagnostic and therapy.

Elabela/toddler is the second endogenous ligand recently identified after Apelin, that binds to the G protein-coupled receptor APJ. Elabela is a 54-amino acid peptide initially identified in fish and human genomes and classified as noncoding. This precursor can be cleaved to shorter sequences (32, 21, and 11 amino acids), which bind and activate APJ, and can be blocked by APJ antagonists. Contrary to Apelin and APJ, widely distributed in organs and tissues, Elabela expression is more restricted, and different studies have revealed the potential role of Elabela in cancers. This review summarizes the current studies focusing on the role of Elabela in different cancers.

Furin Prodomain ppFurin Enhances Ca2+ Entry Through Orai and TRPC6 Channels' Activation in Breast Cancer Cells.

The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we showed that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced store-operated calcium entry (SOCE) and reduced the cell malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments revealed that the ppFurin-expressing cells are unable to adequately process the proprotein convertase (PC) substrates vascular endothelial growth factor C (proVEGF-C) and insulin-like growth factor-1 receptor (proIGF-1R). Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 and BT20 cells significantly enhanced SOCE and induced constitutive Ca2+ entry. The enhanced SOCE is impaired by inhibition of Orai channels while the constitutive Ca2+ entry is attenuated by silencing or inhibition of TRPC6 or inhibition of Orai channels. Analysis of TRPC6 activation revealed its upregulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. In addition, while ppFurin had no effect on MDA-MB-435 cell viability, in MDA-MB-231 cells ppFurin expression reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide and similar results were observed in BT20 cells. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization, leading to breast cancer cells' malignant phenotype repression and reduction of their resistance to treatments.

Hypoxia-induced apelin expression regulates endothelial cell proliferation and regenerative angiogenesis.

Apelin has been identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ. This peptide exerts a variety of cardiovascular effects and particularly acts as an activator of angiogenesis. Importantly, hypoxia has been reported to regulate apelin expression but the molecular mechanism underlying hypoxia-induced apelin expression and the relationship with the physiological response of the apelin/APJ system are still not established. Here, we demonstrate that apelin expression is induced by hypoxia in cultured endothelial and vascular smooth muscle cells as well as in lung from mice exposed to acute hypoxia. Transient transfection experiments show that hypoxia-inducible transcriptional activation of apelin requires an intact hypoxia-responsive element (+813/+826) located within the first intron of the human apelin gene. Chromatin immunoprecipitation assay reveals that hypoxia-inducible factor-1alpha binds to the endogenous hypoxia-responsive element site of the apelin gene. Moreover, overexpression of hypoxia-inducible factor-1alpha increases the transcriptional activity of a reporter construct containing this hypoxia-responsive element, whereas small interfering RNA-mediated hypoxia-inducible factor-1alpha knockdown abolishes hypoxia-induced apelin expression. Finally, microinterfering RNA-mediated apelin or APJ receptor knockdown inhibits both hypoxia-induced endothelial cell proliferation in vitro and hypoxia-induced vessel regeneration in the caudal fin regeneration of Fli-1 transgenic zebrafish. The hypoxia-induced apelin expression may, thus, provide a new mechanism involved in adaptive physiological and pathophysiological response of vascular cells to low oxygen level.

Proprotein convertases: Key players in inflammation-related malignancies and metastasis.

Many cancers occur from locations of inflammation due to chronic irritation and/or infection. Tumor microenvironment contains various different inflammatory cells and mediators that orchestrate diverse neoplastic processes, including proliferation, survival, adhesion and migration. In parallel, tumor cells have adapted some of the signaling molecules used by inflammatory cells, such as selectins and chemokines as well as their receptors for invasion, extravasation and subsequently metastasis. Expression and/or activation of the majority of these molecules is mediated by the proprotein convertases (PCs); proteases expressed by both tumor cells and inflammatory cells. This review analyzes the potential role of these enzymatic system in inflammation-associated cancer impacting on the malignant and metastatic potential of cancer cells, describing the possible use of PCs as a new anti-inflammatory therapeutic approach to tumor progression and metastasis.

Patients Lung Derived Tumoroids (PLDTs) to model therapeutic response.

Preclinical lung cancer models are essential for a basic understanding of lung cancer biology and its translation into efficient treatment options for affected patients. Lung cancer cell lines and xenografts derived directly from human lung tumors have proven highly valuable in fundamental oncology research and anticancer drug discovery. Both models inherently comprise advantages and caveats that have to be accounted for. Recently, we have enabled reliable in vitro culture techniques from lung cancer biopsies as Patients Lung Derived Tumoroids (PLDTs). This breakthrough provides the possibility of high-throughput drug screening covering the spectrum of lung cancer phenotypes seen clinically. We have adapted and optimized our in vitro three-dimensional model as a preclinical lung cancer model to recapitulate the tumor microenvironment (TME) using matrix reconstitution. Hence, we developed directly PLDTs to screen for chemotherapeutics and radiation treatment. This original model will enable precision medicine to become a reality, allowing a given patient sample to be screened for effective ex vivo therapeutics, aiming at tailoring of treatments specific to that individual. Hence, this tool can enhance clinical outcomes and avoid morbidity due to ineffective therapies.

The proprotein convertase furin is a pro-oncogenic driver in KRAS and BRAF driven colorectal cancer.

Mutations in KRAS and/or BRAF that activate the ERK kinase are frequently found in colorectal cancer (CRC) and drive resistance to targeted therapies. Therefore, the identification of therapeutic targets that affect multiple signaling pathways simultaneously is crucial for improving the treatment of patients with KRAS or BRAF mutations. The proprotein convertase furin activates several oncogenic protein precursors involved in the ERK-MAPK pathway by endoproteolytic cleavage. Here we show that genetic inactivation of furin suppresses tumorigenic growth, proliferation, and migration in KRAS or BRAF mutant CRC cell lines but not in wild-type KRAS and BRAF cells. In a mouse xenograft model, these KRAS or BRAF mutant cells lacking furin displayed reduced growth and angiogenesis, and increased apoptosis. Mechanistically, furin inactivation prevents the processing of various protein pecursors including proIGF1R, proIR, proc-MET, proTGF-ß1 and NOTCH1 leading to potent and durable ERK-MAPK pathway suppression in KRAS or BRAF mutant cells. Furthermore, we identified genes involved in activating the ERK-MAPK pathway, such as PTGS2, which are downregulated in the KRAS or BRAF mutant cells after furin inactivation but upregulated in wild-type KRAS and BRAF cells. Analysis of human colorectal tumor samples reveals a positive correlation between enhanced furin expression and KRAS or BRAF expression. These results indicate that furin plays an important role in KRAS or BRAF-associated ERK-MAPK pathway activation and tumorigenesis, providing a potential target for personalized treatment.

ELA/APELA precursor cleaved by furin displays tumor suppressor function in renal cell carcinoma through mTORC1 activation.

Apelin is a well-established mediator of survival and mitogenic signaling through the apelin receptor (Aplnr) and has been implicated in various cancers; however, little is known regarding Elabela (ELA/APELA) signaling, also mediated by Aplnr, and its role and the role of the conversion of its precursor proELA into mature ELA in cancer are unknown. Here, we identified a function of mTORC1 signaling as an essential mediator of ELA that repressed kidney tumor cell growth, migration, and survival. Moreover, sunitinib and ELA showed a synergistic effect in repressing tumor growth and angiogenesis in mice. The use of site-directed mutagenesis and pharmacological experiments provided evidence that the alteration of the cleavage site of proELA by furin induced improved ELA antitumorigenic activity. Finally, a cohort of tumors and public data sets revealed that ELA was only repressed in the main human kidney cancer subtypes, namely clear cell, papillary, and chromophobe renal cell carcinoma. Aplnr was expressed by various kidney cells, whereas ELA was generally expressed by epithelial cells. Collectively, these results showed the tumor-suppressive role of mTORC1 signaling mediated by ELA and established the potential use of ELA or derivatives in kidney cancer treatment.

Opposing function of the proprotein convertases furin and PACE4 on breast cancer cells' malignant phenotypes: role of tissue inhibitors of metalloproteinase-1.

Proteolytic cleavage of various cancer-related substrates by the proprotein convertases (PC) was reported to be important in the processes of neoplasia. These enzymes are inhibited by their naturally occurring inhibitors, the prosegments (ppPC), and by the engineered general PC inhibitor, the serpin variant alpha1-PDX. In the present study, we sought to compare the effect of these PC inhibitors on malignant phenotypes of breast cancer cells. Overexpression in a stable manner of alpha1-PDX and the prosegment ppPACE4 in MDA-MB-231 breast cancer cells resulted in increased matrix metalloproteinase (MMP)-9 (but not MMP-2) activity and a reduced secretion of tissue inhibitor of metalloproteinase 1 (TIMP-1). This was associated with significant enhancement in cell motility, migration, and invasion of collagen in vitro. In contrast, ppFurin expression in these cells decreased MMP-9 activity and diminished these biological functions, but had no significant effect on TIMP-1 secretion. Taken together, these data showed the specific and opposing roles of Furin and PACE4 in the regulation of MMP-9/TIMP-1-mediated cell motility and invasion.

Inactivation of Proprotein Convertases in T Cells Inhibits PD-1 Expression and Creates a Favorable Immune Microenvironment in Colorectal Cancer.

Proprotein convertases (PC) activate precursor proteins that play crucial roles in various cancers. In this study, we investigated whether PC enzyme activity is required for expression of the checkpoint protein programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTL) in colon cancer. Although altered expression of the PC secretory pathway was observed in human colon cancers, only furin showed highly diffuse expression throughout the tumors. Inhibition of PCs in T cells using the general protein-based inhibitor α1-PDX or the pharmacologic inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethylketone repressed PD-1 and exhausted CTLs via induction of T-cell proliferation and apoptosis inhibition, which improved CTL efficacy against microsatellite instable and microsatellite stable colon cancer cells. In vivo, inhibition of PCs enhanced CTL infiltration in colorectal tumors and increased tumor clearance in syngeneic mice compared with immunodeficient mice. Inhibition of PCs repressed PD-1 expression by blocking proteolytic maturation of the Notch precursor, inhibiting calcium/NFAT and NF-κB signaling, and enhancing ERK activation. These findings define a key role for PCs in regulating PD-1 expression and suggest targeting PCs as an adjunct approach to colorectal tumor immunotherapy. SIGNIFICANCE: Protein convertase enzymatic activity is required for PD-1 expression on T cells, and inhibition of protein convertase improves T-cell targeting of microsatellite instable and stable colorectal cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/5008/F1.large.jpg.

Inhibitory feature of the proprotein convertases prosegments.

The proprotein convertases (PCs) are serine proteases involved in various physiological processes and their overactivity or inactivity has been linked to different disorders. PCs are responsible for the proteolytic processing of various polypeptide precursors. Here, we discuss the effect of their N-terminal prosegments on various PC substrates processing and functions.